Solution comprising treosulfan The invention relates to a solution comprising treosulfan, which is in particular useful for lyophilisation, and the use of such a solution for preparing a corresponding lyophilisate, which lyophilisate has very favourable characteristics for use as a pharmaceutical composition and in particular can be quickly reconstituted to form ready-to-use solutions and shows a high stability and purity.

Treosulfan, chemical name (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 has been disclosed in DE 1 188 583 and DE 1 193 938 and is for example effected by reacting L-l , 4-dibromobutane-2 , 3-diol and the silver salt of methanesulfonic acid.

Treosulfan is a dihydroxy derivative of busulfan and acts as an antineoplastic agent in view of its ability to alkylate the DNA. It is in use for the treatment of ovarian cancer either as such or in combination with further chemotherapeutics for example melphalan and dacarbazine (Baynes et al . , Blood 96(11): 170a, Abstr. No. 731, 2000). For the treatment of ovarian cancer the monotherapy with treosulfan involves administering to the patient an amount of 8 g/m ² body surface area, whereas the combination therapy with treosulfan and cisplatin involves administering treosulfan in an amount of 5 g/m ² .

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

Furthermore, EP 1 227 808 Al discloses the use of treosulfan in conditioning therapy before bone marrow or blood stem cell transplantation to a patient. In such conditioning therapy, the administration of treosulfan can effectively be combined with either administration of further agents, e.g. cyclophosphamid, carboplatin, thiotepa, melphalan, fludarabin, immune suppressive antibodies, or irradiation of the body. In comparison to the use of busulfan, serious side effects can predominantly or entirely be avoided. High dosages of treosulfan can even be used without causing serious liver, lung, kidney or CNS toxicities. The conditioning phase comprises a period of 2 to 7 days with a total dose of treosulfan of at least 20 g/m ² body surface area before allogenic transplantation of bone marrow or haematopoietic stem cells.

Treosulfan is commercially available as capsules for oral use and a sterile powder consisting of crystalline treosulfan for preparing a solution for infusion. The solution is administered intravenously within about 15 to 30 minutes. For preparing a solution for infusion, the commercial sterile powder is dissolved in e.g. in water to a concentration of 50 mg/ml and the obtained solution is diluted with e.g. isotonic NaCl solution. However, the water used as solvent has to be warmed to 30°C for the reconstitution step. Moreover, the powder has to be completely removed from the walls of the vial. This step is important to avoid formation of powder particles which are sticking to the wall. Such sticky particles of treosulfan are difficult to be dissolved and they protract the complete dissolution. The whole process for preparing the solution for infusion from the sterile powder, including the preparation of the vial, the necessary warming of water and the complete dissolution of the powder, takes about 10 minutes. Moreover, the use of warm solvent enhances the risk of undesired degradation.

WO 2015/107534 refers to two allegedly novel and distinct polymorphic forms of treosulfan, designated as form I and form II as well as lyophilized formulations which are said to typically include treosulfan of form I. These lyophilized formulations are prepared by freeze-drying a solution of treosulfan in water. However, the obtained lyophilisates suffer from a couple of disadvantages. In particular, they require long times for their reconstitution and their content of methanesulfonic acid and water, in particular after storage, is undesirably high and hence their purity and stability is not satisfactory. The solubility of treosulfan in water was found by the present inventors to be only about 50 mg/ml at 18°C and this rather low solubility prolongs the lyophilisation process. Finally, the known process also leads to lyophilisates with properties varying to a large degree and hence lacks the desired reproducibility which is very problematic bearing in mind that they are intended to be used as pharmaceutical compositions.

It is, therefore, an object of the present invention to avoid the disadvantages of the known solutions of treosulfan and to provide solutions which can comprise treosulfan in a higher concentration and allow to prepare in an economic manner lyophilisates which have favorable characteristics and in particular can be quickly reconstituted to form ready-to-use solutions and show a high stability and purity.

This object is achieved by the solution comprising treosulfan according to claims 1 to 3.

The invention also relates to the use of the solution according to claim 4 and the process for preparing a lyophilisate of treosulfan according to claims 5 to 16.

