Composition containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium

Abstract

The present invention relates to a stable formulation and a lyophilized product containing high-content disodium salt, sulfate salt and citrate salt of 5,10-methylene(6R)-tetrahydrofolic acid.

Description

Technical Field

[0001] The present invention relates to a stable formulation and a lyophilized product containing a high concentration of disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (5,10-CH 2 -(6R)-THF * Na 2 ), sulfate and citrate.

Background Art

[0002] Background of the Invention : 5,10-Methylenetetrahydrofolic acid is known as a pharmaceutical used in combination with 5-fluorouracil (5-FU) in the treatment of solid tumors (Non-Patent Document 1: Seley, K. L. Drugs 4(1), 99, 2001). The active isomer, 5,10-methylene-(6R)-tetrahydrofolic acid (hereinafter abbreviated as 5,10-CH 2 -(6R)-THF), exerts a chemotherapeutic effect together with the base analog 5-FdUMP, which is a metabolite of 5-FU, by inhibiting the enzyme thymidylate synthase (TS). TS catalyzes the conversion of deoxyuridylic acid (dUMP) to deoxythymidylic acid (dTMP), which is an essential component for DNA synthesis. Inactivation of TS occurs by forming a covalent ternary inhibitory complex among TS, the base analog 5-FdUMP, which is a metabolite of 5-FU, and 5,10-CH 2 -(6R)-THF.

[0003] The cytotoxic effect of 5-FU can be enhanced by increasing the intracellular concentration of 5,10-CH 2 -(6R)-THF, resulting in increased stability of the ternary inhibitory complex. This directly inhibits DNA synthesis and repair, ultimately leading to cell death and tumor growth delay. To increase the intracellular concentration of 5,10-CH 2 -(6R)-THF, the use of stable high-content products is desired.

[0004] However, 5,10-CH 2-(6R)-THF has undesirable properties that limit its pharmaceutical use. For example, 5,10-CH 2 -(6R)-THF is very susceptible to oxidation and chemical decomposition, resulting in unacceptably high impurity levels.

[0005] 5,10-CH 2 The susceptibility of (6R)-THF to oxidation and chemical decomposition is particularly high when it is present in an aqueous solution, or in an amorphous form where the compound has a large surface area (e.g., in the form of a lyophilized product for pharmaceutical use), or in a re-dissolved form such as an injection solution. It is well known that for pharmaceutical use, each composition needs to meet several requirements, including high chemical stability and isomer stability, such that the composition can be effectively stored for an acceptable period without showing significant changes in the physicochemical properties, ease of handling and processing, etc.

[0006] 5,10-Methylenetetrahydrofolic acid is an addition product of tetrahydrofolic acid and formaldehyde (see, for example, Non-Patent Document 2: Poe, M. et al. Biochemistry 18(24), 5527, 1979; Non-Patent Document 3: Kallen, R.G. Methods in Enzymology 18B, 705, 1971), and is known to be highly susceptible to oxidation by air and unstable in neutral and / or acidic environments, which may cause chemical degradation and / or hydrolysis (see, for example, Non-Patent Document 4: Odin, E. et al., Cancer Investigation 16(7), 447, 1998; Non-Patent Document 5: Osborn, M.J. et al., J. Am. Chem. Soc. 82, 4921, 1960; Non-Patent Document 6: Hawkes, J., and Villota, R. Food Sci. Nutr. 28, 439, 1989).

[0007] As attempts to stabilize compositions of 5,10-methylenetetrahydrofolic acid, for example, (i) strict exclusion of oxygen in the atmosphere by using special technical devices for reconstitution of solid preparations, and injection of 5,10-methylenetetrahydrofolic acid in an oxygen-free environment (see, for example, Non-Patent Document 4: Odin, E. et al., Cancer Investigation 16(7), 447, 1998, Patent Document 1: U.S. Patent No. 4,564,054), (ii) addition of reducing agents such as L(+)-ascorbic acid or its salts, reduced glutathione, beta-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc. as antioxidants for very sensitive 5,10-methylenetetrahydrofolic acid, especially tetrahydrofolic acid, etc. can be mentioned.

