EA043302B1 - TREOSULPHAN LYOPHYLISATE - Google Patents

Description

The invention relates to a lyophilisate containing a new treosulfan crystal form B, the lyophilisate having very favorable characteristics for use as a pharmaceutical composition and in particular can be quickly reconstituted to form ready-to-use solutions and exhibit high stability and purity.

Treosulfan, whose chemical name is (2S,3S)-(-)1,4-di(mesyloxy)-2,3-butanediol or L-threitol-1,4-di(methanesulfonate), has the following chemical formula:

The chemical synthesis of treosulfan is described 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 and the silver salt of methanesulfonic acid.

Treosulfan is a dihydroxy derivative of busulfan and acts as an antitumor agent given its ability to alkylate DNA. It is used to treat ovarian cancer alone or in combination with other chemotherapeutic agents such as melphalan and dacarbazine (Baynes et al., Blood 96 (11): 170a, Abstr. no. 731, 2000). For the treatment of ovarian cancer, treosulfan monotherapy involves administering treosulfan to a patient in an amount of 8 g/m of body surface area, while combination therapy with treosulfan and cisplatin involves administering treosulfan in an amount of 5 g/m ² .

Treosulfan has also been used to treat advanced, inoperable non-small cell lung carcinomas (Pawel et al., Onkologie 21: 316-319; 1998).

In addition, EP 1227808 A1 discloses the use of treosulfan in preparatory (conditioning) therapy before bone marrow or blood stem cell transplantation in a patient. In such conditioning therapy, the administration of treosulfan can be effectively combined with the administration of additional agents such as cyclophosphamide, carboplatin, thiotepa, melphalan, fludarabine, immunosuppressive antibodies, or body irradiation. Compared with the use of busulfan, serious side effects can be largely or completely avoided. It is even possible to use high doses of treosulfan without causing serious toxicity to the liver, lungs, kidneys, or CNS. The conditioning phase includes a period of 2 to 7 days with a total dose of treosulfan of at least 20 g/m ² body surface area before allogeneic bone marrow or hematopoietic stem cell transplantation. Treosulfan is commercially available as oral capsules and as a sterile powder of treosulfan for infusion. The solution is administered intravenously over about 15-30 minutes. Treosulfan in these products is a crystalline form showing an X-ray powder diffraction (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 ± 0.2 degrees 2Θ. This crystalline form is designated form A and its XRPD diagram is shown in FIG. 2.

To prepare a solution for infusion, a commercial sterile powder is dissolved, for example, in water to a concentration of 50 mg/ml, and the resulting solution is diluted, for example, with isotonic NaCl solution. However, the water used as a solvent must be heated to 30° C. during the reduction step. In addition, it is necessary to completely remove the powder from the walls of the vial. This step is important to avoid sticking of the powder particles to the wall. These sticky Form A particles of treosulfan are difficult to dissolve and delay complete dissolution. The whole process of preparing a solution for infusion from a sterile powder, including the preparation of a vial, the necessary heating of water and complete dissolution of the powder, takes about 10 minutes. In addition, the use of a warm solvent increases the risk of unwanted degradation.

WO 2015/107534 refers to two putatively new and distinct polymorphs of treosulfan, designated Form I and Form II. There is no indication in this document of how Form II can be obtained, and therefore there is no possibility of disclosing Form II. The process for preparing Form I is only described in a very general manner and is said to simply involve recrystallization from organic solvents or mixtures thereof, with some preferred organic solvents indicated. No description of a specific method of obtaining Form I in this document is not disclosed. The X-ray powder diffraction pattern shown for Form I strongly resembles that of crystalline Form A, the commercially available product shown in FIG. 2 below, suggesting that these shapes are virtually identical. Finally, WO 2015/107534 also describes lyophilized formulations which are said to typically include treosulfan in form I.

However, the known lyophilizates suffer from several disadvantages. In particular, the known lyophilisates require a long time to reconstitute them, and their content of methanesulfonic acid and water, in particular after storage, is undesirably high, and hence their purity and stability are unsatisfactory. Moreover, the optimized lyophilization process that appears to have been used results in samples with very different properties and therefore lacking the desired reproducibility, which is very problematic given that these lyophilisates are intended to be used as pharmaceutical compositions.

Methanesulfonic acid (MSA) is a decomposition product of treosulfan as shown in the following reaction scheme.

Treosulfan OH o O, CH,

,.S О λ ^сн > О ОН ⁰

MSA

OH ,s <

c< o

Treosulfan monoepoxide

- ΜΞΑ

Treosulfan diepoxide

Therefore, its presence indicates the decomposition of treosulfan. Due to its high acidity, it accelerates the hydrolysis of the treosulfan ester groups and thus enhances the degradation process. For this reason, the amount of methanesulfonic acid should be as small as possible.

Thus, the aim of the present invention is to eliminate the disadvantages of known products containing treosulfan.

This goal is achieved using treosulfan lyophilisate according to claims 1-10 of the formula.

The invention also relates to a method for producing a treosulfan lyophilisate according to claims 11-21 and a treosulfan lyophilisate for use in medicine according to claims 22-24.

The lyophilisate according to the invention is characterized in that it comprises a crystalline form B of treosulfan showing an X-ray powder diffraction pattern having characteristic peaks at 20.87 and 23.47 ± 0.20 degrees 2Θ.

Preferably, crystalline form B shows 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 substantially as shown in FIG. 1.

