5-methyl-(6s)-tetrahydrofolic acid and crystalline salts of l-leucine ethyl ester

Abstract

The present invention relates to crystalline salts comprising 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester and / or hydrates and / or solvates thereof and processes for obtaining the same, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is from 1:0.3 to 1:3.0 (in mol / mol).

Description

[0001] The present invention relates to a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and / or a hydrate and / or solvate thereof, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is from 1 :0.3 to 1 :3.0 (in mol / mol).

[0002] Tetrahydrofolate salts are mainly used as calcium salts of 5-formyltetrahydrofolic acid (folinic acid and levoleucovorin), calcium salts of 5-methyltetrahydrofolic acid or sulphate salts of 5,10-methylenetetrahydrofolic acid The most prominent field of application is for the treatment of megaloblastic folic acid deficiency, as a detoxifying agent to increase the compatibility of folic acid antagonists, especially aminopterin and methotrexate, in cancer therapy ("antifolate rescue"), for increasing the therapeutic effect of fluorinated pyrimidines and for the treatment of autoimmune diseases, such as psoriasis and rheumatoid arthritis, for increasing the compatibility of certain antiparasitic agents, such as trimethoprim-sulphamethoxazole, and for reducing the toxicity of bis-deazatetrahydrofolic acid in chemotherapy.

[0003] Calcium salts of 5-methyl-(6S)-tetrahydrofolic acid are used in particular as pharmaceutical and food additives, as vitamin preparations, for the prevention of neural tube defects, for the treatment of depression and for influencing homocysteine levels.

[0004] 5-Methyl-(6S)-tetrahydrofolic acid and its salts are known to be very unstable. In particular, they are extremely susceptible to oxidation (see also in this regard A. L. Fitzhugh, Pteridines 4(4), 187-191 (1993)), so that it is difficult to produce them at a level of purity acceptable as pharmaceutical active ingredients or food additives.

[0005] In order to overcome the instability of 5-methyltetrahydrofolic acid and its salts, various methods have been employed, such as the complete exclusion of oxygen or the addition of antioxidants such as ascorbic acid or reduced L-glutathione.

[0006] US 6,441,168 B1 discloses alkaline earth metal salts, in particular calcium salts, of 5-methyltetrahydrofolic acid, the crystallization thereof and the use thereof. A disadvantage of such a crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid is that it exists in up to four polymorphic modifications in its crystal form. Therefore, the production process of the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid has to be controlled very precisely. In addition, the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid of US 6,441,168 B1 usually contains at least one but at most four equivalents of water per equivalent of 5-methyl-(6S)-tetrahydrofolic acid in the crystal lattice of all its polymorphic forms.

[0007] US 2016207925 A1 claims protection for lyophilized, spray-dried, or boiled-down compositions comprising L-asparagine or L-arginine and 5-methyl-(6S)-tetrahydrofolate. However, the disclosed compositions are simple non-stoichiometric mixtures and exist in an amorphous state.

[0008] New crystalline forms of pharmaceutically useful compounds offer opportunities to improve the performance characteristics of drugs and / or vitamins / medicinal foods. This expands the library of materials that formulation scientists can use to design new dosage forms with improved properties.

[0009] The technical problem of this invention is to provide a crystal form containing 5-methyl-(6S)-tetrahydrofolate, which overcomes the disadvantages of crystalline calcium salts of 5-methyl-(6S)-tetrahydrofolate known in the art.

[0010] In addition, new crystal forms often exhibit different physical and / or biological properties that can help in the manufacture or formulation of active compounds to achieve the purity levels and homogeneity required for regulatory approval.

[0011] To ensure the stability of tetrahydrofolate, the long-standing goal has been to provide compounds with low water absorption during storage and that can be thoroughly dried during manufacturing. Furthermore, there is a strong need for drugs that do not absorb large amounts of water under environmental conditions. In particular, substances whose water content does not change with variations in relative humidity are required, as large fluctuations in water content due to changes in relative humidity make it difficult to achieve high precision in dosage forms.

[0012] This technical problem was solved by a crystalline salt and / or hydrate and / or solvate of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is 1:0.3 to 1:3.0 (mol / mol).

[0013] The solid form of the present invention has improved pharmacological properties, thus providing the possibility of enhancing and designing improved pharmaceutical products. Compared to the crystalline polymorphs of the calcium salt of 5-methyl-(6S)-tetrahydrofolate known in the art, the crystalline salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester have significantly lower water absorption rates, resulting in significantly improved control over the target dosage form level in pharmaceutical products, as changes in the amount of water adsorbed under varying relative humidity conditions are significantly less pronounced.

