Stable lyophilisate comprising 5, 10-methylene-(6R)-tetrahydrofolic acid

Abstract

The present invention relates to a stable lyophilisate comprising a high content of 5, 10-methylene-(6R)-tetrahydrofolic acid, the composition being free of any foreign stabilizers or additional chemotherapeutic agents.

Description

Technical Field

[0001] The present invention relates to a stable lyophilisate containing a high content of 5,10-methylene-(6R)-tetrahydrofolate, wherein the composition does not contain any external stabilizer or additional chemotherapeutic agent. Background Art

[0002] 5,10-methylenetetrahydrofolate is known as a drug for solid tumor treatment in combination with 5-fluorouracil (Seley, KL Drugs 4 (1), 99, 2001). The active isomeric form 5,10-methylene-(6R)-tetrahydrofolate (hereinafter referred to as 5,10-CH2-(6R)-THF) achieves its chemotherapeutic effect by inhibiting the enzyme thymidylate synthase (TS) together with the base analog and 5-FU metabolite 5-FdUMP. TS catalyzes the conversion of deoxyuridylic acid (dUMP) to deoxythymidylic acid (dTMP), which is an important building block for DNA synthesis. Inactivation of TS occurs by forming a covalent ternary inhibition complex between TS, the base analog 5-FdUMP and 5,10-CH2-(6R)-THF.

[0003] The enhancement of the cytotoxic effect of 5-FU can be achieved by increasing the intracellular concentration of 5,10-CH2-(6R)-THF, thereby increasing the stability of the ternary inhibitory complex. This leads to a direct inhibition of DNA synthesis and repair, which ultimately leads to cell death and tumor growth delay. In order to obtain high intracellular concentrations of 5,10-CH2-(6R)-THF, it is necessary to apply a corresponding stable high-content product.

[0004] However, there are undesirable properties associated with 5,10-CH2-(6R)-THF that limit its pharmaceutical use. For example, 5,10-CH2-(6R)-THF is highly susceptible to oxidation and chemical degradation, resulting in insufficient stability and undesirably high impurity levels.

[0005] 5,10-Methylenetetrahydrofolate is an addition product of tetrahydrofolate and formaldehyde (see, e.g., Poe, M. et al. Biochemistry 18 (24), 5527, 1979; Kallen, RG Methods in Enzymology 18B, 705, 1971), and is known for its extremely high sensitivity to air oxidation and its instability that may lead to chemical degradation and / or hydrolysis in neutral and / or acidic environments (see, e.g., Odin, E. et al., Cancer Investigation 16 (7), 447, 1998; Osborn, MJ et al., J. Am. Chem. Soc. 82, 4921, 1960; Hawkes, J., and Villota, R. Food Sci. Nutr. 28, 439, 1989). The susceptibility to oxidation, chemical degradation and insufficient stability of 5,10-CH2-(6R)-THF are particularly evident in aqueous solution, or when the compound is present in its amorphous form with a large surface area (e.g., in its pharmaceutical form as a lyophilisate) or in a reconstituted form such as a solution for injection. It is well known that in order to be suitable for pharmaceutical use, the corresponding composition needs to meet several requirements, including high chemical and isomeric stability, so that effective storage can be achieved within an acceptable period of time without showing significant changes in the physicochemical properties of the composition, ease of handling and processing, etc.

[0006] Attempts to stabilize compositions of 5,10-methylenetetrahydrofolate include, for example: (i) strict exclusion of atmospheric oxygen by using special technical devices for reconstitution of solid preparations and injections of 5,10-methylenetetrahydrofolate in an air-free environment (see, for example, Odin, E. et al., Cancer Investigation 16 (7), 447, 1998; U.S. Pat. No. 4,564,054); (ii) addition of reducing agents such as L(+)-ascorbic acid or its salts, reduced γ-glutathione, β-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc. as antioxidants for the highly sensitive 5,10-methylenetetrahydrofolate, and in particular for tetrahydrofolate; (iii) stabilization by means of cyclodextrin inclusion complexes (see, for example, EP 0 579 996); (iv) addition of citrate while adjusting the pH to alkaline values ​​(see, for example, EP 1 641 996); 460); or (v) in various crystalline forms, such as sulfate (see, e.g., EP 0 537 492) or hemisulfate (see, e.g., EP 2 837 631).

[0007] As described above, lyophilisates of 5,10-CH2-(6R)-THF were previously prepared from aqueous solutions which, in addition to the active compound, ie, 5,10-CH2-(6R)-THF, also contained dicarboxylic acids and / or tricarboxylic acids such as citric acid and / or other stabilizers, see, for example, WO2019034673, US 2007 / 0099866 and US10059710 B2. The solutions disclosed therein for the purpose of preparing lyophilisates contain up to 2-3% by weight of 5,10-CH2-(6R)-THF.

