Lyophilized treosulfan

Abstract

A lyophilized product of treosulfan is described, which has favorable characteristics in terms of short reconstitution time and high purity and stability, and is particularly useful in the treatment of cancer and for conditioning therapy before bone marrow or blood stem cell transplantation. The lyophilized product according to the present invention is characterized by containing crystalline form B of treosulfan, which exhibits an X-ray powder diffraction pattern with characteristic peaks at 20.87 and 23.47±0.20 degrees 2θ. Preferably, crystalline form B exhibits an X-ray powder diffraction pattern with 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. More preferably, crystalline form B exhibits an X-ray powder diffraction pattern essentially as shown in FIG. 1.

Description

[Technical Field]

[0001] The present invention relates to a lyophilisate containing a new crystalline form B of treosulfan, which has very good characteristics for use as a pharmaceutical composition, in particular it can be quickly reconstituted to form a ready-to-use solution and exhibits high stability and purity. Treosulfan, chemically named (2S,3S)-(-)1,4-di(mesyloxy)-2,3-butanediol or L-threitol-1,4-di(methanesulfonate), has the following chemical formula: [ka] [Background technology]

[0002] The chemical synthesis of treosulfan is disclosed in German Patent Nos. 1 188 583 and 1 193 938 and is carried out, for example, by reacting L-1,4-dibromobutane-2,3-diol and the silver salt of methanesulfonic acid.

[0003] Treosulfan is a dihydroxy derivative of busulfan and acts as an antitumor agent due to its ability to alkylate DNA. It is used by itself or in combination with additional chemotherapy drugs, such as melphalan and dacarbazine, for the treatment of ovarian cancer (Baynes et al., Blood 96(11): 170a, Abstr. No. 731, 2000). For the treatment of ovarian cancer, monotherapy with treosulfan requires patients to undergo chemotherapy for 1 m of body surface area. 2 The combination therapy with treosulfan and cisplatin involves administering a dose of 8 g per minute. 2 This involves administering treosulfan in an amount of 5 g per dose.

[0004] Treosulfan has also been used in the treatment of advanced unresectable non-small cell lung cancer (Pawel et al., Onkologie 21:316-319; 1998).

[0005] Furthermore, European Patent Application Publication No. 1 227 808 discloses the use of treosulfan in conditioning therapy prior to bone marrow or hematopoietic stem cell transplantation in patients. In such conditioning therapy, administration of treosulfan can be effectively combined with the administration of additional drugs, such as cyclophosphamide, carboplatin, thiotepa, melphalan, fludarabine, immunosuppressive antibodies, or body irradiation. Compared to the use of busulfan, serious side effects can be largely or completely avoided. Even high dosages of treosulfan can be used without causing significant liver, lung, kidney, or CNS toxicity. The conditioning phase is administered to patients with a body surface area of ​​1 m2 prior to allogeneic transplantation of bone marrow or hematopoietic stem cells. 2 A total dose of at least 20 g of treosulfan per day for a period of 2 to 7 days.

[0006] Treosulfan is commercially available as capsules for oral use and as a sterile powder consisting of treosulfan for preparing a solution for infusion. The solution is administered intravenously within approximately 15-30 minutes. The treosulfan in these products is a crystalline form that exhibits a powder X-ray diffraction pattern (XRPD) with 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 ± 0.2 2θ degrees. This crystalline form, designated Form A below, has an XRPD pattern shown in Figure 2.

[0007] To prepare a solution for infusion, commercially available sterile powder is dissolved in water, for example, to a concentration of 50 mg / ml, and the resulting solution is then diluted with, for example, an isotonic NaCl solution. However, the water used as the solvent must be warmed to 30°C for the reconstitution step. Furthermore, the powder must be completely removed from the vial walls. This step is important to avoid the formation of powder particles adhering to the walls. Such adhered particles of treosulfan Form A are difficult to dissolve, and they prolong complete dissolution. The entire process of preparing a solution for infusion from sterile powder, including vial preparation, necessary warming of the water, and complete dissolution of the powder, takes approximately 10 minutes. Furthermore, the use of a warm solvent increases the risk of undesired degradation. International Application WO 2015 / 107534 refers to two allegedly novel and distinct polymorphs of treosulfan, designated Form I and Form II. The document lacks any suggestion as to how Form II can be obtained, and thus lacks an enabling disclosure. The process for preparing Form I is described only very generally, allegedly involving recrystallization from an organic solvent or mixture thereof, with reference to several preferred organic solvents. No specific process for preparing Form I is disclosed. The X-ray powder diffraction pattern provided for Form I closely resembles that of crystalline Form A of the commercial product, depicted in Figure 2 below, suggesting that these forms are indeed identical. Finally, International Application WO 2015 / 107534 also describes a lyophilized formulation allegedly containing, typically, Form I of treosulfan. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] German Patent No. 1188583 [Patent Document 2] German Patent No. 1193938 [Patent Document 3] European Patent Application Publication No. 1227808 [Patent Document 4] International Publication No. 2015 / 107534 [Non-patent literature]

