Stable pharmaceutical composition containing 5,10-methylene-(6R)-tetrahydrofolic acid and NaCl

Abstract

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

Description

Technical Field

[0001] The present invention is directed to a stable pharmaceutical composition containing 5,10-methylene-(6R)-tetrahydrofolic acid and NaCl.

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

Background Art

[0003] 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). Active 5,10-CH 2 -(6R)-THF is a base analog that exerts a chemotherapeutic effect together with 5-FdUMP, a metabolite of 5-FU, by inhibiting the enzyme thymidylate synthase (TS). TS catalyzes the conversion of deoxyuridylic acid (dUMP) to deoxythymidylate (dTMP), an essential component for DNA synthesis. Inactivation of TS occurs by the formation of a covalent ternary inhibitory complex (also called an inhibitory ternary complex) between TS, the base analog 5-FdUMP, and 5,10-CH 2 -(6R)-THF. 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 complex. This directly inhibits DNA synthesis and repair, ultimately leading to cell death and tumor growth delay.

[0004] However, 5,10-CH2 -(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. 5,10-CH 2 The susceptibility of -(6R)-THF to oxidation and chemical decomposition is particularly high when the compound exists in an amorphous form and has a large surface area, such as when present in a re-dissolved form such as a lyophilisate for pharmaceutical use or an injection solution. It is well known that in order to be suitable for pharmaceutical use, each composition must meet several requirements including high stability so that it can be effectively stored for an acceptable period without showing significant changes in the physicochemical properties, ease of handling and processing, etc. of the composition.

[0005] 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 to cause chemical degradation and / or hydrolysis due to instability in a neutral and / or acidic environment (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).

[0006] 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, β - mercaptoethanol, thioglycerol, N - acetyl - L - cysteine, etc. as antioxidants for very sensitive 5,10 - methylenetetrahydrofolic acid, especially tetrahydrofolic acid, (iii) stabilization by cyclodextrin inclusion compounds (see, for example, Patent Document 2: European Patent No. 0579996), (iv) addition of citric acid while adjusting the pH to a basic value (see, for example, Patent Document 3: European Patent No. 1641460), or (v) addition of dicarboxylic acids (see, for example, Patent Document 4: European Patent No. 18752161.2), or (vi) formation of various crystalline forms such as sulfates (see, for example, Patent Document 5: European Patent No. 0537492) or hemisulfates (see, for example, Patent Document 6: European Patent No. 2837631) can be mentioned.

[0007] From a clinical perspective, it would be advantageous if stable forms, especially lyophilized products and solutions of 5,10 - CH 2 -(6R)-THF with a high content of the active ingredient and without any kind of stabilizer were available. For example, citric acid is associated with various undesirable effects such as QT c elongation (Non - Patent Document 7: Laspina et al. Transfusion 42(2002) p.899; Non - Patent Document 8: Toyoshima et al. Clinical Nutrition(2006) 25, 653 - 660), induction of hypocalcemia (Non - Patent Document 9: Payne et. Al. J. Physiol.(1964), 170, pp.613 - 620), etc.

[0008] The teachings of the prior art do not readily enable the preparation of compositions that do not contain such stabilizing agents as 5,10-CH 2 -(6R)-THF, and thus there remains a great need for stable liquid and solid pharmaceutical compositions of 5,10-CH 2 -(6R)-THF that do not contain foreign stabilizing compounds such as stabilizers, buffers, reducing agents, etc. The stabilized versions of 5,10-CH 2 -(6R)-THF known in the prior art are diluted in the final dosage form of the drug by the stabilizing additive, and thus it is a problem that usually the content of the active pharmaceutical compound is less than 50%.

[0009] There remains a high need for stable pharmaceutical compositions, particularly lyophilized products, that contain a high content of 5,10-CH 2 -(6R)-THF.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

[0012] Surprisingly, a specific solid composition containing 5,10 - methylene-(6R)-tetrahydrofolic acid and NaCl overcomes the aforementioned known drawbacks and enables the preparation of a pharmaceutical composition with high purity, high stability, a high content of 5,10 - methylene-(6R)-tetrahydrofolic acid, and a low content of oxidation products or other chemical decomposition products. The advantageous stability characteristics of the solid composition of the present invention enable effective use in pharmaceutical applications.