[0008] As an example of the effectiveness of reducing agents (antioxidants) in preventing oxidation of 5,10-methylenetetrahydrofolic acid molecules, Adventrx Pharmaceuticals conducted stability studies on its candidate drug CoFactor (registered trademark), that is, the calcium salt of the diastereomer mixture 5,10-methylene-(6R,S)-tetrahydrofolic acid, and these studies are published in Patent Document 2: International Publication No. 2007 / 064968, etc. The chemical stability of the diastereomer mixture 5,10-methylene-(6R,S)-tetrahydrofolic acid is assumed to be the same as that of the pure diastereomer 5,10-CH 2 -(6R)-THF of the present invention.

[0009] In the studies by Adventrx, the stability of 5,10-methylenetetrahydrofolic acid when used as it is (unformulated), with only trisodium citrate, or in combination with both trisodium citrate and ascorbic acid was compared (see Figure 1).

[0010] Linear regression analysis of the stability profiles of the isolated lyophilized products showed that the degradation of 5,10-methylene-(6R,S)-tetrahydrofolic acid was linear over time (see Figure 2). The degradation rates (the slopes of the best-fit lines) of each formulation (reconstituted lyophilized product) were in the order from the fastest to the slowest as follows: unformulated > formulated with sodium citrate only > formulated with both sodium citrate and the reducing agent ascorbic acid (Figure 2). Unformulated 5,10-methylene-(6R,S)-tetrahydrofolic acid had a purity decrease of 2.2% per hour and reached a purity of 84% after 7 hours, while in the formulation containing 250% w / w sodium citrate, the purity decreased by 1.4% per hour and reached a purity of 89% after 7 hours. The formulation containing both 250% w / w sodium citrate and 175% w / w ascorbic acid showed much higher stability, with a purity decrease of only 0.5% per hour and reaching a purity of about 96% after 7 hours.

[0011] This study revealed that the addition of a reducing agent has a great stabilizing effect on the formulation of 5,10-methylenetetrahydrofolic acid. However, as a result, the active ingredient content in the lyophilized formulation decreases to less than 20% w / w (Figure 3). Patent Document 2: The solution for preparing a lyophilized product disclosed in International Publication No. 2007 / 064968 contains up to about 4% of 5,10-CH 2 -THF.

[0012] The stabilization of 5,10-methylenetetrahydrofolic acid is also achieved by the formation of various crystalline forms such as sulfates (see, for example, Patent Document 3: European Patent No. 0537492) or hemisulfates (see, for example, Patent Document 4: European Patent No. 2837631). However, such salt forms of 5,10-methylenetetrahydrofolic acid have low water solubility and are not easily useful for pharmaceutical purposes.

[0013] 5,10-CH containing tricarboxylic acids such as dicarboxylic acids and / or citric acid and / or other stabilizers 2The lyophilized product of (6R)-THF is also disclosed, for example, in Patent Document 5: International Publication No. 2019 / 034673, Patent Document 6: US Patent Publication No. 2007 / 0099866, and Patent Document 7: US Patent No. 10059710. The solutions disclosed therein for the purpose of preparing the lyophilized product contain up to 2 to 3% by weight of 5,10-CH 2 -(6R)-THF.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0016] From a clinical perspective, the formulations of 5,10-CH 2 -(6R)-THF known in the art do not have a satisfactory combination of water solubility, high content of the active ingredient, and high stability. Therefore, there remains a great need for a stable and soluble pharmaceutical composition containing a high content of 5,10-CH 2 -(6R)-THF.

Means for Solving the Problems

[0017] Summary of the Invention : Now, surprisingly, a lyophilized composition comprising the disodium salt of 5,10-CH 2 -(6R)-methylenetetrahydrofolic acid (hereinafter referred to as 5,10-CH 2 -(6R)-THF*Na 2 ) in combination with a citrate and an alkali metal sulfate has been found to have comparable or even higher stability than compositions containing 5,10-methylenetetrahydrofolic acid, a citrate, and a reducing agent such as L-(+)-ascorbic acid.

