Stable pharmaceutical composition comprising 5, 10-methylene-(6R)-tetrahydrofolic acid and NACl

Abstract

The present invention relates to a solid pharmaceutical composition comprising 5, 10-methylene-(6R)-tetrahydrofolic acid and NaCl, as well as to a process for obtaining the composition, and to the use of such a product.

Description

[0001] The present invention relates to a stable pharmaceutical composition comprising 5,10-methylene-(6R)-tetrahydrofolate and NaCl.

[0002] As used herein, 5,10-CH 2 -(6R)-THF refers to 5,10-methylenetetrahydrofolate in its naturally occurring isomeric form (N-[4-[(6aR)-3-amino-1,2,5,6,6a,7-hexahydro-1-oxoimidazolo[1,5-f]pteridin-8(9H)-yl]benzoyl]-L-glutamate) wherein the chiral center at C6 of the pteridine ring and the α-carbon of the glutamic acid moiety are in their naturally occurring configuration. Background of the Invention

[0004] 5,10-Methylenetetrahydrofolate is known as a drug used in combination with 5-fluorouracil (5-FU) for the treatment of solid tumors (Seley, KL Drugs 4 (1), 99, 2001). 2 -(6R)-THF, together with the base analog and 5-FU metabolite 5-FdUMP, achieves its chemotherapeutic effect by inhibiting thymidylate synthase (TS). TS catalyzes the conversion of deoxyuridine (dUMP) to deoxythymidylate (dTMP), which is a basic building block for DNA synthesis. The inactivation of TS is achieved by the presence of TS, the base analog 5-FdUMP and 5,10-CH 2 -(6R)-THF forms a covalent ternary inhibitory complex (often referred to as an inhibitory ternary complex). 2 The intracellular concentration of (6R)-THF can achieve an enhancement of the cytotoxic effect of 5-FU, thereby increasing the stability of the ternary complex. This leads to a direct inhibition of DNA synthesis and repair, ultimately resulting in cell death and delayed tumor growth.

[0005] However, with 5,10-CH 2 -(6R)-THF has undesirable properties that limit its pharmaceutical use. 2 -(6R)-THF is highly susceptible to oxidation and chemical degradation leading to undesirably high impurity levels. 2 -(6R)-THF 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 an injection solution) has a high surface area for 5,10-CH 2-(6R)-THF is particularly susceptible to oxidation and chemical degradation. It is well known that in order to be suitable for pharmaceutical use, the corresponding composition needs to meet several requirements, including high stability (so that effective storage within an acceptable period of time can be achieved without showing significant changes in the physicochemical properties of the composition), easy 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 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). Attempts to stabilize 5,10-methylenetetrahydrofolate compositions include, 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, 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, especially for tetrahydrofolate; (iii) stabilization by inclusion of the compound in cyclodextrins (see, for example, EP 0 579 996); (iv) addition of citrate while adjusting the pH to alkaline values ​​(see, for example, EP 1 641 460); or (v) addition of dicarboxylic acids (see, for example, EP 18752161.2); or (vi) in various crystalline forms, such as sulfates (see, e.g., EP 0 537 492) or hemisulfates (see, e.g., EP 2 837 631).

[0007] From a clinical point of view, it is important to obtain a stable form, especially 5,10-CH 2 -(6R)-THF lyophilizates and solutions would be advantageous. For example, citric acid has been associated with various undesirable effects such as QTc 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.

[0008] The teachings of the prior art do not readily allow for the preparation of such stable stabilizer-free 5,10-CH2-(6R)-THF compositions, and therefore there remains a great need for stable liquid and solid pharmaceutical compositions, in particular lyophilizates of 5,10-CH2-(6R)-THF, which do not contain adventitious stabilizing compounds, such as stabilizers, buffers, reducing agents, etc. A resulting problem is that the stabilized forms of 5,10-CH2-(6R)-THF known in the prior art typically contain less than 50% of the active pharmaceutical compound due to dilution by stabilizing additives in the final dosage form of the pharmaceutical product.

[0009] There is still a great need for high 5,10-CH 2 -Stable pharmaceutical compositions of (6R)-THF, in particular lyophilizates. Summary of the invention

[0010] It has now been surprisingly found that certain solid compositions comprising 5,10-methylene-(6R)-tetrahydrofolate and NaCl overcome the known disadvantages discussed previously and allow the preparation of pharmaceutical compositions of high purity, high stability, high content of 5,10-methylene-(6R)-tetrahydrofolate and low content of oxidation products or other chemical degradation products. The favorable stability characteristics of the solid compositions of the present invention will allow their effective use in medical applications.

