Hemisulfate salt of 5,10-methylene-(6R)-tetrahydrofolic acid

Abstract

The present invention provides a highly stable hemisulfate salt of 5,10-methylene-(6R)-tetrahydrofolic acid. A lyophilisate is obtained by i) dissolving the hemisulfate salt of 5,10-methylene-(6R)-tetrahydrofolic acid in water; ii) freezing the water; and iii) subsequently removing the frozen water under vacuum. Preferably, a buffer, adjuvant, other therapeutic agent, or surfactant is added to the lyophilisate in step i).

Description

Technical Field

[0001] The present invention is directed to the hemisulfate salt of 5,10-methylene-(6R)-tetrahydrofolic acid, which is preferably in a substantially crystalline form, and to pharmaceutical compositions and their use in therapy, preferably chemotherapy.

Background Art

[0002] 5,10-Methylene-5,6,7,8-tetrahydrofolate (5,10-CH2-THF), a reduced folate, is known for its effectiveness as a cytostatic agent in the treatment of solid tumors and has preferably been administered in combination with fluoropyrimidines, such as 5-fluorouracil (5-FU) (Seley, K. L. IDrugs 4(1), 99, 2001 (Non-Patent Document 1)). 5,10-CH2-THF, a base analog and a 5-FU metabolite, together with 5-FdUMP, a 5-FU metabolite, achieves its chemotherapeutic effect by inhibiting the enzyme thymidylate synthase (TS). TS catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), an essential building block of DNA synthesis. Inactivation of TS occurs by the formation of a covalent ternary inhibitory complex between TS, the base analog 5-FdUMP, which is a metabolite of 5-FU, and 5,10-CH2-THF. Enhancement of the cytotoxic effect of 5-FU can be achieved by increasing the intracellular concentration of 5,10-CH2-THF, which results in increased stability of the ternary complex. This causes direct inhibition of DNA synthesis and repair, ultimately leading to cell death and delayed tumor growth.

[0003] However, there are undesirable properties associated with 5,10-CH2-THF, which currently limit its pharmaceutical use. In order to be acceptable for pharmaceutical use, the active substance (e.g., 5,10-CH2-THF) must meet several requirements, including (i) high (chemical, isomeric, crystalline) stability of the active substance itself and its pharmaceutical composition such that effective storage over an acceptable period can be achieved without significant changes in the physicochemical properties of the active substance, (ii) high (chemical, isomeric, crystalline) purity of the active substance, and (iii) ease of handling and processing of the active substance such that it can be transferred to suitable formulations, etc.

[0004] 5,10-CH2-THF is an addition product of tetrahydrofolic acid (THF) and formaldehyde (see, e.g., Poe, M. et al. Biochemistry 18(24), 5527, 1979 (Non-Patent Document 2); Kallen, R. G. Methods in Enzymology 18B, 705, 1971 (Non-Patent Document 3)), and its extremely high sensitivity to oxidation by air and instability in neutral and / or acidic environments that can potentially lead to chemical decomposition and / or hydrolysis are known (see, e.g., Odin, E. et al., Cancer Investigation 16(7), 447, 1998 (Non-Patent Document 4); Osborn, M. J. et al., J. Am. Chem. Soc. 82, 4921, 1960 (Non-Patent Document 5); Hawkes, J., and Villota, R. Food Sci. Nutr. 28, 439, 1989 (Non-Patent Document 6)). Attempts to stabilize 5,10-CH2-THF include, for example, (i) strict exclusion of atmospheric oxygen by the use of special technical devices for the reconstitution of solid preparations and injection of 5,10-CH2-THF in an oxygen-free environment (see, e.g., Odin, E. et al., Cancer Investigation 16(7), 447, 1998 (Non-Patent Document 7); U.S. Pat. No. 4,564,054 (Patent Document 1)); (ii) addition of antioxidants for highly sensitive 5,10-CH2-THF, particularly for THF, such as reducing agents, for example, L(+)-ascorbic acid or its salts, reduced glutathione, beta-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc.; (iii) stabilization by cyclodextrin inclusion compounds (see, e.g., EP0579996B1 (Patent Document 2)); (iv) addition of citrate while adjusting the pH to a basic value (see, e.g., EP1641460B1 (Patent Document 3)); or (v) formation of various salts, for example, sulfates (see, e.g., EP0537492B1 (Patent Document 4)).

[0005] Nevertheless, there remains a significant need for stabilized 5,10-CH2-THF compounds that exhibit high (chemical, isomeric, and / or crystalline) purity and / or possess high stability both as a compound and when formulated into a pharmaceutical composition, can be more efficiently manufactured, purified, and isolated, and / or can tolerate formulation with acceptable operability (e.g., acceptable solubility, flowability, and particle size in pharmaceutically acceptable solvents) and / or negligible degradation or changes in the physical and chemical properties of the compound, preferably at a high molar percentage (to minimize the amount of the substance that must be formulated and administered to produce a therapeutically effective dose).

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0006]

PATENT DOCUMENT 1

PATENT DOCUMENT 2

PATENT DOCUMENT 3

PATENT DOCUMENT 4

NON-PATENT DOCUMENTS

[0007]

NON-PATENT DOCUMENT 1

NON-PATENT DOCUMENT 2

NON-PATENT DOCUMENT 3

NON-PATENT DOCUMENT 4

NON-PATENT DOCUMENT 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0008] Furthermore, the existence of stable solid (polymorphic) forms of (known) chemical compounds having these suitable properties cannot be expected. It is impossible to predict what the properties of such solid forms may be, i.e., whether it is a salt, anhydrous, hydrated or solvated form, let alone the detailed conditions (e.g., crystallization conditions and variables such as solvents, temperature, pH, etc.) under which specific polymorphs can be isolated. The selection and control of such parameters are extremely important for obtaining the desired solid form with high purity, stability and processability. These are important factors that directly affect the properties and performance of the product and their further uses. It is impossible to predict which of the many variables (i.e., solution pH, temperature, pressure, time, solution composition, type and concentration of additives) will be the determining factors.

