Composition comprising disodium 5, 10-methylene-(6R)-tetrahydrofolate

Abstract

The present invention relates to stable formulations and freeze-dried products comprising high levels of disodium, sulphate and citrate salts of 5, 10-methylene-(6R)-tetrahydrofolate.

Description

[0001] The present invention relates to stable formulations and freeze-dried products containing high contents of disodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na2), sulfate and citrate. Background of the Invention

[0003] 5,10-methylenetetrahydrofolate is known to be an agent used in combination with 5-fluorouracil (5-FU) to treat solid tumors (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 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 a basic building block for DNA synthesis. The inactivation of TS occurs by forming a covalent ternary inhibition complex between TS, the base analog 5-FdUMP (which is a metabolite of 5-FU) and 5,10-CH2-(6R)-THF.

[0004] 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 delayed tumor growth. In order to achieve high intracellular concentrations of 5,10-CH2-(6R)-THF, it is desirable to use a corresponding stable, high-content product.

[0005] However, undesirable properties associated with 5,10-CH2-(6R)-THF limit its pharmaceutical use. For example, 5,10-CH2-(6R)-THF is highly susceptible to oxidation and chemical degradation resulting in undesirably high impurity levels.

[0006] 5,10-CH2-(6R)-THF is particularly susceptible to oxidation and chemical degradation in aqueous solution, or when the compound is in its amorphous form with a large surface area (e.g., in its pharmaceutical form as a lyophilizate), or in a reconstituted form (e.g., a solution for injection). As is well known, in order to be suitable for pharmaceutical use, the corresponding composition needs to meet a number of requirements, including high chemical and isomeric stability (so that effective storage can be achieved within an acceptable period of time without exhibiting significant changes in the physicochemical characteristics of the composition), ease of handling and processing, etc.

[0007] 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 extreme sensitivity to air oxidation and instability in neutral and / or acidic environments (potentially leading to chemical degradation and / or hydrolysis) (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).

[0008] Attempts to stabilize compositions of 5,10-methylenetetrahydrofolate have included, for example, (i) strict exclusion of atmospheric oxygen by using special technical equipment for reconstitution of solid formulations and injection of 5,10-methylenetetrahydrofolate in an airless environment (see, e.g., 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, particularly for tetrahydrofolate.

[0009] As an example of the effectiveness of reducing agents (antioxidants) in preventing oxidation of the 5,10-methylenetetrahydrofolate molecule, Adventrx Pharmaceuticals Inc. The stability of the diastereomeric mixture 5,10-methylene-(6R,S)-tetrahydrofolate (i.e., the calcium salt of the diastereomeric mixture 5,10-methylene-(6R,S)-tetrahydrofolate) was studied, which is disclosed in WO 2007 / 064968. The chemical stability of the diastereomeric mixture 5,10-methylene-(6R,S)-tetrahydrofolate is believed to be similar to that of the pure diastereoisomer 5,10-CH2-(6R)-THF of the present invention.

[0010] The Adventrx study compared the stability of 5,10-MTHF by itself (unformulated), or 5,10-MTHF formulated with trisodium citrate alone, or with both trisodium citrate and ascorbic acid (see Figure 1 ).

[0011] Linear regression analysis of the stability curves of the isolated freeze-dried products 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 of degradation rate from fastest to slowest: unformulated > formulated with trisodium citrate only > formulated with both trisodium citrate and reducing agent ascorbic acid ( Figure 2 ). Thus, unformulated 5,10-methylene-(6R,S)-tetrahydrofolate was found to lose 2.2% purity per hour, resulting in a purity of 84% after 7 hours, whereas the formulation containing 250% w / w trisodium citrate lost 1.4% purity per hour, resulting in a purity of 89% after 7 hours. The formulation containing both 250% w / w trisodium citrate and 175% w / w ascorbic acid had a much higher stability, losing only 0.5% purity per hour, resulting in a purity of about 96% after 7 hours.

[0012] Thus, this study shows that the addition of a reducing agent has a major stabilizing effect on the formulation of 5,10-methylenetetrahydrofolate. However, as a result, the content of active ingredient in the lyophilized formulation is reduced to less than 20% w / w ( Figure 3 ). The solution disclosed in WO 2007 / 064968 for the purpose of preparing the lyophilisate contains up to about 4% by weight of 5,10-CH2-THF.