[0009] [Non-Patent Document 1] Baynes et al., Blood 96(11): 170a, Abstr. No. 731, 2000 [Non-patent document 2] Pawel et al., Onkologie 21:316-319; 1998 Summary of the Invention [Means for solving the problem]

[0010] However, known lyophilisates have several drawbacks. In particular, known lyophilisates require a long time for their reconstitution, and their content of methanesulfonic acid and water, especially after storage, is undesirably high, so their purity and stability are unsatisfactory. Furthermore, the seemingly optimized lyophilisation processes used result in samples with highly variable properties, thus lacking the desired reproducibility, which is very problematic considering that these lyophilisates are intended to be used as pharmaceutical compositions. Methanesulfonic acid (MSA) is a decomposition product of treosulfan, as shown by the following reaction scheme: [ka]

[0011] Its presence therefore indicates the decomposition of treosulfan. Due to its strong acidity, it accelerates the hydrolysis of the ester group of treosulfan, thus enhancing the decomposition process. For this reason, the amount of methanesulfonic acid should be kept as low as possible.

[0012] It is therefore an object of the present invention to avoid the drawbacks of known products containing treosulfan.

[0013] This object is achieved by the lyophilized treosulfan according to claims 1-10.

[0014] The present invention also relates to processes for preparing a lyophilized product of treosulfan according to claims 11 to 21 and to lyophilized products of treosulfan for use in medicine according to claims 22 to 24. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1. [Figure 2] Figure 2. [Figure 3] Figure 3. DETAILED DESCRIPTION OF THE INVENTION

[0016] The lyophilisate according to the invention is characterised by containing crystalline form B of treosulfan which exhibits an X-ray powder diffraction pattern with characteristic peaks at 20.87 and 23.47±0.20 degrees 2θ. Preferably, crystalline form B exhibits an X-ray powder diffraction pattern with 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.

[0017] More preferably, crystalline form B exhibits an X-ray powder diffraction pattern essentially as shown in FIG.

[0018] It is further preferred that crystalline form B exhibits an X-ray powder diffraction pattern that is free of peaks in at least one, preferably all, of the following regions a to f, expressed in degrees 2θ: [Table 1]

[0019] Crystalline form B is also characterized by the space group and parameters a, b, c, α, β, γ of the unit cell, as well as the volume of the unit cell, preferably obtained by single crystal X-ray diffraction (SCXRD) analysis, the structural data of which, together with further information particularly regarding the quality of the fit compared to that of the commercially available form A, are given in the table below. [Table 2] a, b and c = unit cell edge lengths α, β and γ = angles between the edges of the unit cell V = volume of unit cell Z / Z' = number of molecules in the unit cell R1 and wR2 = confidence values T = temperature at which the analysis was performed

[0020] As can be seen from these data, form B has two molecules per unit cell (space group P21) and 561.5 Å 3 whereas form A has four molecules per unit cell (space group P212121) and a volume of 1127.22 Å. 3 It has a volume of

[0021] The lyophilisate according to the invention in particular comprises at least 96% by weight, preferably at least 97% by weight, more preferably at least 98% by weight, even more preferably at least 99% by weight of crystalline form B, relative to the combined amount of crystalline form B and crystalline form A.

[0022] Thus, the lyophilisate according to the invention contains a very small amount of the conventional crystalline form A and a very large amount of crystalline form B. A high polymorphic purity is particularly advantageous for the use of the lyophilisate according to the invention as a pharmaceutical composition.

[0023] In a further preferred embodiment, the lyophilisate according to the invention 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, even more preferably at least 95% by weight of crystalline form B, relative to the amount of lyophilisate.

[0024] In yet another preferred embodiment, the lyophilisate according to the invention comprises less than 20% by weight, in particular less than 15% by weight, preferably less than 10% by weight, more preferably less than 5% by weight of amorphous phase relative to the amount of lyophilisate.

[0025] The small amount of amorphous phase avoids several significant drawbacks associated with this phase. First, the amorphous phase tends to lead to uncontrolled crystallization. Furthermore, it decomposes more quickly, has a higher residual water content after drying, exhibits poor flowability and wettability, and is more easily electrostatically charged. All of these properties are undesirable for lyophilized products used for pharmaceutical purposes.

[0026] The lyophilizate according to the present invention surprisingly exhibits a combination of advantageous properties that are assumed to be caused by the high amount of treosulfan crystalline form B. In particular, it requires only a very short time for complete dissolution in the media typically used for reconstitution to obtain a ready-to-use injection or infusion solution. Isotonic saline and water for injection are typically used as such media. Other pharmaceutically acceptable solutions, such as Ringer's lactate solution or phosphate buffer, are also possible for reconstitution. The very short reconstitution time is highly advantageous, as it allows medical personnel to prepare a fresh, ready-to-use solution immediately before intended administration to a patient, without having to wait a long time for complete dissolution. Likewise, such a short reconstitution time reduces the risk of undesired degradation reactions of treosulfan.

[0027] Furthermore, the lyophilisate according to the invention also has a high purity and stability, as reflected by a very high content of the active ingredient treosulfan and a very low content of the degradation product methanesulfonic acid.