[0008] However, both the lyophilisates containing dicarboxylic acids and / or tricarboxylic acids such as citric acid and / or other stabilizers, and the crystalline salt forms of 5,10-methylenetetrahydrofolate are not easily used for pharmaceutical purposes due to their low water solubility, and the stabilized forms of 5,10-methylenetetrahydrofolate known in the art generally contain less than 50% of the active drug compound 5,10-CH2-(6R)-THF due to dilution by stabilizing additives in the final dosage form.

[0009] As an example, Adventrx Pharmaceuticals is developing a novel novel molecule for its drug candidate CoFactor ® Stability studies were conducted on the calcium salt of the diastereoisomer mixture 5,10-methylene-(6R,S)-tetrahydrofolate, in particular ( ia ) is disclosed in WO 2007 / 064968. The chemical stability of the diastereomeric mixture 5,10-CH2-(6R)-THF is assumed to be similar to that of the pure diastereomeric 5,10-CH2-(6R)-THF of the present invention. This study compared the stability of unformulated 5,10-methylene-(6R,S)-tetrahydrofolate with that formulated with trisodium citrate alone or with both ascorbic acid and trisodium citrate; both compounds are well-known reducing agents (see Figure 1 ).

[0010] Linear regression analysis of the stability curves of the isolated lyophilizates showed that the degradation of 5,10-methylene-(6R,S)-tetrahydrofolate was linear over time (see Figure 2 The degradation rate (slope of the best fit line) of each formulation (reconstituted lyophilisate) showed the following order, from fastest to slowest degradation rate: non-formulated > formulated with trisodium citrate only > formulated with both ascorbic acid and trisodium citrate ( Figure 2). Thus, it was found that non-formulated 5,10-methylene-(6R,S)-tetrahydrofolate lost 2.3% of its purity per hour, resulting in a purity of 84% after 7 hours, whereas a formulation containing trisodium citrate + ascorbic acid had a much higher stability, resulting in a purity of about 95% after 7 hours.

[0011] Furthermore, the solution disclosed in WO 2007 / 064968 for the purpose of preparing the most stable lyophilisate contains less than 5% by weight of 5,10-methylene-(6R,S)-tetrahydrofolate, and the resulting lyophilisate contains less than 20% by weight of 5,10-methylene-(6R,S)-tetrahydrofolate (see Figure 3 ).

[0012] Furthermore, stabilizers such as citric acid, which are used in WO 2007 064 968 to prepare the most stable lyophilisates, have been associated with various undesirable effects, such as QT c Prolongation (Laspina et al. Transfusion 42 (2002) p. 899, Toyoshima et al. Clinical Nutrition (2006) 25, 653-660), induction of hypocalcemia (Payne et. Al. J. Physiol. (1964), 170, pp. 613-620), etc. From a clinical point of view, the availability of pharmaceutical dosage forms such as stable solutions and lyophilisates of 5,10-CH2-(6R)-THF having a high content of active ingredient and not containing any type of stabilizer would therefore be advantageous.

[0013] Therefore, there is still a great need for stable pharmaceutical dosage forms with high content of 5,10-methylene-(6R)-tetrahydrofolate. Summary of the invention

[0014] It has now been found that surprisingly stable lyophilisates comprising 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF) can be prepared from a highly concentrated aqueous solution of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na), which solution additionally contains at least 40 mol-%, preferably about 40 mol-% to 200 mol-%, even more preferably about 50 mol-% to about 100 mol-%, of an alkali metal sulfate, but contains no reducing agent or other stabilizing agents.

[0015] Thus, the lyophilisates of the present invention overcome the known disadvantages discussed previously and allow for the preparation of stable solid-state pharmaceutical compositions and dosage forms of high purity and low levels of oxidation products or other chemical degradation products.

[0016] The favorable stability and concentration properties of the lyophilisates of the invention will allow an effective and safe use in medical applications.

[0017] Thus, in a first aspect, the present invention relates to a pharmaceutical dosage form which is a stable lyophilisate, which dosage form comprises 5,10-methylene-(6R)-tetrahydrofolate and does not contain a stabilizer or any additional chemotherapeutic agent.