[0018] Therefore, without adding a reducing agent (antioxidant) such as L-(+)-ascorbic acid and without excluding oxygen in the air, 5,10-CH 2 -(6R)-THF*Na 2, Solutions of alkali metal sulfates and citrates remain very stable over several hours, and these solutions according to the invention are then converted in the same way into lyophilizates with equally good stability. These lyophilizates contain 5,10-CH 2 -(6R)-THF*Na 2 , contain alkali metal sulfates and citrates and no other stabilizers, thus overcoming the known drawbacks discussed above and enabling the preparation of a solid pharmaceutical composition with high purity and low content of either oxidation products or other chemical decomposition products.

[0019] Thus, in a first aspect, the present invention relates to a lyophilizate composition comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (5,10-CH 2 -(6R)-THF*Na 2 ), a citrate and an alkali metal sulfate.

[0020] A second aspect of the present invention is directed to a method for producing the lyophilizate composition according to the first aspect, the method comprising the following steps: i. Dissolving (6S)-tetrahydrofolic acid in water with NaOH, ii. Adjusting the pH of the solution to 8.6 ± 0.5, iii. Adding 110 - 120 mol% of formaldehyde, iv. Stirring the reaction mixture until the reaction is complete, v. Adding about 40 - 200 mol% of an alkali metal sulfate and about 200 - 400 mol% of a citrate, vi. Filtering the reaction mixture to obtain a clear solution, and vii. Lyophilizing the obtained clear solution, This method does not include the step of adding additional reducing agents or antioxidants.

[0021] A third aspect of the present invention relates to the lyophilizate composition according to the first aspect for use in the treatment of cancer or cancer therapy in a human patient.

[0022] In a fourth aspect, the present invention further relates to a method for treating cancer or a cancer therapy in a human patient, comprising administering the lyophilized composition according to the first aspect to a human patient in need thereof.

[0023] In a fifth aspect, the present invention further relates to the use of the lyophilized composition according to the first aspect for the manufacture of a medicament for treating cancer in a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1

[0025]

Figure 2

[0026]

Figure 3

[0027]

Figure 4

[0028]

Figure 5

[0029]

Figure 6

[0030]

Figure 7

[0031] Definitions : As used herein, the terms "stabilizers" or "stabilizing agents" refer to buffering agents such as citrate (or citric acid and its salts); dicarboxylates such as succinate, malate, 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-hydroxylalkylammonium; glutamate; borate; lactate; and combinations thereof. The terms "stabilizer" or "stabilizing agent" further relate to reducing agents such as L-(+)-ascorbic acid or its salts, reduced glutathione, β-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc., which can act as antioxidants against labile 5,10-methylenetetrahydrofolic acid, especially tetrahydrofolic acid.

[0032] As used herein, the term "buffer" refers 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; phosphates; carbonates; ammonium; mono-, di-, and tri-alkylammonium; mono-, di-, and tri-hydroxyalkylammonium; glutamate; borate; lactate, and combinations thereof.

[0033] As used herein, the terms "reducing agent" or "antioxidants" relate to L-(+)-ascorbic acid or its salts, reduced glutathione, β-mercaptoethanol, thioglycerol, and N-acetyl-L-cysteine.

[0034] As used herein, the term "solvent" relates to solvents that can be used in lyophilization methods. The "Solutions" referred to herein include aqueous solutions and solutions of organic solvents. Typically, "aqueous solutions" mean solutions in water, physiological saline, water containing a small amount of buffer, water containing an isotonic amount of NaCl, or a mixture of water and an organic solvent. Typical organic solvents include DMSO, acetonitrile, acetone, methanol, or ethanol.