Means for Solving the Problems

[0009] Surprisingly, it has now been found that the conversion of the (6R)-isomer [(6R)-5,10-CH2-THF] of 5,10-CH2-THF to its hemisulfate salt imparts excellent stability to the compound and its pharmaceutical compositions, thereby overcoming the aforementioned drawbacks. The advantageous stability properties of (6R)-5,10-CH2-THF hemisulfate enable its effective use in the pharmaceutical applications of this compound.

[0010] In a first aspect, the present invention is directed to the hemisulfate of (6R)-5,10-CH2-THF (hereinafter also referred to as the hemisulfate of the present invention or the compound of the present invention).

[0011] Preferably, the hemisulfate of (6R)-5,10-CH2-THF is in a chemically and / or isomerically and / or crystallographically pure form, and more preferably, the hemisulfate of (6R)-5,10-CH2-THF is in a substantially crystalline form.

[0012] In certain embodiments, the hemisulfate of (6R)-5,10-CH2-THF is in an anhydrous form, and thus, in preferred embodiments, the hemisulfate of (6R)-5,10-CH2-THF is in a crystalline anhydrous form.

[0013] Preferably, the hemisulfate of (6R)-5,10-CH2-THF is in a crystalline form characterized by one or more X-ray pattern peak positions at diffraction angles two-theta (2θ) of 4.7°, 17.9° and 23.3° represented by 2θ±0.2° 2θ (CuKα radiation).

[0014] In certain embodiments, the hemisulfate of (6R)-5,10-CH2-THF is characterized by an FT-Raman spectrum comprising peaks at wavenumbers (±2 cm -1 represented) of 1672, 1656, 1603, 1553, 1474, 1301, 637, 624 and 363 cm -1 .

[0015] In a further aspect, the present invention is directed to a pharmaceutical composition comprising the hemisulfate of (6R)-5,10-CH2-THF and a pharmaceutically acceptable carrier or diluent, optionally further comprising at least one additional therapeutic agent (a bactericide, an antibiotic, an antiviral agent, a preservative, an antitumor agent, an anticancer compound, such as a chemotherapeutic agent, an antifungal agent, and / or an anti-inflammatory agent, or other bioactive or therapeutic agent suitable for human use, particularly an anticancer compound, such as a chemotherapeutic agent, such as 5-FU and derivatives, and an antifolate agent, such as methotrexate, pemetrexed, but not limited thereto).

[0016] In a further aspect, the present invention is directed to the use of the hemisulfate of (6R)-5,10-CH2-THF (or a pharmaceutical composition thereof) in therapy, preferably in cancer chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1

Figure 2a

Figure 2b

Figure 2c

Figure 2d

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0018] Detailed Description of the Invention In a first aspect, the present invention is directed to the hemisulfate salt of (6R)-5,10-CH2-THF (also referred to as the compound of the present invention or the hemisulfate salt of the present invention). In one embodiment, the hemisulfate salt of (6R)-5,10-CH2-THF is in a substantially crystalline form, and more particularly, in a crystalline anhydrous form.

[0019] As used herein, (6R)-5,10-CH2-THF refers to 5,10-CH2-THF (the chiral centers at C6 of the pteridine ring and the α-carbon of the glutamic acid moiety being in their naturally occurring configurations) in its naturally occurring isomeric form (5,10-methylene-(6R)-tetrahydrofolic 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). Thus, as used herein, the terms "isomeric purity" and "stereoisomeric purity" each refer to the amount of (6R)-5,10-CH2-THF in a sample that may contain one or more other isomers of the same compound. As used herein, the terms "isomerically pure" and "stereoisomerically pure" each mean a compound of the invention having an isomeric excess of the desired (6R)-5,10-CH2-THF isomer that is greater than about 80%, preferably greater than about 90%, preferably greater than about 95%, more preferably greater than about 97%, even more preferably greater than about 99% or more, and most preferably up to 100%, where the remainder may be one or more other isomers.

[0020] As used herein, the term "crystalline form" (or "polymorph" or "crystal form") refers to a solid state form consisting of a specific regular three-dimensional arrangement of structural units. Thus, different crystalline forms of the same compound result from different packings of the molecules in the solid state, which gives rise to different crystal symmetries and / or unit cell parameters. Usually, different solid or crystal forms have one or more different physical and / or chemical properties, such as different dissolution profiles, different thermodynamic and chemical stabilities, different melting point temperatures and / or different X-ray diffraction patterns, and can thus be distinguished by X-ray diffraction, infrared (IR) spectroscopy, differential scanning calorimetry (DSC), Raman spectroscopy, solid state NMR, as well as melting point, density, hardness, optical and electrical properties, stability and / or dissolution profile, etc. A mostly irregular or irregular three-dimensional arrangement is usually described by the term "amorphous".

[0021] The term "(of the present invention) 'crystalline compound'" refers to a solid form of a compound of the present invention containing a recognizable amount of the crystal form(s) or polymorph(s) of the compound of the present invention, preferably more than 50%, 60%, 70%, 80%, 90% or 95% of one (or more) crystal form(s) or polymorph(s) of the compound of the present invention. The amount, degree and nature of the crystallinity of the crystalline compound of the present invention can be measured by one or more technical means including optical microscopy, electron microscopy, X-ray powder diffraction, solid state NMR spectroscopy or polarized light microscopy.

[0022] As used herein, the expression "(of the present invention)'hemisulfate'" includes all its specific embodiments and is preferably provided in a chemically and / or (stereo) isomerically and / or crystalline pure form. In one particular embodiment, it is in a substantially crystalline form, more specifically, a crystalline anhydrous form (hereinafter also referred to as crystalline form type 1).