[0011] It has been surprisingly found that 5,10-CH 2 Adding NaCl to the lyophilized solution of -(6R)-THF can reduce the active ingredient 5,10-CH 2 The purity of -(6R)-THF is maintained at a very high level, while the amount of by-products is kept at an acceptably low level. The lyophilized product obtained contains a high content of 5,10-CH 2 -(6R)-THF, and exhibits stability for months or longer without significant loss of active ingredients, i.e., the amount of active ingredients is maintained at 95% or higher, more preferably at 98% or higher, including most preferably about 99%, 99.5% or 99.8% for several months, even without adding any stabilizer, buffer, reducing agent, etc. 2The lyophilisate of -(6R)-THF can be produced, stored and used without significant decomposition prior to reconstitution.

[0012] It was also found that the corresponding active ingredients and the previously known 5,10-CH 2 Compared with a freeze-dried product of -(6R)-THF (e.g. a freeze-dried product containing citrate and adjusted to an alkaline pH), the freeze-dried product of the present invention has a different water content.

[0013] Lyophilization or freeze drying is a dehydration process that works by freezing an aqueous solution containing dissolved material therein and then reducing the surrounding pressure to allow the frozen water to sublime directly from the solid phase to the gas phase. There are generally four stages in the complete freeze drying process: pretreatment, freezing, primary drying, and secondary drying.

[0014] Pretreatment includes any method of treating the material before freezing. This may include the addition of other components. 2 In the preparation of the stable freeze-dried product of -(6R)-THF and NaCl, a pretreatment may be performed, but is not necessary.

[0015] Freezing is usually accomplished by placing an aqueous solution of the material into a freeze drying flask that is either mechanically refrigerated or cooled with dry ice or liquid nitrogen. On a larger scale, freezing aqueous solutions is often accomplished using a freeze dryer. In this step, it is important to cool the material below its triple point, which is the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in subsequent steps. 2 In the case of a stable freeze-dried product of -(6R)-THF and NaCl, freezing is preferably accomplished at a temperature of -45°C to -70°C.

[0016] In the preparation of 5,10-CH 2 The stable freeze-dried product of -(6R)-THF and NaCl can be annealed at a storage temperature of about -5°C to -2°C for 1 to 2 hours, but this is not necessary.

[0017] During the primary drying stage, the pressure is reduced (to the millibar range) and enough heat is supplied to the material to cause the ice to sublime. During this initial drying stage, about 95% of the water in the material is sublimated. This stage can be slow (days in industry) because if too much heat is added, the structure of the material can be altered. During the primary drying stage, the pressure is controlled by applying a partial vacuum. The vacuum speeds up the sublimation, making it useful as a targeted drying process. 2In the preparation of a stable lyophilisate of -(6R)-THF and NaCl, the primary drying phase is started at a freezing temperature of preferably between -45° C. and -70° C. Then, during the primary drying phase, after an optional start time of preferably 10 to 120 minutes at freezing temperature, the temperature is increased over time to preferably about 0° C. During the primary drying phase, the pressure is maintained at preferably about 50 μbar to 200 μbar.

[0018] The secondary drying stage aims to remove the unfrozen water molecules, since the ice was removed in the primary drying stage. In this stage, the temperature is raised to a higher temperature than in the primary drying stage, even above 0°C, to disrupt any physicochemical interactions formed between the water molecules and the frozen material. Typically, the pressure is also reduced in this stage to facilitate desorption (usually in the range of microbars or fractions of a Pascal). 2 In the preparation of a stable lyophilisate of -(6R)-THF and NaCl, the secondary drying is preferably carried out at a temperature of up to about 25°C to 30°C and a pressure of about 50 microbar to 200 microbar.

[0019] In the presence of 5,10-CH 2 In the preparation of a stable lyophilisate of -(6R)-THF and NaCl, the primary and secondary drying stages can be combined by following a temperature ramp from freezing temperature up to about 25°C to 30°C and a pressure ramp from about 50 microbars to 200 microbars. The temperature ramp can contain multiple holding steps, in which the temperature is kept constant for a period of time. Preferably, the holding steps, if any, are at freezing temperature, at about 0°C and about 25°C to 30°C.

[0020] After the freeze-drying process is complete, the vacuum is usually broken with an inert gas (such as nitrogen) before sealing the material. 2 The final residual water content of the freeze-dried product of -(6R)-THF and NaCl is usually less than 5%, preferably at most 3%, even more preferably at most 2%, and most preferably less than 1%. In a particularly preferred embodiment, the lyophilized product containing 5,10-CH 2 The freeze-dried product of -(6R)-THF and NaCl is essentially anhydrous.