[0028] In a preferred embodiment, the lyophilisate according to the invention comprises at least 95% by weight, in particular at least 95% by weight, preferably at least 98% by weight, more preferably at least 99% by weight of treosulfan.

[0029] Furthermore, the lyophilisates according to the invention have only a low methanesulfonic acid content, in particular less than 0.2% by weight, preferably less than 0.1% by weight, more preferably less than 0.05% by weight of methanesulfonic acid. The particularly low amount of methanesulfonic acid is one reasonable explanation for the high storage stability of the lyophilisates according to the invention, since this acid accelerates the hydrolysis of the ester group of treosulfan and thus promotes its degradation.

[0030] The lyophilisate contains in particular less than 0.2% by weight, preferably less than 0.1% by weight, more preferably less than 0.05% by weight of methanesulfonic acid, even after storage for 3 months at 40° C. and 75% relative humidity, which is an indication of the excellent storage stability of the lyophilisate according to the invention and makes it highly suitable for use as a pharmaceutical composition or component thereof.

[0031] A further advantage of the lyophilisate according to the invention is that it can be reconstituted using a solvent having a temperature of about 20° C., thus eliminating the need for preheated solvents. Furthermore, the tedious preparation of removing sticky agglomerates of commercial Form A from the vial wall prior to reconstitution is also unnecessary.

[0032] Furthermore, the lyophilisates according to the invention have only a low water content, in particular containing water in an amount of less than 1% by weight, preferably less than 0.5% by weight, more preferably less than 0.1% by weight, as determined by Karl Fischer titration.

[0033] The present invention also relates to a process for preparing a lyophilisate, which comprises the step of lyophilising an aqueous solution containing treosulfan.

[0034] The aqueous solution to be subjected to lyophilization is also referred to below as the "pre-lyophilization solution".

[0035] The aqueous solution preferably comprises water as solvent or a mixture of water and at least one organic solvent, in particular acetic acid.

[0036] The amount of water in the solvent is particularly 80 to 100% by weight, preferably 90 to 100% by weight The amount of acetic acid in the solvent is particularly 1 to 20% by weight, preferably 2 to 10% by weight.

[0037] Even when using a pre-lyophilization solution containing acetic acid, the lyophilizates obtained according to the invention surprisingly contain only very small amounts of acetic acid, in particular less than 1.0% by weight, preferably less than 0.5% by weight, more preferably less than 0.2% by weight.

[0038] The aqueous pre-lyophilization solution usually contains treosulfan at a concentration of 50 to 150 mg / g, particularly 50 to 100 mg / g, more preferably 50 mg / g to 80 mg / g.

[0039] The pre-lyophilization solution may contain additives such as solubilizers, e.g., polysorbates, cyclodextrins, sodium dodecyl sulfate, poloxamers, etc.; chelating agents, e.g., sodium EDTA, DTPA, cartelidol, etc.; antioxidants, e.g., butylated hydroxytoluene, butylated hydroxyanisole, methionine, glutathione, sodium metabisulfite, alpha-tocopherol, sodium thioglycolate, cysteine, ascorbic acid, etc.; pH adjusters and buffers, e.g., sodium hydroxide, hydrochloric acid, citric acid, acetic acid, etc. The composition may also contain: sodium citrate, arginine, aspartic acid, sodium bicarbonate, sodium citrate, disodium citrate, trisodium citrate, maleic acid, sulfuric acid, hydrogen phosphate, etc.; bulking agents, for example, amino acids such as alanine and arginine; sugar derivatives, for example, sucrose, glucose, mannitol, trehalose, mannose, etc.; or polymers, for example, polyethylene glycol, gelatin, dextran, etc.; stabilizers and tonicity adjusters, for example, sodium chloride, magnesium chloride, sodium sulfate, etc.

[0040] Before the aqueous solution is subjected to the freeze-drying process, it is usually filtered using a conventional filter, for example a 0.22 μm filter, in order to obtain a sterile solution.

[0041] Freeze-drying of the pre-lyophilized solution is typically carried out using a freeze-dryer commonly used for pharmaceutical purposes. Generally, the solution is filled into a suitable container, such as a vial, and the container is placed in a conventional freeze-dryer with a coolable and heatable surface, which can expose the solution to various temperatures during the freeze-drying process. To achieve drying, the solution is typically frozen and exposed to reduced atmospheric pressure. This results in a large amount of solvent sublimation from the frozen solution, which precipitates, for example, on the cooling zone of the freeze-dryer provided for this purpose. This is typically followed by secondary drying at a higher temperature. After freeze-drying is complete, the resulting freeze-dried product is typically brought to room temperature, and the container containing the freeze-dried product is sealed under sterile conditions.

[0042] In a preferred embodiment, the process according to the invention 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 3 K / min or less; (c) drying the frozen solution obtained in step (b) to obtain the lyophilisate; Includes.

[0043] Since higher cooling rates than those used by conventional processes require the use of very sophisticated equipment, it is surprising and highly advantageous that the process according to the invention allows the use of such rather low cooling rates, and therefore the process according to the invention is very economical.