[0018] A second aspect of the present invention relates to a method for preparing a stable lyophilisate according to the first aspect, the method comprising the following steps:

[0019] i. Dissolve (6S)-tetrahydrofolate in aqueous NaOH solution,

[0020] ii. Adjust the pH of the solution to 8.6 ± 0.5,

[0021] iii. Add 100-120 mol% formaldehyde,

[0022] iv. stirring the reaction mixture until the reaction is complete,

[0023] v. adding a solution of an alkali metal sulfate to a final molar ratio of sulfate:5,10-methylene-(6R)-tetrahydrofolate of about 0.4:1 to about 1:2,

[0024] vi. filtering the reaction mixture to obtain a clear solution of 5,10-CH2-(6R)-THF*Na, and

[0025] vii. Freeze-drying the clear solution obtained in step vi.

[0026] In a third aspect the present invention further relates to a reconstituted solution of the pharmaceutical dosage form according to the first aspect for use in the treatment of cancer or in cancer therapy in a human patient.

[0027] In a fourth aspect, the present invention further relates to a method of treating cancer or cancer therapy in a human patient, the method comprising administering the pharmaceutical dosage form according to the first aspect or a reconstituted solution thereof to a human patient in need thereof.

[0028] In a fifth aspect, the present invention further relates to the use of the pharmaceutical dosage form according to the first aspect, or a reconstituted solution thereof, for the preparation of a medicament for the treatment of cancer in a human patient.

[0029] The lyophilisates of the present invention exhibit stability for more than several months without significant loss of active ingredients, for example, maintaining the amount of active ingredients at or above 95%, and more preferably at or above 98%, including most preferably about 99%, 99.5% or 99.8% over several months. This makes it possible to prepare, store and use the lyophilisates of the present invention without significant decomposition prior to reconstitution. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Adapted from Table 2 in WO 2007 / 064968 and demonstrating the stability (% normalized purity) over time of non-formulated and various formulated forms of 5,10-methylene-(6R,S)-tetrahydrofolate. It can be seen that each formulation has different stability characteristics. Therefore, Unprepared 5,10-methylene-(6R,S)-tetrahydrofolate degrades rapidly over time at neutral pH. 24 hours after dissolution in water, the purity of non-formulated 5,10-methylene-(6R,S)-tetrahydrofolate was only 44.9% of the starting purity. The reference formulation formulated with trisodium citrate alone (adjusted pH>7.5) showed a slower degradation after dissolution in water.

[0031] However, compared to the initial purity, the purity after 24 hours was still only 65%, indicating that degradation was not effectively inhibited by adding trisodium citrate and adjusting pH. The two test formulations #1 and #2 (i.e., 5,10-methylene-(6R,S)-tetrahydrofolate formulated with both ascorbic acid and trisodium citrate) were the most stable formulations (purity of about 89% after 24 hours).

[0032] Figure 2 Adapted from WO 2007 / 064968 Figure 1 , and the diagram confirms the Figure 1 The tabulation results.

[0033] Figure 3 is a table adapted from Example 1 of WO 2007 / 064968 showing the Figure 1 and Figure 2 The compositions of the unformulated and formulated forms of 5,10-methylene-(6R,S)-tetrahydrofolate are shown in FIG.

[0034] Figure 4The purity analysis of four identical solutions of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate of the present invention tested under four different conditions is shown: 5°C without N2 coverage, 5°C with N2 coverage, 4 hours at 5°C followed by 3 hours at room temperature with N2 coverage, and 4 hours at 5°C followed by 3 hours at room temperature without N2 coverage. The results for a total time period of 7 hours are shown. As can be seen from the graph, the solution is very stable under storage conditions, changing from an initial purity between 96.6-97% to a purity (area %) of 96.4-96.5%. It can also be seen that the effect of N2 coverage is minimal.

[0035] Figure 5 As shown in this article Figure 4 Analysis of the same four solutions of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate shown in FIG. Figure 5 In Figure 3, the development of the main impurity 10-formyl-(6R)-tetrahydrofolate (10-FTHFA) in a solution prepared as in Example 3 is shown over a period of 7 hours when stored at 2-8° C. It can be seen that the level of this impurity is practically constant over time.

[0036] Figure 6 The long-term stability of lyophilisates containing 5,10-methylene-(6R)-tetrahydrofolate at 5°C, 25°C and at 40°C is shown. As can be seen from the figure, the lyophilisates are very stable, especially at lower temperatures such as 5°C, where the purity drops to 99.3% within 24 months, or at 25°C where the purity drops to 96.6% within 24 months (relative to the initial purity).

[0037] Figure 7 Shown is the analysis of the main impurity, 10-formyl-(6R)-tetrahydrofolate (10-FTHFA), of lyophilisates containing 5,10-methylene-(6R)-tetrahydrofolate at 5° C., 25° C. and at 40° C. It can be seen that the level of this impurity is practically constant over time.

[0038] definition

[0039] As used herein, the term "sulfate" shall mean an inorganic, water-soluble sulfate salt, such as an alkali metal sulfate or an alkali metal hydrogen sulfate.

