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

Abstract

The present invention relates to crystalline salts of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and / or hydrates and / or solvates thereof and to a process for obtaining the same, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-isoleucine ethyl ester is from 1:0.3 to 1:2.0 (in mol / mol).

Description

[0001] The present invention relates to a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid (N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo-(6S)-pteridinyl)methyl]amino]benzoyl]-L- glutamic acid) and L-isoleucine ethyl ester and to a process for obtaining it.

[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 bisdesazatetrahydrofolic 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 in a purity level which is acceptable as a pharmaceutical active ingredient or food additive.

[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 crystalline calcium salts 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 a lyophilized, spray-dried or boiled down composition comprising L-asparagine or L-arginine and 5-methyl-(6S)-tetrahydrofolic acid. However, the disclosed composition is a simple non-stoichiometric mixture and exists in an amorphous state.

[0008] New crystal forms of pharmaceutically useful compounds offer the opportunity to improve the performance characteristics of pharmaceuticals and / or vitamins / medical foods. It enlarges the repertoire of materials that formulation scientists can use to design new dosage forms with improved properties.

[0009] The technical problem addressed by the present invention is to provide a crystal form comprising 5-methyl-(6S)-tetrahydrofolic acid which overcomes the disadvantages of the known crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid.

[0010] In addition, the new crystal form usually exhibits desired different physical and / or biological properties which can help in the manufacture or formulation of the active compound, to reach the purity level and homogeneity required for regulatory approval.

[0011] For the stability of tetrahydrofolic acid it has always been the aim to provide a compound which has a low water uptake upon storage and which can be dried sufficiently during the manufacturing process. In addition, there is a great need for pharmaceuticals which do not take up a large amount of water under ambient conditions. It is particularly desirable that the substance does not change its water content when the relative humidity of the environment changes, because a large change in the water content due to a change in the relative humidity of the environment makes it more difficult to achieve a very high precision with regard to the dosage form.

[0012] This technical problem is solved by a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and / or a hydrate and / or a solvate thereof, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-isoleucine ethyl ester is in the range of 1 :0.3 to 1 :2.0 (in mol / mol).

[0013] The solid forms of the present application have improved pharmacological properties, thus providing an enhanced possibility to modulate and design improved pharmaceutical products. The water sorption of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester is significantly lower compared to the crystalline polymorphs of the calcium salt of 5-methyl-(6S)-tetrahydrofolate known in the art, leading to a significantly improved control of the target dosage level in a pharmaceutical product, as the variation in the amount of water adsorbed under varying relative humidity conditions is significantly less pronounced.

[0014] Another advantageous aspect of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester is that a high chemical and optical purity of 5-methyl-(6S)-tetrahydrofolate can be achieved in one single crystallization step. Thus, starting from tetrahydrofolate benzenesulfonic acid salt showing a diastereomeric purity of 95%, a single crystallization of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester can be finally obtained showing a diastereomeric purity of more than 99%. On the other hand, when crystallizing the calcium salt of 5-methyl-(6S)-tetrahydrofolate, a starting material with a diastereomeric purity of at least 97.0% is required to obtain a final product showing a diastereomeric purity of more than 99%.

[0015] Generally, the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester has a molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester of 1 :0.3 to 1 :2.0 (in mol / mol).

[0016] Preferably, the crystalline salt has a molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester of 1 :0.5 to 1 :1.5 (in mol / mol).

[0017] Even more preferably, the crystalline salt has a molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester of 1 :0.75 to 1 :1.5 (in mol / mol).

[0018] In another preferred embodiment, the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester is 1 :0.75 to 1 :1.25 (in mol / mol).

[0019] Most preferably, the ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester is about 1 :1 (in mol / mol).

[0020] Preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern containing at least one characteristic peak (expressed in 2Q ± 0.2° 2Q (Cu Ka radiation)) selected from the group consisting of peaks at 5.8, 6.9, 14.0, 17.5 and 22.2, hereinafter referred to as (Form A).