[0014] Another advantage of the crystalline salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester is that high chemical and optical purity of 5-methyl-(6S)-tetrahydrofolate can be obtained in a single crystallization step.

[0015] Advantageously, the drug exhibits high kinetic solubility when administered orally, leading to improved and faster bioavailability. Consequently, the drug can act more readily.

[0016] 5-Methyl-(6S)-tetrahydrofolate is poorly soluble in water. The thermodynamically stable form of its calcium salt (crystal form III) is known to exhibit a water solubility of approximately 2.5 mg / ml at room temperature, while the metastable crystal form I has a solubility of approximately 10 mg / ml. Under certain pH conditions, particularly when the ambient pH is below the equilibrium pH of a given salt, the salt may potentially disproportionate into a free acid, resulting in a significant decrease in solubility. Therefore, the thermodynamic solubility of the claimed salt at approximately neutral to lower pH values ​​is unattainable due to the slow disproportionation of the salt (formation of a poorly soluble free acid). However, bioavailability is governed by kinetics. Drug products in solid form undergo dissolution after administration, and after the first dissolution step, the drug is diluted and distributed via body fluids. Therefore, kinetic solubility is a key parameter affecting bioavailability, as the initially dissolved drug substance is readily diluted and transported. Surprisingly, the salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine showed approximately twice the kinetic solubility compared to the known calcium salts (metastable form I). It is speculated that the difference in kinetic solubility of the salts of this invention relative to the thermodynamically stable form of the calcium salts (form III) is even greater. Therefore, much higher drug concentrations can be temporarily achieved.

[0017] Furthermore, the crystalline salt containing 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester exists in its crystalline form as a well-defined polymorphic modified form. Therefore, the method for producing the crystalline salt containing 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester does not require very precise control of the crystallization conditions.

[0018] Preferably, the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is 1:0.5 to 1:2.5 (in mol / mol).

[0019] Even more preferably, the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is 1:0.75 to 1:1.25 (in mol / mol).

[0020] Preferably, the ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is about 1:1 (in mol / mol) and / or its hydrate and / or solvate.

[0021] Preferably, the salt of the present invention is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, and has a PXRD pattern containing at least one characteristic peak (expressed as 2θ±0.2°2θ (CuKα radiation)) selected from the following peaks located at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7.

[0022] Most preferably, the salt of the present invention is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, and has a PXRD pattern containing at least three characteristic peaks (expressed as 2θ±0.2°2θ (CuKα radiation)) selected from the following at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7. Even more preferably, the salt of the present invention is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester and has a PXRD pattern containing characteristic peaks (expressed as 2θ±0.2°2θ (CuKα radiation)) at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7.

[0023] Preferably, the salt of the present invention is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, and has a PXRD pattern containing at least one characteristic peak (expressed as 2θ±0.2°2θ (CuKα radiation)) selected from the following peaks located at 5.6, 6.9, 8.4, 12.9, 13.8, 14.1, 15.0, 17.5, 18.2, 19.1, 19.7, 21.2, 21.4, 23.7, 26.1 and 27.6.

[0024] Most preferably, the salt of the present invention is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, and has substantially the following properties: Figure 1 The PXRD pattern shown.

[0025] Even more preferably, the crystalline salt described above has a chemical and / or stereoisomeric purity of at least 99 wt% or higher.

[0026] Another aspect of the present invention is a method for obtaining a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester, the method comprising the following steps:

[0027] i) Optionally, a mixture of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester is provided in a suitable solvent or solvent mixture.

[0028] ii) Add a base, optionally in a suitable solvent or solvent mixture, to dissolve the compound;

[0029] iii) Heat the composition to at least 60°C, and optionally perform clarification filtration;

[0030] iv) Crystallize the mixture and cool it to a temperature of 1°C to 30°C, optionally adding more solvent or a mixture of solvents; and

[0031] v) Separate the obtained solid material and optionally dry the product.

[0032] Preferably, the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester in step ii) is 1:1 to 1:3.

[0033] More preferably, the solvent is water.

[0034] In steps iii) and / or iv), seed crystals may be added.

[0035] Preferably, L-leucine ethyl ester is used as L-leucine ethyl ester hydrochloride.

[0036] Furthermore, pharmaceutical compositions, food additives, and / or formulations comprising crystalline salts containing 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, and optionally one or more acceptable excipients, are part of this invention.

[0037] The pharmaceutical composition may be in the form of tablets, capsules, oral liquid preparations, powders, lyophilized preparations, granules, lozenges, reconfigurable powders, injectable or infusionable solutions or suspensions or suppositories.