[0013] Stabilization of 5,10-methylenetetrahydrofolate is also achieved by forming various crystalline forms such as the sulfate (see, e.g., EP 0 537 492) or hemisulfate (see, e.g., EP 2 837 631). However, such salt forms of 5,10-methylenetetrahydrofolate are not easily usable for pharmaceutical purposes due to their low water solubility.

[0014] Freeze-dried products of 5,10-CH2-(6R)-THF containing dicarboxylic acids and / or tricarboxylic acids such as citric acid and / or other stabilizers have also been disclosed in, for example, WO 2019 / 034673, US2007 / 0099866 and US10059710 B2. The solutions disclosed therein for the purpose of preparing freeze-dried products contain up to 2-3% by weight of 5,10-CH2-(6R)-THF.

[0015] From a clinical point of view, the 5,10-CH2-(6R)-THF formulations known in the art do not have a satisfactory combination of water solubility, high content of active ingredient and high stability. Therefore, there is still a great need for stable and soluble pharmaceutical compositions with high content of 5,10-CH2-(6R)-THF. Summary of the invention

[0016] Surprisingly, it has now been found that a lyophilized composition comprising the disodium salt of 5,10-CH2-(6R)-methylenetetrahydrofolate (hereinafter denoted as 5,10-CH2-(6R)-THF*Na2) in combination with citrate and an alkali metal sulfate has a stability comparable to or even higher than a composition comprising 5,10-methylenetetrahydrofolate, citrate and a reducing agent such as L-(+)-ascorbic acid.

[0017] Thus, even without the addition of a reducing agent (antioxidant) such as L-(+)-ascorbic acid and without the exclusion of atmospheric oxygen, the solution of 5,10-CH2-(6R)-THF*Na2, alkali metal sulfate and citrate remains highly stable for several hours, which solution is then converted into a lyophilisate having similarly good stability according to the invention. These lyophilisates contain 5,10-CH2-(6R)-THF*Na2, alkali metal sulfate and citrate without other stabilizing agents, thus overcoming the known disadvantages discussed above and allowing the preparation of solid pharmaceutical compositions of high purity and low content of oxidation products or other chemical degradation products.

[0018] Thus, in a first aspect, the present invention relates to a lyophilized composition comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na2), a citrate and an alkali metal sulfate.

[0019] A second aspect of the present invention relates to a method for preparing a freeze-dried composition according to the first aspect, the method comprising the following steps:

[0020] i. dissolving (6S)-tetrahydrofolate in water with NaOH,

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

[0022] iii. Add 110-120 mol% formaldehyde,

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

[0024] v. adding about 40-200 mol-% of an alkali metal sulfate and about 200-400 mol-% of a citrate,

[0025] vi. filtering the reaction mixture to obtain a clear solution, and

[0026] vii. freeze-drying the clear solution obtained,

[0027] The process does not involve the addition of any other reducing agents or antioxidants.

[0028] In a third aspect, the present invention also relates to the lyophilized composition according to the first aspect for use in cancer treatment or cancer therapy in a human patient.

[0029] In a fourth aspect, the present invention also relates to a method of cancer treatment or cancer therapy in a human patient, comprising administering the lyophilized composition according to the first aspect to a human patient in need thereof.

[0030] In a fifth aspect, the present invention also relates to the use of the lyophilized composition according to the first aspect for the preparation of a medicament for treating cancer in a human patient.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a table adapted from WO 2007 / 064968, which shows the stability (normalized purity %) of unformulated and various formulated forms of 5,10-methylene-(6R,S)-tetrahydrofolate over time. Unformulated 5,10-methylene-(6R,S)-tetrahydrofolate was found to lose 2.2% of its purity per hour, resulting in a normalized purity of 45% after 24 hours. The reference formulation containing 250% w / w trisodium citrate was found to lose 1.4% of its purity per hour, resulting in a normalized purity of 65% after 24 hours. Formulations #1 and #2 containing both 250% w / w trisodium citrate and 175% w / w ascorbic acid had much higher stability, losing only 0.5% of purity per hour, resulting in a normalized purity of about 90% after 24 hours.