[0035] As used herein, the phrase "composition containing 5,10-CH 2 -(6R)-THF*Na 2 relates to both solutions and solid compositions such as lyophilized products, and includes, for example, lyophilized products reconstituted for medical use. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention: It is well known in the art that 5,10-methylenetetrahydrofolic acid is extremely sensitive to oxidation. Furthermore, it is known that solutions of 5,10-methylenetetrahydrofolic acid are more chemically labile than its solid form. Heretofore, this problem has been addressed in the art by adding an antioxidant, i.e., a reducing agent such as ascorbic acid, to a 5,10-methylenetetrahydrofolic acid solution already containing citric acid. Thereby, as shown in FIGS. 1 and 2 of the present specification, it has been shown that the stability of dissolved 5,10-methylenetetrahydrofolic acid is increased, but at the expense of a decrease in the relative content of 5,10-methylenetetrahydrofolic acid.

[0037] Surprisingly, an aqueous solution of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (hereinafter denoted as 5,10-CH 2 -(6R)-THF*Na 2 ), in combination with a citrate and an alkali metal sulfate, has been found to have stability equal to or greater than that of an aqueous solution containing 5,10-methylenetetrahydrofolic acid, a citrate, and a reducing agent such as L-(+)-ascorbic acid. The solution according to the present invention is similarly converted into a lyophilizate having good stability. Since the alkali metal sulfate is not itself a reducing agent, it is normally considered to be chemically inert. Therefore, the discovered stability is surprising.

[0038] The lyophilizate of the present invention contains more than 20% w / w of 5,10-CH 2 -(6R)-THF*Na 2 , for example more than 25% w / w, for example more than 30% w / w, for example more than 35% w / w or for example about 40% w / w of 5,10-CH 2 -(6R)-THF*Na 2 .

[0039] The lyophilized powder has a higher stability than the aqueous solution and can be reconstituted to a set concentration with a diluent for administration. The lyophilized product reconstituted in this way can be administered intramuscularly or intravenously.

[0040] To facilitate the formation of an acceptable lyophilized cake, bulking agents such as mannitol can be added to the solution before the lyophilization method. The structure and porosity of the lyophilized cake are important because good pore formation can promote drying and moisture movement during the drying cycle.

[0041] Also, electrolytes, saccharides and / or dextrose, polyols such as glycerol and mannitol, and sodium chloride can be added to adjust the osmotic pressure. Adjustment of the osmotic pressure can be carried out before or after lyophilization. The reconstituted lyophilized solution preferably has an osmotic pressure in the range of 250 - 350 mOsm. However, an osmotic pressure in the range of 200 - 600 mOsm is also acceptable and varies depending on the dosage and injection / infusion time.

[0042] The pH of the solution is typically in the range of 8.0 - 9.0, preferably in the range of 8.4 - 8.8, and can be adjusted during pharmaceutical manufacture using, for example, small amounts of hydrochloric acid or sodium hydroxide.

[0043] Stability is an important property and a component in pharmaceutical formulation research and drug development. Stability tests are carried out in both the solution state and the solid state. It is an established fact that the stability in the solution state and the solid state can be qualitatively and quantitatively different. Extensive studies have been carried out on the stability of the active pharmaceutical ingredient and its pharmaceutical compositions by exposing them to various stress factors such as high temperature and high humidity. These studies also provide information on degradation products and are useful for developing meaningful specifications as well as understanding the inherent stability of the pharmaceutical composition. The most common pathways of drug degradation include hydrolysis, oxidation, photochemical decomposition, etc.

[0044] The purpose of the stability test is to provide evidence of how the quality of the product changes over time under the influence of various environmental factors such as temperature, humidity, light, etc., and to establish the appropriate shelf life and recommended storage conditions of the pharmaceutical product in order to ensure the safety of patients.

[0045] In a first aspect, the present invention relates to a lyophilized composition comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid (5,10-CH 2 -(6R)-THF*Na 2 ), citrate and an alkali metal sulfate.

[0046] In a preferred embodiment of the first aspect, the present invention relates to a stable lyophilized product comprising more than 20% w / w of 5,10-CH 2 -(6R)-THF*Na 2 , for example more than 25% w / w, for example more than 30% w / w, for example more than 35% w / w or for example about 40% w / w of 5,10-CH 2 -(6R)-THF*Na 2 .

[0047] The lyophilized product of the present invention is substantially amorphous, but has improved stability, for example improved storage stability. The lyophilized product of the present invention can be reconstituted into an aqueous pharmaceutical formulation by methods known in the art and administered to patients in need thereof.