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 from a to f, expressed in degrees 2Θ:

Region Degrees 2Θ A 19.00-19.50 b 20.00 - 20.65 With 21.50 -23.21 d 23.75 -24.95 e 27.40 -28.35 f 30.00 -30.60

The crystalline form B is preferably also characterized by the space group and the unit cell parameters a, b, c, α, β γ and the unit cell volume obtained by single crystal X-ray diffraction (SCXRD) analysis, the structural data for which are given in the following table along with additional information, especially on the quality of fit compared to Commercial Form A data.

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Form A Form B (according to the invention) space group orthorhombic, Ρ2ι2ι2ι monoclinic, P21 A 5.5306(5) A 5.5692(9) A b 8.9220(8) A 8.9469(15) A With 22.8442(18) A 11.322(2) A A 90° 90° β 90° 95.497(16)° At 90° 90° V 1127.22(17) A ³ 561.54(17) A ³ Z/Z' 4/1 2/1 Finite R-indices (observational data, I > 2σ(Ι)) RI = 0.0256, wR2 = 0.0462 RI = 0.0798, wR2 = 0.1956 R-indices (all data) RI=0.0280, wR2=0.0471 RI=0.0912, wR2=0.2065 Approximation accuracy 0.966 1.163 T 173(2)K 173(2)K

a, b and c are the lengths of the unit cell edges;

α, β and γ - angles between the edges of the unit cell;

V is the unit cell volume;

Z/Z' - the number of molecules in the unit cell;

R1 and wR2 - confidence values;

T is the temperature at which the analysis was carried out.

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

The lyophilizate according to the invention contains, in particular, at least 96 wt.%, preferably at least 97 wt.%, more preferably at least 98 wt.% and even more preferably at least 99 wt.% of the crystalline form B relative to the combined the amount of crystalline form B and crystalline form A.

Thus, the lyophilisate according to the invention contains only very small amounts of the usual crystalline form A and very large amounts of crystalline form B. The high polymorphic purity is particularly advantageous for the use of the lyophilisate according to the invention as a pharmaceutical composition.

In a further preferred embodiment, the lyophilizate according to the invention contains at least 75 wt.%, in particular at least 80 wt.%, preferably at least 85 wt.%, more preferably at least 90 wt.% and even more preferably at least least 95 wt.% crystalline form In relation to the amount of lyophilisate.

In another preferred embodiment, the lyophilizate according to the invention contains less than 20 wt.%, in particular less than 15 wt.%, preferably less than 10 wt.% and more preferably less than 5 wt.% of the amorphous phase in relation to the amount of lyophilisate.

A small amount of amorphous phase avoids several significant disadvantages associated with this phase. First of all, the amorphous phase tends to crystallize out of control. In addition, it decomposes faster, has a higher residual moisture content after drying, exhibits poorer fluidity and wettability, and becomes electrostatically charged more easily. All these properties are undesirable for a lyophilisate used for pharmaceutical purposes.

The lyophilisate according to the invention unexpectedly exhibits a combination of beneficial properties which are believed to be due to the high amount of treosulfan crystal form B. In particular, it takes a very short time for it to completely dissolve in the media commonly used for reconstitution into ready-to-use solutions for injection or infusion. Typically, isotonic saline and water for injection are used as such media. Other pharmaceutically acceptable solutions are also possible for reconstitution, such as Ringer's lactate solutions or phosphate buffers. The very short recovery time is very advantageous as it allows the clinic staff to prepare fresh, ready-to-use solutions immediately prior to intended administration to patients, without the need for long waiting times for complete dissolution. Likewise, with such a short recovery time, the risk of unwanted degradation reactions of treosulfan is reduced.

In addition, the lyophilizate according to the invention also has high purity and stability, as evidenced by its very high content of the active ingredient treosulfan and its very low content of the decomposition product of methanesulfonic acid.

In a preferred embodiment, the lyophilizate according to the invention contains at least 95 wt.%, in particular at least 95 wt.%, preferably at least 98 wt.% and more preferably at least 99 wt.% treosulfan.

In addition, the lyophilisate according to the invention has only a small content of methanesulfonic acid and comprises, in particular, less than 0.2 wt.%, preferably less than 0.1 wt.% and more preferably less than 0.05 wt.% methanesulfonic acid. A particularly small amount of methanesulfonic acid is one of the reasonable explanations for the high storage stability of the lyophilizate according to the invention, since this acid accelerates the hydrolysis of the ester groups of treosulfan and therefore contributes to its decomposition.

Even after storage of the lyophilisate for three months at 40° C. and 75% relative humidity, it contains, in particular, less than 0.2 wt.%, preferably less than 0.1 wt.% and more preferably less than 0.05 wt.% methanesulfonic acids. This is an indication of the excellent storage stability of the lyophilisate according to the invention, which makes it very suitable for use as a pharmaceutical composition or as a component thereof.

An additional advantage of the lyophilizate according to the invention is that it can be reconstituted using a solvent having a temperature of about 20° C., which makes it possible to dispense with the use of preheated solvents. In addition, no bulky formulation is required to remove commercial Form A sticky clusters from the vial walls prior to reconstitution.

In addition, the lyophilisate according to the invention has only a low water content and comprises water in an amount in particular less than 1 wt.%, preferably less than 0.5 wt.% and more preferably less than 0.1 wt.%, as determined by Karl titration Fisher.

The invention also relates to a method for producing a lyophilisate. The method according to the invention includes freeze-drying an aqueous solution containing treosulfan.

The freeze-dried aqueous solution is hereinafter also referred to as the pre-lyophilized solution.

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

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.

Even when using a pre-lyophilized solution which contains acetic acid, the lyophilizate obtained according to the invention unexpectedly 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 wt.% acetic acid.