[0021] Stability is a key characteristic and element in pharmaceutical formulations and their research and during drug development. Chemical stability studies are conducted in both solution and solid states. It is a recognized fact that solution state and solid state stability are qualitatively and quantitatively different. In addition, in the solid state, the stability of crystalline materials and amorphous materials such as freeze-dried materials may be different. The chemical 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 to formulate meaningful specifications and the intrinsic stability of pharmaceutical compositions. The most common drug degradation pathways include hydrolysis, oxidation and photochemical degradation.

[0022] 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 appropriate shelf life and recommended storage conditions for drug products to ensure patient safety.

[0023] One embodiment relates to a 5,10-CH 2 -(6R)-THF and NaCl. A preferred embodiment relates to a pharmaceutical composition comprising 5,10-CH 2 Another preferred embodiment relates to a solid pharmaceutical composition comprising 5,10-CH 2 -(6R)-THF and NaCl in the absence of other chemotherapeutic agents.

[0024] In a preferred embodiment, the solid pharmaceutical composition of the present invention comprises 5,10-CH 2 In a further preferred embodiment, the 5,10-CH 2 The purity of -(6R)-THF is greater than 99%, more preferably greater than 99.5%.

[0025] One embodiment relates to a 5,10-CH 2 The freeze-dried product of -(6R)-THF and NaCl is further characterized by containing one or more stabilizing agents, such as stabilizers, buffers, reducing agents, etc., as defined below.

[0026] One embodiment relates to a 5,10-CH 2 -A freeze-dried product of (6R)-THF and NaCl, further characterized in that it contains neither citric acid nor citrate ions.

[0027] The lyophilisate of the present invention is a substantially amorphous solid composition having enhanced stability, such as enhanced storage stability.

[0028] The freeze-dried product of the present invention has a high content of 5,10-CH 2 -(6R)-THF, typically at least 25% w / w, such as at least 30% w / w, such as at least 35% w / w, such as at least 40% w / w

[0029] The lyophilisate of the present invention may further preferably be reconstituted into an aqueous pharmaceutical formulation for administration to a patient in need thereof.

[0030] A further aspect relates to a method for preparing the freeze-dried product of the present invention, comprising the following steps

[0031] (i) dissolving 5,10-methylene-(6R)-tetrahydrofolate or a salt thereof in a solvent or a solvent system;

[0032] (ii) adding NaCl and optional excipients to improve cake formation and / or adjust the osmotic pressure mass concentration of the solution;

[0033] (iii) freezing the solution; and

[0034] (iv) subsequently removing the frozen solvent under vacuum;

[0035] or

[0036] (i) dissolving 5,10-methylene-(6R)-tetrahydrofolate or a salt thereof in a solvent or a solvent system;

[0037] (iia) adding NaOH to raise the pH to about 11, and optionally adding excipients for improving cake formation and / or adjusting the osmolarity of the solution;

[0038] (iib) adding HCl to lower the pH to about 9.3, and optionally adding excipients for improving cake formation and / or adjusting the osmolarity of the solution;

[0039] (iii) freezing the solution; and

[0040] (iv) Thereafter the frozen solvent was removed under vacuum.

[0041] Prior to performing step (iii), the solution of step (ii), (ii)a or (iib) may optionally be filtered through a sterile filter.

[0042] Preferably, the pH of the solution after step (ii) or (iib) is above 6, more preferably about 8-14, even more preferably about 9.3.

[0043] In step (i), (ii), (iia) or (iib), an excipient may be optionally added to improve cake formation and / or adjust the solution osmotic pressure mass concentration and / or matrix stabilizer. Such optional excipients may be one or more bulking agents selected from arginine, glycine, histidine, dextran and / or polyethylene glycol.

[0044] The structure and porosity of the lyophilized cake are important because good pore formation can promote the transportation of water during drying and drying cycles. In addition, electrolytes or sodium chloride can be added to the composition 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 (re-dissolved) 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 it will depend on the volume to be administered and the injection / infusion time.

[0045] A further aspect relates to a reconstituted pharmaceutical composition of the lyophilisate of the present invention comprising 5,10-CH 2 -(6R)-THF and NaCl and a pharmaceutically acceptable carrier or diluent (such as sterile water or a liquid pharmaceutically acceptable vehicle), optionally further comprising an excipient and / or a matrix stabilizer for improving cake formation and / or adjusting the molar osmotic pressure mass concentration of the solution.