[0044] Furthermore, it is preferred that the cooling rate is at most 2 K / min, preferably at most 1.5 K / min, more preferably at most 1.3 K / min. In an alternative embodiment, the cooling rate in step (b) is in particular between 0.05 and 1.5, preferably between 0.1 and 1.3 K / min.

[0045] The first temperature in the process according to the invention is in particular between 15°C and 95°C, preferably between 20°C and 50°C, more preferably between 25°C and 35°C.

[0046] The refrigeration temperature used in the process is in particular -40°C or lower, preferably between -60°C and -40°C, more preferably between -50°C and -40°C.

[0047] The frozen solution is maintained at the frozen temperature, in particular for at least 1 hour, preferably for 1 to 10 hours, more preferably for 2 to 8 hours.

[0048] In a further preferred embodiment of the process, the drying in step (c) comprises primary drying 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 said frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar, more preferably 0.3 to 0.5 mbar.

[0049] In an alternative further preferred embodiment of the process, the drying in step (c) comprises primary drying carried out by subjecting the frozen solution to a temperature of 0°C or higher, preferably a temperature between 0°C and 60°C, more preferably a temperature between 20°C and 60°C, even more preferably a temperature between 30°C and 50°C, and subjecting said frozen solution to a pressure between 0.03 and 1.0 mbar, preferably between 0.1 and 0.6 mbar, more preferably between 0.3 and 0.5 mbar.

[0050] Primary drying is preferably carried out for at least 5 hours, in particular for at least 10 hours.

[0051] It is also preferred that after primary drying, secondary drying is carried out by subjecting the product of primary drying to a temperature of at least 30°C, preferably 30-50°C, and by subjecting the product of primary drying to a pressure of 0.03-1.0 mbar, preferably 0.1-0.6 mbar, more preferably 0.3-0.5 mbar.

[0052] The secondary drying is preferably carried out for at least 2 hours, in particular for at least 4 hours.

[0053] The process according to the invention makes it possible to prepare lyophilisates with excellent properties in a highly reproducible manner, which is a substantial advantage compared to conventional processes which give products with substantially different properties.

[0054] The lyophilisates according to the invention also prove to be particularly useful in medicine. The invention therefore also relates to the lyophilisates according to the invention for use as a medicament. In a further embodiment, the invention also relates to the lyophilisates according to the invention for use in the treatment of cancer, in particular ovarian cancer. In yet another embodiment, the invention also relates to the lyophilisates according to the invention for use in conditioning therapy before transplantation of bone marrow or blood stem cells.

[0055] In a further aspect, the present invention also relates to the use of the lyophilisate according to the invention for the treatment of cancer or for conditioning therapy before bone marrow or blood stem cell transplantation.

[0056] In yet another aspect, the present invention also relates to a method for treating a patient suffering from cancer or for pre-treating a patient prior to a bone marrow or blood stem cell transplant, comprising administering to the patient a solution made from a lyophilisate according to the invention.

[0057] The invention is explained in more detail below with reference to non-limiting examples which also include methods which are particularly suitable for determining the above-mentioned properties of the lyophilisate and crystalline form B according to the invention and treosulfan. [Example]

[0058] Methods and Apparatus Below, the methods used to obtain the X-ray powder diffraction (XRPD) patterns, for the investigation by single crystal X-ray diffraction (SCXRD), for determining the amount of crystalline form B and crystalline form A, and for determining the amount of amorphous phase, as well as for determining the amounts of treosulfan, acetic acid, methanesulfonic acid and water are given.

[0059] Additionally, the general procedures for preparing glass vials and for determining reconstitution behavior, as well as the equipment used for lyophilization, are provided below.

[0060] General Procedure - Glass Vial Preparation Glass vials for lyophilization were rinsed with purified water before use and depyrogenated for 2 hours at 300°C. Lyophilization stoppers were autoclaved (121°C, 20 minutes, 2 bar) and dried at 110°C for 7 hours.

[0061] freeze dryer The freeze-drying was carried out using a 0.4 m 2 The freeze dryer was run in a freeze dryer GT2 (manufacturer: Hof Sonderanlagenbau, Lohra, Germany) with a shelf area of ​​1000 sq m and an 8 kg ice condenser capacity.

[0062] X-ray powder diffraction (XRPD) Each sample was carefully hand-ground with a pestle in a mortar and then introduced into a standard glass capillary tube (Φ = 0.7 mm). X-ray powder diffraction patterns were 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 while rotating the sample. Data were collected over a 2θ range of 3 to 50 degrees. The tube voltage and current were set to 40 kV and 40 mA, respectively.

[0063] Single Crystal X-ray Diffraction (SCXRD) 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).