[0038] The pharmaceutical composition may further comprise at least one additional therapeutic agent, and is preferably a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, periodontal, topical or rectal administration.

[0039] The present invention also covers the use of crystalline salts comprising 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester as ingredients for the production of pharmaceuticals and / or as food additives.

[0040] Crystalline salts containing 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester can be used to treat low homocysteine, anemia, neural tube defects, cardiovascular disease, depression, cognitive impairment, Alzheimer's disease and osteoporosis and / or for dietary management of low plasma and / or low red blood cell and / or low cerebrospinal fluid and / or low peripheral or central nervous system folate.

[0041] In summary, the performance characteristics provided by the salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester of the present invention are advantageous for use in pharmaceuticals or as food additives. In particular, the low variation in water content in environments with relative humidity between 20% and 75% is unpredictable to those skilled in the art.

[0042] Furthermore, its kinetic solubility is greater, which was something that those skilled in the art could not have foreseen. Example

[0043] Powder X-ray diffraction

[0044] Stoe Stadi P equipped with a Mythen 1K detector; Cu-Kα1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; bent germanium monochromator; 0.02°2θ step size, 48 s step time, 1.5–50.5°2θ scan range; detector mode: step scan; 1°2θ detector step; standard sample preparation: 10–20 mg sample placed between two acetate / salt foils; sample holder: Stoe transmission sample holder; sample rotation during measurement. All sample preparation and measurements were performed in ambient air.

[0045] TG-FTIR

[0046] Thermogravimetric measurements were performed using a Netzsch Thermo-Microbalance TG 209 (sample disk with pinholes, N2 atmosphere, heating rate 10 K / min) coupled to a Bruker FTIR Spectrometer Vector 22.

[0047] DVS

[0048] DVS measurements are typically performed using the SPS11-100n “Sorptions Prüfsystem” from ProUmid (formerly known as “Projekt Messtechnik”), August-Nagel-Str. 23, 89079Ulm (Germany).

[0049] Example 1: Preparation of salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester without sowing

[0050] A mixture of 5.0 g of 5-methyl-(6S)-tetrahydrofolate monohydrate ([6S]-diastereomer: 98.4%) and 13 ml of water was heated to approximately 80 °C under an argon atmosphere, and 1.98 ml of an aqueous sodium hydroxide solution (30% w / w) was added to form a solution. This solution was heated to approximately 80 °C, and a solution of 4.20 g of L-leucine ethyl ester hydrochloride in 13 ml of water was added. The heating bath was removed to allow the solution to cool to approximately 25 °C over approximately two hours. During cooling, the solution gradually became a concentrated suspension. This suspension was diluted with 15 ml of water and stirred overnight at ambient temperature. The reactor containing the suspension was further cooled to approximately 1 °C in an ice / water bath over approximately half an hour. The suspension was then filtered through a porous glass filter, and the solid product was washed with 5 ml of cold water. The solid product was dried overnight in a vacuum desiccator at approximately 35 °C / 10 mbar and passed through… 1 ¹H NMR analysis identified it as a 1:1 salt of 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester. Powder X-ray diffraction was performed, yielding essentially the following results: Figure 1 The PXRD pattern of the crystalline L-leucine ethyl ester salt is shown. HPLC analysis showed a purity of 98.7% area, with optical purity increased to 99.7% [6S] diastereomer.

[0051] Example 2: Preparation of salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester under sowing conditions

[0052] A mixture of 5.0 g of 5-methyl-(6S)-tetrahydrofolate monohydrate ([6S] diastereomer: 98.4%) and 20 ml of water was heated to approximately 80 °C under a nitrogen atmosphere, and 1.9 ml of an aqueous sodium hydroxide solution (32% w / w) was added to form a solution. This solution was heated to approximately 61 °C, and a solution of 4.20 g of L-leucine ethyl ester hydrochloride in 20 ml of water was added. The heating bath was removed to allow the solution to cool to approximately 32 °C over approximately half an hour. During cooling, the solution was seeded at approximately 60 °C with a small amount of crystalline L-leucine ethyl ester salt prepared according to Example 1, and the solution gradually became a concentrated suspension. The suspension was diluted with 12 ml of water at approximately 49 °C. The reactor containing the suspension was further cooled in an ice / water bath at approximately 32 °C, resulting in a clear solution. After stirring overnight at ambient temperature, 0.15 mL of a 37% (w / w) concentrated hydrochloric acid aqueous solution was added, followed by 0.5 mL of hydrochloric acid in the form of a 2.00 mol aqueous solution. At ambient temperature, a small amount of the salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester prepared according to Example 1 was seeded into the solution, forming a weak suspension. The suspension was stirred at ambient temperature, and an additional 0.35 mL of hydrochloric acid was added in the form of a 2 mol aqueous solution. The suspension was cooled to approximately 2°C, and an additional 0.30 mL of hydrochloric acid was added in the form of a 2 mol aqueous solution. The suspension was continued to be stirred at approximately 2°C for approximately 15 minutes, and filtered through a porous glass filter. The solid product was dried in a vacuum desiccator at approximately 40°C / 10 mbar and passed through… 1 H-NMR analysis revealed it to be 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester salt. Powder X-ray diffraction yielded essentially the following results: Figure 1 The PXRD pattern of L-leucine ethyl ester salt is shown; however, the PXRD pattern indicates that the sample contains a small amount of NaCl. HPLC analysis showed a purity of 98.39% area and an optical purity of 99.7% [6S] diastereomer.