The aqueous pre-lyophilized solution usually contains treosulfan at a concentration of 50 to 150 mg/g, in particular 50 to 100 mg/g and more preferably 50 to 80 mg/g.

The pre-lyophilized solution may also include additives such as solubilizers such as polysorbate, cyclodextrins, sodium dodecyl sulfate, poloxamer and the like; chelating agents such as sodium EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), calteridol, and the like; antioxidants, eg butylated hydroxytoluene, butylated hydroxyanisole, methionine, glutathione, sodium metabisulphite, alpha-tocopherol, sodium thioglycolate, cysteine, ascorbic acid, and the like; pH adjusting agents and buffering agents 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, and the like .; excipients, for example amino acids such as alanine, arginine, and the like; sugar derivatives such as sucrose, dextrose, mannitol, trehalose, mannose, and the like; or polymers, such as polyethylene glycol, gelatin, dextran, and the like; stabilizers and tonicity agents such as sodium chloride, magnesium chloride, sodium sulfate, and the like.

Before subjecting the aqueous solution to the freeze-drying process, it is usually filtered using conventional filters, such as a 0.22 µm filter, to obtain a sterile solution.

Freeze drying of the pre-lyophilized solution is usually carried out using freeze drying machines commonly used in pharmaceutical applications. Usually,

- 4 043302 the solution is poured into suitable containers, such as vials, and the containers are placed in a conventional freeze dryer with cooled and heated surfaces on which the solution can be exposed to the different temperatures of the freeze drying process. For drying, the solution is usually frozen and subjected to reduced atmospheric pressure. As a result, a significant sublimation of the solvent from the frozen solution occurs, which is deposited, for example, in the colder areas of the freeze dryer provided for this. This is usually followed by secondary drying at higher temperatures. After the freeze-drying is completed, the resulting lyophilisate is usually allowed to warm to room temperature and the containers containing the lyophilisate are sealed under sterile conditions.

In a preferred embodiment, the method according to the invention includes:

(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 the freezing temperature at a cooling rate of not more than 3 K/min, and (c) drying the frozen solution obtained in step (b) to obtain a lyophilizate.

The method according to the invention unexpectedly and very advantageously allows the use of such a rather low cooling rate, since the higher cooling rates used in conventional processes require the use of very sophisticated equipment. Therefore, the method according to the invention is very economical.

Moreover, it is preferable that the cooling rate in step (b) is not more than 2 K/min, preferably not more than 1.5 K/min, and more preferably not more than 1.3 K/min. Alternatively, the cooling rate in step (b) is in particular 0.05 to 1.5 and preferably 0.1 to 1.3 K/min.

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

The freezing temperature according to the method is in particular -40°C or less, preferably -60 to -40°C, and more preferably -50 to -40°C.

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

In another preferred embodiment of the method, the drying in step (c) comprises a primary drying, which is carried out by exposing the frozen solution to a temperature of -25°C or higher, preferably a temperature of -15 to 0°C, and exposing 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.

In an alternative further preferred embodiment of the method, the drying in step (c) comprises a primary drying, which is carried out by exposing the frozen solution to a temperature of 0°C or higher, preferably a temperature of 0 to 60°C, more preferably a temperature of 20 to 60°C, even more preferably at a temperature of 30 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 or more, preferably 0.3 to 0.5 mbar .

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

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

Secondary drying is preferably carried out for at least 2 hours, in particular at least 4 hours.

The method according to the invention makes it possible to obtain lyophilizates with excellent properties with high reproducibility, which is a significant advantage compared to conventional methods, which give products that differ significantly in their properties.

The lyophilizate according to the invention has also proved to be particularly useful in medicine. Therefore, the invention also relates to a lyophilisate according to the invention for use as a medicament. In a further embodiment, the invention also relates to a lyophilisate 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 a lyophilisate according to the invention for use in conditioning therapy prior to bone marrow or blood stem cell transplantation.

In a further aspect, the invention also relates to the use of the lyophilizate according to the invention for the treatment of cancer or for conditioning therapy prior to bone marrow or blood stem cell transplantation.

In a further aspect, the invention also relates to a method for treating patients suffering from cancer or a method for conditioning patients prior to bone marrow or blood stem cell transplantation, which comprises administering to patients a solution prepared from a lyophilisate according to the invention.

The invention is explained in more detail below with reference to non-limiting examples, which also include methods that are particularly suitable for determining the aforementioned properties of the lyophilisate according to the invention and treosulfan crystal forms B and A.

Examples

Methods and equipment.

The following methods were used to obtain X-ray powder diffraction (XRPD) patterns, for single crystal X-ray diffraction (SCXRD) studies, to quantify crystal form B and crystalline form A and the amount of amorphous phase, and to quantify treosulfane, acetic acid, methanesulfonic acid and water.

Moreover, the general procedure for preparing glass vials and characterizing recovery, as well as the device used for freeze drying, are also listed below.

The general procedure is the preparation of glass vials.

Glass vials for lyophilization were rinsed with purified water before use and depyrogenated for 2 h at 300°C. Stoppers for lyophilization autoclaved (121°C, 20 min, 2 bar) and dried for 7 h at 110°C.

Freeze dryer.

Freeze drying was carried out in a GT 2 freeze dryer (manufacturer: Hof Sonderanlagenbau (Lohra, Germany)) with a shelf area of 0.4 m ² and an ice condenser capacity of 8 kg, including means for measuring differential pressure.

X-ray powder diffraction (XRPD).