[0013] 5,10 - CH 2 When NaCl is added to a lyophilized solution of 5,10 - CH 2-(6R)-THF was found to be maintained at a significantly high level of purity, while at the same time the amount of by-products was maintained at an acceptably low level. The obtained lyophilized product contains 5,10-CH 2 -(6R)-THF at a high content and shows stability over several months or more without significant loss of the active ingredient. That is, the obtained lyophilized product contains 5,10-CH 2 -(6R)-THF at a high content and shows stability over several months or more without significantly losing the active ingredient. That is, the amount of the active ingredient is maintained at 95% or about 95%, more preferably 98% or about 98%, for several months without adding stabilizers, buffers, reducing agents, etc., and most preferably about 99%, 99.5% or 99.8%. This enables the production, storage and use of the lyophilized product of 5,10-CH 2 -(6R)-THF without significant decomposition before reconstitution.

[0014] Furthermore, the lyophilized product of the present invention was found to have a water content different from that of the known lyophilized products of the corresponding active ingredient and 5,10-CH 2 -(6R)-THF (for example, those containing citric acid while adjusting the pH to a basic value).

[0015] Lyophilization or freeze-drying is a dehydration method that functions by freezing an aqueous solution containing a dissolved substance and then reducing the surrounding pressure to directly sublime the frozen water from the solid phase to the gas phase. A complete lyophilization method usually has four stages: pretreatment, freezing, primary drying and secondary drying.

[0016] Pretreatment includes any method of treating the substance before freezing. This may include the addition of other components. In the preparation of a stable lyophilized product containing 5,10-CH 2 -(6R)-THF and NaCl, pretreatment is possible but not essential.

[0017] Freezing is often carried out by placing an aqueous solution of the substance in a freeze-drying flask and cooling it by mechanical cooling or by using dry ice or liquid nitrogen. On a larger scale, freezing of the aqueous solution is usually carried out using a freeze dryer. In this step, it is important to cool the substance to a temperature below the triple point, which is the lowest temperature at which the solid and liquid phases of the substance can coexist. This ensures that sublimation rather than melting occurs in the next step. 5,10-CH 2 When preparing a stable lyophilized product containing 5,10-CH-(6R)-THF and NaCl, freezing is preferably carried out at a temperature of -45 °C to -70 °C.

[0018] 5,10-CH 2 When preparing a stable lyophilized product containing 5,10-CH-(6R)-THF and NaCl, annealing at a shelf temperature of about -5 °C to -2 °C for 1 to 2 hours is possible but not essential.

[0019] In the primary drying phase, the pressure is reduced (to the range of a few millibars) and sufficient heat is supplied to the material for the ice to sublimate. In this initial drying phase, about 95% of the moisture in the material sublimates. This phase can be slow (taking several days in the industry) because if too much heat is applied, the structure of the material may change. In the primary drying phase, a partial vacuum is applied to control the pressure. Vacuum accelerates sublimation and is useful as a planned drying method. 5,10-CH 2 In the preparation of a stable lyophilized product containing 5,10-CH-(6R)-THF and NaCl, the primary drying phase preferably starts at a freezing temperature between -45 °C and -70 °C. Next, during the primary drying phase, after an optional start period of preferably 10 to 120 minutes at the freezing temperature, the temperature preferably rises with time to about 0 °C. During the primary drying phase, a pressure of preferably about 50 μbar to 200 μbar is maintained.

[0020] The purpose of the secondary drying stage is to remove unfrozen water molecules since ice has been removed in the primary drying stage. In this stage, the temperature is higher than that in the primary drying stage and can exceed 0 °C in order to break the physicochemical interactions formed between water molecules and the frozen substances. Usually, in this stage, the pressure is also reduced (typically in the microbar range, i.e., a fraction of a Pascal) to promote desorption. 5,10-CH 2 In the production of a stable lyophilized product containing 5,10-CH-(6R)-THF and NaCl, secondary drying is preferably carried out at a temperature from about 25 °C to 30 °C and a pressure from about 50 μbar to 200 μbar.