[0033] Figure 2 Adapted from WO 2007 / 064968 Figure 1 The chart shows the Figure 1 Tabulation results.

[0034] Figure 3 is a table adapted from Example 1 of WO 2007 / 064968, showing Figure 1 and Figure 2 The compositions of the unformulated and formulated forms of 5,10-methylene-(6R,S)-tetrahydrofolate are shown in . It can be seen that the most stable compositions (Formulations #1 and #2) contain less than 20% of the active ingredient 5,10-methylene-(6R,S)-tetrahydrofolate.

[0035] Figure 4The purity analysis of three solutions containing 75 mg / mL of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate is shown, tested under three different conditions: 25°C, 5°C and 5°C with the addition of 1 mg / mL sodium ascorbate as an antioxidant. The results are shown for a total period of 7 hours. As can be seen from the graph, the solution is very stable under storage conditions. At 5°C, the purity does not change practically, remaining at about 97% (area %). It can also be seen that the effect of ascorbate is minimal. At 25°C, the purity changes from an initial purity of about 96% to a purity of about 95.5% (area %) within 7 hours.

[0036] Figure 5 Shown in this article Figure 4 Analysis of the same three solutions of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate shown. Figure 5 In Figure 1, the development of the main impurity 10-formyl-(6R)-tetrahydrofolate (10-FTHFA) in solution is shown over a period of 7 hours. It can be seen that the level of this impurity is practically constant over time.

[0037] Figure 6 The stability of a lyophilized composition comprising disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate at 5°C, 25°C and 40°C is shown. As can be seen from the graph, the lyophilized composition is very stable under storage conditions. Even at room temperature, when stored for 24 months, the purity is virtually unchanged (99.8% relative purity % w / w).

[0038] Figure 7 The development of the main impurity 10-formyl-(6R)-tetrahydrofolate (10-FTHFA) in a lyophilized composition comprising disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate upon storage at 5° C., 25° C. and 40° C. is shown. As can be seen, the level of this impurity is practically constant over time.

[0039] definition

[0040] As used herein, the term "stabilizer" or "stabilizing agent" refers to buffers such as citrate (or citric acid and its salts); dicarboxylates such as succinate, malate and maleate; tris(hydroxymethyl)aminomethane (TRIS); N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid (TES); 3-(N-morpholino)propanesulfonic acid (MOPS); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); MES; MOPSO; HEPES; phosphates; carbonates; ammonium; mono-, di- and tri-alkylammonium; mono-, di- and tri-hydroxyalkylammonium; glutamate; borate; lactate and combinations thereof. The term "stabilizer" or "stabilizing agent" also relates to reducing agents, such as L-(+) ascorbic acid or its salts, reduced γ-glutathione, β-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc., which can act as antioxidants for sensitive 5,10-methylenetetrahydrofolate, especially tetrahydrofolate.

[0041] 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-aminoethane-sulfonic acid (BES); MES; MOPSO; HEPES; phosphates; carbonates; ammonium; mono-, di- and tri-alkylammonium; mono-, di- and tri-hydroxyalkylammonium; glutamate; borate; lactate and combinations of these.

[0042] In the present context, the term "reducing agent" or "antioxidant" refers to L-(+) ascorbic acid or its salts, reduced γ-glutathione, β-mercaptoethanol, thioglycerol and N-acetyl-L-cysteine.

[0043] 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, a saline solution, water containing a small amount of buffer, water containing an isotonic amount of NaCl, or a mixture of water and an organic solvent, etc. Typical organic solvents include DMSO, acetonitrile, acetone, methanol or ethanol.

[0044] In the present context, the phrase "a composition comprising 5,10-CH2-(6R)-THF*Na2" relates to both solutions and solid compositions, such as lyophilisates, including lyophilisates that have been reconstituted, such as for use in medical treatment.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] It is well known in the art that 5,10-methylenetetrahydrofolate is extremely sensitive to oxidation. It is also known that solutions of 5,10-methylenetetrahydrofolate are more susceptible to chemical degradation than its solid form. Previously, this problem has been addressed in the art by adding an antioxidant (i.e., a reducing agent such as ascorbate) to a 5,10-methylenetetrahydrofolate solution that already contains citric acid. As described herein Figure 1 and Figure 2 As shown, this shows an increase in the stability of dissolved 5,10-MTHF, but at the expense of a decrease in the relative content of 5,10-MTHF.