The corresponding sample was introduced into a standard glass capillary (0 = 0.7 mm) after careful manual grinding with a pestle in a mortar. The X-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance diffractometer (Cu-Ka1 = 1.54059 A, Johansson primary beam monochromator, position sensitive detector) in transmission mode with sample rotation. Data were collected over a range of 3 to 50 degrees 2Θ. The tube voltage and current were set to 40 kV and 40 mA respectively.

Single crystal X-ray diffraction (SCXRD).

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-Κα = 0.71073 A).

Quantification of Forms B and A by XRPD and Rietveld Analysis To quantify the crystalline form B and A of treosulfan, an appropriate sample was introduced into a standard glass capillary (0=0.7 mm) after thorough manual grinding with a pestle and mortar. The X-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance diffractometer (Cu-Ka1 = 1.54059 A, Johansson primary beam monochromator, position sensitive detector) in transmission mode with sample rotation. Data were collected over a range of 4 to 50 degrees Θ 2 for 4 hours. The tube voltage and current were set to 40 kV and 40 mA, respectively. The data obtained were subjected to a quantitative Rietveld analysis using the TOPAS program.

Amorphous phase quantification by XRPD and Rietveld analysis with internal standard.

To determine the amount of amorphous phase, the corresponding sample was mixed with 25 wt.% CaF ₂ (Aldrich Chemistry, Lot #MKBP1959V, calcium fluoride anhydrous, 99.99%) as an internal standard. After thorough manual grinding with a pestle in a mortar, the mixture was introduced into a standard glass capillary (0 = 1.0 mm). The X-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance diffractometer (Cu-Ka1 = 1.54059 A, Johansson primary beam monochromator, position sensitive detector) in transmission mode with sample rotation. Data were collected over a range of 4 to 50 degrees Θ 2 for 12 hours. The tube voltage and current were set to 30 kV and 30 mA, respectively. The data obtained were subjected to a quantitative Rietveld analysis using the TOPAS program.

Crystal Form A and Crystal Form B were the only crystalline phases that could be identified.

Determination of the amount of treosulfan by the RP-HPLC method.

The amount of treosulfan in the respective sample was determined using reverse phase high performance liquid chromatography (RP-HPLC) as follows:

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Equipment for HPLC Agilent Technologies Column Luna C 18(2), 5 µm, 250 x 4.6 mm (phenomenex) Mobile phase A 720 ml diluent + 30 ml methanol Isocratic, 25 min. Consumption 0.8 ml/min Column temperature 40°C Injection volume 20 µl Diluent 697 mg K ₂ HP0 ₄ /1000 ml, pH 4.5 (H ₃ PO ₄ ) detection Refractometric detector control solution 50 mg/ml treosulfan in diluent Sample solution 50 mg/ml treosulfan in solvent for recovery

Determination of the amount of methanesulfonic acid using HILIC.

The amount of methanesulfonic acid (MSA) was determined using hydrophilic interaction liquid chromatography (HILIC) as follows:

HPLC Equipment Column Nucleodur HILIC (250 x 4.6 mm, 3 µm) eluent 10 mmol ammonium formate in H ₂ O/acetonitrile (7:93) (v/v) Consumption 1.4 ml/min Column temperature 45°C Injection volume 20 µl Detector 35°C Cycle time 1.5 times the retention time of methanesulfonic acid detection Refractometric detector Control solution 1 Dissolve the methanesulfonic acid in HPLC water to a final concentration of 2.0 mg/mL. Control Solution 2 Dilute the control solution with eluent 1 to 0.1 mg/mL. Control solution 2 is used to quantify methanesulfonic acid in the test solution. Test solution The assay sample was dissolved in HPLC water to a final concentration of 20 mg/mL.

Determination of the residual content of acetic acid using headspace gas chromatography (HS-GC).

The amount of residual acetic acid was determined using HS-GC after esterification to ethyl acetate.

For sample preparation, the lyophilisate from one vial 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 tube. The GC tube was tightly crimped. All samples were prepared in duplicate.

To prepare the standards, a stock solution of 1 mg/mL acetic acid was prepared and diluted to 5 individual standards containing 25 to 0.5 µg/mL in water. Each stock solution (500 µl) was mixed with 100 µl saturated NaHSO4 solution and 50 µl ethanol in a GC tube.

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Standards: c (HO Ac) mg/ml Stock solution µl Water µl NaHSO ₄ µl Ethanol µl 0.05% 0.025 125 375 100 50 0.02% 0.01 50 450 100 50 0.01% 0.005 25 475 100 50 0.005% 0.0025 12.5 488 100 50 0.001% 0.0005 2.5 498 100 50

The standards have been prepared in duplicate.

The GC method for quantification of residual solvents was used to quantify acetic acid in its ethyl ester form (see Ph. Eur. 2.4.24 Identification and control of residual solvents: System A). The chromatographic conditions used for the quantitative determination of ethyl acetate are in accordance with USP 467 for the determination of residual solvents.

The following gas chromatograph was used:

Manufacturer Hewlett Packard Type 6890 headspace sampler 7694, Agilent Technologies Detector Flame ionization detector (FID) Source N2 Nitrogen generator G1000E, Parker Source H2 Hydrogen generator PGH2100

The gas chromatograph and headspace dispenser were operated under the following conditions:

6890N 7694 Headspace Sampler Program GC thermostat: Loop size: 1 ml Initial temperature 35°C Thermostat 85°C Start time 20 minutes Transmission line 120°С Consumption 25 °C/min A loop 100°C ultimate temperature 250°C Balance time 30 min final time 15 minutes bottle pressure 14psi Sample Injector Separation, 160 °C Pressurization 0.15 min No separation Loop filling 0.5 min Detector FID 270 °C Input 0.5 min Auxiliary nitrogen carrier gas Nitrogen Flow 3.7 ml/min

restorative behavior.