[0021] 5,10-CH 2 In the production of a stable lyophilized product containing 5,10-CH-(6R)-THF and NaCl, the primary drying stage and the secondary drying stage can be combined by following a temperature gradient from the freezing temperature to a temperature from about 25 °C to 30 °C and a pressure gradient from about 50 μbar to 200 μbar. The temperature gradient may include a plurality of holding steps where the temperature is maintained for a certain period of time. When there are holding steps, they are preferably carried out at the freezing temperature, about 0 °C, and about 25 °C to 30 °C.

[0022] After the lyophilization process is completed and before the material is sealed, the vacuum is usually broken with an inert gas such as nitrogen. At the end of the operation, 5,10-CH 2 The final residual moisture content of the lyophilized product containing 5,10-CH-(6R)-THF and NaCl is usually less than 5%, preferably at most 3%, more preferably at most 2%, and most preferably less than 1%. In a particularly preferred embodiment, 5,10-CH 2 The lyophilized product containing 5,10-CH-(6R)-THF and NaCl is essentially anhydrous.

[0023] Stability is not only important in the research and development of pharmaceutical formulations but also a crucial property and element of pharmaceutical formulations. The study of chemical stability is 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 differ qualitatively and quantitatively. Also, in the solid state, the stability of crystalline substances and amorphous substances such as lyophilized products may vary. Extensive studies have been conducted on the chemical stability of active ingredients and their 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 pharmaceutical compositions. The most common pathways in drug degradation include hydrolysis, oxidation, photochemical decomposition, etc.

[0024] The purpose of stability testing is to provide evidence of how the quality of a 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 for pharmaceuticals in order to ensure patient safety.

[0025] One embodiment is directed to a pharmaceutical composition containing 5,10-CH 2 -(6R)-THF and NaCl. A preferred embodiment is directed to a solid pharmaceutical composition containing 5,10-CH 2 -(6R)-THF and NaCl. Another preferred embodiment is directed to a solid pharmaceutical composition containing 5,10-CH 2 -(6R)-THF and NaCl and containing no further chemotherapeutic agents.

[0026] In a preferred embodiment, the solid pharmaceutical composition of the present invention is a lyophilized product containing 5,10-CH 2 -(6R)-THF and NaCl and containing no further chemotherapeutic agents. In a more preferred embodiment, the 5,10-CH 2 -(6R)-THF used has a purity exceeding 99%, more preferably exceeding 99.5%.

[0027] One embodiment is 5,10-CH 2Directed to a lyophilizate characterized by containing (6R)-THF and NaCl, and further containing one or more stabilizers such as stabilizers, buffers, reducing agents, etc. as defined below.

[0028] One embodiment is 5,10-CH 2 Directed to a lyophilizate characterized by containing (6R)-THF and NaCl and further not containing citric acid and citrate ions.

[0029] The lyophilizate of the present invention is a substantially amorphous solid composition and has improved stability, for example, improved storage stability.

[0030] The lyophilizate of the present invention is 5,10-CH 2 The content of -(6R)-THF is high, typically at least 25% w / w, for example at least 30% w / w, for example at least 35% w / w, for example at least 40% w / w.

[0031] The lyophilizate of the present invention is more preferably reconstitutable into an aqueous pharmaceutical formulation for administration to a patient in need thereof.

[0032] A further aspect is a method for preparing the lyophilizate of the present invention, which is directed to a method comprising the following steps. (i) Dissolving 5,10-methylene-(6R)-tetrahydrofolic acid or a salt thereof in a solvent or solvent system; (ii) Adding NaCl and optionally an excipient for improving cake formation and / or adjusting the osmotic pressure of the solution. (iii) Freezing the solution, and (iv) Then removing the frozen solvent under vacuum; Or, (i) Dissolving 5,10-methylene-(6R)-tetrahydrofolic acid or a salt thereof in a solvent or solvent system; (iia) Adding NaOH to raise the pH to about 11 and optionally adding excipients to improve cake formation and / or adjust the osmotic pressure of the solution. (iib) Adding HCl to lower the pH to about 9.3 and optionally adding excipients to improve cake formation and / or adjust the osmotic pressure of the solution. (iii) Freezing the solution, and (iv) Then removing the frozen solvent under vacuum.