[0048] In one embodiment, the present invention discloses a lyophilized composition according to the first aspect, wherein the molar ratio of the alkali metal sulfate to 5,10-CH 2 -(6R)-THF*Na 2 is about 40 mol% to about 400 mol%, preferably about 50 mol% to about 100 mol%.

[0049] In another embodiment, the present invention discloses a lyophilized composition according to the first aspect, wherein the molar ratio of 5,10-CH 2 -(6R)-THF*Na 2 to citrate is about 200 to 400 mol%, preferably about 350 mol%.

[0050] The second aspect of the present invention is directed to a method for preparing a lyophilized composition according to the first aspect, the method comprising the following steps: i. Dissolving (6S)-tetrahydrofolic acid in water with NaOH; ii. Adjusting the pH of the solution to 8.6 ± 0.5; iii. Adding 110 - 120 mol% of formaldehyde; iv. Stirring the reaction mixture until the reaction is complete; v. Adding about 40 - 200 mol% of an alkali metal sulfate and about 200 - 400 mol% of a citrate; vi. Filtering the reaction mixture to obtain a clear solution; and vii. Lyophilizing the obtained clear solution. This process does not include the step of adding an additional reducing agent or antioxidant.

[0051] In one embodiment, the alkali metal sulfate added in step v is in the form of sodium sulfate. In one embodiment, the citrate added in step v is in the form of sodium citrate. In another embodiment, the citrate is added as citric acid.

[0052] In a third aspect, the present invention further relates to a lyophilized composition comprising 5,10-CH 2 -(6R)-THF*Na 2 according to the first aspect for use in the treatment of cancer or cancer therapy in a human patient.

[0053] In a preferred embodiment, the present invention relates to a stable lyophilized product or a reconstituted aqueous solution thereof comprising 5,10-CH 2 -(6R)-THF*Na 2 according to the first aspect for use in the treatment of cancer or cancer therapy in a human patient.

[0054] In a fourth aspect, the present invention further relates to a method of treating cancer or cancer therapy in a human patient, comprising administering to the human patient in need thereof the lyophilized composition according to the first aspect.

[0055] In a preferred embodiment, the present invention relates to a method of treating cancer in a human patient, comprising administering to the human patient in need thereof a lyophilized composition comprising 5,10-CH 2 -(6R)-THF*Na 2 or a reconstituted aqueous solution thereof according to the first aspect.

[0056] In a fifth aspect, the present invention further relates to the use of a lyophilized composition comprising 5,10-CH 2 -(6R)-THF*Na 2 according to the first aspect for the manufacture of a medicament for the treatment of cancer in a human patient.

[0057] A further aspect is directed to a reconstituted pharmaceutical composition of the lyophilized product of the present invention, comprising 5,10-CH 2 -(6R)-THF*Na 2 and a pharmaceutically acceptable carrier or diluent such as sterile water or a liquid pharmaceutically acceptable vehicle, and optionally further comprising at least one additional therapeutic agent including, but not limited to, a bactericide, an antibiotic, an antiviral agent, a preservative, an antineoplastic agent, an anticancer compound such as a chemotherapeutic agent, an antifungal agent and / or an anti-inflammatory agent, or other bioactive agent or therapeutic agent suitable for use in humans, particularly an anticancer compound such as a chemotherapeutic agent, for example 5-FU and derivatives and antifolates, for example methotrexate, pemetrexed.

[0058] The lyophilized product of the present invention is in a substantially undissolved anhydrous form, which includes compounds that are completely water-free and compounds that may contain trace amounts of water. The amount of such residual (non-stoichiometric) water may be any amount, but is typically in the range of 0 wt.-% H 2 O to 3 wt.-% H 2 O, preferably 0 wt.-% H 20 to 1 wt.-% H 2 is in the range of O, for example, 0.05 wt.-% H 2 0 to 1 wt.-% H 2 is in the range of O.