[0023] As used herein, the term "crystalline purity" means the percentage of a specific crystalline form of a compound in a sample, which form may include the amorphous form of the compound, one or more other crystalline forms of the compound (other than the above specific crystalline form of the compound), or mixtures thereof. As used herein, the term "substantially crystalline form" represents a crystalline purity of at least about 80%, preferably at least about 90%, preferably at least about 95%, preferably about 97% crystalline purity, more preferably about 99% or higher crystalline purity, and most preferably about 100% crystalline purity. Crystalline purity is measured by X-ray powder diffraction (XRPD), infrared Raman spectroscopy, and other solid state methods.

[0024] As used herein, the term "chemical purity" means the percentage of a specific compound in a sample. As used herein, "substantial chemical purity" refers to a chemical purity of about 80%, preferably about 90%, more preferably about 95%, more preferably about 97%, more preferably about 98%, most preferably 99% or higher or up to 100% chemical purity, as measured by HPLC, for the compounds of the present invention. Chemical impurities may include unreacted starting materials (including solvents), decomposition products of (6R)-5,10-CH2-THF (such as THF), and the like.

[0025] As described above, the crystalline form (and its purity) of the hemisulfate salt of the present invention can be identified, characterized, and distinguished from other salt forms, such as other sulfate forms, by the unique solid state characteristics, such as those shown by the data provided herein with respect to X-ray powder diffraction (XRPD), infrared Raman spectroscopy, and other solid state methods.

[0026] Accordingly, in certain embodiments, the present invention provides a crystalline form of the anhydrous hemisulfate salt of (6R)-5,10-CH2-THF (hereinafter also referred to as crystalline form type 1), characterized by providing the following: (i) An X-ray powder diffraction (XRPD) pattern giving lattice spacings calculated at 4.7°, 17.9° and 23.3°, preferably 4.7°, 16.6°, 17.9°, 18.4°, 18.9°, 20.2°, 23.3°, 23.5°, 24.3° and 24.7° (represented by 2θ±0.2° 2θ (CuKα line)); and / or (ii) An FT-Raman spectrum containing peaks at wavenumbers of 1672, 1656, 1603, 1553, 1474, 1301, 637, 624 and 363 (represented by ±2 cm -1 ), and / or (iii) An IR spectrum having one or more absorption bands according to Table 3.

[0027] In a preferred embodiment, the hemisulfate (type 1) of the present invention is characterized by at least two of the following 10 XRPD peaks (represented by 2θ±0.2° 2θ (CuKα line)) at 4.7°, 16.6°, 17.9°, 18.4°, 18.9°, 20.2°, 23.3°, 23.5°, 24.3° and 24.7°, preferably 4.7°, 17.9° and 23.3°, and at least two of the following 9 FT-Raman peaks of 1672, 1656, 1603, 1553, 1474, 1301, 637, 624 and 363 (represented by ±2 cm -1 ).

[0028] In other embodiments, the hemisulfate (type 1) of (6R)-5,10-CH2-THF of the present invention gives an FT-Raman spectrum substantially according to FIG. 1 and / or peaks reported in Table 1 and / or an X-ray powder diffraction (XRPD) pattern substantially according to FIG. 2(a) and / or peaks reported in Table 2.

[0029]

Table 1

[0030]

Table 2

[0031]

Table 3

[0032] The compounds of the present invention are most efficiently characterized and distinguished from related compounds by X-ray powder diffraction patterns, as measured according to procedures known in the art (see, for example, J. Haleblian, J. Pharm. Sci. 64:1269, 1975; J. Haleblain and W. McCrone, J. Pharm. Sci. 58:911, 1969). FIG. 2(d), which shows the X-ray diffraction pattern of the hemisulfate of (6R)-5,10-CH2-THF prepared in the example compared with the X-ray diffraction pattern of the sulfate of (6R)-5,10-CH2-THF, clearly demonstrates the differential patterns of these two salts.

[0033] The relative intensities of the peaks can vary depending on the sample preparation technique, sample loading procedure, and particularly the apparatus used, but the compounds of the present invention can be identified by differential peaks and peak positions characteristic of a particular polymorph (with small variations in peak assignment of about ±0.5° two-theta (2θ), preferably ±0.2° two-theta (2θ) (CuKα radiation)).

[0034] The compounds of the present invention are in a non-solvated anhydrous form, which includes compounds that contain no water and compounds that may contain trace amounts of water. Such potential residual (non-stoichiometric) water content can be any amount of water, but typically ranges from 0 wt% H2O to 3 wt% H2O, preferably from 0 wt% H2O to 1 wt% H2O.

[0035] The hemisulfate compounds of the present invention can be stored in solid form, such as in powder or lyophilized form, or as a liquid.

[0036] In certain embodiments, the compounds of the invention are preferably prepared by adding an aqueous solution of formaldehyde of (6S)-THF to an aqueous solution of sulfuric acid (or an aqueous solution of acetic acid and sulfuric acid) to cause crystallization of the hemisulfate salt of (6R)-5,10-CH2-THF. This crystallization reaction is carried out at an elevated temperature, for example, a temperature exceeding 35°C. In particular, the method for producing the crystalline hemisulfate salt of (6R)-5,10-CH2-THF includes the steps of (i) reacting a solution of (6S)-tetrahydrofolic acid with an aqueous solution of formaldehyde to obtain (6R)-5,10-CH2-THF in the solution (by a known procedure), (ii) adding the (6R)-5,10-CH2-THF in the resulting solution to an aqueous solution of sulfuric acid (or alternatively, an aqueous solution of acetic acid and sulfuric acid) at a temperature above 35°C, preferably 35°C to 70°C, more preferably 40°C to 60°C, and most preferably 40°C to 50°C to cause crystallization of the hemisulfate salt of (6R)-5,10-CH2-THF, and (iii) isolating the resulting crystalline hemisulfate salt of (6R)-5,10-CH2-THF, for example, by filtration.

[0037] Step (i) can be carried out according to a known procedure as described in the examples.

[0038] In step (ii), the resulting clear solution can be added to the sulfuric acid solution (or an aqueous solution of acetic acid and sulfuric acid) at a temperature of about 40 to 50°C, enabling selective crystallization of the desired product. Optionally, after the addition is complete, the resulting reaction mixture can be stirred at a temperature of about 40 to 50°C for up to 5 hours, and subsequently, the crystallization product can be filtered or centrifuged at the same temperature, optionally washed with water, and dried.

