Freeze-dried substance of quarsulfan

Abstract

Describes a lyophilized product of treosulfan, which has advantageous properties in terms of short reconstitution time, high purity and stability, and which is particularly useful in the treatment of cancer and in conditioning treatment before bone marrow or blood stem cell transplantation.

Description

[0001] The present invention relates to a lyophilizate of a new crystalline form B of treosulfan, which has very advantageous properties for use as a pharmaceutical composition and in particular can be rapidly reconstituted to form a ready-to-use solution and exhibits high stability and high purity.

[0002] Treosulfan, with the chemical name (2S,3S)-(-)-1,4-bis(methanesulfonyloxy)-2,3-butanediol or L-threitol-1,4-bis(methanesulfonate), has the following chemical formula:

[0003]

[0004] The chemical synthesis of treosulfan has been disclosed in DE 1 188 583 and DE 1 193 938 and is carried out, for example, by reacting L-1,4-dibromobutane-2,3-diol with the silver salt of methanesulfonic acid.

[0005] Treosulfan is a dihydroxy derivative of busulfan and, in view of its ability to alkylate DNA, it can be used as an anti-tumor agent. It is used alone or in combination with other chemotherapies such as melphalan and dacarbazine for the treatment of ovarian cancer (Baynes et al., Blood 96(11): 170a, abstract, No. 731, 2000). For the treatment of ovarian cancer, the use of treosulfan as a single therapy involves administering to the patient an amount of 8 g / m 2 body surface area, while the combination therapy using treosulfan and cisplatin involves administering treosulfan in an amount of 5 g / m 2 of body surface area.

[0006] Treosulfan has also been used for the treatment of advanced unresectable non-small cell lung cancer (Pawel et al., Onkologie 21: 316 - 319; 1998).

[0007] Furthermore, EP 1227808A1 discloses the use of treosulfan in a conditioning therapy before transplanting bone marrow or blood stem cells into a patient. In such a conditioning therapy, the administration of treosulfan can be effectively combined with the administration of any one of other agents such as cyclophosphamide, carboplatin, thiotepa, melphalan, fludarabine, immunosuppressive antibodies or irradiation of the human body. Compared with the use of busulfan, serious side effects can be mainly or completely avoided. Even high doses of treosulfan can be used without causing serious liver, lung, kidney or central nervous system toxicity. Before allogeneic transplantation of bone marrow or hematopoietic stem cells, the conditioning phase includes a time period of 2 to 7 days and the total dose of treosulfan is at least 20 g / m 2 body surface area.

[0008] Troxipide is commercially available as a capsule for oral administration and as a sterile powder consisting of troxipide for the preparation of an infusion solution. The solution is administered intravenously within about 15 to 30 minutes. Troxipide in these products is a crystalline form exhibiting a powder X-ray diffraction pattern (XRPD) having characteristic peaks at 2θ of 7.69±0.2, 15.43±0.2, 18.74±0.2, 19.14±0.2, 19.77±0.2, 20.15±0.2, 20.28±0.2, 21.24±0.2, 21.74±0.2, 22.07±0.2, 22.96±0.2, 23.24±0.2, 24.36±0.2, 25.29±0.2, 28.05±0.2, 28.28±0.2, 28.97±0.2, 30.10±0.2 and 40.55±0.2 degrees. This crystalline form is hereinafter referred to as Form A, and its XRPD pattern is shown in Figure 2 as shown.

[0009] To prepare a solution for infusion, the commercial sterile powder is dissolved in, for example, water to a concentration of 50 mg / ml, and the resulting solution is diluted with, for example, isotonic NaCl solution. However, the water used as the solvent must be warmed to 30 °C for the reconstitution step. In addition, the powder must be completely removed from the walls of the vial. This step is important for avoiding the formation of powder particles adhering to the walls. Such sticky particles of Form A of troxipide are difficult to dissolve, and they prolong the time for complete dissolution. The entire process of preparing an infusion solution from the sterile powder, including the preparation of the vial, the necessary warming of the water, and the complete dissolution of the powder, takes about 10 minutes. In addition, the use of a warmed solvent increases the risk of undesired degradation.

[0010] WO 2015 / 107534 relates to two allegedly novel and unique polymorphs of troxipide, designated Form I and Form II. The document lacks any description of how to obtain Form II and thus lacks an enabling disclosure of Form II. The method for preparing Form I is described only in a very general way and is said to involve only recrystallization from an organic solvent or a mixture thereof, with some preferred organic solvents mentioned. No disclosure of a specific process for preparing Form I is provided. The X-ray powder diffraction pattern given for Form I is very similar to the X-ray powder diffraction pattern of the commercially available Form A of the product, which is shown in Figure 2 as shown below, indicating that these crystalline forms are actually the same. Finally, WO 2015 / 107534 also describes a freeze-dried formulation, which is said to generally comprise troxipide in Form I.

[0011] However, the known lyophilizates have several disadvantages. In particular, the known lyophilizates require a long time for their reconstitution, and their content of mesylate and water, especially after storage, is undesirably high, and thus their purity and stability are unsatisfactory. In addition, the optimized lyophilization methods that have apparently been used result in a large variation in the properties of the samples and thus a lack of the desired reproducibility, which is highly problematic considering that these lyophilizates are intended to be used as pharmaceutical compositions.

[0012] Mesylate (MSA) is a degradation product of treosulfan, as shown in the following reaction scheme.