[0064] Determination of the amount of Form B and Form A by XRPD and Rietveld analysis To determine the amounts of treosulfan crystalline forms B and A, each sample was carefully hand-ground with a pestle in a mortar and then introduced into a standard glass capillary tube (Φ = 0.7 mm). X-ray powder diffraction patterns were recorded at room temperature using a Bruker D8 Advance Diffractometer (Cu-Kα = 1.54059 Å, Johansson primary beam monochromator, position-sensitive detector) in transmission mode while rotating the sample. Data were collected over a 4-h period in the 2θ range of 4 to 50 degrees. The tube voltage and current were set at 40 kV and 40 mA, respectively. The obtained data were subjected to quantitative Rietveld analysis using TOPAS software.

[0065] Determination of the amount of amorphous phase by XRPD and Rietveld analysis with an internal standard To determine the amount of amorphous phase, each sample was mixed with 25 wt% CaF2 (Aldrich Chemistry, Lot# MKBP1959V, anhydrous calcium fluoride, 99.99%) as an internal standard. After careful manual grinding with a pestle in a mortar, the mixture was introduced into a standard glass capillary tube (Φ = 1.0 mm). X-ray powder diffraction patterns were 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 while rotating the sample. Data were collected over a 12-h period in the 2θ range of 4–50 degrees. The tube voltage and current were set at 30 kV and 30 mA, respectively. The obtained data were subjected to quantitative Rietveld analysis using TOPAS software.

[0066] Forms A and B were the only crystalline phases that could be identified.

[0067] Determination of the amount of treosulfan by RP-HPLC The amount of treosulfan in each sample was determined using reverse phase high pressure liquid chromatography (RP-HPLC) as follows: [Table 3]

[0068] Determination of the amount of methanesulfonic acid by HILIC The amount of methanesulfonic acid (MSA) was determined using hydrophilic interaction liquid chromatography (HILIC) as shown below: [Table 4]

[0069] Determination of residual acetic acid content by headspace gas chromatography (HS-GC) The amount of residual acetic acid was determined by HS-GC after esterification to ethyl acetate.

[0070] For sample preparation, one vial of lyophilisate was reconstituted with water, using 20 ml of water per 1 g of lyophilisate. 500 μl of the reconstituted sample was mixed with 100 μl of saturated NaHSO4 solution and 50 μl of ethanol in a GC vial. The GC vial was tightly crimped. All samples were prepared in duplicate.

[0071] For standard preparation, a 1 mg / ml stock solution of acetic acid was prepared and diluted in water to five individual standards containing 25 μg / ml to 0.5 μg / ml. Each stock solution (500 μl) was mixed with 100 μl of saturated NaHSO4 solution and 50 μl of ethanol in a GC vial. [Table 5]

[0072] Standards were prepared in duplicate.

[0073] To determine the amount of acetic acid in the form of ethyl ester, a GC method for the quantification of residual solvents was used (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.

[0074] The following gas chromatographs were used: [Table 6] The gas chromatograph and head sampler were operated under the following conditions: [Table 7]

[0075] Reconstruction Behavior The dissolution behavior of the lyophilizate was determined by adding water for injection or 0.45 wt% aqueous NaCl solution at room temperature to obtain a final concentration of approximately 50 mg / ml. The reconstitution process was monitored in terms of dissolution time and behavior.

[0076] Determining the amount of water by "Karl Fischer titration" Approximately 100 mg of each sample was weighed into a glass vial sealed with a crimp cap. The sample was transferred to a furnace sample processor 774 of a Karl Fischer coulometer type 756 from Metrohm (Filderstadt, Germany) heated to 90°C. The septum of the cap was pierced with a syringe needle, and the generated water vapor was transferred directly to the titration chamber of the Karl Fischer coulometer via dry nitrogen. Measurements were repeated once. An empty glass vial was used for blank correction.

[0077] Example 1 Preparation of lyophilized form B The solutions given in the table below were prepared by weighing 8.0 g of treosulfan into a single-use polypropylene (PP) beaker. The required amount of solvent was added and the treosulfan was dissolved under gentle stirring until a clear solution was obtained. Complete dissolution was checked by visual control. The solution was then filtered using a 0.2 μm filter. The solution was filled into clean, depyrogenated glass vials of nominal volume 20 ml. [Table 8]

[0078] The filled vials were partially stoppered and the samples were placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 9]

[0079] All the obtained lyophilized products were identified as treosulfan crystalline form B by XRPD analysis.

[0080] The lyophilizate cake was well formed and homogeneous with no visible defects. With gentle shaking, the entire lyophilizate cake dissolved in 10 ml of water for injection at room temperature in less than 30 seconds. No preheating of the solvent was required. Removal of sticky particles adhering to the vial wall was also not necessary. The residual moisture content of the lyophilizate was below the limit of quantification of 0.005% by weight. [Table 10]

[0081] Examples 2 and 3 Preparation of Form B Lyophilisate The solutions given in the table below were prepared by dissolving treosulfan in the respective solvents (30 min, 25°C, ultrasonic bath), the resulting solutions were filtered, and the filtered solutions were filled into clean, depyrogenated glass vials (10 vials per formulation), stoppered in the lyophilization position, and sealed in lyophilization bags. [Table 11]

[0082] The sample was placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 12]

[0083] All lyophilisates tested were identified as treosulfan crystalline form B by XRPD analysis.