[0021] More preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (Form A) containing at least three characteristic peaks (expressed in 2Q ± 0.2° 2Q (Cu Ka radiation)) selected from the group consisting of peaks at 5.8, 6.9, 14.0, 17.5 and 22.2. Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (Form A) containing peaks at 5.8, 6.9, 14.0, 17.5 and 22.2 (expressed in 2Q ± 0.2° 2Q (Cu Ka radiation)).

[0022] Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (Form A) containing at least one characteristic peak (expressed in 2Q ± 0.2° 2Q (Cu Ka radiation)) selected from the group consisting of peaks at 5.8, 6.9, 8.5, 12.5, 12.9, 14.0, 14.9, 16.2, 17.5, 17.9, 18.9, 19.2, 22.2, 24.4, 25.4, 25.8, 25.9 and 34.6.

[0023] Most preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (Form A) substantially as shown in Figure 1.

[0024] The salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester can alternatively be characterized by Raman spectroscopy, and thus preferably the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine and has a Raman spectrum (Form A) containing at least one characteristic peak (expressed in wave numbers cm -1 ± 2 cm -1 -1) selected from the group consisting of peaks at 1607, 1571, 1506, 1250 and 650.

[0025] More preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a Raman spectrum (Form A) containing at least three characteristic peaks (expressed in wave numbers cm -1 ± 2 cm -1Raman spectrum (crystal form A) having at least one characteristic peak (expressed in wave numbers cm-1) selected from the group consisting of 2961, 2938, 1607, 1571, 1506, 1468, 1332, 1250, 1153, 922, 860 and 650. -1 ± 2 cm -1 Raman spectrum (crystal form A) having at least one characteristic peak (expressed in wave numbers cm-1) selected from the group consisting of 2961, 2938, 1607, 1571, 1506, 1468, 1332, 1250, 1153, 922, 860 and 650.

[0026] Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a Raman spectrum (crystal form A) having at least one characteristic peak (expressed in wave numbers cm-1) selected from the group consisting of 2961, 2938, 1607, 1571, 1506, 1468, 1332, 1250, 1153, 922, 860 and 650. -1 ± 2 cm -1 Raman spectrum (crystal form A) having at least one characteristic peak (expressed in wave numbers cm-1) selected from the group consisting of 2961, 2938, 1607, 1571, 1506, 1468, 1332, 1250, 1153, 922, 860 and 650.

[0027] Most preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a Raman spectrum (crystal form A) substantially as shown in Figure 4.

[0028] Another aspect of the application is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester having a PXRD pattern having at least one characteristic peak (expressed in 2-theta ± 0.2° 2-theta (Cu Ka radiation)) selected from the group consisting of 5.3, 7.0, 14.0, 17.7 and 24.5, hereinafter referred to as crystal form B.

[0029] More preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (crystal form B) having at least three characteristic peaks (expressed in 2-theta ± 0.2° 2-theta (Cu Ka radiation)) selected from the group consisting of 5.3, 7.0, 14.0, 17.7 and 24.5. Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (crystal form B) having peaks (expressed in 2-theta ± 0.2° 2-theta (Cu Ka radiation)) at 5.3, 7.0, 14.0, 17.7 and 24.5.

[0030] Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester and has a PXRD pattern (crystal form B) having at least one characteristic peak (expressed in 2-theta ± 0.2° 2-theta (Cu Ka radiation)) selected from the group consisting of 5.3, 7.0, 8.4, 13.0, 13.2, 14.0, 14.7, 15.9, 17.4, 17.7, 18.1, 18.7, 20.7, 21.2, 22.9, 24.5 and 25.4.

[0031] Most preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and has a PXRD pattern substantially as shown in Figure 2 (Form B).

[0032] While Form A is suitable for tablet formulation, Form B is stable in aqueous environment and thus very suitable for ointment, suspension or cream formulations which exhibit high water activity.