[0040] In the present context, the term "buffer" relates to citrate (or citric acid and its salts), dicarboxylates such as succinate, malate and maleate; tris(hydroxymethyl)aminomethane (TRIS); N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); 3-(N-morpholino)propanesulfonic acid (MOPS); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); MES; MOPSO; HEPES; phosphate; carbonate; ammonium; mono-, di- and tri-alkylammonium; mono-, di- and tri-hydroxyalkylammonium; glutamate; borate; lactate, and combinations of these.

[0041] Herein, the term "reducing agent" refers to L-(+) ascorbic acid or a salt thereof, reduced γ-glutathione, β-mercaptoethanol, thioglycerol and N-acetyl-L-cysteine.

[0042] In this article, the term "solvent" refers to a solvent that can be used in a freeze-drying process. "Solution" mentioned herein includes aqueous solutions and solutions in organic solvents. Typically, "aqueous solution" means a solution in water, saline solution, water containing a small amount of buffer, water containing isotonic NaCl, or a mixture of water and an organic solvent, etc. Typical organic solvents include DMSO, acetonitrile, acetone, methanol or ethanol. DETAILED DESCRIPTION

[0043] As previously mentioned, it has been discovered that surprisingly stable lyophilisates comprising 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF) can be prepared from a highly concentrated aqueous solution of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na), which solution additionally contains at least 40 mol-%, preferably about 40 mol-% to 200 mol-%, even more preferably about 50 mol-% to about 100 mol-%, of an alkali metal sulfate, but contains no antioxidants or other stabilizing agents.

[0044] As discussed above, the highly concentrated solutions used to prepare the lyophilisates of the present invention comprise 5,10-CH2-(6R)-THF*Na and an alkali metal sulfate. These solutions are of high purity and are advantageous from a manufacturing perspective because they maintain chemical stability for at least 7 hours at 5±3°C or at least 3 hours at room temperature (see Figure 4 ). For example, the 75 mg / mL solution was clear and remained clear whether it was stored at 2-8°C or at RT, i.e., no precipitation occurred.

[0045] The pH of the solution is typically in the range of 8.0 to 9.0, preferably in the range of 8.4 to 8.8, and can be adjusted during drug preparation with, for example, small amounts of hydrochloric acid or sodium hydroxide.

[0046] According to the present invention, the highly concentrated solution discussed above can be freeze-dried (lyophilized) into a stable, non-sticky powder and stored. The lyophilized powder can be reconstituted with a diluent to a set concentration for administration. The reconstituted lyophilized material can be administered intramuscularly or intravenously.

[0047] Prior to the freeze drying process, a bulking agent such as mannitol may be added to the concentrated solution to facilitate the formation of an acceptable lyophilized cake. Other excipients may be added if desired.

[0048] In addition, electrolytes, sugars and / or polyols, such as glucose, glycerol, mannitol and sodium chloride, can be added to adjust the osmotic mass molar concentration. The osmotic mass molar concentration adjustment can be completed before (i.e., concentrated solution) or after the reconstruction of the lyophilized product of the present invention. The reconstructed lyophilized product solution preferably has an osmotic mass molar concentration within the range of 250-350mOsm. However, the osmotic mass molar concentration of 200-600mOsm is also permissible and will depend on the volume to be administered and the injection / infusion time.

[0049] Lyophilization or freeze drying is a dehydration process that works by freezing an aqueous solution containing dissolved substances and then reducing the surrounding pressure, causing the frozen water to sublime directly from the solid phase to the gas phase. There are usually four stages in the complete freeze drying process: pretreatment, freezing, primary drying and secondary drying.

[0050] Pretreatment includes any method of treating a substance before freezing. This may include the addition of other components. In the preparation of the stable lyophilisate of the present invention, pretreatment is possible, but not necessary.

[0051] Usually freeze is completed by placing the aqueous solution of material in a freeze drying bottle, which is cooled by mechanical refrigeration or by using dry ice or liquid nitrogen. On a larger scale, a freeze dryer is usually used to freeze the aqueous solution. In this step, it is important that the material is cooled to below its triple point, which is the lowest temperature at which the solid phase and liquid phase of the material can coexist. This ensures that sublimation will occur rather than melting in subsequent steps. In the preparation of stable lyophilized material of the present invention, freezing is preferably completed at a temperature of -45 ° C to -70 ° C.

[0052] In the preparation of the stable lyophilisates of the present invention, annealing at a shelf temperature of about -5°C to -2°C for 1 to 2 hours is possible, but not necessary.

