Concentrated solutions comprising 5, 10-methylene-(6R)-tetrahydrofolic acid

Abstract

The present invention relates to a stable, high content solution comprising 5, 10-methylene-(6R)-tetrahydrofolic acid (5, 10-CH2-(6R)-THF), which solution is free of foreign stabilizing agents.

Description

Technical Field

[0001] The present invention relates to a liquid composition containing a high content of 5,10-methylene-(6R)-tetrahydrofolic acid, wherein the composition does 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 (which is a metabolite of 5-FU) 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 causes 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 corresponding high-content products.

[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 undesirably high impurity levels.

[0005] The susceptibility to oxidation and chemical degradation of 5,10-CH2-(6R)-THF is particularly high 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] 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). 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] It has been previously found (see, for example, WO 2019 / 034673) that the addition of dicarboxylic acids to 5,10-CH2-(6R)-THF helps to maintain the purity of the active ingredient 5,10-CH2-(6R)-THF at a very high level during the freeze-drying process, while at the same time maintaining the amount of by-products at an acceptably low level. It has also been found that the addition of a reducing agent such as L-(+)-ascorbic acid) has a positive effect on the stability of 5,10-CH2-(6R)-THF.

[0008] The addition of citrate while adjusting the pH to alkaline values ​​(see, for example, EP 1 641 460) has in particular such a stabilizing effect, which is described in detail in US 10059710 B2. Thus, the stabilization of 5,10-CH2-(6R)-THF and its hemisulfate salt with citrate at slightly acidic to alkaline pH values ​​is described as being due to the synergistic effect of citrate buffer solutions in this pH range.

[0009] It should be mentioned that US10059710 B2 also mentions a citrate-free formulation of 5,10-CH2-(6R)-THF, however, no guidance is provided for the preparation of such a formulation. On the contrary, US10059710 B2 teaches the skilled person that citrate (or other buffering agents) are required in order to prepare a stable composition of 5,10-CH2-(6R)-THF.

[0010] From a clinical perspective, the availability of stable solutions and lyophilisates of 5,10-CH2-(6R)-THF with a high active ingredient content and without any type of stabilizer would be advantageous. For example, citric acid has 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.

[0011] The prior art, including the teachings of US10059710 B2 (supra), does not readily allow for the preparation of such stabilizer-free compositions of 5,10-CH2-(6R)-THF, and thus there remains a great need for stable liquid and solid pharmaceutical compositions of 5,10-CH2-(6R)-THF which do not contain exogenous stabilizing compounds such as stabilizers, buffers, reducing agents, etc.

[0012] The problem that arises is that the stabilized forms of 5,10-CH2-(6R)-THF known in the prior art usually contain less than 50% of the active drug compound due to dilution by stabilizing additives in the final dosage form of the drug. As an example, Adventrx Pharmaceuticals conducted stability studies on its drug candidate CoFactor®, which is the calcium salt of the diastereomeric mixture 5,10-methylene-(6R,S)-tetrahydrofolate, which is disclosed in particular in WO 2007 / 064968. The chemical stability of the diastereomeric mixture 5,10-methylene-(6R,S)-tetrahydrofolate was 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 that of 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 ).

[0013] Linear regression analysis of the stability curves of the isolated lyophilizates showed that 5,10-methylene-(6R,S)-tetrahydrofolate degraded linearly with 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.

[0014] However, 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 ).

[0015] Furthermore, known stabilizers such as citric acid have been associated with various undesirable effects, such as QT cProlongation (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.

[0016] Therefore, there is still a great need for stable pharmaceutical compositions with high content of 5,10-methylene-(6R)-tetrahydrofolate. Summary of the invention

[0017] It has now surprisingly been found that a highly concentrated, stable solution comprising 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF) having a pH between about 8 and 9.5 can be prepared from 5,10-CH2-(6R)-THF or a salt thereof in the absence of exogenous stabilizing compounds such as stabilizers, buffers, reducing agents, and the like.

[0018] Thus, the concentrated solutions and lyophilisates of the present invention overcome the known disadvantages discussed previously and allow the preparation of liquid and solid pharmaceutical compositions of high purity and low content of oxidation products and other chemical degradation products. The favorable stability properties of the solutions of the present invention will allow the effective and safe use of 5,10-CH2-(6R)-THF in pharmaceutical applications.

[0019] In a first aspect, the present invention relates to a concentrated aqueous solution comprising 5,10-methylene-(6R)-tetrahydrofolate (5,10-CH2-(6R)-THF), which concentrated solution does not additionally contain any stabilizing agents, such as stabilizers, buffers, reducing agents, etc., as defined below.