[0046] A further aspect relates to a reconstituted pharmaceutical composition of the lyophilisate of the present invention comprising 5,10-CH 2 -(6R)-THF and NaCl and a pharmaceutically acceptable carrier or diluent (e.g., sterile water or a liquid pharmaceutically acceptable vehicle), optionally further comprising an excipient and / or a matrix stabilizer for improving cake formation and / or adjusting the molar osmotic pressure mass concentration of the solution, and the reconstituted pharmaceutical composition optionally further comprises at least one additional 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 and pemetrexed.

[0047] As used herein, the term "liquid pharmaceutically acceptable vehicle" refers to propylene glycol, polyethylene glycol, ethanol, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), glycofurol, isopropyl glycerol (glycerol acetone acetal), glycerol formal, acetone, tetrahydrofurfuryl alcohol, monoethylene glycol dimethyl ether, diethylene glycol dimethyl ether, dimethyl isosorbide or ethyl lactate, mixtures thereof or aqueous mixtures thereof.

[0048] 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; 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-hydroxy-alkylammonium; glutamate; borate; lactate, and combinations of these. 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, in particular for tetrahydrofolate.

[0049] In the present context, the term "solvent" relates to a solvent that can be used in a freeze-drying process. Similarly, the term "solvent system" as used herein relates to a mixture of solvents that can be used in a freeze-drying process.

[0050] The “solution” mentioned in the present invention includes aqueous solutions and solutions in organic solvents.

[0051] In general, "aqueous solution" means a solution in water, 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.

[0052] A further aspect relates to the use of the reconstituted pharmaceutical composition of the invention in therapy, preferably in cancer chemotherapy.

[0053] A further aspect of the invention relates to the use of the pharmaceutical compositions of the invention, including solid pharmaceutical compositions and reconstituted solutions thereof, 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 compositions of the invention include fluorinated nucleic acids, such as 5-fluorouracil or a prodrug or analog thereof.

[0054] The reconstituted pharmaceutical composition of the present invention can be used for therapy, in particular for cancer chemotherapy, i.e., for a method of treating cancer, which method comprises administering to a subject in need of such treatment a therapeutically effective amount of 5,10-CH 2 -(6R)-THF.

[0055] Another embodiment relates to the use of the reconstituted pharmaceutical composition of the present invention in therapy, in particular in cancer chemotherapy, comprising administering to a subject in need of such treatment a therapeutically effective amount of 5,10-CH 2 -(6R)-THF.

[0056] Another embodiment relates to a reconstituted pharmaceutical composition of the present invention for use in the treatment of cancer, in particular a cancer form selected from colon cancer, gastric cancer, breast cancer, intestinal cancer, gallbladder cancer, lung cancer (especially adenocarcinoma), colorectal cancer (CRC) (including metastatic CRC), head and neck cancer, liver cancer and pancreatic cancer.

[0057] Another embodiment relates to the use of the reconstituted pharmaceutical composition of the invention for the preparation of a medicament for the treatment of cancer, in particular a cancer form selected from colon cancer, gastric cancer, breast cancer, intestinal cancer, gallbladder cancer, lung cancer (especially adenocarcinoma), colorectal cancer (CRC) (including metastatic CRC), head and neck cancer, liver cancer and pancreatic cancer.

[0058] In another embodiment, the reconstituted pharmaceutical composition of the present invention is used for therapy, preferably for chemotherapy, i.e. for the treatment of cancer. Examples of cancers to be treated include, but are not limited to, breast cancer, esophageal cancer, gastric cancer, gallbladder cancer, bile duct cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and mesothelioma cancer.

[0059] In a preferred embodiment, the cancer is selected from a variety of cancer forms including colon cancer, gastric cancer, breast cancer, intestinal cancer, gallbladder cancer, lung cancer (particularly adenocarcinoma), colorectal cancer (CRC) (including metastatic CRC), head and neck cancer, liver cancer and pancreatic cancer.

[0060] The reconstituted pharmaceutical composition of the present invention is in a form suitable for parenteral administration, such as intravenous or intramuscular injection, subcutaneous injection or intra-arterial administration.

[0061] For parenteral administration, the fluid unit dosage form generally comprises a reconstituted lyophilisate (preferably a reconstituted pharmaceutical composition of the present invention), optionally other therapeutic agents and a pharmaceutically acceptable carrier or diluent to form, for example, a water-based solution or an oil-based suspension. For parenteral solutions, the lyophilisate of the present invention can be filter sterilized during its preparation (e.g., before filling into a suitable vial or ampoule).