Example

[0059] HPLC : For the measurement of purity / content and decomposition products, the HPLC-UV gradient method (HPLC-UV Gradient Method) was used. Column type: ODS, mobile phase A: aqueous buffer, mobile phase B: aqueous buffer / methanol, run time: 30 minutes, sample solvent: aqueous buffer.

[0060] Water content : The measurement of water content was carried out according to Ph.Eur. 2.5.32 / USP <921 / Method Ic>.

[0061] Osmotic pressure : The measurement of osmotic pressure was carried out according to Ph.Eur. 2.2.35 (osmometer) / USP <785>.

[0062] Example 1: Preparation of an aqueous solution containing 5,10-methylene(6R)-tetrahydrofolic acid disodium, citrate and sodium sulfate (a) (6S)-tetrahydrofolic acid (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 pH 11 by slowly adding 32% aqueous NaOH solution, then adjusted to pH 8.3 with 1.0 M HCl, and cooled to about 0 °C. Formaldehyde solution (1.44 g, 110 mol%) was added all at once, 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 5,10-methylene-(6R)-tetrahydrofolic acid disodium (5,10-CH 2 -(6R)-THF*Na 2A transparent solution of ( ) was obtained. The thus-obtained disodium 5,10-methylene-(6R)-tetrahydrofolate solution can be purified by the following steps (b) and (c), if necessary, by precipitation of the sulfate salt of 5,10-CH₂-(6R)-THF followed by redissolution with sodium hydroxide.

[0063] (b) (Optional) 55 mL of 1M H 2 SO 4 (0.055 mol, 344 mol%) was heated to 60 °C under nitrogen, and the solution obtained in step (a) above was added dropwise over 15 minutes. The resulting reaction mixture (slurry) was stirred at 50 °C for 2 hours. Next, the reaction mixture was suction filtered at 50 °C, and 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-CH 2 -(6R)-THF sulfate as light gray crystals (yield 86%).

[0064] (c) (Optional) The product of step (b) was dissolved in 50 mL of 0.1N NaOH under nitrogen, and the pH was adjusted to 8.6 ± 0.50 with 1.5 NaOH.

[0065] (d) To the solution of step (c) or step (a) was added a solution prepared by adding 21 g of citric acid to 20 mL of water, and the sulfate content was adjusted to 200 mol% with an aqueous sodium sulfate solution if necessary. Next, the total volume was adjusted to 100 mL with sterile filtered water and filtered using a 0.22 μm filter, preferably a hydrophilic PVDF type such as Millipore Durapore® 0.22 μm. While keeping the solution as cold as possible, the filtrate was filled into vials (2 mL or 160 mg of 5,10-CH 2 -(6R)-THF*Na 2 ) per vial).

[0066] Example 2: Preparation of a lyophilized composition containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citric acid and sodium sulfate The vial of Example 1 was frozen and then lyophilized. The vial was sealed and crimped under slightly reduced pressure with nitrogen in the headspace. The resulting lyophilized product contained more than 30% w / w of 5,10-CH 2 -(6R)-THF*Na 2 was included.

[0067] Example 3: Stability test a) Stability of a solution containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citrate and sulfate at 25 °C To measure the stability of a sterile filtered solution containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citrate and sulfate at 25 °C, the solution prepared in step (d) of Example 1 was stored at 25 °C. The purity of 5,10-CH 2 -(6R)-THF was measured periodically by HPLC (area %). The results are shown in Figure 4. During the test, the color of the test solution darkened from yellow to dark yellow. The samples stored at room temperature were not shielded from light. At room temperature, the purity remained stable at 96% during the 7-hour storage period. The trend of the analysis results during the stability test is summarized in Figure 4. The content of 10-formyl-(6R)-tetrahydrofolic acid (10-FTHFA), the main degradation product when stored at 25 °C, was measured periodically by HPLC. The results are shown in Figure 5.