[0020] In a second 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 treating cancer in a human patient.

[0021] In a third aspect, the present invention further relates to a method of treating cancer in a human patient, 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.

[0022] In a fourth aspect, the present invention further relates to a composition comprising 5,10-CH2-(6R)-THF, which composition is obtainable by reconstitution or dilution of the concentrated aqueous solution according to the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] However, the purity after 24 hours was still only 65% ​​compared to the initial purity, 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.

[0025] Figure 2 Adapted from WO 2007 / 064968 Figure 1 , and the diagram confirms the Figure 1 The tabulation results.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] definition

[0030] As used herein, the term "stabilizer" or "stabilizing agent" relates 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-aminoethanesulfonic 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 of these. The term "stabilizer" or "stabilizing agent" further 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, and in particular for tetrahydrofolate.

[0031] 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.

[0032] 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.

[0033] 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

[0034] Surprisingly, it has now been found that highly concentrated solutions of 5,10-CH2-(6R)-THF in pure water or brine can be prepared, which have similar stability to hydrolysis and oxidation as the solutions containing the above-mentioned various stabilizers described above (see, for example, WO2019034673, US 2007 / 0099866 and US10059710 B2). The solutions of the present invention are free of stabilizers (hereinafter referred to as "free of stabilizers") and contain greater than 40 mg / ml 5,10-CH2-(6R)-THF, such as greater than 45 mg / ml, greater than 55 mg / ml, greater than 60 mg / ml, greater than 65 mg / ml, such as preferably 70 mg / ml 5,10-CH2-(6R)-THF. Solutions of higher concentrations can be prepared, but they become very viscous.

[0035] The highly concentrated solution according to the present invention remains stable for at least 8 days at 5 ± 3 ° C. For longer term storage, the aqueous solution can be filled in a container and freeze-dried (lyophilized) into a stable, non-sticky, stabilizer-free powder. The powder 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 lyophilisate can be administered intramuscularly or intravenously.

[0036] The solution of the present invention may contain additional excipients. For example, electrolytes, sugars and / or polyols such as glucose, glycerol, mannitol and sodium chloride may be added to adjust the osmotic mass molar concentration. The osmotic mass molar concentration adjustment may be completed before or after the solution prepared for administration. The solution of reconstruction (dilution) preferably has an osmotic mass molar concentration within the range of 250-350 mOsm. However, the osmotic mass molar concentration of 200-600 mOsm is also permissible and will depend on the volume to be administered and the injection / infusion time.

[0037] 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.

[0038] The high stability obtained for concentrated solutions of 5,10-CH2-(6R)-THF at pH 8.6 is very surprising in view of the above described prior art, in which the presence of stabilizers like citrate and / or ascorbic acid would have been mandatory.

[0039] Therefore, a first aspect of the present invention relates to a concentrated aqueous solution comprising 5,10-methylene-(6R)-tetrahydrofolate and containing no stabilizer.

[0040] In a preferred embodiment, the present invention relates to a concentrated solution of 5,10-CH2-(6R)-THF containing greater than 45 mg / ml 5,10-CH2-(6R)-THF, such as greater than 50 mg / ml, greater than 55 mg / ml, greater than 60 mg / ml, greater than 65 mg / ml, such as preferably 70 mg / ml 5,10-CH2-(6R)-THF, which solution does not additionally contain any stabilizing agents, such as stabilizers, buffers, reducing agents, etc., as defined below.

[0041] In a preferred embodiment, the composition according to the first aspect is prepared from 5,10-CH2-(6R)-THF hemisulfate.

[0042] In another embodiment, the composition according to the first aspect does not contain a compound 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.

[0043] In a second aspect, the present invention further relates to the concentrated solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, for use in treating cancer in a human patient.

[0044] In a third aspect, the present invention further relates to a method of treating cancer in a human patient comprising administering the concentrated solution according to the first aspect, or a reconstituted or diluted aqueous solution thereof, to a human patient in need thereof.

[0045] One embodiment is directed to a concentrated solution comprising 5,10-CH2-(6R)-THF that is free of tricarboxylic acid or a salt thereof.

[0046] One embodiment is directed to a concentrated solution comprising 5,10-CH2-(6R)-THF that is free of a dicarboxylic acid or a salt thereof.

[0047] One embodiment is directed to a concentrated solution comprising 5,10-CH2-(6R)-THF that does not contain a dicarboxylic acid or a salt thereof, or a reducing agent such as L-(+) ascorbic acid.

[0048] One embodiment is directed to a concentrated solution comprising 5,10-CH2-(6R)-THF having a purity greater than 98%.