[0084] For reconstitution tests, the vials were punctured, opened, and 10 ml of 0.45 wt % aqueous NaCl solution (room temperature) was added using a 10 ml pipette. The lyophilized cakes of Examples 2 and 3 were reconstituted within just 1 minute. No preheating of the solvent was required. Removal of sticky particles adhering to the vial walls was also not required.

[0085] Only very low amounts of residual moisture were determined in all lyophilized products. Furthermore, all samples were free of impurities and showed similar high treosulfan contents. The acetic acid content was below the detection limit of 0.003 wt% for HS-GC analysis. [Table 13]

[0086] Example 4 Preparation of lyophilized form B The solutions given in the table below were prepared by weighing 10 g of treosulfan into a 150 ml polypropylene (PP) beaker. The solvent was added and the treosulfan was allowed to dissolve under stirring at ambient temperature of 22° C. The resulting solution was filled into clean, depyrogenated glass vials of nominal volume of 20 ml. [Table 14]

[0087] The vials were stoppered in the freeze-drying position and sealed in freeze-drying bags. The samples were placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 15] "atm." means atmospheric pressure

[0088] All lyophilisates tested were identified as treosulfan crystalline form B by XRPD analysis.

[0089] The resulting lyophilized cake was acceptable. For reconstitution testing, the vial was punctured open and 20 ml of 0.45 wt % aqueous NaCl solution (approximately 22°C) was added. The lyophilized cake was reconstituted within 1.5 minutes. No preheating of the solvent was required. Removal of adherent particles adhering to the vial wall was also not necessary.

[0090] All samples were free of impurities and showed very high treosulfan content. The acetic acid content was very low. [Table 16]

[0091] Example 5 Preparation of lyophilized form B 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 clean depyrogenated glass vials. [Table 17]

[0092] The vials were stoppered in the freeze-drying position and sealed in freeze-drying bags. The samples were placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 18] "atm." means atmospheric pressure

[0093] The resulting lyophilized product was identified as treosulfan crystalline form B by XRPD analysis.

[0094] The resulting lyophilized cake was homogeneous and free of any defects. For reconstitution testing, the vial was punctured open and 100 ml of 0.45 wt% aqueous NaCl solution (room temperature) was added to a final treosulfan concentration of 50 mg / ml. The lyophilized cake was reconstituted within only 30 seconds. No preheating of the solvent was required. No removal of sticky particles adhering to the vial wall was required.

[0095] All lyophilisates showed very high amounts of treosulfan and very low residual water content. [Table 19]

[0096] The lyophilisate samples were stored at 80°C for 96 hours. The stored samples still showed a very high treosulfan content of >99.4% by weight. The methanesulfonic acid content was less than 0.05% at the beginning of the test and 0.05% after storage, demonstrating the high stability of the lyophilisate. [Table 20]

[0097] Example 6 Preparation of lyophilized form B Solutions of the compositions given in the table below were prepared by measuring 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 resulting solution was filtered and the filtered solution was immediately filled into clean, depyrogenated glass vials warmed to 20°C. [Table 21]

[0098] The vials were stoppered in the freeze-drying position and sealed in freeze-drying bags. The samples were placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 22]

[0099] The resulting lyophilisate was identified as treosulfan form B by XRPD analysis.

[0100] All lyophilisates showed very high treosulfan content and very low residual water content. In addition, the amount of methanesulfonic acid was also very low. [Table 23]

[0101] Lyophilized samples were stored at 60°C for 30 days, at 70°C for 18 days, and at 80°C for 5 days. Regardless of storage conditions, at the end of the study, all samples were completely dissolved in 20 ml of 0.45 wt% aqueous NaCl solution within 1.5 minutes. No preheating of the solvent was required. Removal of stubborn particles adhering to the vial walls was also not required.

[0102] Furthermore, all samples still showed very high contents of treosulfan. [Table 24]

[0103] Lyophilized samples were also stored at 40°C and 75% relative humidity (rH) for 3 months. All stored samples still showed very high treosulfan content and very low methanesulfonic acid content, indicating their excellent stability. [Table 25]

[0104] Example 7 Preparation of lyophilized form B Pre-lyophilization solutions of the compositions given in the table below were prepared by measuring 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 for 30 minutes at 30° C. The resulting solution was filtered and the filtered solution was immediately filled into clean, depyrogenated glass vials warmed to 30° C. [Table 26]

[0105] The vials were stoppered in the freeze-drying position and sealed in freeze-drying bags. The samples were placed in a freeze-dryer and freeze-dried according to the following freeze-drying cycle: [Table 27]

[0106] The resulting lyophilized cake was homogeneous and free of any defects. For reconstitution testing, the vial was punctured open and 100 ml of 0.45 wt % aqueous NaCl solution (room temperature) was added to obtain a final treosulfan concentration of 50 mg / ml. The lyophilized cake was reconstituted within only 30 seconds. No preheating of the solvent was required. No removal of sticky particles adhering to the vial wall was required.