[0062] In the case of combination therapy with a reconstituted pharmaceutical composition of the invention and at least one other therapeutic agent, the active agent may be administered as part of the same pharmaceutical composition, or the at least one other therapeutic agent may be administered separately, i.e. as separate (and possibly different) pharmaceutical compositions, optionally administered simultaneously or sequentially via different routes of administration.

[0063] The active agent(s) used in the treatments described herein, namely 5,10-CH 2 The dosage of -(6R)-THF (and optionally at least one other therapeutic agent) will depend on various factors, including the age and health of the subject to be treated, the type and severity of the disease to be treated, and the frequency of administration. Those skilled in the art of cancer treatment and chemotherapy will be able to determine the dosage of the active pharmaceutical ingredient 5,10-CH based on known protocols for evaluating toxicity and efficacy. 2 -Therapeutically effective amounts and regimens of (6R)-THF alone or in combination with at least one other therapeutic agent as defined above.

[0064] The term "therapeutically effective amount" refers to the amount of active compound that elicits a biological or medical response in a tissue, system, animal, individual or human that is sought by a skilled practitioner (e.g., a researcher, veterinarian, physician or other clinician or caregiver), including (i) preventing disease; and / or (ii) inhibiting disease (e.g., preventing further development of pathology and / or symptoms); and / or (iii) ameliorating disease (e.g., reversing pathology and / or symptoms). Similarly, the term "treating" as used herein refers to (i) preventing disease; and / or (ii) inhibiting disease (e.g., preventing further development of pathology and / or symptoms); and / or (iii) ameliorating disease (e.g., reversing pathology and / or symptoms).

[0065] Depending on the method of administration, the selected pharmaceutical composition may contain 0.1 wt % to 99 wt %, preferably 10 wt % to 60 wt % of the active pharmaceutical ingredient (i.e. 5,10-CH 2 -(6R)-THF, optionally in combination with at least one other therapeutic agent).

[0066] 5,10-CH 2 Typical doses of (6R)-THF can range from 5 mg / m 2 Up to 1.5g / m 2 , preferably 30 mg / m 2 Up to 500 mg / m 2 (for colorectal cancer) or 10 mg / m 2 Up to 1000 mg / m 2 (for methotrexate therapy), more preferably about 60 mg / m 2About 300 mg / m 2 (for colorectal cancer treatment) or 50 mg / m 2 Up to 500 mg / m 2 (For methotrexate therapy).

[0067] As used herein, the term "purity" means the percentage of a particular compound in a sample. As used herein, the term "substantially pure" refers to a compound having a purity of about 80%, preferably about 90%, more preferably about 95%, more preferably about 97%, more preferably about 98% purity, and most preferably 99% or higher, such as 99.5, 99.6, 99.7, 99.8, 99.9 or up to 100% purity, as determined by HPLC. Impurities may include unreacted starting materials and solvents, 5,10-CH 2 -Degradation products of (6R)-THF (e.g. 5,10-methylene-(6R)-tetrahydropteroic acid and other degradation products), etc.

[0068] The term "pharmaceutically acceptable" as used herein means that the carrier is approved or approved for use in animals, more specifically in humans, i.e., it is non-toxic to the subject or patient. In addition, the selected carrier will not interfere with the effectiveness of the biological activity of the active ingredient. The term "carrier" refers to any auxiliary material necessary for the selected specific mode of administration, and includes, for example, solvents (diluents), excipients, or other additives administered together with the freeze-dried product of the present invention. Typical diluent pharmaceutical carriers used include sterile liquids, such as aqueous solutions and oils (e.g., petroleum, animal, plant or synthetic sources), such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Typical aqueous liquids used include water, saline solutions, dextrose and glycerol aqueous solutions, etc. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are well known in the art and are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin, 18th ed., Mack Publishing Co., Easton, PA (1990).

[0069] Throughout the specification and following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated compounds, or classes or groups of compounds, or groups of steps but not the exclusion of any other integers or steps or integers or groups of steps.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Contains two tables: Table 1 (top) and Table 2 (bottom).

[0072] Table 1 shows the long-term stability of the freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 2.

[0073] Table 2 similarly presents the long-term stability of the freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 3.

[0074] Figure 2 Contains Table 3

[0075] Table 3 shows the long-term stability of the freeze-dried material containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 2 by following the development of the content of one of the main degradation products (5,10-methylene-(6R)-tetrahydropteroic acid) in the freeze-dried material containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 3.