[0049] One embodiment is directed to a concentrated solution comprising 5,10-CH2-(6R)-THF that is free of stabilizers.

[0050] The concentrated solutions of the present invention are preferably reconstituted (diluted) into aqueous pharmaceutical formulations for administration to a patient in need thereof.

[0051] Another aspect relates to a method for preparing the concentrated aqueous solution of the present invention, comprising the following steps:

[0052] (i) dissolving 45-70 mg / ml 5,10-methylene-(6R)-tetrahydrofolate or a salt thereof in water or optionally saline, such as 0.4-0.95% NaCl aqueous solution, such as 0.45% NaCl or 0.9% NaCl;

[0053] (ii) optionally, in particular (ia) adding excipients to adjust the osmolarity of the solution.

[0054] In an embodiment, the salt of 5,10-methylene-(6R)-tetrahydrofolate used in step (i) is a hemisulfate or sulfate salt of 5,10-methylene-(6R)-tetrahydrofolate.

[0055] Optionally a base, like aqueous NaOH or KOH, is added in step (i) to complete the dissolution of the 5,10-methylene-(6R)-tetrahydrofolate compound.

[0056] The solution of step (i) may optionally be filtered through a sterile filter, optionally under an inert atmosphere, prior to step (ii).

[0057] In a separate embodiment, the pH of the solution in step (i) is above 6, typically about 8-14, preferably about 9-10 or 8.5-9.5.

[0058] In a fourth aspect, the present invention further relates to a composition comprising 5,10-CH2-(6R)-THF, which composition can be obtained by reconstitution or dilution of the composition according to the first aspect. Adjustment of the osmolality (if necessary) can be done at the time of dissolution (reconstitution) of the lyophilisate.

[0059] Another aspect relates to a reconstituted pharmaceutical concentrate solution of the present invention, which comprises 5,10-CH2-(6R)-THF 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 agents or therapeutic agents suitable for human use, in particular anticancer compounds, such as chemotherapeutic agents, for example 5-FU (5-fluorouracil) and its derivatives, and folic acid antagonists, for example methotrexate, pemetrexed.

[0060] "Liquid pharmaceutically acceptable solvent" refers to propylene glycol, polyethylene glycols, ethanol, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), polyethylene glycol tetrahydrofurfuryl ether (glycofurol), solketal, glycerol formal, acetone, tetrahydrofurfuryl alcohol, monoethylene glycol dimethyl ether, diethylene glycol dimethyl ether, dimethyl isosorbide or ethyl lactate, and mixtures thereof, including aqueous mixtures thereof.

[0061] Another aspect relates to the use of the reconstituted concentrated solution comprising 5,10-CH2-(6R)-THF according to the invention in therapy, preferably in cancer chemotherapy.

[0062] Yet another aspect relates to a reconstituted concentrated solution comprising 5,10-CH2-(6R)-THF according to the invention for use in treating cancer, preferably in human patients.

[0063] In a preferred embodiment, the reconstituted concentrated solution comprising 5,10-CH2-(6R)-THF according to the invention is administered in combination with 5-fluorouracil (5-FU) or in a dosage regimen that additionally includes the administration of fluorouracil (5-FU).

[0064] Another aspect of the invention relates to the use of the concentrated solution of the invention comprising 5,10-CH2-(6R)-THF, including its reconstituted (diluted) solution, in combination with at least one or more additional therapeutic agents. In this context, preferred additional therapeutic agents include chemotherapeutic agents and other anticancer drugs. Particularly preferred drugs that can be combined with the pharmaceutical composition of the invention include fluorinated nucleic acids, such as 5-fluorouracil or its prodrugs or analogs. Example

[0065] HPLC

[0066] 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.

[0067] Water content

[0068] The determination of the water content was carried out according to Ph. Eur. 2.5.32 / USP <921 / Method Ic>.

[0069] Osmolality

[0070] According to Ph. Eur. 2.2.35 (Osmometer) / USP <785> The osmolality was determined.

[0071] Example 1 Preparation of a concentrated solution of 5,10-CH2-(6R)-THF in 0.9% NaCl

[0072] The following describes the preparation of 50 ml of 70 mg / ml 5,10-CH2-(6R)-THF in saline solution. Example 2 below relates to a similar preparation of a concentrated solution in pure water. The preparation procedure can be adjusted to any suitable volume as required.

[0073] The preparation process should not take longer than 30 minutes. 5,10-CH2-(6R)-THF solutions should always be prepared using cold solutions (2-8 °C), and the solution must also be kept cold during mixing by keeping the reaction vessel cool. The temperature in the solution should not be higher than 8 °C. After the preparation is completed, the 5,10-CH2-(6R)-THF solution is transferred directly to a refrigerator / freezer until the intended use itself, or until the solution is to be lyophilized.

