Concentrated solution comprising sodium 5, 10-methylene-(6R)-tetrahydrofolate

Abstract

The present invention relates to liquid compositions comprising high levels of sodium salts and sulfates of 5, 10-methylene-(6R)-tetrahydrofolate, the formulations and lyophilisate thereof being free of any foreign stabilizers.

Description

Technical Field

[0001] The present invention relates to a liquid composition containing high content of sodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na) and sulfate, wherein the formulation and lyophilizate thereof do not contain any external stabilizer. 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-methylene-(6R,S)-tetrahydrofolate is assumed to be similar to that of the pure diastereoisomer 5,10-CH2-(6R)-THF of the present invention. This study compared the stability of unformulated 5,10-methylene-(6R,S)-tetrahydrofolate with 5,10-methylene-(6R,S)-tetrahydrofolate formulated with trisodium citrate alone or with both ascorbic acid and trisodium citrate; both compounds are well-known antioxidants (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 stable solutions and lyophilisates of 5,10-CH2-(6R)-THF having a high content of active ingredients and not containing any type of stabilizer would be advantageous.

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

[0014] Surprisingly, it has now been found that aqueous solutions can be prepared which contain a high content of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (hereinafter denoted as 5,10-CH2-(6R)-THF*Na) in combination with greater than 40 mol-%, preferably from about 40 mol-% to 200 mol-%, even more preferably from about 50 mol-% to about 100 mol-% of an inorganic, water-soluble sulfate salt, such as an alkali metal sulfate or alkali metal hydrogensulfate (hereinafter simply referred to as "sulfate" or "alkali metal sulfate").

[0015] The obtained solution contains more than 60 mg 5,10-CH2-(6R)-THF*Na per milliliter, such as more than 65 mg / ml, more than 70 mg / ml, more than 75 mg / ml, such as preferably at least 80 mg 5,10-CH2-(6R)-THF*Na per milliliter. Solutions with higher concentrations can be prepared, but they become very viscous. In the obtained solution, 5,10-CH2-(6R)-THF*Na accounts for more than about 60% w / w (or more than 15 mol-%) of the solid material, preferably more than 80% w / w (or more than 30 mol-%).

[0016] The solutions of the invention can be further converted into stable lyophilisates containing 5,10-CH2-(6R)-THF*Na and sulfate in high yields, which do not contain any stabilizing agents. These lyophilisates were found to have surprisingly high stability, thereby overcoming the known disadvantages discussed previously and allowing the preparation of stable solid pharmaceutical compositions of high purity and low content of oxidation products or other chemical degradation products.

[0017] The favorable stability and concentration characteristics of the concentrated aqueous solutions of the present invention will allow for effective and safe use in medical applications.

[0018] In a first aspect, the present invention relates to a concentrated aqueous solution comprising the sodium salt of 5,10-CH2-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na) and an alkali metal sulfate, the concentrated aqueous solution additionally not containing any stabilizing agents, such as buffers, reducing agents, etc., as defined herein.

[0019] A second aspect of the present invention relates to a method for preparing a concentrated aqueous solution according to the first aspect, the process comprising the following steps:

[0020] i. Dissolve (6S)-tetrahydrofolate in aqueous NaOH solution,

[0021] ii. Adjust the pH of the solution to 8.6 ± 0.5,

[0022] iii. Add 100-120 mol% formaldehyde,

[0023] iv. stirring the reaction mixture until the reaction is complete,

[0024] v. adding a solution of an alkali metal sulfate, and

[0025] vi. Filter the reaction mixture to obtain a clear solution of 5,10-CH2-(6R)-THF*Na and alkali metal sulfate.

[0026] In a third aspect, the present invention further relates to the concentrated aqueous solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, 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 concentrated aqueous solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, to a human patient in need thereof.

[0028] In a fifth aspect, the present invention further relates to the use of the concentrated aqueous solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, for the preparation of a medicament for treating cancer in a human patient.