[0107] All lyophilizates showed very low amounts of residual water and very low amounts of methanesulfonic acid, the latter even below the limit of detection (LOD) of 0.01 wt %. [Table 28]

[0108] The obtained lyophilizates were also subjected to XRPD analysis using Rietveld refinement to determine their crystallinity and the amount of Form A, Form B and amorphous phase. Crystalline forms A and B were the only crystalline phases that could be detected. The results are given in the table below. [Table 29]

[0109] The XRPD pattern of the lyophilisate is shown in FIG.

[0110] Example 8 Preparation of crystalline form B 99.8 mg of treosulfan was weighed into a 4.0 ml vial equipped with a PTFE (polytetrafluoroethylene) seal and a stir bar. 1.5 ml of a mixture of 80 wt % water and 20 wt % isopropanol preheated to 65°C was then added. The resulting solution was completely withdrawn with a 5 ml syringe and filtered through a 0.2 μm filter into a 4.0 ml vial. The syringe, the 4.0 ml vial, and the filter were warmed to 65°C before use. The solvent was evaporated to dryness from the open vial at room temperature, resulting in the formation of crystals.

[0111] The XRPD pattern of the resulting crystals of Form B is shown in FIG.

[0112] Additionally, suitable single crystals of Form B were selected under a microscope and analyzed by single crystal X-ray diffraction (SCXRD). The data obtained are presented above in the previous section of the Examples.

[0113] Example 9 Preparation of Crystalline Form A (Reference) Approximately 5 g of treosulfan was dissolved in approximately 80 g of 2-propanol with 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 approximately 40° C.

[0114] The XRPD pattern of the dried crystals is shown in Figure 2 and confirms that it is treosulfan crystalline form A. Crystalline form A exhibits an XRPD pattern with 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.

[0115] Additionally, suitable single crystals of Form A were selected under a microscope and analyzed by single crystal X-ray diffraction (SCXRD). The data obtained are presented above in the previous section of the Examples. According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) A lyophilized product of treosulfan, including crystalline form B of treosulfan, which exhibits an X-ray powder diffraction pattern with characteristic peaks at 20.87 and 23.47±0.20 degrees 2θ. (Section 2) Item 2. The freeze-dried product according to item 1, wherein the crystalline form B exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ degrees of 20.87, 23.47, 26.20, 29.65, 30.81, 34.54, 35.30, 36.87, and 46.24±0.2. (Section 3) 3. The lyophilized product according to item 1 or 2, wherein the crystalline form B exhibits an X-ray powder diffraction pattern essentially as shown in FIG. (Section 4) 4. The freeze-dried product according to any one of items 1 to 3, wherein the crystalline form B exhibits an X-ray powder diffraction pattern that has no peaks in at least one, preferably all, of the following regions a to f, expressed in 2θ degrees: Table 30 (Section 5) 5. The freeze-dried product according to any one of items 1 to 4, comprising at least 96% by weight, particularly at least 97% by weight, preferably at least 98% by weight, more preferably at least 99% by weight of crystalline form B, based on the combined amount of crystalline form B and crystalline form A. (Section 6) 5. The lyophilizate according to any one of items 1 to 4, comprising at least 75% by weight, particularly 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 crystalline form B, based on the amount of the lyophilizate. (Section 7) 7. The freeze-dried product according to any one of items 1 to 6, comprising an amorphous phase in an amount of less than 20% by weight, particularly less than 15% by weight, preferably less than 10% by weight, more preferably less than 5% by weight, based on the amount of the freeze-dried product. (Section 8) 8. The freeze-dried product according to any one of items 1 to 7, comprising at least 95% by weight, particularly at least 96% by weight, preferably at least 98% by weight, more preferably at least 99% by weight of treosulfan. (Section 9) 9. The freeze-dried product according to any one of items 1 to 8, which contains less than 0.2% by weight, preferably less than 0.1% by weight, more preferably less than 0.05% by weight, of methanesulfonic acid. (Section 10) 10. The freeze-dried product according to any one of items 1 to 9, which contains less than 1% by weight, preferably less than 0.5% by weight, more preferably less than 0.1% by weight of water. (Section 11) 11. A process for preparing the lyophilized product according to any one of items 1 to 10, comprising the step of lyophilizing an aqueous solution containing treosulfan. (Section 12) 12. The process of claim 11, wherein the aqueous solution comprises water and optionally one or more organic solvents. (Section 13) 13. The process according to claim 12, wherein the organic solvent is acetic acid. (Section 14) (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 3 K / min or less; (c) drying the frozen solution obtained in step (b) to obtain the lyophilisate; 14. The process according to any one of items 11 to 13, comprising: (Section 15) Item 15. The process according to item 14, wherein the cooling rate in step (b) is 2 K / min or less, preferably 1.5 K / min or less, more preferably 1.3 K / min or less, or the cooling rate in step (b) is 0.05 to 1.5 K / min, preferably 0.1 to 1.3 K / min. (Section 16) 16. The process according to any one of items 11 to 15, wherein the first temperature is 15°C to 95°C, preferably 20°C to 50°C, more preferably 25°C to 35°C. (Section 17) 17. The process according to any one of items 11 to 16, wherein the freezing temperature is -40°C or lower, preferably -60°C to -40°C, more preferably -50°C to -40°C. (Section 18) 18. The process according to any one of the above items 11 to 17, wherein the frozen solution is maintained at the frozen temperature for at least 1 hour, preferably 1 to 10 hours, more preferably 2 to 8 hours. (Section 19) wherein the drying in step (c) comprises primary drying 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 a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar, more preferably 0.3 to 0.5 mbar; or 19. The process according to any one of the above paragraphs 14 to 18, wherein the drying in step (c) comprises primary drying carried out by subjecting the frozen solution to a temperature of 0°C or higher, preferably 0°C to 60°C, more preferably 20°C to 60°C, even more preferably 30°C to 50°C, and a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar, more preferably 0.3 to 0.5 mbar. (Section 20) 20. The process of claim 19, wherein the primary drying is carried out for at least 5 hours, preferably at least 10 hours. (Section 21) 21. The process according to paragraph 19 or 20, wherein secondary drying is carried out after the primary drying by subjecting the product of the primary drying to a temperature of at least 30°C, preferably 30-50°C, and by subjecting the product of the primary drying to a pressure of 0.03-1.0 mbar, preferably 0.1-0.6 mbar, more preferably 0.3-0.5 mbar. (Section 22) 11. The lyophilized product according to any one of items 1 to 10, for use as a medicine. (Section 23) 11. The lyophilized product according to any one of items 1 to 10, for use in treating cancer, particularly ovarian cancer. (Section 24) 11. The lyophilized product according to any one of items 1 to 10, for use in conditioning therapy before bone marrow or blood stem cell transplantation.