[0074] The saline solution procedure begins by preparing the following solution:

[0075] Add 3750 mg of 5,10-CH2-(6R)-THF hemisulfate to a 50 ml volumetric flask or container that allows for good mixing. Add 25 ml of Solution A to the flask and stir efficiently under an inert atmosphere. Slowly and carefully adjust the pH to 8.6 ± 0.20 with Solution B. The pH should not exceed pH 9.2. If necessary, the pH can be adjusted to 8.6 ± 0.20 with 0.1 M HCl.

[0076] Adjust the total volume to 50 ml with 0.9% NaCl and measure the pH, which should be 8.6 ± 0.20. Sterile filter using a 0.22 μm filter. A hydrophilic PVDF type such as Millipore Durapore ® The resulting solution contained 70 mg / ml 5,10-CH2-(6R)-THF in saline solution, pH 8.6 ± 0.20.

[0077] Example 2 Preparation of a concentrated solution of 5,10-CH2-(6R)-THF in water

[0078] For a general description, see Example 1 above.

[0079] The pure aqueous solution process begins by preparing the following solutions:

[0080] Add 3750 mg of 5,10-CH2-(6R)-THF hemisulfate to a 50 ml volumetric flask or container that allows for good mixing. Add 25 ml of Solution A to the flask and stir efficiently under an inert atmosphere. Slowly and carefully adjust the pH to 8.6 ± 0.20 with Solution B. If necessary, the pH can be adjusted to 8.6 ± 0.20 with 0.1 M HCl.

[0081] Adjust the total volume to 50 ml with sterile filtered water and measure the pH, which should be 8.6 ± 0.20. Sterile filter using a 0.22 μm filter. A hydrophilic PVDF type such as Millipore Durapore ® The resulting solution contained 70 mg / ml 5,10-CH2-(6R)-THF and had a pH of 8.6±0.20. Example 3: Stability Test

[0082] The stability of the solution produced in Example 2 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.

[0083] As part of the stability analysis, the development of the main impurity 10-formyl-(6R)-tetrahydrofolate in the solution produced as in Example 2 over a 7-hour period was also measured when stored at 2-8°C (see Figure 5 ). It can be seen that the level of this impurity is virtually constant.

[0084] Example 4 Preparation of lyophilized material without stabilizer

[0085] If desired, the concentrated solution of Example 1 or 2 can be converted into a stable lyophilisate as follows: Fill vials (2 mL or 150 mg / vial 5,10-CH2-(6R)-THF) with the filtered solution from Example 1 or Example 2 at a temperature of 2-8°C while keeping the solution as cold as possible.

[0086] Freeze-dry the vials and seal them under slight vacuum with nitrogen in the headspace. The vials were compressed. The resulting lyophilisate contained 70-80% w / w 5,10-CH2-(6R)-THF.

Claims

1. A concentrated aqueous solution comprising 5,10-methylene-(6R)-tetrahydrofolic acid and containing no stabilizer.

2. The concentrated aqueous solution according to claim 1, which is prepared from 5,10-methylene-(6R)-tetrahydrofolate hemisulfate.

3. The concentrated aqueous solution according to claim 1 or 2, wherein the excluded stabilizer includes 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.

4. The concentrated aqueous solution according to claims 1 to 3, containing at least 50 mg / ml of 5,10-methylene-(6R)-tetrahydrofolate.

5. A reconstituted product obtained by diluting the concentrated aqueous solution according to any one of claims 1 to 4 in water or a liquid pharmaceutically acceptable solvent. The reconstituted product according to claim 5 , wherein the water is sterile water for injection.

7. The reconstituted product according to any one of claims 5 or 6, further comprising a pharmaceutically acceptable carrier.

8. The reconstituted product according to any one of claims 5 to 7, further comprising an additional pharmaceutically acceptable active ingredient.

9. The reconstituted product according to any one of claims 5 to 8, further comprising a buffer and / or one or more osmolality correction excipients.

10. The reconstituted product according to any one of claims 5 to 9 for use in the treatment of cancer or cancer therapy.

11. The reconstituted product according to any one of claims 5 to 10, further comprising a pharmaceutically acceptable carrier.

12. The reconstituted product according to any one of claims 5 to 11, further comprising a further pharmaceutically acceptable active ingredient.

13. The reconstituted product according to any one of claims 5 to 12, further comprising a buffer and / or one or more osmolality correction excipients.

14. The reconstituted product according to any one of claims 5 to 13 for use in the treatment of cancer or cancer therapy.

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