[0076] Figure 3 Contains two scatter plots: Chart 1 (top) and Chart 2 (bottom)

[0077] The Figure contains a graph comparing the effect of NaCl relative to citrate on the stability of lyophilisates at different storage temperatures. The effect of NaCl is comparable to that of citrate, as demonstrated by the development of the 5,10-methylene-(6R)-tetrahydrofolate content over time.

[0078] Graph 1 shows the long-term stability of the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 2, Example 3 and Reference Example 8 at 5°C.

[0079] Graph 2 similarly shows the long-term stability of the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 2, Example 3 and Reference Example 8 at 25°C.

[0080] Figure 4 Contains a scatter plot: Chart 3

[0081] The Figure contains a graph comparing the effect of NaCl versus citrate on freeze-dried product stability at elevated storage temperatures. The effect of NaCl is comparable to that of citrate, as demonstrated by the development of 5,10-methylene-(6R)-tetrahydrofolate content over time.

[0082] Graph 3 shows the long-term stability of the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 2, Example 3 and Reference Example 8 at 40°C.

[0083] Figure 5 Contains two scatter plots: Chart 4 (top) and Chart 5 (bottom)

[0084] The Figure contains a graph comparing the effect of NaCl relative to citrate on the stability of lyophilisates at different storage temperatures. The effect of NaCl is comparable to that of citrate, as demonstrated by the development over time of the content of one of the main degradation products, 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA).

[0085] Graph 4 shows the content development of 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA), one of the main degradation products, in the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 3 and Reference Example 8 at 5°C.

[0086] Graph 5 similarly shows the development of the content of 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA), one of the main degradation products, in the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 3 and Reference Example 8 at 25°C.

[0087] Figure 6 Contains a scatter plot: Chart 6

[0088] The Figure contains a graph comparing the effect of NaCl versus citrate on the stability of lyophilisates at elevated storage temperatures. The effect of NaCl is comparable to that of citrate, as evidenced by the development over time of the content of one of the main degradation products, 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA).

[0089] Graph 6 shows the content development of 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA), one of the main degradation products, in the freeze-dried products containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl prepared according to Example 3 and Reference Example 8 at 40°C. Example

[0090] HPLC

[0091] To measure purity / content and degradation products, a 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

[0092] Water content

[0093] The determination of the water content was carried out according to Ph. Eur. 2.5.32 / USP <921 / Method Ic>.

[0094] Osmolarity concentration

[0095] According to Ph.Eur.2.2.35 (osmometer) / USP <785> The molar osmotic pressure and mass concentration were measured.

[0096] Example 1: Freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl

[0097] Under nitrogen, 210 g of pure water and 16.5 g of sodium hydroxide 2M were cooled to 3 ± 2 °C (to obtain pH 14.0). 4.57 g of NaCl was added, followed by 5.70 g of 5,10-methylene-(6R)-tetrahydrofolate hemisulfate (pH starts to drop) and rinsed with 2.5 g of pure water. The pH was maintained at 9.3 ± 0.1 by adding 2M sodium hydroxide. 18.74 g of pure water was added. In total, 20.8 g of 2M sodium hydroxide was required to maintain the pH at 9.3 ± 0.1.

[0098] 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials) per vial. The vials were immediately frozen with liquid nitrogen and stored at <10 -1 Lyophilized at 1000 mbar.

[0099] The obtained vial contained 102 mg of 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as free acid) and a lyophilisate of NaCl, wherein the w / w percentage of 5,10-methylene-(6R)-tetrahydrofolic acid in the lyophilisate was 40% of the total solids in the lyophilisate.

[0100] When reconstituted, the osmolarity of the solution was 346 mOsmol / kg.

[0101] Example 2: Freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl

[0102] Under nitrogen, 210 g of pure water and 16.5 g of sodium hydroxide 2M are cooled to 3 ± 2 °C (to obtain pH 14.0). 4.10 g of NaCl and 5.70 g of 5,10-methylene-(6R)-tetrahydrofolate hemisulfate are added (pH starts to drop) and rinsed with 2.5 g of pure water. The pH is maintained at 9.3 ± 0.1 by adding 2M sodium hydroxide. 18.8 g of pure water are added. In total, 21.2 g of 2M sodium hydroxide are required to maintain the pH at 9.3 ± 0.1.

[0103] 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials) per vial. The vials were immediately frozen with liquid nitrogen and stored at <10 -1 Lyophilized at 1000 mbar.

[0104] The obtained vial contained 102 mg of 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as free acid) and a freeze-dried product of NaCl. 5,10-methylene-(6R)-tetrahydrofolic acid showed a purity of 93.2% w / w (as free acid) as measured by HPLC.