[0013]

[0014] Thus, its presence indicates the degradation of treosulfan. Due to its strong acidity, it accelerates the hydrolysis of the ester groups of treosulfan and thus enhances the degradation process. For this reason, the amount of mesylate should be as low as possible.

[0015] Accordingly, an object of the present invention is to avoid the disadvantages of the known products containing treosulfan.

[0016] This object is achieved by a lyophilizate of treosulfan according to claims 1 to 10.

[0017] The present invention also relates to a method for preparing a lyophilizate of treosulfan according to claims 11 to 21, and to the use of a lyophilizate of treosulfan according to claims 22 to 24 in medicaments.

[0018] The lyophilizate according to the present invention is characterized in that it contains crystalline form B of treosulfan, which exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ of 20.87 ± 0.20 and 23.47 ± 0.20 degrees.

[0019] Preferably, crystalline form B exhibits an X-ray powder diffraction pattern having peaks at 2θ of 20.87 ± 0.20, 23.47 ± 0.20, 26.20 ± 0.20, 29.65 ± 0.20, 30.81 ± 0.20, 34.54 ± 0.20, 35.30 ± 0.20, 36.87 ± 0.20 and 46.24 ± 0.20 degrees.

[0020] More preferably, crystalline form B exhibits an X-ray powder diffraction pattern substantially as Figure 1 shown.

[0021] Even more preferably, crystalline form B exhibits an X-ray powder diffraction pattern having no peaks in at least one and preferably all of the following regions a to f expressed in degrees 2θ:

[0022] Region Degree 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

[0023] The characteristics of polymorph B are preferably also the space group of the unit cell and the parameters a, b, c, α, β, γ, and the unit cell volume obtained by single crystal x-ray diffraction (SCXRD) analysis. These structural data are given in the following table, and further information is also given, especially further information related to the goodness of fit compared to the structural data of the commercial A-type.

[0024]

[0025]

[0026] a, b, and c = the lengths of the edges of the unit cell

[0027] α, β, and γ = the angles between the edges of the unit cell

[0028] V = the volume of the unit cell

[0029] Z / Z' = the number of molecules in the unit cell

[0030] R1 and wR2 = confidence values

[0031] T = the temperature at which the analysis is carried out

[0032] From these data, it can be seen that polymorph B has two molecules per unit cell (space group P21) and a volume of while polymorph A has four molecules per unit cell (space group P212121) and a volume of

[0033] The lyophilizate according to the invention particularly comprises at least 96% by weight, preferably at least 97% by weight, more preferably at least 98% by weight, and even more preferably at least 99% by weight of polymorph B relative to the combined amount of polymorph B and polymorph A.

[0034] Therefore, the lyophilizate according to the invention contains only a very small amount of the conventional polymorph A and a very large amount of polymorph B. The high polymorph purity is particularly advantageous for using the lyophilizate according to the invention as a pharmaceutical composition.

[0035] In another preferred embodiment, the lyophilizate according to the invention comprises at least 75% by weight, especially 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 polymorph B relative to the amount of the lyophilizate.

[0036] In yet another preferred embodiment, the lyophilizate according to the invention comprises less than 20% by weight, especially less than 15% by weight, preferably less than 10% by weight, and more preferably less than 5% by weight of the amorphous phase relative to the amount of the lyophilizate.

[0037] A small amount of amorphous phase avoids several significant drawbacks associated with this phase. First, the amorphous phase tends to cause uncontrolled crystallization. Additionally, it degrades faster, has a higher residual moisture content after drying, exhibits poor flowability and wettability, and is more prone to static electricity. All of these properties are undesirable for lyophilizates used for pharmaceutical purposes.

[0038] The lyophilizate according to the invention surprisingly shows a combination of advantageous properties, which are believed to be caused by a large amount of crystalline form B of treosulfan. Specifically, it requires only a very short time to completely dissolve in the medium commonly used for reconstitution to obtain a ready-to-use injection or infusion solution. Isotonic saline solution and water for injection are commonly used as such a medium. Other pharmaceutically acceptable solutions are also possible for reconstitution, such as Ringer's lactate solution or phosphate buffer. The very short period for reconstitution is highly advantageous because it enables clinical staff to freshly prepare the ready-to-use solution directly before the scheduled administration to the patient without having to consider a long waiting time for complete dissolution. Also, at such a short reconstitution time, the risk of undesirable degradation reactions of treosulfan is reduced.

[0039] Furthermore, the lyophilizate according to the invention also has high purity and stability, which is reflected by its very high content of the active ingredient treosulfan and very low content of the degradation product methanesulfonic acid.

[0040] In a preferred embodiment, the lyophilizate according to the invention contains at least 95% by weight, in particular at least 95% by weight, preferably at least 98% by weight, and more preferably at least 99% by weight of treosulfan.

[0041] In addition, the lyophilizate according to the invention has only a low content of methanesulfonic acid and particularly contains 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. The particularly low amount of methanesulfonic acid is a reasonable explanation for the high storage stability of the lyophilizate according to the invention, since this acid accelerates the hydrolysis of the ester group of treosulfan and thus promotes its degradation.

[0042] Even after the lyophilizate is stored at 40 °C and 75% relative humidity for three months, it particularly contains 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. This is an indication of the excellent storage stability of the lyophilizate according to the invention, making it very suitable for use as a pharmaceutical composition or a component thereof.