[0053] Example 3: Washing of the salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester

[0054] Approximately 420 mg of the solid material obtained in Example 2 was weighed into a filtration centrifuge apparatus, and 2.0 mL of water was added. The mixture was then centrifuged under ambient conditions. This washing step was repeated twice using 0.5 mL of water. The wet filter cake was then transferred to a porous glass filter and air-dried by drawing ambient air (approximately 22°C / approximately 21% RH) through the glass filter for approximately 10 minutes. 1H-NMR analysis of the dried material identified it as a 1:1 salt of 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester. TG-FTIR analysis showed that the sample contained only about 0.7% water. Powder X-ray diffraction was performed, yielding essentially the following results: Figure 1 The PXRD pattern of L-leucine ethyl ester salt shown in Table 1 shows a peak at the 2-θ angle.

[0055] Table 1: 2-θ angle, d-spacing, and qualitative strength of the 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester salt according to Example 3. Vs = very strong strength, s = strong strength, m = moderate strength, w = weak strength, vw = very weak strength. It should be noted that the strength values ​​can vary significantly due to the preferred orientation effect.

[0056]

[0057]

[0058] Example 4: Hygroscopicity and Water Content (DVS Experiment)

[0059] The water content of the 5-methyl-(6S)-tetrahydrofolate calcium salt sample was measured and found to be 12.4%. TG-FTIR analysis of the 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester sample according to Example 3 showed that this sample contained only about 0.7% water. The samples of 5-methyl-(6S)-tetrahydrofolate calcium salt and the 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester sample according to Example 3 (approximately 20 mg each) were examined by dynamic water vapor adsorption analysis (DVS) within a relative humidity range of 0 to 75% RH. DVS measurements were performed as follows: the samples were placed on the aluminum support at the top of a microbalance and allowed to equilibrate at 50% RH, followed by a pre-defined humidity program.

[0060] (1) Keep at a constant relative humidity (RH) of 50% for two hours, then

[0061] (2) Reduce to 0% RH at a rate of 5% per hour.

[0062] (3) Keep RH at 0% for five hours.

[0063] (4) Increase RH to 75% at a rate of 5% per hour.

[0064] (5) Maintain RH at 75% for five hours.

[0065] (6) Reduce to 0% RH at a rate of 5% per hour.

[0066] (7) Keep RH at 0% for five hours.

[0067] (8) Increase RH to 75% at a rate of 5% per hour.

[0068] (9) Reduce to 50% RH at a rate of 5% per hour.

[0069] (10) Keep the RH at 50% for about one hour.

[0070] Comparing the results of 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester salt with those of the calcium salt showed that the water content of 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester salt varied by approximately 2.6% within the test range, while the water content of the calcium salt varied by approximately 7.4%. The results are as follows... Figure 2 As shown.

[0071] Example 5: Kinetic solubility of crystalline salts of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester

[0072] 43.1 mg of anhydrous 5-methyl-(6S)-tetrahydrofolate L-leucine ethyl ester salt according to Example 3 was weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified / deionized water (e.g., water used for chromatography) was added to the solid using an adjustable-volume pipette. The mixture was vigorously stirred at room temperature for 1 minute. After one minute, a turbid solution was observed, indicating that most of the sample had dissolved. The solution was filtered by centrifugation, and 1.50 mL of the aqueous solution was transferred to a tare glass vial (approximately 10 mL in volume). The water was dried in an air desiccator at 40°C for approximately 15 hours, then at 50°C for approximately 8 hours, followed by drying at 50°C under vacuum (10 to 20 mbar) for approximately 13 hours. Solubility was determined by gravimetric analysis of the solid residue. The solubility was 15.1 mg of 5-methyl-(6S)-tetrahydrofolate / mL.