The solution according to the invention is characterized in that it comprises treosulfan and a mixture of water and acetic acid.

It is preferred that the mixture comprises 2 to 50 % by weight, in particular 2 to 25 % by weight and preferably 2 to 10 % by weight of acetic acid.

The use of a mixture of acetic acid and water as solvent for treosulfan surprisingly results in a number of significant advantages .

It was found that the solubility of treosulfan in such a mixture is higher than in either water or acetic acid. The solubility at 18°C of treosulfan in water is about 50 mg/ml and in acetic acid is about 18 mg/ml.

It was, therefore, unexpected to find that the addition of acetic acid to water actually leads to an increase of the solubility of treosulfan substantially above 50 mg/ml. Due to the higher concentration of treosulfan, the use of the resulting solution allows to reduce the duration of the lyophilisation process and makes the process more economic.

The solution according to the invention comprises treosulfan in an amount of in particular 70 to 250 mg/g, preferably 70 to 155 mg/g and more preferably 70 to 125 mg/g solution.

The solution may also include additives such as solubilizers, e.g. polysorbate, cyclodextrins , sodium dodecyl sulfate, and poloxamer; chelating agents, e.g. sodium EDTA, DTPA, and calteridol; antioxidants, e.g. butylated hydroxy toluene, butylated hydroxy anisole, methionine, glutathione, metabisulfite sodium, alpha-tocopherol , thioglycolate sodium, cysteine, and ascorbic acid; pH adjusting agents and buffering agents, e.g. sodium hydroxide, hydrochloric acid, citric acid, sodium acetate, arginine, aspartic acid, sodium bicarbonate, sodium citrate, disodium citrate, trisodium citrate, maleic acid, sulfuric acid, and hydrogen phosphate; bulking agents, e.g. amino acids such as alanine and arginine; sugar derivatives, e.g. sucrose, dextrose, mannitol, trehalose, and mannose; polymers, e.g. polyethylene glycol, gelatin, and dextran; stabilizers and tonicity adjusting agents e.g. sodium chloride, magnesium chloride, and sodium sulfate.

The solution according to the invention surprisingly allows the economic preparation of a lyophilisate of treosulfan which shows a combination of very advantageous properties. The invention is therefore also directed to the use of the solution according to the invention for preparing a lyophilisate of treosulfan.

The invention is also directed to a process for preparing a lyophilisate of treosulfan which process is characterized in that the solution according to the invention is freeze-dried.

Before subjecting the solution to the freeze-drying process, it is usually filtered employing conventional filters, e.g. a 0.22 ym filter, to obtain a sterile solution.

The freeze-drying of the solution is typically effected by using freeze-drying machines normally employed for pharmaceutical purposes. As a rule, the solution is filled into suitable containers, such as vials, and the containers are placed in a conventional freeze-dryer with coolable and heatable surfaces on which the solution can be exposed to the various temperatures of the freeze-drying process. To achieve the drying, the solution is usually frozen and exposed to a decreased atmospheric pressure. As a result, sublimation of the solvent from the frozen solution takes place to a great extent, which precipitates for example on cooler regions of the freeze-dryer provided for this. This is then usually followed by a secondary drying at higher temperatures. After completion of the freeze drying, the lyophilisate obtained is normally allowed to come to room temperature and the containers including the lyophilisate are sealed under sterile conditions.

In a preferred embodiment, the process according to the invention comprises

(a) providing the solution having a first temperature,

(b) freezing the solution, wherein the solution is cooled from the first temperature to a freezing temperature at a cooling rate of not more than 3 K/min, and (c) drying the frozen solution obtained in step (b) to give the lyophilisate .

It is surprising and very advantageous that the process according to the invention allows to use such a rather low cooling rate since higher cooling rates as employed by conventional processes require the use of very sophisticated equipment. The process according to the invention is therefore very economic.

Moreover, it is preferred 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. 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.

The first temperature in the process 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.