[0043] Another advantage of the lyophilizate according to the invention is that it can be reconstituted using a solvent at a temperature of about 20 °C, thus eliminating the need to use a preheated solvent. Additionally, there is no need for the cumbersome preparation of removing the sticky clusters of commercial type A from the vial wall before reconstitution.

[0044] Furthermore, the lyophilizate according to the present invention has a low water content, and the amount of water contained is in particular less than 1% by weight, preferably less than 0.5% by weight, and more preferably less than 0.1% by weight, which is determined by Karl Fischer titration.

[0045] The present invention also relates to a method for preparing a lyophilizate. The method according to the present invention comprises lyophilizing an aqueous solution containing trospium chloride.

[0046] The aqueous solution to be subjected to lyophilization is hereinafter also referred to as "pre-lyophilized solution".

[0047] Preferably, the aqueous solution contains water or a mixture of water and at least one organic solvent as the solvent. The organic solvent is in particular acetic acid.

[0048] The amount of water in the solvent is in particular 80 to 100% by weight, and preferably 90 to 100% by weight. The amount of acetic acid in the solvent is in particular 1 to 20% by weight, and preferably 2 to 10% by weight.

[0049] Even when using a pre-lyophilized solution containing acetic acid, the lyophilizate obtained according to the present invention surprisingly contains only a very small amount of acetic acid, and in particular less than 1.0% by weight, preferably less than 0.5% by weight, and more preferably less than 0.2% by weight of acetic acid.

[0050] The pre-lyophilized aqueous solution generally contains trospium chloride at a concentration of 50 to 150 mg / g, in particular 50 to 100 mg / g, and more preferably 50 mg / g to 80 mg / g.

[0051] The pre-lyophilized solution may further include additives such as solubilizers such as polysorbate, cyclodextrin, sodium dodecyl sulfate, poloxamer, etc.; chelating agents such as sodium EDTA, DTPA, calixarene, etc.; antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, methionine, glutathione, sodium metabisulfite, α-tocopherol, sodium thioglycolate, cysteine, ascorbic acid, etc.; pH regulators and buffers such as sodium hydroxide, hydrochloric acid, citric acid, sodium acetate, arginine, aspartic acid, sodium bicarbonate, sodium citrate, disodium citrate, trisodium citrate, maleic acid, sulfuric acid, hydrogen phosphate, etc.; fillers such as amino acids such as alanine and arginine, etc.; sugar derivatives such as sucrose, dextrose, mannitol, trehalose, mannose, etc.; or polymers such as polyethylene glycol, gelatin, dextran, etc.; stabilizers and tonicity regulators such as sodium chloride, magnesium chloride, sodium sulfate, etc.

[0052] Before subjecting the aqueous solution to the lyophilization process, it is usually filtered using a conventional filter such as a 0.22 μm filter to obtain a sterile solution.

[0053] The lyophilization of the pre-lyophilized solution is typically achieved by using a lyophilizer 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 lyophilizer having a coolable and heatable surface on which the solution can be exposed to various temperatures of the lyophilization process. To effect drying, the solution is typically frozen and exposed to a reduced atmospheric pressure. As a result, sublimation of the solvent from the frozen solution occurs to a large extent, which deposits, for example, on the colder areas of the lyophilizer provided therefor. This is usually followed by a secondary drying at a higher temperature. After the lyophilization is completed, the obtained lyophilizate is usually allowed to reach room temperature, and the container containing the lyophilizate is sealed under aseptic conditions.

[0054] In a preferred embodiment, the method according to the invention comprises

[0055] (a) providing an aqueous solution having a first temperature,

[0056] (b) freezing the aqueous solution, wherein the aqueous solution is cooled from the first temperature to a freezing temperature at a cooling rate not exceeding 3 K / min, and

[0057] (c) drying the frozen solution obtained in step (b) to obtain a lyophilizate.

[0058] Surprisingly and very advantageously, the method according to the invention allows the use of a rather low cooling rate, since the higher cooling rates employed by conventional methods require the use of very complex equipment. Thus, the method according to the invention is very economical.

[0059] Furthermore, preferably, the cooling rate in step (b) is not greater than 2 K / min, preferably not greater than 1.5 K / min, and more preferably not greater than 1.3 K / min. In an alternative embodiment, the cooling rate in step (b) is in particular from 0.05 to 1.5, and preferably from 0.1 to 1.3 K / min.

[0060] The first temperature in the method of the invention is in particular from 15 °C to 95 °C, preferably from 20 °C to 50 °C, and more preferably from 25 °C to 35 °C.

[0061] The freezing temperature used in the method is in particular -40 °C or lower, preferably from -60 °C to -40 °C, and more preferably from -50 °C to -40 °C.

[0062] The frozen solution is held at the freezing temperature for in particular at least 1 hour, preferably from 1 to 10 hours, and more preferably from 2 to 8 hours.

[0063] In another preferred embodiment of the method, the drying in step (c) comprises 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.

[0064] In another alternative preferred embodiment of the method, the drying in step (c) comprises primary drying, which is 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, and 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.

[0065] The primary drying is preferably carried out for at least 5 hours, and in particular at least 10 hours.

[0066] It is also preferred that after the primary drying, secondary drying is carried out by subjecting the primarily dried product to a temperature of at least 30 °C, preferably 30 to 50 °C, and subjecting the primarily dried product 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.

[0067] The secondary drying is preferably carried out for at least 2 hours, and in particular at least 4 hours.

[0068] The method according to the invention allows the preparation of lyophilizates with excellent properties in a highly reproducible manner, which is a significant advantage compared to conventional methods that provide products that are significantly different in their properties.