[0039] In a further aspect, the present invention is directed to a pharmaceutical composition (also referred to as the pharmaceutical composition of the present invention) comprising a therapeutically effective amount of the hemisulfate salt of (6R)-5,10-CH2-THF of the present invention and a pharmaceutically acceptable carrier for administration to a patient. As used herein, the phrase "pharmaceutically acceptable" indicates that the carrier is approved or permitted for use in animals, more particularly in humans, i.e., is not toxic to the host or patient. Further, a preferred carrier does not interfere with the effectiveness of the biological activity of the active ingredient. The phrase "carrier" represents any auxiliary material necessary for a particular mode of preferred administration and includes, for example, solvents (diluents), additives or other excipients with which the compounds of the present invention are administered. Commonly used diluent pharmaceutical carriers include sterile liquids such as aqueous solutions and oils (e.g., petroleum, animal, vegetable or synthetic sources), such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Commonly used aqueous liquids include water, aqueous saline solutions, aqueous dextrose and glycerol solutions, etc. Suitable pharmaceutical additives include citric acid, ascorbic acid, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Optionally, the composition can include excipients such as wetting or emulsifying agents, pH buffering agents or binding agents. Examples of suitable pharmaceutical carriers are known in the art and are described, for example, in "Remington’s Pharmaceutical Sciences" by E.W. Martin (18th ed., Mack Publishing Co., Easton, PA (1990)).

[0040] Optionally, the pharmaceutical composition of the present invention can further comprise at least one additional therapeutic agent. In certain embodiments, said at least one additional therapeutic agent can be selected from bactericides, antibiotics, antiviral agents, preservatives, antitumor agents, anticancer compounds such as chemotherapeutic agents, antifungal agents, and / or anti-inflammatory agents, or other bioactive or therapeutic agents suitable for human use, particularly anticancer compounds such as chemotherapeutic agents. Anticancer agents, such as chemotherapeutic agents, can include, but are not limited to, specific binding members, proteins, nucleic acids or nucleic acid analogs (e.g., but not limited to, antisense molecules, ribozymes and siRNA), lipids, steroids, macromolecules, small molecules or metals. One or more anticancer agents can include, for example, one or more of the following (non-limiting) chemotherapeutic agents: nucleic acids, particularly fluorinated nucleic acids (e.g., 5-fluorouracil or its analogs or prodrugs), antifolates (e.g., pemetrexed, raltitrexed, lometrexol), topoisomerase inhibitors (e.g., irinotecan, topotecan), antimetabolites (e.g., methotrexate, gemcitabine, tiazofurin), 5-FU modulators, alkylating agents (e.g., cyclophosphamide, carmustine), nucleic acid biosynthesis inhibitors (e.g., mitomycin, anthracyclines (e.g., epirubicin, doxorubicin)), platinum derivatives (cisplatin, oxaliplatin, carboplatin), microtubule disrupting agents (e.g., paclitaxel, docetaxel, vinorelbine, vincristine), hormone blockers (e.g., tamoxifen), inhibitors of kinases including but not limited to receptor or non-receptor tyrosine kinases (e.g., Iressa, Tarceva, SU5416, PTK787, Gleevec), proteasome inhibitors (e.g., bortezomib), immunomodulators (e.g., levamisole), anti-inflammatory agents, angiogenesis inhibitors, cytokines (e.g., interleukins, tumor necrosis factor), and agents that inhibit the activity of cytokines, hormones, or receptors for cytokines or hormones (e.g., the anti-VEGF antibody bevacizumab or "Avastin").The anticancer agent may also include monoclonal antibodies, such as monoclonal antibodies that bind to cytokines, hormones or hormone receptors (not limited to these) (for example, antibodies that block the activation of EGF or VEGF growth factors, such as Avastin, Erbitux, Herceptin), etc.

[0041] The compound of the present invention or its pharmaceutical composition can be used for treatment, particularly in cancer chemotherapy, that is, in a method for treating cancer, which includes administering a therapeutically effective amount of the hemisulfate of the present invention or its pharmaceutical composition to a subject in need of such treatment.

[0042] Accordingly, in a further aspect, the present invention is further directed to the use of the hemisulfate of the present invention (or its pharmaceutical composition) in treatment, preferably in chemotherapy, that is, in the treatment of cancer. Examples of cancers to be treated by the present invention include, but are not limited to, breast cancer, esophageal cancer, gastric cancer, gallbladder cancer, cholangiocarcinoma, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and mesothelioma.

[0043] Suitable pharmaceutical compositions of the present invention can be adapted for oral, parenteral or rectal administration and can themselves be in the form of tablets, capsules, oral liquid preparations, powders, lyophilized products, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions, or suppositories. Preferably, the pharmaceutical composition is in a form suitable for parenteral administration such as intravenous, intramuscular, subcutaneous, intra-arterial.

[0044] For parenteral administration, liquid unit dosage forms usually contain a compound of the invention, optionally an additional therapeutic agent, and a pharmaceutically acceptable carrier or diluent to form an aqueous-based solution or an oil-based suspension (or their lyophilized products). Depending on the presence of other therapeutic agents, the carrier, and the concentration used, the compound may be suspended or dissolved in the carrier. For parenteral solutions, the compound can be dissolved for injection, filter sterilized, and then filled and sealed in appropriate vials or ampoules. Optionally, adjuvants such as local anesthetics, preservatives, and buffers can be dissolved in the vehicle. If desired, the resulting solution can be subjected to lyophilization (i.e., the composition can be frozen after filling into vials and water can be removed under vacuum). For parenteral suspensions, the compound is suspended (instead of dissolved) in the vehicle, and preferred sterilization includes exposure to ethylene oxide prior to suspension in a sterile vehicle (such as a vial or ampoule). Optionally, a surfactant or wetting agent can be included in the composition to promote uniform distribution of the compound.