[0033] The solution of step (ii), (iia), or (iib) can optionally be filtered through a sterile filter before performing step (iii).

[0034] The pH of the solution after step (ii) or (iib) is preferably 6 or higher, more preferably about 8 - 14, and even more preferably about 9.3. In step (i), (ii), (iia), or (iib), optionally, excipients and / or matrix stabilizers for improving cake formation and / or adjusting the osmotic pressure of the solution may be added. Such optional excipients may be one or more bulking agents selected from arginine, glycine, histidine, dextran, and / or polyethylene glycol.

[0035] Good pore formation can promote drying and moisture movement during the drying cycle, so the structure and porosity of the lyophilized cake are important. Also, electrolytes or sodium chloride can be added to the composition to adjust the osmotic pressure. Adjustment of the osmotic pressure can be done before or after lyophilization. The reconstituted (redissolved) lyophilized solution preferably has an osmotic pressure in the range of 250 - 350 mOsm. However, an osmotic pressure of 200 - 600 mOsm is also acceptable and depends on the dosage and injection / infusion time.

[0036] A further aspect is 5,10-CH 2It is directed to a reconstituted pharmaceutical composition of the lyophilized product of the present invention, which contains (6R)-THF and NaCl, and a pharmaceutically acceptable carrier or diluent such as sterile water or a liquid pharmaceutically acceptable vehicle, and optionally further contains excipients and / or matrix stabilizers for improving cake formation and / or adjusting the osmotic pressure of the solution.

[0037] A further aspect is 5,10-CH 2 It is directed to a reconstituted pharmaceutical composition of the lyophilized product of the present invention, which contains (6R)-THF and NaCl, and a pharmaceutically acceptable carrier or diluent such as sterile water or a liquid pharmaceutically acceptable vehicle, and optionally further contains excipients and / or matrix stabilizers for improving cake formation and / or adjusting the osmotic pressure of the solution. The reconstituted pharmaceutical composition further optionally contains at least one additional therapeutic agent such as 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.

[0038] As used herein, the term "liquid pharmaceutically acceptable vehicle" refers to propylene glycol, polyethylene glycol, ethanol, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), glycolfural, isopropylidene glycerol (sorbitol ketal), glycerol formal, acetone, tetrahydrofurfuryl alcohol, monoglyme, diglyme, dimethyl isosorbide or ethyl lactate, mixtures thereof or aqueous mixtures thereof.

[0039] As used herein, the terms "stabilisers" or "stabilising agents" refer to buffers such as citrates (or citric acid and its salts), succinates, malates and maleates (dicarboxylates), TRIS; N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); 3-(N-morpholino)propanesulfonic acid (MOPS); N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid (BES); MES; MOPSO; HEPES; phosphates; carbonates; ammonium; mono-, di- and tri-alkylammonium; mono-, di- and tri-hydroxyalkylammonium; glutamates; borates; lactates and combinations thereof. The terms "stabilisers" or "stabilising agents" 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 sensitive 5,10-methylenetetrahydrofolate, especially tetrahydrofolate.

[0040] As used herein, the term "solvent" relates to solvents that can be used in freeze-drying processes. Similarly, the term "solvent system" as used herein relates to mixtures of solvents that can be used in freeze-drying processes.

[0041] The "Solutions" referred to in the present invention include aqueous solutions and solutions in organic solvents.

[0042] Typically, "aqueous solutions" mean solutions of 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.

[0043] A further aspect relates to the use of the reconstituted pharmaceutical composition of the invention in therapy, preferably in cancer chemotherapy.

[0044] A further aspect of the present invention relates to the use of the pharmaceutical compositions of the present invention (including solid pharmaceutical compositions and their reconstitution solutions) in combination with at least one or more additional therapeutic agents. Preferred additional therapeutic agents include, in this context, chemotherapeutic agents and other anti-cancer agents. Particularly preferred agents that can be combined with the pharmaceutical compositions of the present invention include fluorinated nucleic acids such as 5-fluorouracil or their prodrugs or analogs.