[0069] The lyophilizates according to the invention have also proven to be particularly useful in medicine. Accordingly, the invention also relates to lyophilizates according to the invention for use as medicaments. In another embodiment, the invention also relates to lyophilizates according to the invention for use in the treatment of cancer and in particular ovarian cancer. In yet another embodiment, the invention also relates to lyophilizates according to the invention for use in conditioning treatment before bone marrow or blood stem cell transplantation.

[0070] On the other hand, the invention also relates to the use of lyophilizates according to the invention for the treatment of cancer or for use in conditioning treatment before bone marrow or blood stem cell transplantation.

[0071] In yet another aspect, the invention also relates to a method for treating a patient suffering from cancer or for conditioning a patient before bone marrow or blood stem cell transplantation, the method comprising administering to the patient a solution prepared from a lyophilizate according to the invention.

[0072] The present invention will be explained in more detail below with reference to non - limiting examples, which also include methods particularly suitable for determining the above - mentioned properties of the lyophilizates and polymorph B and polymorph A of tropisetron according to the present invention. Example

[0073] Methods and apparatus

[0074] Hereinafter, methods for obtaining X - ray powder diffraction (XRPD) patterns, for performing studies by single - crystal X - ray diffraction (SCXRD), for determining the amounts of polymorph B and polymorph A and the amount of the amorphous phase, and for determining the amounts of tropisetron, acetic acid, methanesulfonic acid, and water are given.

[0075] In addition, the general procedures for preparing glass vials and for determining the reconstitution behavior, as well as the apparatus for freeze - drying, are also shown below.

[0076] General procedure - Preparation of glass vials

[0077] The glass vials for freeze - drying are rinsed with pure water before use and depyrogenized at 300 °C for 2 hours. The freeze - drying stoppers are autoclaved (121 °C, 20 min, 2 bar) and dried at 110 °C for 7 hours.

[0078] Freeze dryer

[0079] Freeze - drying is carried out in a freeze - dryer GT2 (manufacturer: Hof Soderalagenbau (Lohra, Germany)) with a shelf area of 0.4 m 2 and an ice condenser capacity of 8 kg for differential pressure measuring devices.

[0080] X-ray powder diffraction (XRPD)

[0081] After careful manual grinding in a mortar with a pestle, the corresponding samples are introduced into standard glass capillaries In transmission mode, the X - ray powder diffraction patterns are recorded with sample rotation at room temperature using a Bruker D8 Advance diffractometer ( Johansson primary beam monochromator, position - sensitive detector). Data are collected in the range of 2θ from 3 to 50 degrees. The tube voltage and current are set to 40 kV and 40 mA, respectively.

[0082] Single crystal x-ray diffraction (SCXRD)

[0083] Using an instrument equipped with a molybdenum anode Single crystal X-ray diffraction data were recorded on a Rigaku Xcalibur, Sapphire2, large Be window diffractometer of the X-ray generator.

[0084] Determination of the amounts of Form B and Form A by XRPD and Rietveld analysis

[0085] To determine the amounts of the B and A crystal forms of tropisetron, after careful manual grinding in a mortar with a pestle, the corresponding samples were introduced into a standard glass capillary tube. At room temperature, X-ray powder diffraction patterns were recorded in transmission mode with sample rotation using a Bruker D8 Advance diffractometer equipped with a Johansson primary beam monochromator and a position-sensitive detector. Data were collected in the range of 2θ from 4 to 50 degrees over a period of 4 h. The tube voltage and current were set at 40 kV and 40 mA, respectively. The data obtained were subjected to quantitative Rietveld analysis using TOPAS software.

[0086] Determination of the amount of the amorphous phase by XRPD and Rietveld analysis using an internal standard

[0087] To determine the amount of the amorphous phase, the corresponding sample was mixed with 25 wt% of CaF2 (Aldrich Chemistry, Lot# MKBP1959V, anhydrous calcium fluoride, 99.99%) as an internal standard. After careful manual grinding in a mortar with a pestle, the mixture was introduced into a standard glass capillary tube. At room temperature, X-ray powder diffraction patterns were recorded in transmission mode with sample rotation using a Bruker D8 Advance diffractometer equipped with a Johansson primary beam monochromator and a position-sensitive detector. Data were collected in the range of 2θ from 4 to 50 degrees over a period of 12 h. The tube voltage and current were set at 30 kV and 30 mA, respectively. The data obtained were subjected to quantitative Rietveld analysis using TOPAS software.

[0088] The A and B crystal forms are the only identifiable crystalline phases.

[0089] Determination of the amount of treosulfan by RP-HPLC

[0090] The amount of tropisetron in the corresponding samples was determined using reversed-phase high-performance liquid chromatography (RP-HPLC) as shown below:

[0091]

[0092] Determination of the amount of methanesulfonic acid by HILIC

[0093] The amount of methanesulfonic acid (MSA) was determined using hydrophilic interaction liquid chromatography (HILIC) as shown below:

[0094]

[0095] Headspace gas chromatography (HS-GC) for determination of residual acetic acid content

[0096] After esterification to ethyl acetate, the amount of residual acetic acid was determined by HS-GC.

[0097] For sample preparation, 20 ml of water was used per 1 g of lyophilizate, and the lyophilizate in one vial was reconstituted with water. 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 crimped tightly. All samples were prepared in duplicate.