[0047] Surprisingly, it has now been found that aqueous solutions comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (hereinafter denoted 5,10-CH2-(6R)-THF*Na2) with citrate and alkali metal sulfate have a stability comparable to or even higher than an aqueous solution comprising 5,10-methylenetetrahydrofolic acid, citrate and a reducing agent such as L-(+)-ascorbic acid, wherein the solution according to the invention is subsequently converted into a freeze-dried product having a similarly good stability. Alkali metal sulfates themselves are not reducing agents and are therefore generally considered to be chemically inert. The stability found is therefore surprising.

[0048] The freeze-dried product of the present invention contains greater than 20% w / w 5,10-CH2-(6R)-THF*Na2, for example greater than 25% w / w, for example greater than 30% w / w, for example greater than 35% w / w, or for example about 40% w / w 5,10-CH2-(6R)-THF*Na2.

[0049] Lyophilized powders have even greater stability than aqueous solutions and can be reconstituted with a diluent to a set concentration for administration. Such reconstituted lyophilisates can be administered intramuscularly or intravenously.

[0050] Prior to the freeze drying process, bulking agents such as mannitol may be added to the solution to facilitate the formation of an acceptable lyophilized cake. The structure and porosity of the lyophilized cake are important because good pore formation can facilitate drying and water transport during the drying cycle.

[0051] In addition, electrolytes, sugars and / or polyols such as dextrose, glycerol, mannitol and sodium chloride can be added to adjust the molar osmotic pressure mass concentration. The molar osmotic pressure mass concentration adjustment can be completed before or after freeze drying. The reconstructed lyophilized solution preferably has a molar osmotic pressure mass concentration within the range of 250-350mOsm. However, the molar osmotic pressure mass concentration of 200-600mOsm is also permissible, and will depend on the volume to be administered and the injection / infusion time.

[0052] 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 the preparation of the drug product with, for example, small amounts of hydrochloric acid or sodium hydroxide.

[0053] Stability is a key characteristic and element in pharmaceutical formulation research and drug development. Stability studies are conducted in both solution and solid states. It is a recognized fact that solution state and solid state stability can be qualitatively and quantitatively different. The stability of drug substances and their pharmaceutical compositions has been extensively studied by exposing them to various stressors (such as high temperature and / or high humidity). These studies also provide information on degradation products, which helps in the formulation of meaningful specifications and the intrinsic stability of pharmaceutical compositions. The most common drug degradation pathways include hydrolysis, oxidation and photochemical degradation.

[0054] The purpose of stability testing is to provide evidence of how product quality changes over time under the influence of various environmental factors (such as temperature, humidity, and light) and to determine the shelf life of the drug product and the recommended storage conditions to ensure patient safety.

[0055] In a first aspect, the present invention relates to a lyophilized composition comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF*Na2), a citrate and an alkali metal sulfate.

[0056] In a preferred embodiment of the first aspect, the present invention relates to a stable freeze-dried product containing greater than 20% w / w 5,10-CH2-(6R)-THF*Na2, for example greater than 25% w / w, for example greater than 30% w / w, for example greater than 35% w / w, or for example about 40% w / w 5,10-CH2-(6R)-THF*Na2.

[0057] The lyophilisate of the present invention is substantially amorphous while having enhanced stability, such as enhanced storage stability. The lyophilisate of the present invention can be reconstituted into an aqueous pharmaceutical formulation by methods known in the art for administration to a patient in need thereof.

[0058] In one embodiment, the present invention discloses a freeze-dried composition according to the first aspect, wherein the molar ratio of alkali metal sulfate to 5,10-CH2-(6R)-THF*Na2 is about 40 mol-% to about 400 mol-%, preferably about 50 mol-% to about 100 mol-%.