[0045] Tablets and capsules for oral administration can be in unit dosage form and can contain conventional additives such as binders, fillers, tablet lubricants, disintegrants, and acceptable wetting agents. Tablets can be coated according to methods well known in ordinary pharmaceutical practice.

[0046] Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or in the form of dry products for reconstitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional excipients such as suspending agents, emulsifying agents, non-aqueous vehicles (which can include edible oils), preservatives, and optionally conventional flavoring or coloring agents.

[0047] In the case of a combination therapy in which the pharmaceutical composition of the present invention comprises the compound of the present invention and at least one additional therapeutic agent, the active agents can be administered as part of the same pharmaceutical composition or the at least one additional therapeutic agent can be administered separately, i.e., as separate (and possibly different) pharmaceutical compositions, optionally by different routes of administration, simultaneously or sequentially.

[0048] The dosage of the active pharmaceutical agent, i.e., the compound of the present invention (and optionally at least one additional therapeutic agent) used in the treatment as described herein, depends on various factors including the age and health status of the subject being treated, the type and severity of the disease being treated, the route and frequency of administration, etc. Specialists in cancer treatment and chemotherapy will be able to determine a therapeutically effective amount and dosing schedule for the compound of the present invention, either alone or in combination with at least one additional therapeutic agent as defined above, based on known protocols for evaluating toxicity and efficacy.

[0049] As used herein, the phrase "therapeutically effective amount" refers to the amount of an active compound that elicits a biological or drug response in a tissue, system, animal, individual or human that is sought by one of ordinary skill in the art and that includes (i) prevention of a disease; and / or (ii) suppression of a disease (e.g., halting further progression of a medical condition and / or symptoms); and / or (iii) amelioration of a disease (e.g., recovery from a medical condition and / or symptoms). Similarly, as used herein, the phrase "treatment" refers to (i) prevention of a disease; and / or (ii) suppression of a disease (e.g., halting further progression of a medical condition and / or symptoms); and / or (iii) amelioration of a disease (e.g., recovery from a medical condition and / or symptoms).

[0050] The pharmaceutical composition can preferably contain from 0.1% to 99% by weight, preferably from 10% to 60% by weight, of the active pharmaceutical agent (i.e., the compound of the present invention optionally combined with at least one additional therapeutic agent), depending on the method of administration.

[0051] Typical dosage ranges of the compounds of the present invention to be used in cancer treatment are, respectively, 10 mg / m 2 ~1 g / m 2 、preferably 50 mg / m 2 ~500 mg / m 2 (for colorectal cancer treatment), 10 mg / m 2 ~200 mg / m 2 (for methotrexate therapy), more preferably about 100 mg / m 2 ~250 mg / m 2 (for colorectal cancer treatment), respectively 50 mg / m 2 ~150 mg / m 2 (for methotrexate therapy) can be in the range.

[0052] Note that although this application relates to the invention described in the claims, the following may also be included as other aspects. 1. Hemisulfate of 5,10 - methylene-(6R)-tetrahydrofolic acid. 2. The hemisulfate described in 1 above, which is substantially in crystalline form. 3. The hemisulfate described in 1 or 2 above, having a crystal purity of at least 80%, preferably 90%, preferably 95%, more preferably 97%, most preferably 99% or more. 4. The hemisulfate described in any one of 1 to 3 above, having a chemical purity of at least 80%, preferably 90%, preferably 95%, more preferably 97%, most preferably 99% or more. 5. The hemisulfate described in any one of 1 to 4 above, which is in anhydrous form. 6. The hemisulfate described in any one of 1 to 5 above, having one or more X - ray pattern peak positions at diffraction angles 2θ of 4.7°, 17.9° and 23.3° represented by 2θ±0.2° 2θ (CuKα line, reflection). 7. The hemisulfate described in any one of 1 to 6 above, having one or more X - ray pattern peak positions at diffraction angles 2θ of 4.7°, 16.6°, 17.9°, 18.4°, 18.9°, 20.2°, 23.3°, 23.5°, 24.3° and 24.7° represented by 2θ±0.2° 2θ (CuKα line, reflection). 8. Having an FT - Raman spectrum containing one or more peaks at wavenumbers (±2 cm -1 represented) of 1672, 1656, 1603, 1553, 1474, 1301, 637, 624 and 363, the hemisulfate described in any one of 1 to 7 above. 9. The hemisulfate described in any one of 1 to 8 above, having an FT - Raman spectrum substantially following Figure 1 and / or an X - ray powder diffraction (XRPD) pattern substantially following Figure 2(a) or 2(b). 10. At least two of the ten XRPD peaks (represented by 2θ±0.2° 2θ (CuKα line)) at 4.7°, 16.6°, 17.9°, 18.4°, 18.9°, 20.2°, 23.3°, 23.5°, 24.3° and 24.7° below, and nine FT - Raman peaks (±2 cm -1 The hemisulfate according to any one of 1 to 9 above, having at least two of those represented by At least two of the following three XRPD peaks (represented by 2θ±0.2° 2θ (CuKα line)) at 11.4.7°, 17.9° and 23.3°, and at least two of the following five FT-Raman peaks of 1672, 1656, 1603, 1553, 1474, 1301, 637, 624 and 363 (±2 cm -1 The hemisulfate according to any one of 1 to 10 above, having at least two of those represented by 12. A pharmaceutical composition comprising the hemisulfate of 5,10-methylene-(6R)-tetrahydrofolic acid according to any one of 1 to 11 above and optionally a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to 12 above, in the form of tablets, capsules, oral liquid preparations, powders, lyophilized products, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions, or suppositories, preferably in the form of a lyophilized product. 14. The pharmaceutical composition according to 12 or 13 above, further comprising at least one additional therapeutic agent. 15. The pharmaceutical composition according to any one of 12 to 14 above, which is a pharmaceutical composition for oral, parenteral or rectal administration. 16. The hemisulfate of 5,10-methylene-(6R)-tetrahydrofolic acid according to any one of 1 to 11 above or the pharmaceutical composition according to any one of 12 to 15 above for use in treatment, preferably cancer chemotherapy. 17. A method for the treatment of cancer, comprising administering to a subject in need of such treatment the hemisulfate of 5,10-methylene-(6R)-tetrahydrofolic acid according to any one of 1 to 11 above or the pharmaceutical composition according to any one of 12 to 15 above. The following examples serve to illustrate the present invention but are not intended to limit its scope.