[0045] The reconstituted pharmaceutical composition of the present invention can be used in the treatment, specifically in cancer chemotherapy, i.e., a method for treating cancer, which method comprises administering a therapeutically effective amount of 5,10-CH 2 -(6R)-THF to a subject in need of such treatment.

[0046] Another embodiment is directed to the use of the reconstituted pharmaceutical composition of the present invention in the treatment, specifically in cancer chemotherapy, which method comprises administering a therapeutically effective amount of 5,10-CH 2 -(6R)-THF to a subject in need of such treatment.

[0047] Another embodiment is directed to the use of the reconstituted pharmaceutical composition of the present invention for the treatment of cancerous forms selected from cancer, particularly 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.

[0048] Another embodiment is directed to the use of the reconstituted pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of cancerous forms selected from cancer, particularly colon cancer, gastric cancer, breast cancer, intestinal cancer, gallbladder cancer, lung cancer (specifically adenocarcinoma), colorectal cancer (CRC) including metastatic CRC, head and neck cancer, liver cancer and pancreatic cancer.

[0049] In another embodiment, the reconstituted pharmaceutical composition of the present invention is used in treatment, preferably chemotherapy, i.e., the treatment of cancer. Examples of cancers to be treated include, but are not limited to, breast cancer, esophageal cancer, gastric cancer, gallbladder cancer, cholangiocarcinoma, colon cancer, rectal cancer, liver cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and mesothelioma cancer.

[0050] In a preferred embodiment, the cancer is selected from various cancer forms including 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.

[0051] The reconstituted pharmaceutical composition of the present invention is in a form suitable for parenteral administration such as intravenous, intramuscular, subcutaneous, or intraarterial.

[0052] In the case of parenteral administration, the liquid unit dosage form typically includes the reconstituted lyophilized product, preferably the reconstituted pharmaceutical composition of the present invention, optionally further therapeutic agents, and a pharmaceutically acceptable carrier or diluent, for example, forming an aqueous solution or an oily suspension. In the case of a parenteral solution, the lyophilized product of the present invention may be filter sterilized during its preparation, for example, before filling into a suitable vial or ampoule.

[0053] In the case of combination therapy of the reconstituted pharmaceutical composition of the present invention with at least one further therapeutic agent, the active agent may be administered as part of the same pharmaceutical composition, or at least one further therapeutic agent may be administered separately, i.e., as a separate (optionally different) pharmaceutical composition, optionally via a different route of administration, simultaneously or sequentially.

[0054] The active agent used in the treatment as described herein, i.e., 5,10-CH 2The dosage of (6R)-THF (and optionally at least one additional therapeutic agent) will depend on various factors including the age and health status of the subject being treated, the type and severity of the disease being treated, and the frequency of administration, etc. Those skilled in the art of cancer treatment and chemotherapy will be able to determine therapeutically effective amounts and regimens for the administration of (6R)-THF alone or in combination with at least one additional therapeutic agent as defined above, based on known protocols for evaluating toxicity and efficacy. 2- For the administration of (6R)-THF alone or in combination with at least one additional therapeutic agent as defined above, therapeutically effective amounts and regimens can be determined.

[0055] The term "therapeutically effective amount" refers to the amount of an active compound that elicits a biological or pharmaceutical response in a tissue, system, animal, individual, or human that is sought by those skilled in the art (e.g., researchers, veterinarians, medical doctors, other clinicians, or caregivers), and includes (i) the prevention of a disease and / or (ii) the suppression of a disease (e.g., preventing further progression of a medical condition and / or symptoms) and / or (iii) the improvement of a disease (e.g., reversing a medical condition and / or symptoms). Similarly, the term "treatment" as used herein refers to (i) the prevention of a disease; and / or (ii) the suppression of a disease (e.g., preventing further progression of a medical condition and / or symptoms); and / or (iii) the improvement of a disease (e.g., reversing a medical condition and / or symptoms).