[0098] To prepare the standards, a stock solution of acetic acid at 1 mg / ml was prepared and diluted in water to 5 individual standards containing from 25 μg / ml to 0.5 μg / ml. 500 μl of each stock solution was mixed with 100 μl of saturated NaHSO4 solution and 50 μl of ethanol in a GC vial.

[0099]

[0100] The standards were prepared in duplicate.

[0101] The amount of acetic acid in its ethyl ester form was determined using a GC method for the quantification of residual solvents (see Ph.Eur. 2.4.24 Identification and control of residual solvents: System A). The chromatographic conditions for the quantification of the amount of ethyl acetate corresponded to the USP 467 method for the determination of residual solvents.

[0102] The following gas chromatograph was used:

[0103]

[0104] The gas chromatograph and headspace sampler were operated under the following conditions:

[0105]

[0106] Reconstitution behavior

[0107] The dissolution behavior of the lyophilizate was determined by adding water for injection or 0.45 wt% aqueous NaCl solution at room temperature to give a final concentration of approximately 50 mg / ml. The reconstitution process was monitored according to the dissolution time and behavior.

[0108] Determination of the amount of water by "Karl Fischer titration"

[0109] Weigh approximately 100 mg of the respective sample into a glass vial sealed with a crimp cap. Transfer the sample to the furnace of a Metrohm (Filderstadt, Germany) Model 756 Karl Fischer coulometer (furnace sample processor 774) heated to 90 °C. Pierce the septum of the cap with an injection needle, and the evolved water vapor is directly transferred to the titration cell of the Karl Fischer coulometer through dry nitrogen. The measurement is repeated once. An empty glass vial is used for blank correction.

[0110] Example 1 - Preparation of the freeze-dried product of Form B

[0111] The solutions given in the following table were prepared by weighing 8.0 g of treosulfan into a disposable polypropylene (PP) beaker. Add the required amount of solvent and dissolve treosulfan with gentle stirring until a clear solution is obtained. Check for complete dissolution by visual control. Thereafter, filter the solution using a 0.2 μm filter. Fill the solution into a clean and pyrogen-free glass vial with a nominal volume of 20 ml.

[0112] Composition of the predried solution,

[0113] Target dose 500 mg treosulfan per vial

[0114]

[0115] Partially stopper the filled vials, load the samples into a freeze dryer, and lyophilize according to the following lyophilization cycle.

[0116] Freeze-drying cycle

[0117]

[0118] All the obtained lyophilized products were identified as crystalline form B of treosulfan by XRPD analysis.

[0119] The lyophilized cakes were well formed and uniform, without visible defects. At room temperature, with gentle shaking, the completely lyophilized cakes were dissolved in 10 ml of water for injection within less than 30 seconds. No preheating of the solvent was required. Nor was it necessary to remove the sticky particles adhering to the vial wall. The water residue of the lyophilized products was below the quantification limit of 0.005 wt%.

[0120] Properties of the freeze-dried product

[0121] Amount of water Reconstitution time Below the limit of quantification <30s

[0122] Examples 2 and 3 - Preparation of the freeze-dried product of Form B

[0123] The solutions given in the following table were prepared by dissolving treosulfan in the respective solvents (30 minutes, 25 °C, ultrasonic bath). The obtained solutions were filtered and the filtered solutions were filled into clean and pyrogen-free glass vials (10 vials for each preparation), which were stoppered in the lyophilization position and sealed in a lyophilization bag.

[0124] Composition of the predried solution, target dose 500 mg treosulfan per vial

[0125]

[0126] The samples were loaded into a freeze dryer and lyophilized according to the following lyophilization cycle.

[0127] Freeze-drying cycle

[0128]

[0129] All the tested lyophilized products were identified as crystalline form B of treosulfan by XRPD analysis.

[0130] For the reconstitution test, the vials were vented and opened, and 10 ml of an aqueous solution of 0.45% by weight of NaCl (room temperature) was added using a 10 ml volumetric pipette. The lyophilized cakes of both Examples 2 and 3 were reconstituted within only 1 min. Preheating of the solvent was not required. Nor was it necessary to remove the sticky particles adhering to the vial walls.

[0131] For all the lyophilized products, only very small amounts of residual water were measured. In addition, all the samples were free of impurities and showed similar and high treosulfan contents. The acetic acid content was below the detection limit of HS-GC analysis of 0.003% by weight.

[0132] Properties of the freeze-dried product

[0133]

[0134] Example 4 - Preparation of the freeze-dried product of Form B

[0135] The solutions given in the following table were prepared by weighing 10 g of treosulfan in a 150 ml polypropylene (PP) beaker. The solvent was added and treosulfan was dissolved with stirring at an ambient temperature of 22 °C. The obtained solution was filled into clean and pyrogen-free glass vials with a nominal volume of 20 ml.

[0136] Composition of the predried solution,

[0137] Target dose approximately 1000 mg treosulfan per vial

[0138]

[0139] Stopper the vial in the freeze-drying position and seal it in a freeze-drying bag. Load the sample into a freeze dryer and lyophilize according to the following freeze-drying cycle.

[0140] Freeze-drying cycle

[0141]

[0142] "atm." represents atmospheric pressure

[0143] All tested lyophilized products were identified as Form B of treosulfan by XRPD analysis.

[0144] The obtained lyophilized cake was acceptable. For the reconstitution test, vent and open the vial and add 20 ml of 0.45 wt% aqueous NaCl solution (approx. 22 °C). The lyophilized cake was reconstituted within 1.5 min. No preheating of the solvent was required. Nor was it necessary to remove the sticky particles adhering to the vial wall.