Examples

[0053] Differential Scanning Calorimetry (Thermal Analysis Q2000): Closed (sealed) gold crucible; sample filled after 3 minutes of equilibration under ambient conditions or in an N2 environment; heating rate of 10 K min-1; -50 °C to 254 °C range. When two heating scans were performed, the sample was rapidly cooled to -50 °C between scans. The transition temperatures listed correspond to peak maxima and minima and do not correspond to onset temperatures.

[0054] FT-Raman Spectroscopy (Bruker RFS100; using OPUS 6.5 software; offline data analysis performed with OPUS 7.0 software): Nd:YAG 1064-nm excitation; nominally 300 mW laser power; Ge detector; 64 - 256 scans; 3500 - 100 cm -1 spectral region used for analysis; 2 cm -1 resolution.

[0055] 1 1H-NMR (Bruker DPX300): 1H-NMR spectra were recorded using a proton frequency of 300.13 MHz, a 30° excitation pulse, and a recycle delay of 1 s. 1 Either 16 or 256 scans were integrated, and deuterated DMSO was used as the solvent. The solvent peak was used for reference, and chemical shifts were reported on the TMS scale.

[0056] 13 13C NMR (Bruker AMX 300): 13 13C NMR spectra were obtained using a Bruker AMX300 spectrometer equipped with a 5 mm TXO probe head. Hemisulfate was dissolved in 0.1 N NaOD. Spectra were measured at 303 K with 4000 scans and a digital resolution of 32768 data points. Chemical shifts were assigned in ppm relative to an internal TSP (((3-trimethylsilyl)-2,2’,3,3’-tetradeuteropropionic acid, sodium salt)) standard.

[0057] Powder X-ray diffraction (Bruker D8 Advance): copper Kα line, 40 kV / 40 mA, LynxEye detector, Bragg-Brentano reflection geometry, 0.02° 2θ step size, 37 s step time, 2θ range of 2.5 - 50°. The powder sample was measured using a silicon single crystal sample holder with a depth of 0.1-mm or 0.5-mm. No special treatment was used to prepare the sample except applying slight pressure to obtain a flat surface. Ambient air atmosphere was used for all measurements, and the sample was rotated during the measurement. The absence of information on the opposite X-ray diffraction data is shown as reflection data.

[0058] Powder X-ray diffraction (Stoe Stadi P.): copper Kα1 line, 40 kV / 40 mA, Mythen1K detector, transmission mode, curved Ge monochromator, 0.02° 2θ step size, 60 s step time, detection step of 1° 2θ in step scan mode, scanning range of 1.5 - 50.5° 2θ. The sample (10 - 20 mg of powder) was measured between two acetate films. No special treatment was used to prepare the sample. Ambient air atmosphere was used for all measurements, and each sample was rotated during the measurement.

[0059] TG-FTIR (Netzsch Thermo-Microbalance TG 209 equipped with Bruker FT-IR Spectrometer IFS 28): Al crucible (with micropores); N2 atmosphere; 10 K / min -1 heating rate; range of 25 °C to 300 °C.

[0060] IR (FT-IR Paragon 1000): The infrared spectrum was recorded in 100 scans from a hemisulfate sample pressed in a bromide disk using a Perkin Elmer Fourier Transform Infrared System.

[0061] Example 1: (6R)-5,10-CH 2 -THF hemisulfate production A solution of (6S)-tetrahydrofolic acid (16 mmol, 7.93 g) in 78.0 g of distilled water was charged into a round-bottom flask under N2 at room temperature. The pH of this solution was adjusted to pH 11 by adding 32% NaOH solution (slowly). As soon as the solution became clear, 1.00 M HCl solution was added to adjust the pH of the solution to 8.3 at 25 °C. The resulting clear solution was cooled to about 0 °C, and at that temperature it showed a pH of 8.8. By adding 1 M HCl, the pH was adjusted to pH = 8.6, and 1.44 g of 36.8% HCHO solution (110 mol%) was added all at once. After the addition was complete, the solution was stirred at 0 °C (ice bath) for 1 hour. Activated carbon (0.2 g, Norit C Extra) was added, and the reaction mixture was stirred at 0 °C for 30 minutes, and then a clear solution was obtained by cold filtering in suction filtration, which was used in step (b) without further purification.

[0062] (b) A mixture of 55 ml of 1 M H2SO4 (0.055 mol; 344 mol%) was charged into a round-bottom flask under N2 at 60 °C. To this solution, a solution as obtained in step (a) was added dropwise over 15 minutes, and the resulting reaction mixture was stirred at 50 °C for 2 hours. Then, the reaction mixture was filtered at 50 °C in suction filtration, 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 (6R)-5,10-CH2-THF hemisulfate (7.36 g, 86% yield) in the form of pale gray crystals. The obtained product had a purity of 98.4% and an isomer purity (6R-isomer) of 97.6% as measured by HPLC. Analysis by XRPD showed crystal form type 1 (see Examples 2 and 3 for complete characterization).

[0063] Example 2: Characterization (a) The FT Raman spectrum of (6R)-5,10-CH2-THF hemisulfate recorded using a nominal laser power level of 300 mW and 64 scans is shown in Figure 1.

[0064] (b) The corresponding powder X-ray diffractogram recorded in the transmission mode is shown in Figure 2.

[0065] (c) The TG-FTIR thermogram of (6R)-5,10-CH2-THF hemisulfate is shown in Figure 3. It was carried out under a N2 flow (to prevent oxidative decomposition). The sample showed a loss of 0.5 wt% H2O from about 40 °C to 210 °C, which is residual water (either due to hygroscopicity or incomplete drying). Decomposition occurs only above 210 °C.