[0029] The concentrated aqueous solution of the first aspect comprises sodium and sulfate salts of 5,10-methylene-(6R)-tetrahydrofolate, has high purity, and maintains chemical stability for at least 7 hours at 5±3°C or at least 3 hours at room temperature, even without purging the solution with nitrogen to minimize degradation by oxidation. Figure 4 The 75 mg / mL highly concentrated solution was clear and remained clear whether it was stored at 2-8°C or at RT, ie, no precipitation occurred. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Table 2 is adapted from WO 2007 / 064968 and demonstrates the stability (% normalized purity) of non-formulated and various formulated forms of 5,10-methylene-(6R,S)-tetrahydrofolate over time. It can be seen that each formulation has different stability characteristics. Thus, non-formulated 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 initial 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 4 The 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] definition

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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

[0041] It has surprisingly been found that aqueous solutions comprising the sodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (expressed as 5,10-CH2-(6R)-THF*Na) and high contents of about 40-200 mol-% of alkali metal sulfate are very stable.

[0042] The solution of the present invention contains more than 60 mg 5,10-CH2-(6R)-THF*Na per ml, such as more than 65 mg / ml, more than 70 mg / ml, more than 75 mg / ml, such as preferably at least 80 mg 5,10-CH2-(6R)-THF*Na per ml. Solutions with higher concentrations can be prepared, but they become very viscous.

[0043] As disclosed above, the highly concentrated solutions according to the present invention are aqueous compositions comprising 5,10-CH2-(6R)-THF*Na and alkali metal sulfate. These compositions have high purity and remain chemically stable at 5±3°C for at least 7 hours or at room temperature for at least 3 hours even without purging the solution with nitrogen to minimize degradation due to oxidation (see Figure 4 ). The 75 mg / mL highly concentrated solution was clear and remained clear whether it was stored at 2-8°C or at RT, ie, no precipitation occurred.

[0044] The aqueous solution according to the present invention can be filled in a container and freeze-dried (lyophilized) into a stable, non-viscous lyophilized powder and stored. The lyophilized material can be reconstituted with a diluent to a set concentration for administration. Alternatively, the aqueous solution can be produced in a "ready-to-use" concentration and filled in a container such as a vial or ampoule. Such a solution or reconstituted lyophilized material can be administered intramuscularly or intravenously.

[0045] The solutions of the invention may contain additional excipients. Bulking agents such as mannitol may be added to the solution prior to the freeze-drying process to facilitate the formation of an acceptable lyophilized cake.

[0046] Also, electrolytes, sugars and / or polyols, such as glucose, glycerol, mannitol and sodium chloride, may be added to the aqueous solution of the present invention to adjust the osmolality.

[0047] 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.

[0048] For longer term storage it may be advantageous to lyophilize the highly concentrated solutions of the invention.

[0049] 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.

[0050] 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.

[0051] In view of the technology described above, the high stability observed for concentrated solutions of 5,10-CH2-(6R)-THF*Na in combination with alkali metal sulfates, where the presence of a stabilizer such as citric acid should be mandatory, is very surprising. Figure 4 and Figure 1-3 The comparison strongly suggests that the high content solutions of the present invention have a CoFactor of approximately 1.000 wt% compared to the ascorbate / citrate stable solutions discussed in particular in WO 2007 / 064968. ® Similar or better stability of the composition.

[0052] In a first aspect, the present invention relates to a concentrated aqueous solution comprising the sodium salt of 5,10-CH2-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na) and an alkali metal sulfate, the concentrated aqueous solution additionally not containing any stabilizing agents, such as buffers, reducing agents, etc., as defined herein.

[0053] The concentrated aqueous solutions of the present invention are preferably reconstituted by dilution into an aqueous pharmaceutical formulation to be administered to a patient in need thereof.

[0054] In one embodiment, the present invention discloses a concentrated aqueous solution according to the first aspect, wherein the molar ratio of alkali metal sulfate:5,10-CH2-(6R)-THF is about 0.4:1 to about 1:2, preferably about 0.5:1 to about 1:1.

[0055] A second aspect of the present invention relates to a method for preparing an aqueous solution comprising a sodium salt of 5,10-CH2-(6R)-tetrahydrofolate and an alkali metal sulfate, the method comprising the following steps:

[0056] i. Dissolve (6S)-tetrahydrofolate in aqueous NaOH solution,

[0057] ii. Adjust the pH of the solution to 8.6 ± 0.5,

[0058] iii. Add 100-120 mol% formaldehyde,

[0059] iv. stirring the reaction mixture until the reaction is complete,

[0060] v. Add a solution of alkali metal sulfate,

[0061] vi. Filter the reaction mixture to obtain a clear solution of 5,10-CH2-(6R)-THF*Na and alkali metal sulfate.