[0105] When reconstituted, the osmolarity of the solution was 320 mOsmol / kg.

[0106] Example 3: Freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl

[0107] Under nitrogen, 210 g of pure water and 16.5 g of sodium hydroxide 2M are cooled to 3 ± 2 °C (to obtain pH 14.0). 4.10 g of NaCl and 5.80 g of 5,10-methylene-(6R)-tetrahydrofolate hemisulfate are added (pH starts to drop) and rinsed with 2.5 g of pure water. The pH is maintained at 9.3 ± 0.1 by adding 2M sodium hydroxide. 18.8 g of pure water are added. In total, 21.2 g of 2M sodium hydroxide are required to maintain the pH at 9.3 ± 0.1.

[0108] 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials) per vial. The vials were immediately frozen with liquid nitrogen and stored at <10 -1 Lyophilized at 1000 mbar.

[0109] The obtained vial contained 97.468 mg of 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as free acid) and a freeze-dried product of NaCl. 5,10-methylene-(6R)-tetrahydrofolic acid showed a purity of 93.2% w / w (as free acid) or 96.7% area, with a sum of by-products of 1.07%, both measured by HPLC. The water content was 0.63 w / w.

[0110] When reconstituted, the solution had an osmolarity of 325 mOsmol / kg and a pH of 8.23.

[0111] Example 4: Freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl

[0112] Under nitrogen, 210 g of pure water and 16.5 g of sodium hydroxide 2M are maintained at 25 ± 2 ° C (to obtain pH 13.2). 4.10 g of NaCl and 5.80 g of 5,10-methylene-(6R)-tetrahydrofolate hemisulfate are added (pH starts to drop) and rinsed with 2.5 g of pure water. The pH is maintained at 9.3 ± 0.1 by adding 2M sodium hydroxide. 17.1 g of pure water is added. In total, 21.2 g of 2M sodium hydroxide is required to maintain the pH at 9.3 ± 0.1.

[0113] 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials) per vial. The vials were immediately frozen with liquid nitrogen and stored at <10 -1 Lyophilized at 1000 mbar.

[0114] The obtained vial contained a freeze-dried product of 5,10-methylene-(6R)-tetrahydrofolate with NaCl, showing a purity of 95.9% area for 5,10-methylene-(6R)-tetrahydrofolate and 1.07% for the sum of by-products, both measured by HPLC. The water content was 0.67% w / w.

[0115] When reconstituted, the solution had an osmolarity of 329 mOsmol / kg and a pH of 9.06.

[0116] Example 5: Long-term stability of a freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl ((6R)-5,10-CH 2 -THF content)

[0117] In order to determine the 5,10-CH 2 Long-term stability of the lyophilisates of -(6R)-THF was measured by HPLC at periodic intervals. 2 -(6R)-THF content and is given by comparison with the initial value (% rel.). The results are shown in Tables 1 and 2 (Graphs 1-3).

[0118] Tables 1 and 2 (Figures 1-3) clearly show that 5,10-CH 2 The freeze-dried product of -(6R)-THF and NaCl is highly stable over a long period of time even at elevated temperatures.

[0119] Example 6: Long-term stability of a freeze-dried product containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl (Stability index: 5,10-methylene-(6R)-tetrahydropteroic acid content)

[0120] In order to determine the (6R)-5,10-CH 2The long-term stability of the lyophilized product of -THF was stored in air at +5°C, +25°C / 60% relative humidity and +40°C / 75% relative humidity. The content of 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA), one of the main degradation products, was measured by HPLC at periodic intervals. The results are shown in Table 3 (Figures 4-6).

[0121] Table 3 (Figures 4-6) confirms that even at elevated temperatures, 5,10-CH 2 The freeze-dried product of (6R)-THF and NaCl was also highly stable over a long period of time, as also confirmed by the very slow increase in the content of one of the major degradation products, 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA).

[0122] Example 7: Reconstitution of a lyophilisate containing 5,10-methylene-(6R)-tetrahydrofolate and NaCl

[0123] The lyophilisates prepared according to Examples 1 to 4 can be easily reconstituted by adding 10 ml water for injection to the vial. The lyophilisates dissolve completely.

[0124] Reference Example 8: A composition containing 5,10-methylene-(6R)-tetrahydrofolic acid and citrate, the pH of which has been adjusted to Reduce alkalinity (EP1641460)

[0125] As a reference, a 5,10-CH 2 -(6R)-THF and citrate, the pH having been adjusted to 8.5-9.5.