[0145] All samples were free of impurities and had a very high content of treosulfan. The acetic acid content was very low.

[0146] Properties of the freeze-dried product

[0147]

[0148] Example 5 - Preparation of the freeze-dried product of Form B

[0149] A predried solution was prepared by mixing 52.5 g of treosulfan and 603.75 g of water with stirring at a temperature of 30 °C. Stirring was continued for 30 minutes until the treosulfan was completely dissolved. After filtration using a 0.2 μm membrane filter, the filtered solution was filled into clean and pyrogen-free glass vials.

[0150] Composition of the predried solution,

[0151] Target dose 5000 mg treosulfan per vial

[0152]

[0153] Stopper the vial in the freeze-drying position and seal it in a freeze-drying bag. Load the sample into a freeze dryer and lyophilize according to the following freeze-drying cycle.

[0154] Freeze-drying cycle

[0155]

[0156] "atm." represents atmospheric pressure

[0157] The lyophilized product obtained by XRPD analysis was identified as Form B of treosulfan.

[0158] The obtained lyophilizate cake is uniform and free of any defects. For the reconstitution test, the vial was vented, opened, and 100 ml of 0.45 wt% aqueous NaCl solution (room temperature) was added to give a final concentration of 50 mg / ml of tropisetron. The lyophilizate cake was reconstituted in only 30 seconds. No preheating of the solvent was required. Nor was it necessary to remove the sticky particles adhering to the vial wall.

[0159] All lyophilizates showed a very high amount of tropisetron and a very low amount of residual water.

[0160] Properties of the freeze-dried product

[0161]

[0162] The lyophilizate samples were stored at 80 °C for 96 hours. The stored samples still showed a very high tropisetron content of > 99.4 wt%. At the start of the test, the mesylate content was below 0.05%, and after storage it was 0.05%, demonstrating that the lyophilizate was very stable.

[0163] Properties of the freeze-dried product after storage at 80 °C for 96 hours

[0164]

[0165] Example 6 - Preparation of the freeze-dried product of Form B

[0166] The solutions of the compositions given in the following table were prepared by weighing water into a glass beaker and adjusting its temperature to 20 °C using a water bath. The corresponding amount of tropisetron was added and the mixture was stirred until completely dissolved. The obtained solution was filtered and the filtered solution was immediately filled into clean and pyrogen-free glass vials, which were tempered at 20 °C.

[0167] Composition of the predried solution,

[0168] Target dose approximately 1000 mg treosulfan per vial

[0169]

[0170] The vials were stoppered in the lyophilization position and sealed in a lyophilization bag. The samples were loaded into a freeze dryer and lyophilized according to the following lyophilization cycle.

[0171] Freeze-drying cycle

[0172]

[0173] The lyophilizate obtained by XRPD analysis was identified as the B form of tropisetron.

[0174] All the lyophilized products showed a very high content of trofosfamide and a very low amount of residual water. In addition, the amount of methanesulfonic acid was also very low.

[0175] Properties of the freeze-dried product

[0176]

[0177] The lyophilized product samples were stored at 60 °C for 30 days, at 70 °C for 18 days, and at 80 °C for 5 days. Regardless of the storage conditions, at the end of the test, all samples completely dissolved in 20 ml of a 0.45% by weight aqueous NaCl solution within 1.5 min. No preheating of the solvent was required. Nor was it necessary to remove the sticky particles adhering to the vial walls.

[0178] In addition, all samples still showed a very high content of trofosfamide.

[0179] Properties of the freeze-dried product after storage at 60 to 80 °C

[0180]

[0181] The lyophilized product samples were also stored at 40 °C and 75% relative humidity (r.H.) for 3 months. All the stored samples still showed a very high content of trofosfamide and a very low amount of methanesulfonic acid, indicating their excellent stability.

[0182] Properties of the freeze-dried product after storage at 40 °C / 75% r.H.

[0183]

[0184] Example 7 - Preparation of the freeze-dried product of Form B

[0185] The pre-lyophilization solutions of the compositions given in the following table were prepared by weighing water into a glass beaker and adjusting its temperature to 30 °C using a water bath. The corresponding amount of trofosfamide was added, and the mixture was stirred at 30 °C for 30 minutes. The resulting solution was filtered, and the filtered solution was immediately filled into clean and pyrogen-free glass vials, which were tempered at 30 °C.

[0186] Composition of the predried solution,

[0187] Target dose approximately 5000 mg treosulfan per vial

[0188]

[0189] The vials were stoppered in the lyophilization position and sealed in a lyophilization bag. The samples were loaded into a freeze dryer and lyophilized according to the following lyophilization cycle.

[0190] Freeze-drying cycle

[0191]

[0192] The obtained lyophilizate cake is uniform and free of any defects. For the reconstitution test, the vial was vented, opened, and 100 ml of an aqueous 0.45 wt% NaCl solution (at room temperature) was added to give a final concentration of 50 mg / ml of trofosfamide. The lyophilizate cake reconstituted in only 30 seconds. No preheating of the solvent was required. Nor was it necessary to remove the sticky particles adhering to the vial wall.

[0193] All lyophilizates showed a very low amount of residual water and a very low amount of mesylate. The latter was even below the detection limit (LOD) of 0.01 wt%.