[0053] In the primary drying stage, the pressure is reduced (to the scope of several millibars), and enough heat is provided to the material for ice sublimation. In this initial drying stage, about 95% of the water in the material is sublimated. This stage can be slow (can be several days in industry), because if too much heat is added, the structure of the material may be changed. In the primary drying stage, the pressure is controlled by applying a partial vacuum. Vacuum accelerates sublimation, so that it can be used as an unhurried drying process. In the preparation of stable lyophilized product of the present invention, the primary drying stage starts at a freezing temperature preferably between -45 ° C to -70 ° C.

[0054] Then during the primary drying phase, after an optional initial period of preferably 10 minutes to 120 minutes at freezing temperature, the temperature is increased over time to preferably about 0° C. During the primary drying phase, a pressure of preferably about 50 μbar to 200 μbar is maintained.

[0055] The secondary drying stage is intended to remove the unfrozen water molecules, as the ice is removed in the primary drying stage. In this stage, the temperature is raised above the temperature in the primary drying stage, and can even be above 0°C, in order to break any physicochemical interactions that have formed between the water molecules and the frozen material. Usually, the pressure is also reduced in this stage to facilitate desorption (typically in the microbar range, or a few fractions of a Pascal). In the preparation of the stable lyophilisate of the present invention, the secondary drying is preferably performed at a temperature of up to about 25°C to 30°C and a pressure of about 50 μbar to 200 μbar.

[0056] In the preparation of the stable lyophilisate of the present invention, the primary and secondary drying stages can be combined by following a temperature ramp from freezing temperature to a maximum of about 25°C to 30°C and a pressure ramp of about 50 μbar to 200 μbar. The temperature ramp may contain a plurality of holding steps, in which the temperature is kept constant for a period of time. Preferably, the holding steps, if any, are at freezing temperature, at about 0°C and about 25°C to 30°C.

[0057] After the freeze drying process is complete, the vacuum is usually broken with an inert gas such as nitrogen before sealing the material. At the end of the operation, the final residual moisture content of the lyophilized material of the present invention is usually less than 5%, preferably at most 3%, even more preferably at most 2%, and most preferably less than 1%. In a particularly preferred embodiment, the lyophilized material of the present invention is substantially anhydrous.

[0058] Stability is a key property and component of pharmaceutical formulation research and drug development. Stability studies are conducted in both solution and solid states. It is an established fact that solution state and solid state stability may differ qualitatively and quantitatively. By exposing the bulk drug and its pharmaceutical composition to various stressors such as high temperature and / or high humidity, the stability of the bulk drug and its pharmaceutical composition has been extensively studied. These studies also provide information about degradation products and contribute to the development of meaningful specifications and the inherent stability of the pharmaceutical composition. The most common pathways of drug degradation include hydrolysis, oxidation and photochemical degradation in particular.

[0059] The purpose of stability testing is to provide evidence on how the quality of a product changes over time under the influence of various environmental factors such as temperature, humidity and light, and to determine the appropriate shelf life and recommended storage conditions for the drug product to ensure patient safety.

[0060] In view of the above-mentioned technology, the high stability observed for the lyophilisate obtained according to the present invention (see Figure 6 and Figure 7 ) is very surprising, where the presence of a stabilizer such as citric acid should be mandatory. Figure 4 and Figure 1-3 The comparison strongly suggests that the high content solution used to prepare the lyophilisate of the present invention has a CoFactor of 1.50% to that of the ascorbate / citrate solution discussed in particular in WO2007 / 064968. ® Similar or better stability of the composition.

[0061] Thus, in a first aspect, the present invention relates to a pharmaceutical dosage form which is a stable lyophilisate, which dosage form comprises 5,10-methylene-(6R)-tetrahydrofolate and does not contain a stabilizer or any additional chemotherapeutic agent.

[0062] In a preferred embodiment, the pharmaceutical dosage form of the present invention comprises the sodium salt of 5,10-CH2-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na) and the alkali metal sulfate in a molar ratio of about 0.4:1 to about 1:2.

[0063] In one embodiment, the invention relates to a pharmaceutical dosage form according to the first aspect, wherein the molar ratio of alkali metal sulfate:5,10-CH2-(6R)-THF is from about 0.4:1 to about 1:2, preferably from about 0.5:1 to about 1:1.

[0064] The lyophilisate of the present invention is substantially amorphous and has enhanced stability, such as enhanced storage stability.