The dissolution pattern of the lyophilisates was determined by adding water for injection or 0.45 wt.% NaCl aqueous solution at room temperature to give a final concentration of about 50 mg/mL. The recovery process was controlled in terms of dissolution time and behavior.

Determination of the amount of water by titration according to Karl Fischer.

About 100 mg of the respective sample was weighed into a glass vial, which was closed with a crimp cap. The sample was transferred to a Karl Fischer Coulometer Furnace Type 756, Sample Processing Furnace 774 from Metrohm (Filderstadt, Germany), which was heated to 90°C. An injection needle was inserted through the septum of the cap, and the resulting water vapor was directed directly into the titration chamber of the Karl Fischer coulometer through dry nitrogen. The measurement was repeated once. Empty glass vials were used to correct for the control.

Example 1 - Preparation of a lyophilisate of crystalline form B.

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The solution shown in the table below was prepared by weighing 8.0 g of treosulfan into a disposable polypropylene (PP) beaker. The required amount of solvent was added and treosulfan was dissolved with gentle stirring until a clear solution was obtained. Complete dissolution was checked by visual inspection. The solution was then filtered through a 0.2 µm filter. The solution was filled into cleaned and depyrogenated glass vials with a nominal volume of 20 ml.

Composition of the pre-lyophilized solution, target dose of 500 mg treosulfan per vial.

Treosulfan concentration Solvent Quantity per vial 50 mg/g 90 wt.% water for injection and 10.0 g 10 wt% acetic acid

The filled vials were partially stoppered and the samples were loaded into a freeze dryer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle.

Stage Temperature shelves Ice condenser temperature Pressure Time interval # Description [°C] [°C] [mbar] [h:min] 1 Loading 25 1000 00:01 2 Freezing temperature change (1.17 K/min) -45 - 1000 01:00 3 Freezing -45 - 1000 05:00 4 Vacuum adjustment -45 -70 0.05 00:30 5 Temperature change during primary drying (0.28 K/min) -20 -70 0.05 01:30 6 Primary drying -20 -70 0.05 109:00 7 Temperature change during secondary drying (0.25 K/min) 40 -70 0.05 04:00 8 Secondary drying 40 -70 0.05 20:00

All lyophilisates obtained were identified by XRPD analysis as treosulfan crystal form B.

The lyophilizate briquettes were well formed and uniform without visible defects. Ready briquettes of the lyophilisate were dissolved in 10 ml of water for injection at room temperature in less than 30 seconds with gentle shaking. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required. The residual amount of water in the lyophilizates was below the limit of quantitation of 0.005 wt%.

Properties of lyophilizates.

Amount of water Time recovery Values below the limit of quantitation less than 30 s

Examples 2 and 3 - preparation of lyophilisate form B.

The solutions presented in the table below were prepared by dissolving treosulfan in an appropriate solvent (30 min, 25°C, ultrasonic bath). The resulting solutions were filtered, and the filtered solutions were placed in cleaned and depyrogenated glass vials (10 vials per composition), which were stoppered in the position for lyophilization and sealed.

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Composition of the pre-lyophilized solution, the target dose of treosulfan is 500 mg per vial.

Example Treosulfan concentration Solvent Quantity per vial 2 70 mg/g 98 wt.% water for injection and 2 wt% acetic acid 7.14 g 3 70 mg/g 94 wt.% water for injection and 6 wt% acetic acid 7.14 g

Samples were loaded into a lyophilizer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle.

Stage Temperature shelves Ice condenser temperature Pressure Time interval # Description [°C] [°C] [mbar] [h:min] 1 Loading 25 - 1000 00:01 2 Freezing temperature change (0.01 K/min) -45 - 1000 12:00 h Freezing -45 - 1000 02:00 4 Annealing temperature change (0.67 K/min) -25 - 1000 00:30 5 Annealing -25 - 1000 05:00 6 Freezing temperature change (0.67 K/min) -45 - 1000 00:30 7 Freezing -45 - 1000 00:30 8 Vacuum adjustment -45 -70°C 0.05 00:30 9 Temperature change during primary drying (0.28 K/min) -20 -70°C 0.05 01:30 10 Primary drying -20 -70°C 0.05 14:00 AND Change temperature during secondary drying (0.25 K/min) 40 -70°C 0.05 04:00 12 Secondary drying 40 -70°C 0.05 20:00

All tested lyophilizates were identified by XRPD analysis as crystalline form B of treosulfan.

For recovery testing, the vials were vented and opened, and 10 ml of 0.45% NaCl aqueous solution (at room temperature) was added using a 10 ml volumetric pipette. The lyophilisate cakes from both Examples 2 and 3 were reconstituted for only 1 minute. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required.

For all lyophilisates, only a very small amount of residual water was determined. Moreover, all samples were free of impurities and showed the same high content of treosulfan.

The content of acetic acid was below the limit of detection by HS-GC analysis of 0.003 wt%.

Properties of lyophilizates.

Example Amount of treosulfan [wt%] Quantity water [wt%] Amount of acetic acid [wt%] Time recovery 2 100 less than 0.1 Below detection limit 1 min 3 100 less than 0.1 Below detection limit 1 min

Example 4 - Preparation of a lyophilisate of crystalline form B.

The solution shown in the table below was prepared by weighing 10 g of treosulfan into a 150 ml polypropylene (PP) beaker. Solvent was added and treosulfan was dissolved with stirring at ambient temperature 22°C. The resulting solution was filled into cleaned and depyrogenated glass vials with a nominal volume of 20 ml.