[0059] In another embodiment, the present invention discloses a freeze-dried composition according to the first aspect, wherein the molar ratio between 5,10-CH2-(6R)-THF*Na2 and citrate is about 200-400 mol%, preferably about 350 mol%.

[0060] A second aspect of the present invention relates to a method for preparing a freeze-dried composition according to the first aspect, the method comprising the following steps:

[0061] i. dissolving (6S)-tetrahydrofolate in water with NaOH,

[0062] ii. Adjust the pH of the solution to 8.6±0.5,

[0063] iii. Add 110-120 mol% formaldehyde,

[0064] iv. stirring the reaction mixture until the reaction is complete,

[0065] v. adding about 40-200 mol-% of an alkali metal sulfate and about 200-400 mol-% of a citrate,

[0066] vi. filtering the reaction mixture to obtain a clear solution, and

[0067] vii. freeze-drying the clear solution obtained,

[0068] The process does not involve the addition of any other reducing agents or antioxidants.

[0069] In embodiments, the alkali metal sulfate added in step v. is in the form of sodium sulfate.

[0070] In an embodiment, the citrate added in step v. is in the form of sodium citrate. In another embodiment, the citrate is added as citric acid.

[0071] In a third aspect, the present invention also relates to a lyophilized composition comprising 5,10-CH2-(6R)-THF*Na2 according to the first aspect for use in cancer treatment or cancer therapy in human patients.

[0072] In a preferred embodiment, the present invention relates to a stable lyophilisate comprising 5,10-CH2-(6R)-THF*Na2 according to the first aspect or a reconstituted aqueous solution thereof for use in cancer treatment or cancer therapy in human patients.

[0073] In a fourth aspect, the present invention also relates to a method for the treatment of cancer or cancer therapy in a human patient, comprising administering the lyophilized composition according to the first aspect to a human patient in need thereof.

[0074] In a preferred embodiment, the present invention relates to a method for treating cancer in a human patient, the method comprising administering to a human patient in need thereof a lyophilized composition comprising 5,10-CH2-(6R)-THF*Na2 according to the first aspect or a reconstituted aqueous solution thereof.

[0075] In a fifth aspect, the present invention also relates to the use of the lyophilized composition comprising 5,10-CH2-(6R)-THF*Na2 according to the first aspect in the preparation of a medicament for cancer treatment in a human patient.

[0076] Other aspects relate to a reconstituted pharmaceutical composition of the lyophilisate of the present invention, which comprises 5,10-CH2-(6R)-THF*Na2 and a pharmaceutically acceptable carrier or diluent, such as sterile water or a liquid pharmaceutically acceptable vehicle, and optionally further comprises at least one other therapeutic agent, including but not limited to bactericides, antibiotics, antiviral agents, preservatives, antitumor drugs, 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, such as 5-FU and derivatives, and antifolates, such as methotrexate, pemetrexed.

[0077] The lyophilisates of the present invention are in substantially unsolvated anhydrous form, including completely anhydrous compounds and compounds that may contain trace amounts of water. Such possible residual (non-stoichiometric) water amounts may be any amount of water, but typically range from 0 wt.-% H2O to 3 wt.-% H2O, preferably between 0 wt.-% H2O and 1 wt.-% H2O, for example between 0.05 wt.-% H2O and 1 wt.-% H2O. Example

[0078] HPLC

[0079] To measure purity / content and degradation products, 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.

[0080] Water content

[0081] The determination of the water content was carried out according to Ph. Eur. 2.5.32 / USP <921 / Method Ic>.

[0082] Osmolarity concentration

[0083] According to Ph.Eur.2.2.35 (osmometer) / USP <785> The molar osmotic pressure and mass concentration were measured.

[0084] Example 1: Preparation of an aqueous solution containing disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate liquid

[0085] (a) (6S)-tetrahydrofolate (16 mmol, 7.93 g) was dissolved in 78.0 g of distilled water at room temperature under nitrogen. The pH of the solution was first raised to 11 by slowly adding 32% NaOH solution, then adjusted to 8.3 with 1.0 M HCl, and cooled to about 0°C. Formaldehyde solution (1.44 g, 110 mol%) was added in one portion, and the solution was stirred at 0°C for 1 hour. Activated carbon (0.2 g, Norit C Extra) was added, the reaction mixture was stirred at 0°C for 30 minutes, and filtered to obtain a clear solution of 5,10-methylene-(6R)-tetrahydrofolate disodium salt (5,10-CH2-(6R)-THF*Na2).