[0068] b) Stability of a solution containing 5,10-methylene(6R)-tetrahydrofolic acid disodium, citrate and sulfate at 5 °C To measure the stability of a sterile filtered solution containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citrate and sodium sulfate at 5 °C, the solution prepared in step d of Example 1 was stored at 5 °C. 5,10-CH 2The purity of (6R)-THF was periodically measured by HPLC (area %). The results are shown in Fig. 4. The content of 10-formyl-(6R)-tetrahydrofolic acid (10FTHFA), the main degradation product, was periodically measured by HPLC. The results are shown in Fig. 5. During the test, the color of the test solution darkened from yellow to dark yellow. The stability test solution was stored in a refrigerator (2 °C to 8 °C) during the investigation and taken out only at the time of analytical sampling. It was not shielded from light during sampling. At 2 °C to 8 °C, there was no significant change in purity, and it remained stable at 97% even during a storage period of 7 hours. The changes in the analytical results during the stability test are summarized in Figs. 4 and 5. A comparative test solution containing sodium ascorbate (1 mg / mL as an antioxidant) and stored at 2 °C to 8 °C was repeatedly subjected to a stability test at 5 °C. No significant difference was observed between the test solution containing sodium ascorbate and the test solution without sodium ascorbate (Figs. 4 and 5). Therefore, it is considered that the test solution with added sodium ascorbate and the test solution without addition are equivalent in terms of stability. The 75 mg / mL solution of 5,10-methylene-(6R)-tetrahydrofolic acid is transparent and remains transparent whether stored at 2 to 8 °C or at room temperature, and no precipitation occurs. The solution is slightly viscous and the osmotic pressure is about 710 mOsmol / kg. In conclusion, the sterile filtered solution containing 75 mg / mL of 5,10-methylene(6R)-tetrahydrofolic acid disodium, citrate, and sulfate is 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.

[0069] c) Stability of the lyophilized composition containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citrate, and sodium sulfate at 5 °C, 25 °C, and 40 °C To determine the time-dependent stability of the lyophilized composition containing 5,10-methylene-(6R)-tetrahydrofolic acid disodium, citrate, and sodium sulfate at various temperatures, the lyophilized product prepared in Example 2 was stored at 5 °C, 25 °C, and 40 °C. 5,10-CH 2The purity of (6R)-THF was measured by HPLC at regular intervals (area %). The results relative to the starting value are shown in Figure 6 as relative purity in % w / w. The content of 10-formyl-(6R)-tetrahydrofolic acid (10-FTHFA), the main degradation product when stored at 5 °C, 25 °C and 40 °C, was measured by HPLC at regular intervals. The results are shown in Figure 7.

Claims

1. A lyophilized composition comprising the disodium salt, citrate, and alkali metal sulfate of 5,10-methylene-(6R)-tetrahydrofolic acid, the composition being free of additional reducing agents or antioxidants.

2. The lyophilized composition according to claim 1, comprising the disodium salt of 5,10-methylene(6R)-tetrahydrofolic acid, sodium sulfate, sodium citrate, water, and any osmotic correction additive.

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

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

5. The lyophilized composition according to any one of claims 1 to 4, which is a stable lyophilized product having a concentration of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid that exceeds 20% w / w, such as exceeding 25% w / w, such as exceeding 30% w / w, such as exceeding 35% w / w, or such as about 40% w / w of the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid.

6. The lyophilized composition according to any one of claims 1 to 5, comprising the disodium salt of 5,10-methylene-(6R)-tetrahydrofolic acid with a purity exceeding 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 osmotic correction excipients.

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

13. A method for preparing the lyophilized composition according to any one of claims 1 to 6, comprising the disodium salt, citrate and alkali metal sulfate of 5,10-methylene-(6R)-tetrahydrofolic acid, the method comprising the following steps: viii. Dissolving (6S)-tetrahydrofolic acid in water at about pH 11; ii. Adjusting the pH of the clear solution to 8.6 ± 0.5; iii. Adding 110 to 120 mol% of formaldehyde; iv. Stirring the reaction mixture until the reaction is complete; v. Adding about 40 to 200 mol% of alkali metal sulfate and 100 to 400 mol% of citrate; vi. Filtering the reaction mixture to obtain a clear solution; ix. Lyophilizing the obtained clear solution; The method does not include the step of adding an additional reducing agent or antioxidant.

14. The method according to 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 as citric acid or sodium citrate.

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