Composition of a pre-lyophilized solution, the target dose is about 1000 mg of treosulfan per vial.

Treosulfan concentration Solvent Quantity per vial 75 mg/g 90 wt.% water for injection and 10 wt% acetic acid 13.3 g

The flasks were stoppered in the position for lyophilization and sealed into bags for lyophilization. Samples were loaded into a lyophilizer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle.

Stage Temperature shelves Ice condenser temperature Pressure Temporal interval # Description [°C] [°C] [mbar] [h:min] 1 Loading 45 - atm. 00:20 2 Freezing temperature change (0.23 K/min) 15 - atm. 02:10 3 Isothermal incubation 15 - atm. 06:00 4 Freezing temperature change (0.3 K/min) -45 - 1000 03:20 5 Freezing -45 - 1000 05:00 6 Annealing temperature change (1.33 K/min) -5 - 1000 00:30

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7 Annealing -5 - 1000 05:00 8 Freezing temperature change (0.4 K/min) -45 - 1000 01:40 9 Freezing -45 - 1000 00:30 10 Vacuum adjustment -45 -70 0.05 00:30 AND Temperature change during primary drying (0.44 K/min) -5 -70 0.05 01:30 12 Primary drying -5 -70 0.05 70:00 13 Temperature change during secondary drying (0.19 K/min) 40 -70 0.05 04:00 14 Secondary drying 40 -70 0.05 20:00

atm. means atmospheric pressure.

All tested lyophilizates were identified by XRPD analysis as crystalline form B of treosulfan.

The resulting lyophilizate cakes were acceptable. For recovery testing, the vials were vented and opened, and 20 ml of 0.45% NaCl aqueous solution (about 22° C.) was added. Lyophilisate briquettes are restored within 1.5 minutes. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required.

All samples were free of impurities and had very high levels of treosulfan. The content of acetic acid was very low.

Properties of lyophilizates.

Amount of treosulfan [wt%] Amount of acetic acid [wt%] Time recovery 100 0.07 1.5 min.

Example 5 - Preparation of a lyophilisate of crystalline form B.

A pre-lyophilized 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 min until complete dissolution of treosulfan. After filtration using a 0.2 μm membrane filter, the filtered solution was filled into cleaned and depyrogenated glass vials.

Composition of the pre-lyophilized solution, target dose of 5000 mg treosulfan per vial.

Treosulfan concentration Solvent Quantity per vial 80 mg/g Water for injections 62.5 g

The vials were stoppered in the lyophilization position and sealed in lyophilization bags. Samples were loaded into a lyophilizer and lyophilized according to the following lyophilization cycle.

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Lyophilization cycle.

Stage Temperature shelves Ice condenser temperature Pressure Time interval # Description [°C] [°C] [mbar] [h:min] 1 Loading thirty atm. 00:01 2 Freezing temperature change (1.0 K/min) -45 - atm. 01:15 3 Freezing -45 - atm. 03:00 4 Annealing temperature change (1.14 K/min) -5 - atm. 00:35 5 Annealing -5 - atm. 02:00 6 Change freezing temperature (1.17 k/min) -45 - atm. 00:35 7 Freezing -45 - atm. 01:00 8 Vacuum adjustment -45 -70 0.38 00:30 9 Temperature change during primary drying (1.0 K/min) 40 -70 0.38 01:25 10 Primary/secondary drying 40 -70 0.38 77:00

atm. means atmospheric pressure.

All resulting lyophilizates were identified by XRPD analysis as crystalline form B of treosulfan.

The resulting briquettes of lyophilisate were homogeneous without defects. For recovery testing, the vials were vented, opened, and 100 ml of 0.45% NaCl aqueous solution (at room temperature) were added to give a final treosulfan concentration of 50 mg/ml. The lyophilisate briquettes recovered in just 30 seconds. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required.

All lyophilizates showed a very high amount of treosulfan and a very low amount of residual water.

Properties of lyophilizates.

The amount of treosulfan Quantity Water Time recovery [wt%] [wt%] 99.97 0.04 30 sec

A sample of the lyophilisate was stored at 80° C. for 96 hours. The stored sample still showed a very high treosulfan content of over 99.4 wt%. The content of methanesulfonic acid was below 0.05% at the beginning of the test and 0.05% after storage, which proves that the lyophilisate is very stable.

Properties of the lyophilisate after storage at 80°C for 96 hours.

Time Amount of treosulfan [wt%] Amount of methanesulfonic acid [wt%] 0 h 100.3 <0.05 96 h >99.4 0.05

Example 6 - Preparation of a lyophilisate of crystalline form B.

A solution of the composition shown in the table below was prepared by weighing water in a beaker and bringing it to 30° C. using a water bath. An appropriate amount of treosulfan was added and the mixture was stirred until complete dissolution. The resulting solution was filtered, and the filtered solution was immediately poured into cleaned and depyrogenated glass vials, which were calcined at 20°C.

Composition of a pre-lyophilized solution, the target dose is about 1000 mg of treosulfan per vial.

Treosulfan concentration Dissolve eh Quantity per vial 56 mg/g Water 17.95 g

The vials were stoppered in the lyophilization position and sealed in lyophilization bags. Samples were loaded into a lyophilizer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle.