Claims

1. A freeze-dried product of treosulfan, wherein the freeze-dried product comprises crystals of treosulfan crystalline form B, wherein the treosulfan crystalline form B exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ degrees of 20.87, 23.47, 26.20, 29.65, 30.81, 34.54, 35.30, 36.87 and 46.24±0.

20.

2. The lyophilized product according to claim 1, wherein the crystalline form B of treosulfan exhibits an X-ray powder diffraction pattern that is free of peaks in at least one of the following regions a to f, expressed in 2θ degrees: Table 30

3. A freeze-dried product according to claim 1 or 2, comprising at least 96% by weight of treosulfan crystalline form B crystals relative to the combined amount of treosulfan crystalline form B crystals and treosulfan crystalline form A crystals, wherein treosulfan crystalline form A exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ degrees of 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.

2.

4. 3. The lyophilisate according to claim 1, comprising at least 75% by weight of the crystalline form B of treosulfan relative to the amount of the lyophilisate.

5. The lyophilisate according to any one of claims 1 to 4, comprising less than 20% by weight of an amorphous phase, relative to the amount of lyophilisate.

6. The lyophilisate according to any one of claims 1 to 5, which contains at least 95% by weight of treosulfan.

7. The lyophilisate according to any one of claims 1 to 6, which contains less than 0.2% by weight of methanesulfonic acid.

8. The lyophilisate according to any one of claims 1 to 7, which contains less than 1% by weight of water.

9. A process for preparing a lyophilisate according to any one of claims 1 to 8, comprising the step of lyophilising an aqueous solution comprising treosulfan.

10. The process of claim 9, wherein the aqueous solution comprises water and optionally acetic acid.

11. 11. The process of claim 9 or 10, (a) providing the aqueous solution having a first temperature; (b) freezing the aqueous solution to obtain a frozen solution, wherein the aqueous solution is cooled from the first temperature to a freezing temperature at a cooling rate of 3 K / min or less; (c) drying the frozen solution obtained in step (b) to obtain the lyophilisate; Including, wherein the first temperature is between 15°C and 95°C; and wherein the freezing temperature is −40° C. or lower; process.

12. 12. The process of claim 11, wherein the cooling rate in step (b) is 2 K / min or less.

13. 13. The process of claim 11 or 12, wherein the frozen solution is maintained at the frozen temperature for at least 1 hour.

14. wherein the drying in step (c) comprises primary drying carried out by subjecting the frozen solution to a temperature of −25° C. or higher and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar; or 14. The process of any one of claims 11 to 13, wherein the drying of step (c) comprises primary drying carried out by subjecting the frozen solution to a temperature of 0°C or higher and a pressure of 0.03 to 1.0 mbar.

15. 15. The process of claim 14, wherein the primary drying is carried out for at least 5 hours.

16. 16. The process of claim 14 or 15, wherein after the primary drying, secondary drying is carried out by subjecting the product of the primary drying to a temperature of at least 30°C and subjecting the product of the primary drying to a pressure of 0.03 to 1.0 mbar.

17. The lyophilisate according to any one of claims 1 to 8 for use as a medicament.

18. The lyophilisate according to any one of claims 1 to 8 for use in the treatment of cancer.

19. The lyophilisate according to any one of claims 1 to 8 for use in conditioning therapy before bone marrow or blood stem cell transplantation.

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