[0056] The selected pharmaceutical composition may contain from 0.1% to 99% by weight, preferably from 10% to 60% by weight, of the active pharmaceutical ingredient (i.e., 5,10-CH 2 -(6R)-THF, optionally in combination with at least one additional therapeutic agent).

[0057] Typical dosage ranges of 5,10-CH 2 -(6R)-THF used in cancer treatment are from 5 mg / m 2 to 1.5 g / m 2 and preferably, in the case of colorectal cancer treatment, 30 mg / m 2~500 mg / m 2 、 in the case of methotrexate therapy, 10 mg / m 2 ~1000 mg / m 2 ; and more preferably, in the case of colorectal cancer treatment, about 60 mg / m 2 ~about 300 mg / m 2 、 in the case of methotrexate therapy, 50 mg / m 2 ~500 mg / m 2 .

[0058] As used herein, the term "purity" means the proportion of a specific 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%, still more preferably about 97%, more preferably about 98%, and most preferably 99% or more, such as 99.5, 99.6, 99.7, 99.8, 99.9 or 100% as measured by HPLC. Impurities include unreacted starting materials and solvents, decomposition products of 5,10-CH 2 -(6R)-THF (such as 5,10-methylene-(6R)-tetrahydrofolic acid and other decomposition products).

[0059] As used herein, the term "pharmaceutically acceptable" indicates that the carrier is approved or recognized for use in animals, more particularly in humans, i.e., it is not toxic to the host or patient. Further, the selected carrier does not interfere with the effectiveness of the biological activity of the active ingredient. The term "carrier" refers to any auxiliary material required for the selected particular mode of administration and includes, for example, solvents (diluents), excipients, or other additives for administering the lyophilized product of the present invention. Pharmaceutically carriers typically used include sterile liquids such as aqueous solutions and oils (e.g., petroleum-derived, animal-derived, plant-derived, or synthetic-derived), such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Aqueous liquids typically used include water, physiological saline, aqueous dextrose, and glycerol solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, dried skim milk, glycerol, propylene, 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" (18th Edition, Mack Publishing Co., Easton, PA (1990)) by E.W. Martin.

[0060] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", are to be construed to mean the inclusion of the stated compound or class or group of compounds, or group of steps, but not the exclusion of any other integer or step, or group of integers or steps.

Brief Description of the Drawings

[0061]

Figure 1

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Example

[0067] HPLC : For the measurement of purity / content and degradation products, the 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

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

[0069] Osmotic pressure : The measurement of osmotic pressure was carried out according to Ph.Eur.2.2.35 (Osmometer) / USP<785>.

[0070] Example 1: Lyophilized product containing 5,10-methylene(6R)-tetrahydrofolic acid and NaCl Under nitrogen, 210 g of purified water and 16.5 g of 2M sodium hydroxide were cooled to 3 ± 2 °C (resultant pH = 14.0). 4.57 g of NaCl and 5.70 g of 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate were added (pH starts to drop), and it was washed with 2.5 g of purified water. By the addition of 2M sodium hydroxide, the pH was maintained at 9.3 ± 0.1. 18.74 g of purified water was added. A total of 20.8 g of 2M sodium hydroxide was required to keep the pH at 9.3 ± 0.1. 5.0 ml per vial of the resulting clear solution was transferred to 10 ml glass vials (36 vials). The vials were immediately frozen in liquid nitrogen and lyophilized at <10 -1 mbar. The resulting vials contained a lyophilizate of 102 mg of 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as the free acid) together with NaCl, whereby the w / w percentage of 5,10-methylene-(6R)-tetrahydrofolic acid in the lyophilizate was 40% of the total solids in the lyophilizate. Upon reconstitution, the osmotic pressure of the solution was 346 mOsmol / kg.