[0194] Properties of the freeze-dried product

[0195]

[0196] The obtained lyophilizates were also subjected to XRPD analysis using Rietveld refinement to determine their crystallinity and the amounts of their Form A, Form B, and amorphous phases. Form A and Form B are the only detectable crystalline phases. The results are given in the table below.

[0197] Results of XRPD analysis

[0198]

[0199] The XRPD pattern of the lyophilizate is shown in Figure 3 in.

[0200] Example 8 - Preparation of Form B

[0201] 99.8 mg of trofosfamide was weighed in a vial (capacity 4.0 ml) equipped with a PTFE (polytetrafluoroethylene) seal and a stirrer. Then 1.5 ml of a mixture of 80 wt% water and 20 wt% isopropanol preheated to 65 °C was added. The resulting solution was completely aspirated with a syringe (capacity 5 ml) and filtered through a 0.2 μm filter into a second vial (capacity 4.0 ml). Before use, the syringe, the second vial, and the filter were tempered at a temperature of 65 °C. The solvent was evaporated to dryness from the open vial at room temperature, which resulted in the formation of crystals.

[0202] The XRPD pattern of the obtained Form B crystals is shown in Figure 1 in.

[0203] In addition, suitable Form B single crystals were selected under a microscope and analyzed by single crystal x-ray diffraction (SCXRD). The data obtained are described in the section preceding the above examples.

[0204] Example 9 - Preparation of Form A (reference)

[0205] Dissolve about 5 g of tratosulfan in about 80 g of 2-propanol with stirring at 65 °C. Then filter the resulting solution through a 0.2 μm filter and cool to 15 °C, which results in the precipitation of crystals. Collect the crystals and dry at about 40 °C.

[0206] The XRPD pattern of the dried crystals is shown in Figure 2 and confirmed that it is polymorph A of tratosulfan. Polymorph A exhibits an XRPD pattern having characteristic peaks at 2θ of 7.69 ± 0.20, 15.43 ± 0.20, 18.74 ± 0.20, 19.14 ± 0.20, 19.77 ± 0.20, 20.15 ± 0.20, 20.28 ± 0.20, 21.24 ± 0.20, 21.74 ± 0.20, 22.07 ± 0.20, 22.96 ± 0.20, 23.24 ± 0.20, 24.36 ± 0.20, 25.29 ± 0.20, 28.05 ± 0.20, 28.28 ± 0.20, 28.97 ± 0.20, 30.10 ± 0.20 and 40.55 ± 0.20 degrees.

[0207] In addition, single crystals of the appropriate form of A were selected under a microscope and analyzed by single crystal x-ray diffraction (SCXRD). The data obtained are described in the section preceding the above examples.

Claims

1. Freeze-dried product of quarsulfan, wherein, The lyophilizate contains at least 75% by weight of polymorph B of triciribine, relative to the amount of the lyophilizate, and the polymorph B exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ of 20.87 ± 0.20 and 23.47 ± 0.20 degrees.

2. The lyophilized product according to claim 1, wherein, The polymorph B exhibits an X-ray powder diffraction pattern having characteristic peaks at 2θ of 20.87 ± 0.2, 23.47 ± 0.2, 26.20 ± 0.2, 29.65 ± 0.2, 30.81 ± 0.2, 34.54 ± 0.2, 35.30 ± 0.2, 36.87 ± 0.2 and 46.24 ± 0.2 degrees.

3. The lyophilized product according to claim 1 or 2, wherein The polymorph B exhibits an X-ray powder diffraction pattern as shown in Figure 1.

4. The lyophilized product according to claim 1 or 2, wherein, The polymorph B exhibits an X-ray powder diffraction pattern having no peaks in at least one of the following regions a to f expressed in degrees 2θ: 。 5. The lyophilized product according to claim 4, wherein, The polymorph B exhibits an X-ray powder diffraction pattern having no peaks in all of the regions a to f expressed in degrees 2θ.

6. The lyophilizate according to claim 1 or 2, which contains at least 96% by weight of polymorph B, relative to the combined amount of polymorph B and polymorph A.

7. The lyophilizate according to claim 6, which contains at least 97% by weight of polymorph B, relative to the combined amount of polymorph B and polymorph A.

8. The lyophilizate according to claim 6, which contains at least 98% by weight of polymorph B, relative to the combined amount of polymorph B and polymorph A.

9. The lyophilizate according to claim 6, which contains at least 99% by weight of polymorph B, relative to the combined amount of polymorph B and polymorph A.

10. The lyophilizate according to claim 1 or 2, which contains at least 80% by weight of polymorph B, relative to the amount of the lyophilizate.

11. The lyophilizate according to claim 10, which contains at least 85% by weight of polymorph B, relative to the amount of the lyophilizate.

12. The lyophilizate according to claim 10, which contains at least 90% by weight of polymorph B, relative to the amount of the lyophilizate.

13. The lyophilizate according to claim 10, which contains at least 95% by weight of polymorph B, relative to the amount of the lyophilizate.

14. The lyophilizate according to claim 1 or 2, which contains less than 20% by weight of an amorphous phase, relative to the amount of the lyophilizate.

15. The lyophilizate according to claim 14, which contains less than 15% by weight of an amorphous phase, relative to the amount of the lyophilizate.

16. The lyophilizate according to claim 14, which contains less than 10% by weight of an amorphous phase, relative to the amount of the lyophilizate.

17. The lyophilizate according to claim 14, which contains less than 5% by weight of an amorphous phase, relative to the amount of the lyophilizate.