[0066] (d) The DSC thermogram of (6R)-5,10-CH2-THF hemisulfate is shown in Figure 4. The sample was equilibrated for 3 minutes under a nitrogen gas flow before the first heating scan, during which 0.6 wt% of its mass was lost. This is consistent with the water content observed in the TG-FTIR thermogram (see Figure 3), confirming that this water is loosely bound. The sample was subsequently heated to 254 °C at 10 K min -1 in a closed gold crucible, quenched cooled to -50 °C, and heated a second time at 10 K min -1 Only the thermal event in the first heating scan is endothermic at about 247.4 °C (ΔH about 60.9 J g -1 ), which is due to melting. This endothermic event may overlap with the onset of exothermic decomposition in some cases. In the second heating scan, a glass transition is observed at about 104 °C (ΔCp = 0.38 J g -1 K -1 ), confirming that melting occurred in the first scan. No other thermal events were observed up to 250 °C.

[0067] (e) The IR spectrum was recorded on a pressed KBr pellet, and the characteristic absorption bands are shown in Table 3.

[0068] (f) The 1 1H NMR spectrum of (6R)-5,10-CH2-THF hemisulfate was recorded in DMSO-d6, and the chemical shifts (δ) (ppm) are shown in Table 8.

[0069]

Table 4

[0070] (g) 13 13C NMR was recorded in 0.1 N NaOD, and the chemical shifts (δ) (ppm) relative to TSP are shown in Table 9.

[0071]

Table 5

[0072] (h) The crystallinity of (6R)-5,10-CH2-THF hemisulfate was confirmed by analysis using optical microscopy. The sample consisted of an aggregate of small birefringent particles.

[0073] Example 3: (6R)-5,10-CH 2 -THF hemisulfate stability test (a) The suspension equilibria of (6R)-5,10-CH2-THF hemisulfate as a starting material at temperatures other than room temperature in various solvents and mixtures are summarized in Table 10:

[0074]

Table 6

[0075] (b) Stability in 85% ethanol at room temperature (6R)-5,10-CH2-THF hemisulfate (3.0 g) was dispersed in 100 ml of 85% EtOH at room temperature, stirred for 5 hours, then filtered, and dried at 30 °C and 8 mbar for 12 hours (overnight). Analysis by XRPD showed that the X-ray pattern characteristic of crystalline form type 1 was maintained without change.

[0076] (c) Stability at high temperature / low pressure (6R)-5,10-CH2-THF hemisulfate (2.17 g) was placed in a drying chamber at 65 °C and 8 mbar for 21 hours. Analysis by XRPD showed that the X-ray pattern characteristic of crystalline form type 1 was maintained without change.

[0077] (d)(6R)-5,10-CH2-THF Hemisulfate and the Long-Term Stability of Its Pharmaceutical Compositions To measure the long-term stability of (6R)-5,10-CH2-THF hemisulfate, the compound of the present invention was stored in air at 25 °C and 60% relative humidity. The content of the remaining (6R)-5,10-CH2-THF hemisulfate was measured by HPLC at regular intervals and determined as a percentage relative to the initial value (%rel.). The results are shown in Table 11.

[0078]

Table 7

[0079] To measure the long-term stability of (6R)-5,10-CH2-THF hemisulfate as a pharmaceutical composition, more specifically as a lyophilized product (such as produced according to Example 5), the lyophilized product was stored in air at 25 °C and 60% relative humidity. The content of the remaining (6R)-5,10-CH2-THF hemisulfate was measured by HPLC at regular intervals and determined by comparison with the initial value (%rel.). The results are shown in Table 12.

[0080]

Table 8

[0081] Tables 11 and 12 clearly show that (6R)-5,10-CH2-THF hemisulfate is highly stable over a long period even at room temperature, both as a pure compound and in the form of a pharmaceutical composition, such as a lyophilized product.

[0082] Example 4: Comparative stability of (6R)-5,10-CH 2 -THF sulfate To compare the long-term stability of the compound (6R)-5,10-CH2-THF hemisulfate of the present invention with the long-term stability of the sulfate of (6R)-5,10-CH2-THF prepared according to EP0537492B1, stability data of the (6R)-5,10-CH2-THF sulfate were generated at various temperatures and humidities.

[0083] (a) Stability of (6R)-5,10-CH2-THF Sulfate (6R)-5,10-CH2-THF sulfate was prepared according to the literature procedure (EP 0 537 492 B1) and stored at -20 °C for 15 months. Subsequently, samples of the product were stored at 5 °C, 25 °C and 60% relative humidity, and 40 °C and 75% relative humidity, respectively. The content of (6R)-5,10-CH2-THF sulfate remaining in the samples was measured by HPLC at regular intervals. The content of (6R)-5,10-CH2-THF sulfate was compared with the initial value at the time of production (% rel.). The results are shown in Tables 13 and 14.

[0084] [Table 9]

[0085] [Table 10]

[0086] Comparison of the data in Tables 13 and 14 with the stability data of the (6R)-5,10-CH2-THF hemisulfate disclosed in Example 3 clearly shows the following: i) There are significant differences in the stability of the (6R)-5,10-CH2-THF hemisulfate compared to the (6R)-5,10-CH2-THF sulfate, and ii) The (6R)-5,10-CH2-THF hemisulfate was extremely stable over a longer period compared to the (6R)-5,10-CH2-THF sulfate.

[0087] (b) Content of the Degradation Product 10-Formyl-(6R)-Tetrahydrofolic Acid (6R)-5,10-CH2-THF sulfate was prepared according to the literature procedure (EP 0 537 492 B1) and stored at -20 °C for 15 months. Subsequently, samples of the product were stored at 5 °C, 25 °C and 60% relative humidity, and 40 °C and 75% relative humidity, respectively. 10-Formyltetrahydrofolic acid (the main degradation product) was measured by HPCL at regular intervals and disclosed as an absolute value (% w / w). The results are shown in Tables 15 - 16.