[0071] Example 2: Lyophilized product containing 5,10-methylene(6R)-tetrahydrofolic acid and NaCl Under nitrogen, 210 g of purified water and 16.5 g of 2M sodium hydroxide were cooled to 3 ± 2 °C. 4.10 g of NaCl and 5.70 g of 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate were added (pH starts to drop), and it was washed with 2.5 g of purified water. By the addition of 2M sodium hydroxide, the pH was maintained at 9.3 ± 0.1. 18.8 g of purified water was added. A total of 21.2 g of 2M sodium hydroxide was required to keep the pH at 9.3 ± 0.1. 5.0 ml of the resulting clear solution was transferred to 10 ml glass vials (36 vials). The vials were immediately frozen in liquid nitrogen and lyophilized at <10 -1 mbar. The resulting vials contained a lyophilizate of 102 mg of 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as the free acid) together with NaCl. 5,10-methylene-(6R)-tetrahydrofolic acid showed a purity of 93.2% w / w (as the free acid) as measured by HPLC. The osmotic pressure of the reconstituted solution is 320 mOsmol / kg.

[0072] Example 3: Lyophilized product containing 5,10-methylene(6R)-tetrahydrofolic acid and NaCl Under nitrogen, 210 g of purified water and 16.5 g of 2M sodium hydroxide were cooled to 3 ± 2 °C (resulting pH = 14.0). 4.10 g of NaCl and 5.80 g of 5,10 - methylene-(6R)-tetrahydrofolic acid hemisulfate were added (the pH starts to drop), and it was washed with 2.5 g of purified water. By adding 2M sodium hydroxide, the pH was maintained at 9.3 ± 0.1. 18.8 g of purified water was added. To keep the pH at 9.3 ± 0.1, a total of 21.2 g of 2M sodium hydroxide was required. 5.0 ml per vial of the resulting clear solution was transferred to 10 ml glass vials (36 vials). The vials were immediately frozen in liquid nitrogen and lyophilized at <10 -1 mbar. The resulting vials contained a lyophilizate of 97.468 mg of 5,10 - methylene-(6R)-tetrahydrofolic acid (calculated as the free acid) together with NaCl. The purity of 5,10 - methylene-(6R)-tetrahydrofolic acid was 93.2% w / w (as the free acid), the area measured by HPLC was 96.7%, and the total of by - products was 1.07%. The water content was 0.63% w / w. The osmotic pressure of the reconstituted solution was 325 mOsmol / kg and the pH was 8.23.

[0073] Example 4: Lyophilized product containing 5,10-methylene(6R)-tetrahydrofolic acid and NaCl Under nitrogen, 210 g of purified water and 16.5 g of 2M sodium hydroxide were maintained at 25 ± 2 °C (resulting pH = 13.2). 4.10 g of NaCl and 5.80 g of 5,10 - methylene-(6R)-tetrahydrofolic acid hemisulfate were added (the pH starts to drop), and it was washed with 2.5 g of purified water. By adding 2M sodium hydroxide, the pH was maintained at 9.3 ± 0.1. 17.1 g of purified water was added. To keep the pH at 9.3 ± 0.1, a total of 21.2 g of 2M sodium hydroxide was required. The resulting clear solution was transferred 5.0 ml per vial to 10 ml glass vials (36 vials). The vials were immediately frozen in liquid nitrogen and <10 -1Freeze-dried at mbar. The resulting vial contained the freeze-dried product of 5,10-methylene-(6R)-tetrahydrofolic acid together with NaCl. The purity of 5,10-methylene-(6R)-tetrahydrofolic acid was 95.9% by area, and the total by-products were 1.07%, both measured by HPLC. The water content was 0.67% w / w. Upon reconstitution, the solution had an osmotic pressure of 329 mOsmol / kg and a pH of 9.06.

[0074] Example 5: Long-term stability of the lyophilized product containing 5,10-methylene-(6R)-tetrahydrofolic acid and NaCl ((6R)-5,10-CH 2 -THF content) To measure the long-term stability of the freeze-dried product of 5,10-CH 2 -(6R)-THF prepared according to Examples 1 to 4, the freeze-dried product was stored in air at +5 °C, +25 °C / relative humidity 60% and +40 °C / relative humidity 75%. The content of the remaining 5,10-CH 2 -(6R)-THF was measured periodically by HPLC and expressed as a comparison (% rel.) with the initial value. The results are shown in Tables 1 and 2 (Graphs 1 to 3). Tables 1 and 2 (Graphs 1 to 3) clearly show that the freeze-dried product of 5,10-CH 2 -(6R)-THF with added NaCl is very stable over a long period even at high temperatures.