[0062] 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).

[0063] In a preferred embodiment of the second aspect, alkali metal sulfate is added in step v. until the final ratio of alkali metal sulfate:5,10-CH2-(6R)-tetrahydrofolate is from about 0.4:1 to about 1:2.

[0064] In a preferred embodiment of the second aspect, about 110 mol % formaldehyde is used.

[0065] In another preferred embodiment, the alkali metal sulfate added in step v. is sodium sulfate.

[0066] 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.

[0067] In a third aspect, the present invention further relates to the concentrated aqueous solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, for use in the treatment of cancer or in cancer therapy in a human patient.

[0068] 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 concentrated aqueous solution according to the first aspect, or a reconstituted or diluted solution thereof, to a human patient in need thereof.

[0069] In a preferred embodiment, the present invention relates to a method of treating cancer in a human patient, the method comprising administering the concentrated aqueous solution according to the first aspect, or a reconstituted or diluted solution thereof, to a human patient in need thereof.

[0070] In a sixth aspect, the present invention further relates to the use of a composition comprising 5,10-CH2-(6R)-THF*Na according to the first aspect and an alkali metal sulfate, or a reconstituted or diluted aqueous solution thereof, for the preparation of a medicament for treating cancer in a human patient.

[0071] Therefore, another aspect relates to a reconstituted pharmaceutical composition of the concentrated aqueous solution of the present invention, which comprises 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 drugs 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

[0072] HPLC

[0073] 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.

[0074] Water content

[0075] The determination of the water content was carried out according to Ph. Eur. 2.5.32 / USP <921 / Method Ic>.

[0076] Osmolality

[0077] According to Ph. Eur. 2.2.35 (Osmometer) / USP <785> The osmolality was determined.

[0078] Example 1: Preparation of a concentrated aqueous solution containing sulfate and sodium 5,10-methylene-(6R)-tetrahydrofolate

[0079] (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.

[0080] (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.

[0081] (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.

[0082] 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.

[0083] Example 2a: Preparation of 5,10-methylene-(6R)-tetrahydrofolate solution with sulfate

[0084] 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.

[0085] Example 2b: Preparation of 5,10-methylene-(6R)-tetrahydrofolate solution with sulfate

[0086] 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.

[0087] Example 3: Preparation of lyophilisate without stabilizer

[0088] 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

[0089] 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.

[0090] 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.

[0091] Table 1: Impurity analysis (major degradation products) in solutions containing sodium 5,10-methylene-(6R)-tetrahydrofolate and sulfate

Claims

1. A concentrated aqueous solution comprising the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate and an alkali metal sulfate, said concentrated aqueous solution being free of citrate or citric acid or any additional chemotherapeutic agent.

2. The concentrated aqueous solution according to claim 1, which is mainly composed of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate, sodium sulfate, water and an optional osmotic mass molar concentration correction additive.

3. The concentrated aqueous solution of claim 1 or claim 2, 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.

4. The concentrated aqueous solution according to any one of claims 1 to 3, containing more than 60 mg of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate per ml, such as more than 65 mg / ml, more than 70 mg / ml, more than 75 mg / ml, or at least 80 mg of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate per ml.

5. The concentrated aqueous solution according to any one of claims 1 to 4, comprising the sodium salt of 5,10-methylene-(6R)-tetrahydrofolate having a purity of greater than 98%.

6. A reconstituted product obtained by diluting the concentrated aqueous solution according to any one of claims 1 to 5 in water or a liquid pharmaceutically acceptable solvent.

7. The reconstituted product according to claim 6, wherein the water is sterile water for injection.

8. The reconstituted product according to any one of claims 6 or 7, further comprising a pharmaceutically acceptable carrier.

9. The reconstituted product according to any one of claims 6 to 8, further comprising another pharmaceutically acceptable active ingredient.

10. The reconstituted product according to any one of claims 6 to 9, further comprising a buffer and / or one or more osmolality correction excipients.

11. The reconstituted product according to any one of claims 6 to 10, for use in the treatment or cancer therapy of cancer.

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