18. The lyophilizate according to claim 1 or 2, which contains at least 95% by weight of triciribine.

19. The lyophilizate according to claim 18, which contains at least 96% by weight of triciribine.

20. The lyophilizate according to claim 18, which contains at least 98% by weight of triciribine.

21. The lyophilizate according to claim 18, which contains at least 99% by weight of triciribine.

22. The lyophilizate according to claim 1 or 2, which contains less than 0.2% by weight of methanesulfonic acid.

23. The lyophilizate according to claim 22, which contains less than 0.1% by weight of methanesulfonic acid.

24. The lyophilizate according to claim 22, which comprises less than 0.05% by weight of mesylate acid.

25. The lyophilizate according to claim 1 or 2, which comprises less than 1% by weight of water.

26. The lyophilizate according to claim 25, which comprises less than 0.5% by weight of water.

27. The lyophilizate according to claim 25, which comprises less than 0.1% by weight of water.

28. A method for preparing the lyophilizate according to any one of claims 1 to 27, the method comprising lyophilizing an aqueous solution comprising treosulfan.

29. The method according to claim 28, wherein, The aqueous solution comprises water and optionally one or more organic solvents.

30. The method according to claim 29, wherein, The organic solvent is acetic acid.

31. The method according to any one of claims 28 to 30, which comprises (a) providing an 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 not exceeding 3 K / min, and (c) drying the frozen solution obtained in step (b) to obtain a lyophilizate.

32. The method according to claim 31, wherein The cooling rate in step (b) is not greater than 2 K / min.

33. The method according to claim 32, wherein, The cooling rate in step (b) is not greater than 1.5 K / min.

34. The method according to claim 32, wherein, The cooling rate in step (b) is not greater than 1.3 K / min.

35. The method according to claim 32, wherein, The cooling rate in step (b) is from 0.05 to 1.5 K / min.

36. The method according to claim 32, wherein, The cooling rate in step (b) is from 0.1 to 1.3 K / min.

37. The method according to any one of claims 28 to 30, wherein The first temperature is from 15 °C to 95 °C.

38. The method according to claim 37, wherein, The first temperature is from 20 °C to 50 °C.

39. The method according to claim 37, wherein The first temperature is from 25 °C to 35 °C.

40. The method according to any one of claims 28 to 30, wherein The freezing temperature is -40 °C or lower.

41. The method according to claim 40, wherein, The freezing temperature is from -60 °C to -40 °C.

42. The method according to claim 40, wherein, The freezing temperature is from -50 °C to -40 °C.

43. The method according to any one of claims 28 to 30, wherein, The frozen solution is held at the freezing temperature for at least 1 hour.

44. The method according to claim 43, wherein, The frozen solution is held at the freezing temperature for 1 to 10 hours.

45. The method according to claim 43, wherein The frozen solution is held at the freezing temperature for 2 to 8 hours.

46. The method according to claim 31, wherein The drying in step (c) comprises primary drying, which is 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.

47. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of -25 °C or higher and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

48. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of -25 °C or higher and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

49. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of -15 °C to 0 °C and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar.

50. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of -15 °C to 0 °C and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

51. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of -15 °C to 0 °C and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

52. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C or higher and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar.

53. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C or higher and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

54. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C or higher and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

55. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C to 60 °C and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar.

56. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C to 60 °C and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

57. The method according to claim 46, wherein The primary drying is carried out by subjecting the frozen solution to a temperature of 0 °C to 60 °C and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

58. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 20 °C to 60 °C and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar.

59. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 20 °C to 60 °C and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

60. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 20 °C to 60 °C and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

61. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 30 °C to 50 °C and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar.

62. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 30 °C to 50 °C and subjecting the frozen solution to a pressure of 0.1 to 0.6 mbar.

63. The method according to claim 46, wherein, The primary drying is carried out by subjecting the frozen solution to a temperature of 30 °C to 50 °C and subjecting the frozen solution to a pressure of 0.3 to 0.5 mbar.

64. The method according to any one of claims 46 - 63, wherein, The primary drying is carried out for at least 5 hours.

65. The method according to claim 64, wherein, The primary drying is carried out for at least 10 hours.

66. The method according to any one of claims 46 - 63, 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.

67. The method according to claim 66, 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 0.1 to 0.6 mbar.

68. The method according to claim 66, 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 0.3 to 0.5 mbar.

69. The method according to claim 66, wherein, After the primary drying, secondary drying is carried out by subjecting the product of the primary drying to a temperature of 30 to 50 °C and subjecting the product of the primary drying to a pressure of 0.03 to 1.0 mbar.

70. The method according to claim 66, wherein After the primary drying, secondary drying is carried out by subjecting the product of the primary drying to a temperature of 30 to 50 °C and subjecting the product of the primary drying to a pressure of 0.1 to 0.6 mbar.

71. The method according to claim 66, wherein, After primary drying, secondary drying is carried out by subjecting the product of primary drying to a temperature of 30 to 50 °C and subjecting the product of primary drying to a pressure of 0.3 to 0.5 mbar.

72. A pharmaceutical composition comprising the lyophilizate according to any one of claims 1 to 27.

73. Use of the lyophilizate according to any one of claims 1 to 27 in the preparation of a medicament for the treatment of cancer.

74. The use according to claim 73, wherein The medicament is used for the treatment of ovarian cancer.

75. Use of the lyophilizate according to any one of claims 1 to 27 in the preparation of a medicament for conditioning treatment before bone marrow or blood stem cell transplantation.

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