Stage Temperature shelves Ice condenser temperature Pressure Temporal interval # Description [°C] [°C] [mbar] [h:min] 1 Loading 20 - atm. 00:01 2 Change cooling temperature (0.3 K/min) 15 - atm. 00:17 3 Isothermal incubation 15 - atm. 06:00 4 Change freezing temperature (0.3 K/min) -45 - 1000 03:20 5 Freezing -45 - 1000 05:00 6 Change annealing temperature (0.1 °C/min) -15 - 1000 00:30 7 Annealing -15 - 1000 05:00 8 Change freezing temperature (0.3 K/min) -45 - 1000 01:40 9 Freezing -45 - 1000 00:30 10 Vacuum adjustment -45 -70 0.05 00:30 eleven Temperature change during primary drying -5 -70 0.05 01:30 12 Primary -5 -70 0.05 70:00

drying 13 Temperature change during secondary drying 40 -70 0.05 04:00 14 Secondary drying 40 -70 0.05 20:00

The resulting lyophilisates were identified as form B of treosulfan by XRPD analysis.

All lyophilizates showed a very high amount of treosulfan and a very low amount of residual water. In addition, the amount of methanesulfonic acid was also very low.

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Properties of lyophilizates.

Amount of treosulfan [wt%] Quantity water [wt%] Amount of methanesulfonic acid [wt%] 99.95 0.09 <0.05

Lyophilisate samples were stored at 60°C for 30 days, 70°C for 18 days and 80°C for 5 days. Regardless of the storage conditions, at the end of the tests, all samples completely dissolved within 1.5 min in 20 ml of 0.45% NaCl aqueous solution. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required.

Moreover, all samples still showed very high levels of treosulfan. Properties of lyophilizates after storage at a temperature of 60 to 80°C.

t [days and] T [°C] Amount of treosulfan [wt%] Recovery time 0 - 99.95 thirty 60 99.82 1.5 min. 18 70 99.94 1.5 min. 5 80 99.94 1.5 min.

Lyophilisate samples were also stored at 40° C. and 75% relative humidity (rH) for 3 months. All stored samples still showed a very high treosulfan content and a very low amount of methanesulfonic acid, indicating their excellent stability.

Properties of lyophilisates after storage at 40°C/75% rH.

Time (months) T/gN [°C/%] Amount of treosulfan [wt%)] Amount of methanesulfonic acid [wt.%o] 3 40/75 >99.95 <0.05%

Example 6 - Preparation of a lyophilisate of crystalline form B.

A pre-lyophilized solution of the composition shown in the table below was prepared by weighing water in a glass beaker and bringing its temperature to 30° C. using a water bath. An appropriate amount of treosulfan was added and the mixture was stirred at 30° C. for 30 minutes. The resulting solution was filtered, and the filtered solution was immediately poured into cleaned and depyrogenated glass vials, which were calcined at 30°C.

Composition of a pre-lyophilized solution, the target dose is about 5000 mg of treosulfan per vial.

Treosulfan concentration Solvent Quantity per vial 80 mg/g Water for injections 62.5 g

The vials were stoppered in the lyophilization position and sealed in lyophilization bags. Samples were loaded into a lyophilizer and lyophilized according to the following lyophilization cycle.

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Lyophilization cycle.

Stage Shelf temperature Ice condenser temperature a Pressure Stage time # Description [°C] [°C] [mbar] [h:min] 1 Loading thirty 1000 00:01 2 Freezing temperature change (1.17 K/min) -45 - 1000 01:04 3 Freezing -45 - 1000 06:00 4 Annealing temperature change (1K/min) -10 - 1000 00:35 5 Annealing -10 - 1000 06:00 6 Freezing temperature change (1K/min) -45 - 1000 00:35 7 Freezing -45 - 1000 03:00 8 Vacuum adjustment -45 <-70 0.33 00:30 9 Change temperature during primary drying (0.94 K/min) 35 <-70 0.33 01:25 1 0 Primary drying 35 <-70 0.33 62:00

The resulting briquettes of lyophilisate were homogeneous without defects. For recovery testing, the vials were vented, opened, and 100 ml of 0.45% NaCl aqueous solution (at room temperature) were added to give a final treosulfan concentration of 50 mg/ml. The lyophilisate briquettes recovered in just 30 seconds. Solvent preheating was not required. Removal of sticky particles adhering to the walls of the vials was also not required.

All lyophilisates showed very low residual water and very low methanesulfonic acid. The latter was even below the limit of detection (LOD) of 0.01 wt%.

Properties of lyophilizates.

Amount of treosulfan [wt%] Amount of water [wt.%] Amount of methanesulfonic acid [wt%] Time recovery 101.79 0.01 <LOD (Lower Limit of Detection) 30 sec

The resulting lyophilizates were also subjected to XRPD analysis using Rietveld refinement to determine their crystallinity, as well as the amount of form A, form B and amorphous phase. Crystal forms A and B were the only crystalline phases that could be detected. The results are presented in the following table.

Results of XRPD analyzes.

Amount of crystalline treosulfan [wt%] Quantity Form A and IN [wt%] Amount of amorphous phase [wt%] Form A Form B 96.3 0.5 99.5 3.7

An XRPD diagram of the lyophilisates is shown in FIG. 3.

Example 8 - preparation of crystalline form B.

99.8 mg of treosulfan was weighed into a vial (volume 4.0 ml) which was sealed with

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PTFE (polytetrafluoroethylene) and stirrer. Then added 1.5 ml of a mixture of 80 wt.% water and 20 wt.% isopropanol, preheated to 65°C. The resulting solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vessel (volume 4.0 ml). The syringe, second vial and filter were calcined at 65°C before use. The solvents were allowed to evaporate from the open vial at room temperature to dryness, resulting in the formation of crystals.

An XRPD diagram of the obtained form B crystals according to the invention is shown in FIG. 1.