[0075] Example 6: Long-term stability of the lyophilized product containing 5,10-methylene-(6R)-tetrahydrofolic acid and NaCl (stability index: content of 5,10-methylene-(6R)-tetrahydrofolic acid) To measure the long-term stability of the freeze-dried product of (6R)-5,10-CH 2 -THF prepared according to Examples 1 to 4, the freeze-dried product was stored in air at +5 °C, +25 °C / relative humidity 60% and +40 °C / relative humidity 75%. The content of 5,10-methylene-(6R)-tetrahydropteroic acid (CH2THPA), one of the main decomposition products, was measured periodically by HPLC. The results are shown in Table 3 (Graphs 4 - 6). Table 3 (Graphs 4 to 6) shows that 5,10-CH 2It is to confirm that the lyophilized product of (6R)-THF and NaCl is very stable even at high temperatures over a long period of time, and it is also clear from the very slow increase in the content of 5,10-methylene-(6R)-tetrahydrofolic acid (CH2THPA), which is one of the main decomposition products.

[0076] Example 7: Reconstitution of the lyophilized product containing 5,10-methylene-(6R)-tetrahydrofolic acid and NaCl The lyophilized products prepared according to Examples 1 to 4 can be easily reconstituted by adding 10 ml of water for injection to the vial. The lyophilized product completely dissolves.

[0077] Reference Example 8: Composition containing 5,10-methylene-(6R)-tetrahydrofolic acid and citrate with the pH adjusted to basic (Patent Document 3: European Patent No. 1641460) For reference, a composition containing 5,10-CH 2 -(6R)-THF and citrate with the pH adjusted to 8.5 to 9.5 disclosed in European Patent No. 1641460 was prepared according to the following procedure. Under nitrogen, 210 g of purified water and 16.5 g of 2M sodium hydroxide were maintained at 3 ± 2 °C (resulting pH = 13.9). 11.50 g of trisodium citrate and 5.80 g of 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate were added (the pH began to drop), and it was washed with 2.5 g of purified water. By adding 2M sodium hydroxide, the pH was maintained at 9.3 ± 0.1. 11.0 g of purified water was added. A total of 13.8 g of 2M sodium hydroxide was required to keep the pH at 9.3 ± 0.1. 5.0 ml per vial of the resulting clear solution was transferred to 10 ml glass vials (36 vials). The vials were immediately frozen in liquid nitrogen and <10 -1 mbar and lyophilized. The obtained vials were lyophilized products containing the citrate of 5,10-methylene-(6R)-tetrahydrofolic acid. The purity of 5,10-methylene-(6R)-tetrahydrofolic acid was 97.8% by area, and the total of by-products was 0.81%.

Claims

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

2. The solid pharmaceutical composition according to claim 1, further characterized by not containing any other chemotherapeutic agent.

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

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

7.

7. The solid pharmaceutical composition according to any one of claims 1 to 6, wherein the purity of the 5,10-methylene-(6R)-tetrahydrofolic acid used is greater than 99%, for example greater than 99.5%.

8. The solid pharmaceutical composition according to any one of claims 1 to 7, characterized by not containing citric acid and citrate ions.

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

10. The solid pharmaceutical composition according to any one of claims 1 to 8, wherein the water content is at most 3%.

11. The solid pharmaceutical composition according to any one of claims 1 to 8, wherein the water content is less than 1%.

12. The solid pharmaceutical composition according to any one of claims 1 to 8, which is essentially 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 adjustment excipients and / or matrix stabilizers.

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

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 lyophilized product according to any one of claims 1 to 14, comprising the following steps: (i) Dissolving 5,10-methylene-(6R)-tetrahydrofolic acid or a salt thereof in a solvent or solvent system; (ii) Adding NaCl; (iii) Freezing the solution; and (iv) Subsequently removing the frozen solvent under vacuum; or (i) Dissolving 5,10-methylene-(6R)-tetrahydrofolic acid or a salt thereof in a solvent or 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) Subsequently removing the frozen solvent under vacuum.

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

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

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

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

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