[0086] The disodium 5,10-methylene-(6R)-tetrahydrofolate solution thus obtained can optionally be purified by the following steps (b) and (c), by precipitation of the sulfate salt of 5,10-CH2-(6R)-THF and subsequent redissolution with sodium hydroxide.

[0087] (b) (optional) 55 ml of 1M H2SO4 (0.055 mol; 344 mol%) were heated to 60°C under nitrogen and the solution obtained in step (a) was added dropwise over 15 minutes. The reaction mixture (slurry) obtained was stirred at 50°C for 2 hours. The reaction mixture was then filtered with suction at 50°C, the precipitate was washed twice with 25 ml of distilled water at room temperature and dried at 30°C and 10 mbar for 12 hours (overnight) to obtain 7.36 g of 5,10-CH2-(6R)-THF sulfate in the form of light grey crystals (86% yield).

[0088] (c) (Optional) Under nitrogen, dissolve the product of step (b) in 50 mL 0.1 N NaOH and adjust the pH to 8.6 ± 0.50 with 1.5 N NaOH.

[0089] (d) 21 g of citric acid in 20 ml of water are added to the solution of step (c) or step (a), and if necessary, the sulfate content is adjusted to up to 200 mol% with aqueous sodium sulfate. The total volume is then adjusted to 100 mL with sterile filtered water and filtered using a 0.22 μm filter, preferably a hydrophilic PVDF type, such as Millipore 0.22 μm. The filtrate was filled into vials (2 ml or 160 mg of 5,10-CH2-(6R)-THF*Na2 per vial) while keeping the solution as cool as possible.

[0090] Example 2: Preparation of a lyophilized solution containing disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate Composition

[0091] The vials of Example 1 were frozen and subsequently freeze dried. The vials were sealed and crimped under a slight vacuum with nitrogen in the head space. The resulting freeze dried material contained greater than 30% w / w 5,10-CH2-(6R)-THF*Na2.

[0092] Example 3: Stability Test

[0093] a) Stability of solutions containing disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sulfate at 25°C

[0094] To determine the stability of a sterile filtered solution containing 5,10-methylene-(6R)-tetrahydrofolate, citrate and sulfate at 25°C, the solution prepared as in step (d) of Example 1 was stored at 25°C. The purity of 5,10-CH2-(6R)-THF was measured by HPLC at periodic intervals (% area). The results are shown in Table 1. Figure 4 shown.

[0095] During the study period, the color of the test solution changed from yellow to dark yellow. No light protection was used for samples stored at room temperature. Purity remained stable at 96% over a 7-hour storage period at room temperature.

[0096] Figure 4 The progression of analytical results during stability testing is summarized.

[0097] When stored at 25°C, the content of the major degradation product, 10-formyl-(6R)-tetrahydrofolate (10-FTHFA), was measured by HPLC at periodic intervals. Figure 5 shown.

[0098] b) Stability of solutions containing disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sulfate at 5°C

[0099] To determine the stability of a sterile filtered solution containing disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate at 5°C, the solution prepared as in step d of Example 1 was stored at 5°C. The purity of 5,10-CH2-(6R)-THF was measured by HPLC at periodic intervals (% area). The results are shown in Figure 4 shown.

[0100] The content of the major degradation product 10-formyl-(6R)-tetrahydrofolate (10-FTHFA) was measured at periodic intervals by HPLC. Figure 5 shown.

[0101] During the study, the color of the test solution changed from yellow to dark yellow. During the study, the stability test solution was stored in a refrigerator (2°C to 8°C) and was only taken out for analytical sampling. During sampling, no light protection was used. At 2°C to 8°C, the purity did not show significant changes and remained stable at 97% during the 7-hour storage period.

[0102] Figure 4 and Figure 5 The progression of analytical results during stability testing is summarized.