[0126] Under nitrogen, keep 210 g of pure water and 16.5 g of sodium hydroxide 2M at 3 ± 2 ° C (to obtain pH 13.9). Add 11.50 g of trisodium citrate and 5.80 g of 5,10-methylene-(6R)-tetrahydrofolate hemisulfate (pH starts to drop) and rinse with 2.5 g of pure water. Keep the pH at 9.3 ± 0.1 by adding 2M sodium hydroxide. Add 11.0 g of pure water. In total, 13.8 g of 2M sodium hydroxide is required to keep the pH at 9.3 ± 0.1.

[0127] 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials) per vial. The vials were immediately frozen with liquid nitrogen and stored at <10 -1 Lyophilized at 1000 mbar.

[0128] The obtained vial contained a freeze-dried product of 5,10-methylene-(6R)-tetrahydrofolate and citrate, showing a purity of 97.8% area for 5,10-methylene-(6R)-tetrahydrofolate and 0.81% for the sum of by-products, both measured by HPLC.

Claims

1. A solid pharmaceutical composition comprising 5,10-methylene-(6R)-tetrahydrofolate and NaCl.

2. The solid pharmaceutical composition according to claim 1, further characterized in that it does not contain other chemotherapeutic agents.

3. The solid pharmaceutical composition according to claim 1 or 2, characterized in that: The pharmaceutical composition is in the form of a stable lyophilisate.

4. The solid pharmaceutical composition according to any one of claims 1 to 3, wherein the molar ratio of 5,10-methylene-(6R)-tetrahydrofolate to NaCl is 1:1 to 1:

20.

5. The solid pharmaceutical composition according to any one of claims 1 to 4, wherein the molar ratio of 5,10-methylene-(6R)-tetrahydrofolate to NaCl is 1:5 to 1:

10.

6. The solid pharmaceutical composition according to any one of claims 1 to 5, wherein the molar ratio of 5,10-methylene-(6R)-tetrahydrofolate to NaCl is about 1:

7.

7. The solid pharmaceutical composition according to any one of claims 1 to 6, wherein the 5,10-methylene-(6R)-tetrahydrofolate used has a purity greater than 99%, such as a purity greater than 99.5%.

8. The solid pharmaceutical composition according to any one of claims 1 to 7, characterized in that It contains neither citric acid nor citrate ions.

9. The solid pharmaceutical composition according to any one of claims 1 to 8, having a water content of less than 5%.

10. A solid pharmaceutical composition according to any one of claims 1 to 8, having a water content of at most 3%.

11. A solid pharmaceutical composition according to any one of claims 1 to 8, having a water content of less than 1%.

12. A solid pharmaceutical composition according to any one of claims 1 to 8 which is substantially anhydrous.

13. The solid pharmaceutical composition according to any one of claims 1 to 12, further comprising one or more pharmaceutically acceptable excipients and / or osmotic pressure mass concentration correction excipients and / or matrix stabilizers.

14. The solid pharmaceutical composition according to any one of claims 1 to 13, further comprising a buffer.

15. A reconstituted product obtained by dissolving the solid pharmaceutical composition according to any one of claims 1 to 14 in water or a liquid pharmaceutically acceptable vehicle.

16. The reconstituted product according to claim 15, wherein the water is sterile water for injection.

17. A method for preparing a stable freeze-dried product according to any one of claims 1 to 14, comprising the following steps (i) dissolving 5,10-methylene-(6R)-tetrahydrofolate or a salt thereof in a solvent or a solvent system, (ii) adding sodium chloride, (iii) freezing the solution, and (iv) thereafter removing the frozen solvent under vacuum, or (i) dissolving 5,10-methylene-(6R)-tetrahydrofolate or a salt thereof in a solvent or a solvent system, (iia) adding NaOH to raise the pH to about 11, (iib) adding HCl to lower the pH to about 9.3, (iii) freezing the solution, and (iv) Thereafter the frozen solvent was removed under vacuum.

18. The method of claim 17, wherein the solvent is water or an aqueous solvent system.

19. The method according to claim 17 or 18, wherein in step (ii), (iia) and / or (iib), a molar osmotic pressure mass concentration correction excipient and / or a matrix stabilizer is added.

20. The reconstituted product according to any one of claims 15 to 16 for use in cancer treatment or cancer therapy.

21. The reconstituted product according to any one of claims 15 to 16 for use in the treatment or therapy of cancer according to claim 20, wherein the cancer is colorectal cancer.

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