[0065] A second aspect of the present invention relates to a method for preparing a stable lyophilisate according to the first aspect, the method comprising the following steps:

[0066] i. Dissolve (6S)-tetrahydrofolate in aqueous NaOH solution,

[0067] ii. Adjust the pH of the solution to 8.6 ± 0.5,

[0068] iii. Add 100-120 mol% formaldehyde,

[0069] iv. stirring the reaction mixture until the reaction is complete,

[0070] v. adding a solution of an alkali metal sulfate to a final molar ratio of sulfate:5,10-methylene-(6R)-tetrahydrofolate of about 0.4:1 to about 1:2,

[0071] vi. filtering the reaction mixture to obtain a clear solution of 5,10-CH2-(6R)-THF*Na, and

[0072] vii. Freeze-drying the clear solution obtained in step vi.

[0073] The reaction between (6S)-tetrahydrofolate and formaldehyde is quantitative, but it is recommended to use a slight excess of formaldehyde to ensure that the reaction proceeds to completion. Excessive formaldehyde should be avoided as it leads to elevated impurity levels (see Examples 2a and 2b herein).

[0074] In a preferred embodiment of the third aspect, about 110 mol % formaldehyde is used.

[0075] In another preferred embodiment, the alkali metal sulfate added in step v. is sodium sulfate.

[0076] Once the solution of 5,10-CH2-(6R)-THF*Na has been produced, ie from steps iv.-v., the temperature of the reaction mixture should be kept low, preferably about 0-5°C.

[0077] In embodiments, the lyophilisate according to the first aspect may contain up to about 80% w / w 5,10-CH2-(6R)-THF*Na.

[0078] In a third aspect, the present invention further relates to a reconstituted solution of the pharmaceutical dosage form according to the first aspect for use in the treatment of cancer or in cancer therapy in a human patient.

[0079] In a preferred embodiment, the lyophilisate of the present invention is reconstituted into an aqueous pharmaceutical formulation for administration to a patient in need thereof.

[0080] In a fourth aspect, the present invention further relates to a method of treating cancer or cancer therapy in a human patient, the method comprising administering the pharmaceutical dosage form according to the first aspect or a reconstituted solution thereof to a human patient in need thereof.

[0081] In a fifth aspect, the present invention further relates to the use of the pharmaceutical dosage form according to the first aspect, or a reconstituted solution thereof, for the preparation of a medicament for the treatment of cancer in a human patient.

[0082] A further aspect relates to a reconstituted solution of the pharmaceutical dosage form according to the first aspect, comprising 5,10-CH2-(6R)-THF*Na, an alkali metal sulfate and a pharmaceutically acceptable carrier or diluent, such as sterile water or a liquid pharmaceutically acceptable solvent, optionally further comprising at least one additional therapeutic agent, including but not limited to bactericides, antibiotics, antivirals, preservatives, antineoplastic agents, anticancer compounds, such as chemotherapeutic agents, antifungal agents and / or anti-inflammatory agents or other biologically active or therapeutic agents suitable for human use, in particular anticancer compounds, such as chemotherapeutic agents, for example 5-FU and its derivatives, and folic acid antagonists, for example methotrexate, pemetrexed. Example

[0083] HPLC

[0084] To measure purity / content and degradation products, an HPLC-UV gradient method was used: Column type: ODS, Mobile phase: A: aqueous buffer; Mobile phase: B: aqueous buffer / methanol, Run time: 30 min, Sample solvent: aqueous buffer.

[0085] Water content

[0086] The determination of the water content was carried out according to Ph. Eur. 2.5.32 / USP <921 / Method Ic>.

[0087] Osmolality

[0088] According to Ph. Eur. 2.2.35 (Osmometer) / USP <785> The osmolality was determined.

[0089] Example 1: Preparation of a concentrated aqueous solution containing sulfate and sodium 5,10-methylene-(6R)-tetrahydrofolate

[0090] (a) 7.93 g (16 mmol) (6S)-tetrahydrofolate and 78.0 g distilled water were provided in a round bottom flask at room temperature under N2. The resulting suspension was stirred and the pH was adjusted to pH 11 by slowly adding 32% NaOH solution. As soon as the solution became clear, 1 M HCl solution was gradually added at 25°C to adjust the pH of the reaction mixture to 8.3. The clear solution obtained was cooled to about 0°C, at which temperature it showed a pH of 8.8. The pH was again adjusted to pH = 8.6 with 1 M HCl, and 1.44 g of 36.8% HCHO solution (110 mol %) was added in one go. After the addition was complete, the solution was stirred at 0°C (ice bath) for 1 hour. Activated carbon (0.2 g, Norit C Extra) was added, and the reaction mixture was stirred at 0° C. for 30 minutes and then cold filtered through a suction filter to obtain a clear solution of 5,10-CH 2 -(6R)-THF*Na, which was used in step (b) without further purification.