In addition, a suitable Form B single crystal was selected under a microscope and analyzed by single crystal X-ray diffraction (SCXRD). The data obtained is presented above in the section preceding the examples.

Example 9 - preparation of crystalline form A (reference).

About 5 g of treosulfan was dissolved in about 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 a temperature of about 40°C.

The XRPD diagram of the dried crystals is shown in FIG. 2 and confirms that this is the crystalline Form A of treosulfan. Crystal Form A has 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.

In addition, a suitable Form A single crystal was selected under a microscope and analyzed by single crystal X-ray diffraction (SCXRD). The data obtained is presented above in the section preceding the examples.

Claims (24)

1. Lyophilisate of treosulfan, wherein the lyophilisate contains crystalline form B of treosulfan having an X-ray powder diffraction pattern that has characteristic peaks at 20.87 and 23.47±0.20 degrees 2Θ. 2. The lyophilisate of claim 1, wherein the crystalline Form B has an X-ray powder diffraction pattern that has characteristic peaks at 20.87, 23.47, 26.20, 29.65, 30.81, 34.54, 35.30 , 36.87 and 46.24 ± 0.2 degrees 2Θ. 3. The lyophilisate according to claim 1 or 2, wherein the crystalline form B has an X-ray powder diffraction pattern essentially as shown in FIG. 1. 4. The lyophilisate according to any one of claims 1 to 3, wherein the crystalline form B has an X-ray powder diffraction pattern having no peaks in at least one and preferably in all of the following regions from a to f, expressed in degrees 2Θ: Region Degrees 2Θ a 19.00-19.50 b 20.00 - 20.65 s 21.50-23.21d 23.75 - 24.95 e 27.40-28.35f 30.00 - 30.60 5. Lyophilizate according to any one of claims 1 to 4, which contains at least 96 wt.%, in particular at least 97 wt.%, preferably at least 98 wt.% and more preferably at least 99 wt.% crystalline form B relative to the combined amount of crystalline form B and crystalline form A. 6. Lyophilizate according to any one of claims 1 to 4, which contains at least 75 wt.%, in particular at least 80 wt.%, preferably at least 85 wt.%, more preferably at least 90 wt.% and even more preferably at least 95 wt.% of the crystalline Form In relative to the amount of lyophilisate. 7. A lyophilisate according to any one of claims 1 to 6, which contains less than 20 wt.%, in particular less than 15 wt.%, preferably less than 10 wt.% and more preferably less than 5 wt.% of the amorphous phase relative to the amount of lyophilisate. 8. Lyophilisate according to any one of claims 1 to 7, which contains at least 95 wt.%, in particular at least 96 wt.%, preferably at least 98 wt.% and more preferably at least 99 wt.% treosulfan. 9. Lyophilizate according to any one of claims 1 to 8, which contains less than 0.2 wt.%, preferably less than 0.1 wt.% and more preferably less than 0.05 wt.% methanesulfonic acid. 10. Lyophilisate according to any one of claims 1 to 9, which contains less than 1 wt.%, preferably less than 0.5 wt.% and more preferably less than 0.1 wt.% water. 11. A method for producing a lyophilizate according to any one of claims 1 to 10, which includes sublimation - 17 043302 drying an aqueous solution containing treosulfan. 12. The method of claim 11 wherein the aqueous solution contains water and optionally one or more organic solvents. 13. The method of claim 12 wherein the organic solvent is acetic acid. 14. The method according to any one of claims 11-13, which includes: (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 the freezing temperature at a cooling rate of not more than 3 K/min, and (c) drying the frozen solution obtained in step (b) to obtain a lyophilisate. 15. The method according to claim 14, wherein the cooling rate in step (b) is at most 2 K/min, preferably at most 1.5 K/min, and more preferably at most 1.3 K/min, or a cooling rate of step (b) is from 0.05 to 1.5 and preferably from 0.1 to 1.3 K/min. 16. The method according to any one of claims 11 to 15, wherein the first temperature is 15 to 95°C, preferably 20 to 50°C, and more preferably 25 to 35°C. 17. The method according to any one of claims 11 to 16, wherein the freezing temperature is -40°C or less, preferably -60 to -40°C, and more preferably -50 to -40°C. 18. The method according to any one of claims 11 to 17, wherein the frozen solution is kept at a freezing temperature for at least 1 hour, preferably 1 to 10 hours, and more preferably 2 to 8 hours. 19. The method according to any one of claims 14 to 18, wherein the drying in step (c) comprises a primary drying, which is carried out by exposing the frozen solution to a temperature of -25°C or higher, preferably a temperature of -15 to 0°C, and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar, or drying in step (c) comprises a primary drying which carried out by exposing the frozen solution to a temperature of 0°C or higher, preferably a temperature of 0 to 60°C, more preferably a temperature of 20 to 60°C, even more preferably a temperature of 30 to 50°C, and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar. 20. The method according to claim 19, wherein the primary drying is carried out for at least 5 hours and preferably at least 10 hours. 21. The method according to claim 19 or 20, in which, after the primary drying, a secondary drying is carried out, exposing the primary drying product to a temperature of at least 30°C, preferably 30-50°C, and exposing the primary drying product to a pressure of 0.03 up to 1.0 mbar, preferably from 0.1 to 0.6 mbar and more preferably from 0.3 to 0.5 mbar. 22. Lyophilizate according to any one of claims 1 to 10, used as a medicine. 23. A lyophilisate according to any one of claims 1 to 10, used in the treatment of cancer, and in particular ovarian cancer. 24. Lyophilizate according to any one of claims 1 to 10, used in conditioning therapy before bone marrow or blood stem cell transplantation.