[0073] Reference Example 1: Kinetic solubility of calcium 5-methyl-(6S)-tetrahydrofolate

[0074] 42.5 mg of anhydrous crystalline 5-methyl-(6S)-tetrahydrofolate calcium salt was weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified / deionized water (e.g., water used for chromatography) was added to the solid using an adjustable-volume pipette. The mixture was stirred vigorously at room temperature for 1 minute. After one minute, a suspension was observed. The suspension was filtered by centrifugation, and 1.50 mL of the aqueous solution was transferred to a tare glass vial (approximately 10 mL). The water was dried in an air desiccator at 40 °C for approximately 15 hours, then at 50 °C for approximately 8 hours, followed by drying at 50 °C under vacuum (10 to 20 mbar) for approximately 13 hours. Solubility was determined by gravimetric analysis of the solid residue. The solubility was 9.0 mg 5-methyl-(6S)-tetrahydrofolate / mL.

Claims

1. A crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is from 1 :0.75 to 1 :2.5, on a mol / mol basis, characterized in that said salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester and has a PXRD pattern comprising characteristic peaks selected from the group consisting of peaks at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7, expressed in degrees 2-theta ± 0.2° 2-theta, CuKa radiation.

2. The crystalline salt of claim 1, wherein the ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester is 1 : 1, on a mol / mol basis.

3. The crystalline salt of claim 1 or 2, characterized by said salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester and has a PXRD pattern comprising at least one further characteristic peak selected from the group consisting of peaks at 13.8, 15.0, 18.2, 19.7, 26.1 and 27.6, expressed in degrees 2-theta ± 0.2° 2-theta, CuKa radiation.

4. The crystalline salt of claim 1 or 2, characterized by said salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester and has a PXRD pattern as shown in Figure 1.

5. The crystalline salt of claim 1 or 2, having a chemical purity of at least 99 wt%.

6. The crystalline salt of claim 5, having a stereochemical purity of at least 99 wt%.

7. A process for obtaining a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester according to any one of claims 1 to 6, said process comprising the steps of: i) providing a mixture of 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester, optionally in a suitable solvent or solvent mixture ii) adding a base, optionally in a suitable solvent or solvent mixture, to dissolve the compounds; iii) heating the composition to at least 60°C and optionally performing a clear filtration; iv) crystallizing the mixture and cooling to a temperature of 1 °C to 30°C, optionally adding more solvent or solvent mixture; and v) isolating the obtained solid material and optionally drying the product.

8. The method of claim 7, characterized by The molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-leucine ethyl ester in step ii) is from 1 : 1 to 1 :

3.

9. The method of claim 7 or 8, characterized in that The solvent is water.

10. The method of claim 7 or 8, characterized in that Seed crystals are added in step iii) and / or iv).

11. The method of claim 7 or 8, characterized in that L-leucine ethyl ester is used as L-leucine ethyl ester hydrochloride.

12. A pharmaceutical composition comprising a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L-leucine ethyl ester according to any one of claims 1 to 6 and optionally one or more acceptable excipients.

13. The pharmaceutical composition of claim 12, in the form of a tablet, a capsule, an oral liquid formulation, a powder, a lyophilizate, a granulate, a lozenge, a reconstitutable powder, a solution or suspension for injection or infusion or a suppository.

14. The pharmaceutical composition of claim 12 or 13, further comprising at least one additional therapeutic agent.

15. The pharmaceutical composition of claim 12 or 13, which is a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, periodontal or rectal administration.

16. The pharmaceutical composition of claim 12 or 13, which is a pharmaceutical composition for topical administration.

17. A food additive comprising the crystalline salt comprising 5-methyl-(6S)- tetrahydrofolate and L-leucine ethyl ester according to any one of claims 1 to 6 and optionally one or more acceptable excipients.

18. A formulation comprising the crystalline salt comprising 5-methyl-(6S)- tetrahydrofolate and L-leucine ethyl ester according to any one of claims 1 to 6 and optionally one or more acceptable excipients.

19. Use of the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L- leucine ethyl ester according to any one of claims 1 to 6 for the manufacture of a medicament.

20. Use of the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L- leucine ethyl ester according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of anemia, neural tube defects, cardiovascular diseases, depression, cognitive impairment, and osteoporosis and / or for the dietary management of low plasma and / or low red blood cell and / or low cerebrospinal fluid and / or low peripheral or central nervous system folate.

21. Use according to claim 20, wherein the cognitive impairment is Alzheimer's disease.

22. Use of the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L- leucine ethyl ester according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of homocysteine reduction.

23. Use of the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolate and L- leucine ethyl ester according to any one of claims 1 to 6 as a food additive.

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