[0091] (b) An ice-cold solution of 2.8 grams of Na2SO4 (20 mmol, 1.25 mol%) in 15 ml of distilled water is added to the solution obtained in step (a). The pH is then adjusted to 9.3 ± 0.1 with 1 M NaOH, and the reaction mixture obtained is stirred at 0°C under N2 for 2 hours. Activated carbon (0.2 g, Norit C Extra) is added, and the reaction mixture is stirred at 0°C for 30 minutes, then cold filtered through a suction filter and then sterile filtered through a 0.22 μm filter to obtain a clear solution of an approximately 1:1 molar composition of 5,10-CH2-(6R)-THF*Na and sodium sulfate. The solution contains about 8 grams of 5,10-CH2-(6R)-THF*Na / 100 ml, i.e., a concentration of about 80 mg / ml, which is equivalent to about 7.3 grams of 5,10-CH2-(6R)-THF free acid in 100 ml. The solution should be maintained at 2-8°C.

[0092] (c) Cool the solution from step (b) to 2-8°C and pass it through a 0.22 μm filter while keeping the solution as cold as possible. Fill the filtered solution into glass vials (2 ml or 160 mg 5,10-CH2-(6R)-THF*Na / vial) while keeping the solution as cold as possible.

[0093] The effect of excess formaldehyde on product quality was analyzed in the two following examples, which were performed identically except for the excess formaldehyde. In Example 2a, 110 mol % formaldehyde was used, while in Example 2b, 200 mol % formaldehyde was used. Using 110 mol % formaldehyde in Example 2a provided the purest product.

[0094] Example 2a: Preparation of 5,10-methylene-(6R)-tetrahydrofolate solution with sulfate

[0095] 4.72 g (6S)-tetrahydrofolate was added to 220 ml of water containing 10 g NaOH 2M (initial pH 13.74) under nitrogen. The pH was maintained at 9.3 ± 0.1 until complete dissolution with 22.8 g NaOH 2M. 0.901 g of 36.8% HCHO solution (110 mol%) was then added. The solution was stirred for 30 minutes. An ice-cold solution of 4.5 grams of Na2SO4 (20 mmol, 1.25 mol%) in 15 ml of distilled water was added to the solution, after which the pH was again adjusted to 9.3 with 2M aqueous sodium hydroxide solution (0.05 g). The solution thus obtained contained 5,10-methylene-(6R)-tetrahydrofolate and sulfate with a purity of 94.8% area.

[0096] Example 2b: Preparation of 5,10-methylene-(6R)-tetrahydrofolate solution with sulfate

[0097] 4.72 g (6S)-tetrahydrofolate was added to 220 ml of water containing 10 g NaOH 2M (initial pH 13.83) under nitrogen. The pH was maintained at 9.3 ± 0.1 until complete dissolution with 22.8 g NaOH 2M. 1.639 g formaldehyde solution (36.76%) (200 mol%) was then added. The solution was stirred for 30 minutes. An ice-cold solution of 4.5 grams of Na2SO4 (20 mmol, 1.25 mol%) in 15 ml of distilled water was added to the solution, after which the pH was again adjusted to 9.3 with 2M aqueous sodium hydroxide solution. The solution thus obtained contained 5,10-methylene-(6R)-tetrahydrofolate and sulfate with a purity of 91.5% area.

[0098] Example 3: Preparation of lyophilisate without stabilizer

[0099] The filtered solution from Example 1 was filled into vials (2 ml or 150 mg 5,10-CH2-(6R)-THF / vial) at a temperature of 2-8°C, while keeping the solution as cold as possible. The vials were freeze dried and sealed with nitrogen in the head space under a slight vacuum. The vials were compacted. The resulting lyophilizate contained 70-80% w / w 5,10-CH2-(6R)-THF. Example 4: Stability Test

[0100] The stability of the solution produced in Example 1, step c, was tested under four different conditions: 7 hours at 5°C without N2 cover, 7 hours at 5°C with N2 cover, 4 hours at 5°C followed by 3 hours at room temperature with N2 cover, and 4 hours at 5°C followed by 3 hours at room temperature without N2 cover. The results are shown in Figure 4 As can be seen from the figure, the solution is very stable under storage conditions, changing from an initial purity between 96.6-97% to a purity (area %) of 96.4 – 96.5%. Figure 4 It can also be seen that the effect of N2 coverage is minimal.

[0101] As part of the stability analysis, the development of the main impurity 10-formyl-(6R)-tetrahydrofolate (10-FTHFA) in solution as produced in step c of Example 1 was also measured over 7 hours when stored at 2-8°C (see Tables 1 and Figure 5 ). It can be seen that the level of this impurity is virtually constant.