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

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

In a further preferred embodiment of the process, the drying in step (c) includes a primary drying which is carried out by subjecting the frozen solution to a temperature of -25°C or higher, preferably a temperature of -15°C to 0°C, and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar. In an alternative further preferred embodiment of the process, the drying in step (c) includes a primary drying which is carried out by subjecting the frozen solution to a temperature of 0°C or higher, preferably a temperature of 0°C to 60°C, more preferably a temperature of 20°C to 60°C, even more preferably a temperature of 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.

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

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

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

The lyophilisate obtained by the process according to the invention requires only a very short time for complete dissolution in media usually employed for reconstitution to give ready-to-use injection or infusion solutions. Isotonic saline solution and water for injection are typically employed as such media. Other pharmaceutically acceptable solutions are also possible for the reconstitution, e.g. Ringer's lactate solutions or phosphate buffers. The very short period of time for reconstitution is very favourable since it enables clinic staff to prepare ready-to-use solutions freshly directly before the intended administration to patients, without having to allow for long waiting times for complete dissolution. Likewise, with such short reconstitution times, the risk of undesired degradation reactions of the treosulfan decreases. Moreover, the lyophilisate also has a high purity and stability as reflected by its very high content of the active ingredient treosulfan .

In a preferred embodiment, the lyophilisate comprises at least 95 % by weight, in particular at least 96 % by weight, preferably at least 98 % by weight and more preferably at least 99 % by weight of treosulfan.

It is a further advantage of the lyophilisate obtained by the process according to the invention that it can be reconstituted using solvent having a temperature of about 20°C thus dispensing with the need to employ pre-heated solvents. Furthermore, the cumbersome removal of sticky clusters of the commercial form of treosulfan from the vial wall before reconstitution is also not needed.

Moreover, the lyophilisate has only a small water content and comprises water in an amount of in particular less than 1 %, preferably less than 0.5 % and more preferably less than 0.1 % by weight, as determined by Karl Fischer titration.

The lyophilisate obtained by the process according to the invention surprisingly comprises only a very small amount of acetic acid of in particular less than 1.0 % by weight, preferably less than 0.5 % by weight and more preferably less than 0.2 % by weight. This is also a substantial and surprising advantage as it shows that the presence of acetic acid in the solution for lyophilisation is associated with favorable properties in the lyophilisate and at the same time its amount in the lyophilisate is desirably low.

The process according to the invention also allows to prepare the lyophilisate with favorable properties in a highly reproducible manner which is a substantial advantage in comparison to the conventional processes which give products with substantially varying properties.

The invention is explained in more detail below with reference to non-limiting examples which also include methods which are in particular suitable to determine the above-mentioned properties of the lyophilisate obtained by the process according to the invention .

Examples

Methods and Apparatus

In the following, the methods are given which have been used for determining the amount of treosulfan, acetic acid and water.

Moreover, the general procedure for preparing glass vials and for determining the reconstitution behaviour as well the apparatus used for freeze drying is also indicated below.

General procedure - Preparing glass vials

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

Freeze dryer

Freeze drying was carried out in a freeze dryer GT 2 (Manufacturer: Hof Sonderanlagenbau (Lohra, Germany)) with 0.4 m ² shelf area and 8 kg ice condenser capacity including means for differential pressure measurement. Determination of amount of treosulfan and impurities by RP- HPLC

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

Determination of residual acetic acid content by headspace gas chromatography (HS-GC)

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

For sample preparation, the lyophilisate of one vial was reconstituted with water using 20 ml of water per 1 g of lyophilisate. 500 mΐ of the reconstituted sample were mixed with 100 mΐ saturated NaHSCy-solution and 50 mΐ of ethanol in a GC-vial. The GC vial was tightly crimped. All samples were prepared in duplicates.

For preparation of standards, a stock solution of acetic acid of 1 mg/ml was prepared and diluted to 5 individual standards containing 25 yg/ml to 0.5 yg/ml in water. Each stock solution (500 mΐ) was mixed with 100 mΐ saturated NaHSCg solution and 50 mΐ of ethanol in a GC-vial.

Standards were prepared in duplicates.