[0088] [Table 11]

[0089] [Table 12]

[0090] Example 5: (6R)-5,10-CH 2 -THF hemisulfate pharmaceutical dosage form (a) Lyophilized product for reconstitution for intravenous application To 18.480 kg of water at 4 °C, argon was sparged for 1 hour, and 1.386 kg of 2M NaOH and 968.9 g of sodium citrate trihydrate were added. The mixture was stirred at 4 °C under argon until completely dissolved (pH 13.0). Then, 473.9 g of (6R)-5,10-CH2-THF hemisulfate was added under 4 °C washing water saturated with 210 g of argon (slow dissolution, pH 6.5). Subsequently, the pH was adjusted to 9.3 ± 0.1 (121.8 g) with 2M NaOH. 203.6 g of argon-saturated water at 4 °C was added (total solution 21.844 kg).

[0091] Thereafter, the solution was filtered through a sterile filter. 5.201 g (5 ml) of the sterile-filtered solution was added to each 10 ml vial and then lyophilized at -45 °C.

[0092] Before injection, 10 ml of water (WFI) was added to each vial (293 mosmol / kg).

[0093] (b) Preparation of the lyophilized composition of (6R)-5,10-CH2-THF hemisulfate at substantially neutral pH The following materials (mg / 100 ml) and procedure were used to obtain the lyophilized composition: Materials (mg / 100 ml): 5.530 g of (6R)-5,10-CH2-THF hemisulfate (equivalent to 5.000 g of (6R)-5,10-CH2-THF) 6.000 g of citric acid, anhydrous, powder, USP 4.000 g of ascorbic acid, granular, USP NaOH / HCl for pH adjustment 100 mg of water for injection (WFI), USP q.s. (i) Procedure: Sparge WFI with nitrogen gas NF through a filter for 30 minutes. (ii) Record the tare weight of a 100 ml plastic container. (iii) Weigh citric acid, ascorbic acid and water sparged with about 90 g of N2. (iv) Mix and dissolve. (v) Adjust the pH to 7.0 ± 0.1 with 1N NaOH or HCl. (vi) Cool the solution to 10°C. (vii) Add (6R)-5,10-CH2-THF hemisulfate, mix and dissolve. (viii) Record the pH (7.0 ± 0.2). (ix) Add more water to a final weight of 110 g (or 100 ml). Record the weight. (x) Filter the solution through a 0.2 micron filter while cooling as much as possible. (xi) Fill the vials with the solution while cooling as much as possible (2 ml or 100 mg of 5,10-CH2-THF per vial). (xii) Lyophilize. (xiii) Seal the vials under slightly reduced pressure with nitrogen in the headspace. (xiv) Crimp the vials.

[0094] Example 6: Preclinical / clinical results (a) Results from preclinical studies in animal models conducted in accordance with ICH S9 guidance indicate that (6R)-5,10-CH2-THF hemisulfate is safe when administered at the highest dose levels to rats (100 mg / kg / day) and dogs (50 mg / kg / day). Furthermore, clinical data indicate that (6R)-5,10-CH2-THF hemisulfate administered at a dose of up to 200 mg / m 2 is safe for patients.

[0095] (b) In a randomized, single-blind Phase I / II trial (ISO-CC-002) conducted in 32 patients diagnosed with colorectal cancer, the pharmacokinetic and pharmacodynamic properties of (6R)-5,10-CH2-THF hemisulfate in tumor tissue, adjacent mucosa, and plasma were investigated in comparison with leucovorin. The study was conducted at Sahlgrenska University Hospital in Östra, Sweden. Analysis of the data after completion of the study showed that administration of (6R)-5,10-CH2-THF hemisulfate gave significantly greater exposure and peak plasma concentrations than those obtained after administration of leucovorin. Concentrations of methylenetetrahydrofolate and tetrahydrofolate were also very much higher after administration of (6R)-5,10-CH2-THF hemisulfate than those obtained after administration of leucovorin in both tumor and adjacent mucosa.

Claims

1. i) dissolving the hemisulfate salt of 5,10-methylene-(6R)-tetrahydrofolic acid in water; ii) freezing the water; and iii) then removing the frozen water under vacuum, to obtain a lyophilized product.

2. The lyophilized product according to claim 1, wherein in step i), at least one buffer is added to the water.

3. The lyophilized product according to claim 1 or 2, wherein in step i), at least one adjuvant is added to the water.

4. The lyophilized product according to any one of claims 1 to 3, wherein in step i), at least one further therapeutic agent is added to the water.

5. The lyophilized product according to any one of claims 1 to 4, wherein in step i), at least one surfactant is added to the water.

6. The lyophilized product according to any one of claims 1 to 5, wherein in step i), at least one wetting agent is added to the water.

7. The lyophilized product according to any one of claims 1 to 6, wherein a sterilization filtration step is performed between steps i) and ii).

8. The lyophilized product according to any one of claims 1 to 7, wherein the molar ratio of 5,10-methylene-(6R)-tetrahydrofolic acid to sulfate is 2:

1.

9. The lyophilized product according to any one of claims 1 to 8, wherein NaOH is added in step i).

10. The lyophilized product according to any one of claims 1 to 9, wherein sodium citrate or citric acid is added in step i).

11. The lyophilized product according to any one of claims 1 to 10, wherein the composition has stability such that it maintains a purity of 99% or more of 5,10-methylene-(6R)-tetrahydrofolic acid at +25°C for at least 12 months.

12. A reconstituted product obtained by dissolving the lyophilized product according to any one of claims 1 to 11 in water.

13. The reconstituted product according to claim 12, wherein the water is water for injection.

14. A pharmaceutical composition in the form of the lyophilized product according to any one of claims 1 to 11.

15. A pharmaceutical composition in the form of the reconstituted product according to claim 12 or 13.

16. A pharmaceutical composition comprising the lyophilized product according to any one of claims 1 to 11 or the reconstituted product according to claim 12 or 13, and optionally a pharmaceutically acceptable carrier.

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