[0102] Table 1: Impurity analysis (major degradation products) in solutions containing sodium 5,10-methylene-(6R)-tetrahydrofolate and sulfate

[0103]

[0104] Example 5: Stability test (lyophilisate)

[0105] In order to determine the long-term stability of the lyophilisate of 5,10-CH2-(6R)-THF prepared according to Example 3, the lyophilisate was stored in air at +5°C, +25°C / 60% relative humidity and +40°C / 75% relative humidity. The residual 5,10-CH2-(6R)-THF content was measured at periodic intervals by HPLC and compared with the initial value (% relative). The results are shown in Table 2 and Figure 6 shown.

[0106] Table 2: Long-term stability of the lyophilisate containing 5,10-methylene-(6R)-tetrahydrofolate prepared according to Example 3

[0107]

[0108] Table 2 and Figure 6 It is clearly shown that the lyophilizate of 5,10-CH2-(6R)-THF is highly stable over a long period of time, especially at lower temperatures such as 5°C.

[0109] To determine the long-term stability of the lyophilisate of (6R)-5,10-CH2-THF prepared according to Example 3, the lyophilisate was stored in air at +5°C, +25°C / 60% relative humidity and +40°C / 75% relative humidity. The content of 10-formyltetrahydrofolate (10-FTHFA), one of the main degradation products, was measured at periodic intervals by HPLC. The results are shown in Tables 3 and Figure 7 shown.

[0110] Table 3: Long-term stability of the lyophilisate containing 5,10-methylene-(6R)-tetrahydrofolate prepared according to Example 3

[0111]

[0112] Table 3 and Figure 7 It was demonstrated that the lyophilisate of 5,10-CH2-(6R)-THF was highly stable over a long period of time, especially at lower temperatures such as 5°C, as evidenced by the very slow increase in the content of one of the major degradation products, 10-formyltetrahydrofolate (10-FTHFA).

Claims

1. A pharmaceutical dosage form which is a stable lyophilisate comprising 5,10-methylene-(6R)-tetrahydrofolate and does not contain a stabilizer or any additional chemotherapeutic agent.

2. A stable lyophilisate according to claim 1, wherein the excluded stabilizer is selected from the group consisting of: citrate (or citric acid and its salts); tris(hydroxymethyl)aminomethane (TRIS); N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); 3-(N-morpholino)propanesulfonic acid (MOPS); N,N-bis(2-hydroxy-ethyl)-2-aminoethanesulfonic acid (BES); MES; MOPSO; HEPES; phosphate; acetate; succinate; carbonate; ammonium; monoalkylammonium, dialkylammonium and trialkylammonium; monohydroxyalkylammonium, dihydroxyalkylammonium and trihydroxyalkylammonium; maleate; glutamate; borate; lactate; L-(+) ascorbic acid or its salts; reduced γ-glutathione; β-mercaptoethanol; thioglycerol; N-acetyl-L-cysteine, and combinations of these. 3 . The stable lyophilisate according to claim 1 , comprising a sodium salt and an alkali metal sulfate of 5,10-methylene-(6R)-tetrahydrofolate.

4. The stable lyophilisate according to claim 2 or 3, which is mainly composed of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate, sodium sulfate, water and an optional osmolality correction additive.

5. The stable lyophilisate according to claims 2 to 4, wherein the molar ratio of alkali metal sulfate:sodium salt of 5,10-methylene-(6R)-tetrahydrofolate is from about 0.4:1.0 to about 1:

2.

6. The stable lyophilisate according to any one of claims 1 to 5, wherein the concentration of 5,10-methylene-(6R)-tetrahydrofolate in the lyophilisate is at least 60% w / w, at least 65% w / w, or at least 70% w / w, such as between 70-75% w / w.

7. The stable lyophilisate according to claim 6, having a water content of less than 5%, preferably at most 3%, even more preferably at most 2%, and most preferably less than 1%.

8. The stable lyophilisate according to claim 7, which is anhydrous. 9 . The stable lyophilisate according to claim 6 , comprising 5,10-methylene-(6R)-tetrahydrofolate with a purity of greater than 98%.

10. A reconstituted product obtained by dissolving or diluting the stable lyophilisate according to any one of claims 1 to 9 in water or a liquid pharmaceutically acceptable solvent.

11. The reconstituted product according to claim 10, wherein the water is sterile water for injection.

12. The reconstituted product according to any one of claims 10 or 11, further comprising a pharmaceutically acceptable carrier.

13. The reconstituted product according to any one of claims 10 to 12, further comprising an additional pharmaceutically acceptable active ingredient.

14. The reconstituted product according to any one of claims 10 to 13, further comprising a buffer and / or one or more osmolality correction excipients.

15. The reconstituted product according to any one of claims 10 to 14, for use in the treatment or cancer therapy of cancer.

Images