[0103] The stability test at 5°C was repeated for the comparative test solutions to which sodium ascorbate (1 mg / mL as an antioxidant) was added and stored at 2°C to 8°C. The test solutions to which sodium ascorbate was added showed no significant difference compared to the test solutions to which sodium ascorbate was not added ( Figure 4 and Figure 5 ). Therefore, solutions with and without ascorbate can be considered equivalent in terms of stability.

[0104] The 75 mg / mL 5,10-methylene-(6R)-tetrahydrofolate solution is clear and remains clear whether stored at 2-8°C or at room temperature, i.e., no precipitation occurs. The solution is slightly viscous and has an osmotic pressure mass concentration of approximately 710 mOsmol / kg.

[0105] In summary, a sterile filtered solution containing 75 mg / mL of 5,10-methylene-(6R)-tetrahydrofolate disodium salt, citrate, and sulfate was stable for up to 7 hours when stored at 2°C to 8°C and for up to 4 hours when stored at room temperature.

[0106] c) Stability of a lyophilized composition comprising disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sodium sulfate at 5°C, 25°C and 40°C

[0107] In order to determine the stability of the lyophilized composition comprising disodium 5,10-methylene-(6R)-tetrahydrofolate, citrate and sulfate over time at different temperatures, the lyophilisate prepared in Example 2 was stored at 5°C, 25°C and 40°C. The purity of 5,10-CH2-(6R)-THF was determined by HPLC at periodic intervals (% area). The results relative to the starting value are Figure 6 The relative purity is shown in % w / w.

[0108] The content of the major degradation product, 10-formyl-(6R)-tetrahydrofolate (10-FTHFA), was measured by HPLC at periodic intervals when stored at 5°C, 25°C and 40°C. Figure 7 shown.

Claims

1. A freeze-dried composition comprising disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid, citrate and alkali metal sulfate, the composition not containing any other reducing agent or antioxidant.

2. The lyophilized composition according to claim 1, which consists of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate, sodium sulfate, sodium citrate, water and an optional osmotic pressure mass concentration correction additive.

3. The freeze-dried composition according to claim 1 or claim 2, wherein the molar ratio between the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate and the sulfate is about 40 to 200 mol%.

4. The freeze-dried composition according to any one of claims 1 to 3, comprising 200-400 mol% of citrate.

5. The lyophilized composition according to any one of claims 1 to 4, which is a stable freeze-dried material, wherein the concentration of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate is greater than 20% w / w disodium salt of 5,10-methylene-(6R)-tetrahydrofolate, such as greater than 25% w / w, such as greater than 30% w / w, such as greater than 35% w / w, or such as about 40% w / w disodium salt of 5,10-methylene-(6R)-tetrahydrofolate. 6 . The freeze-dried composition according to claim 1 , comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate in a purity greater than 98%.

7. A reconstituted product obtained by dissolving the lyophilized composition according to any one of claims 1 to 6 in water or a liquid pharmaceutically acceptable vehicle.

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

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

10. The reconstituted product according to any one of claims 7 to 9, further comprising an additional pharmaceutically acceptable active ingredient.

11. The reconstituted product according to any one of claims 7 to 10, further comprising a buffer and / or one or more osmolarity correction excipients.

12. The reconstituted product according to any one of claims 7 to 11 for use in cancer treatment or cancer therapy.

13. A method for preparing a lyophilized composition according to any one of claims 1 to 6, comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolate, a citrate and an alkali metal sulfate, the method comprising the following steps: viii. dissolving (6S)-tetrahydrofolic acid in water at a pH of about 11, ii. Adjust the pH of the clear solution to 8.6 ± 0.5, iii. Add 110-120 mol% formaldehyde, iv. stirring the reaction mixture until the reaction is complete, v. adding about 40-200 mol % of an alkali metal sulfate and 100-400 mol % of a citrate, vi. filtering the reaction mixture to obtain a clear solution, and ix. freeze-drying the obtained clear solution, The method does not involve the addition of any other reducing agents or antioxidants.

14. The method of claim 13, wherein the alkali metal sulfate is added in the form of sodium sulfate.

15. The method according to claim 13 or 14, wherein the citrate is added in the form of citric acid or sodium citrate.

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