The GC method for quantification of residual solvents was used to determine the amount of acetic acid in form of its ethyl ester (see Ph.Eur. 2.4.24 Identification and control of residual solvents: System A). The chromatographic conditions used to quantify the amount of ethyl acetate correspond to the USP 467 method for the determination of residual solvents. The following gas chromatograph was used:

The gas chromatograph and the head sampler were operated at the following conditions:

Reconstitution behavior

The dissolution behavior of the lyophilisates was determined by adding water for injection or 0.45 % by weight of aqueous NaCl solution at room temperature to give a final concentration of about 50 mg/ml. The reconstitution process was monitored with regard to dissolution time and behavior. Determination of amount of water by "Karl Fischer titration" About 100 mg of the respective sample was weighed into a glass vial which was sealed with a crimp cap. The sample was transferred into the furnace of a Karl Fischer coulometer type 756, furnace sample processor 774, of Metrohm (Filderstadt , Germany) which was heated to 90 °C. The septum of the cap was penetrated by an injection needle, and the generated water vapour was directly transferred into the titration chamber of the Karl Fischer coulometer via dry nitrogen. The measurement was repeated once. Empty glass vials were used for blank correction .

Examples 1 and 2 - Solutions with 2 and 6 % by weight of acetic acid and lyophilisates

The solutions as given in the table below were prepared by dissolving treosulfan in the respective solvent mixture (30 min, 25°C, ultra-sonic bath) . The obtained solutions were filtered and the filtered solutions were filled in cleaned and depyrogenized glass vials (10 vials per formulation) which were stoppered in lyophili zation position and sealed in lyophilization bags.

Composition of solution for lyophilisation, target dose 500 mg treosulfan per vial

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

Lyophilization cycle

For reconstitution testing, the vials were vented and opened and 10 ml of 0.45 % by weight aqueous NaCl solution (room temperature) were added using a 10 ml volumetric pipette. The lyophilisate cakes of both examples 1 and 2 reconstituted within 1 min only. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vial was also not necessary. For all lyophilisates , only a very low amount of residual water was determined. Moreover, all samples were free of impurities and showed a similar and high treosulfan content . The acetic acid content was below the detection limit (LOD) of the RP-HPLC analysis of 0.003 % by weight.

Properties of lyophilisates

Example 3 - Solution with 10 % by weight of acetic acid and lyophilisate

The solution as given in the table below was prepared by weighing 10 g of treosulfan in a 150 ml polypropylene (PP) beaker. The solvent was added and the treosulfan was dissolved under stirring at an ambient temperature of 22 °C. The obtained solution was filled into cleaned and depyrogenized glass vials of a nominal volume of 20 ml.

Composition of solution for lyophilisation, target dose about 1000 mg treosulfan per vial

The vials were stoppered in lyophilization position and sealed in lyophilisation bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle

"atm." means atmospheric pressure The obtained lyophilisate cakes were acceptable. For reconstitution testing, the vials were vented and opened and 20 ml of 0.45 % by weight aqueous NaCl solution (about 22°C) were added. The lyophilisate cakes reconstituted within 1.5 min. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vials was also not necessary.

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

Properties of lyophilisates

Solution with 10 % by weight of acetic acid and lyophilisate

The calculated amount of a solvent mixture of 90 % by weight water and 10 % by weight acetic acid was weighed into a 250 ml glass bottle and the bottle was placed in a water bath kept at 50°C. 12 g treosulfan were added to give a final concentration of treosulfan of 125 mg/g. The bottle was left in the water bath for 30 minutes with regular shaking. The obtained solution was filed into cleaned and depyrogenized glass vials of a nominal volume of 20 ml. Composition of solution for lyophilization, target dose lOOOmg treosulfan per vial

The vials were stoppered in lyophilization position and sealed in lyophilization bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.

Lyophilization cycle

The obtained lyophilisate cakes were acceptable. For reconstitution testing, the vials were vented, opened and 20 ml of 0.45 % by weight aqueous NaCl solution (room temperature) were added using a multistep pipette. The lyophilisate cakes reconstituted within 1.5 minutes under shaking. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vials was also not necessary.

All lyophilisates showed a very high amount of treosulfan and a very low amount of residual water and acetic acid.

Properties of lyophilisates