[0033] Another aspect of the present application is a crystalline salt of 5-methyl-(6S)- tetrahydrofolic acid and L-isoleucine ethyl ester having a PXRD pattern with at least one characteristic peak (expressed in 2Θ ± 0.2° 2Θ (Cu Ka radiation)) selected from the group consisting of peaks at 7.0, 8.8, 14.0, 17.6, 20.2, 23.4 and 26.9, hereinafter referred to as Form C.

[0034] More preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and has a PXRD pattern (Form C) with at least three characteristic peaks (expressed in 2Θ ± 0.2° 2Θ (Cu Ka radiation)) selected from the group consisting of peaks at 7.0, 8.8, 14.0, 17.6, 20.2, 23.4 and 26.9. Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and has a PXRD pattern (Form C) with characteristic peaks (expressed in 2Θ ± 0.2° 2Θ (Cu Ka radiation)) at 7.0, 8.8, 14.0, 17.6, 20.2, 23.4 and 26.9.

[0035] Even more preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester having a PXRD pattern (Form C) with at least one characteristic peak (expressed in 2Θ ± 0.2° 2Θ (Cu Ka radiation)) selected from the group consisting of peaks at 5.3, 7.0, 8.8, 11.2, 13.0, 14.0, 15.1, 15.9, 16.8, 17.4, 17.6, 20.2, 20.5, 20.8, 21.2, 21.4, 23.4, 24.5, 26.9 and 32.2.

[0036] Most preferably, the salt is a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester and has a PXRD pattern (Form C) substantially as shown in Figure 3.

[0037] Even more preferably, the above crystalline salt has a chemical and / or stereochemical purity of at least 99 wt% or more.

[0038] It is also advantageous to have a crystalline form of 5-methyl-(6S)-tetrahydrofolic acid which has a very low water content and especially a low tendency to absorb / desorb water, for example when handling the substance for compounding in a temperature / humidity controlled environment or in tropical countries where the relative humidity is usually very high. The salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester usually shows a water content below 1 %, whereas the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid usually has a water content of more than 10 %. The results are illustrated graphically in Example 9 or Figure 5.

[0039] In addition, besides the very low water content of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester, this salt surprisingly shows a particularly low tendency to absorb / desorb water over a broad humidity range. Thus, when comparing the water content of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester (crystalline form A) with the water content of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid in the most relevant range of relative humidity of 20-75 % r.h., the water content of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester (crystalline form A) varies by less than 0.8 %, in contrast to which the water content of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid varies by more than 6 % according to the prior art. The results are illustrated graphically in Example 9 or Figure 5. This result is very surprising for the person skilled in the art and cannot be anticipated when considering the teaching of US 6,441,168 B1. Furthermore, the crystalline form of the present application clearly solves the technical problem to which the present application relates.

[0040] In addition, the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester shows a much better stability than the previously known calcium salt. Thus the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester still shows a relative content of 5-methyl-(6S)-tetrahydrofolic acid of more than 98 % w / w (measured relative to the initial content at the beginning) when stored at 25 °C / 60 % rh for more than 12 months, whereas the relative content of 5-methyl-(6S)-tetrahydrofolic acid in the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid has dropped below 98 % w / w before 9 months. The results are illustrated graphically in Example 10 (Table 4b) or Figure 6. In parallel, the content of the pyrazino-s-triazine derivative [MeFox] of the main degradation product 4a-hydroxy-5-methyl-THF is below 0.05 % when stored at 25 °C / 60 % rh for more than 12 months, whereas the MeFox content of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid has increased to more than 0.6 % before 9 months. The results are illustrated graphically in Example 10 (Table 5) or Figure 7.

[0041] The surprising high stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester is even more particularly emphasized when comparing the stability values at 40°C / 75%rh. Even under these elevated conditions, the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester still shows a relative content of 5-methyl-(6S)-tetrahydrofolate of more than 99% (measured relative to the initial content at the start) when stored for more than 12 months, whereas the relative content of 5-methyl-(6S)-tetrahydrofolate in the calcium salt of 5-methyl-(6S)-tetrahydrofolate has dropped to less than 99% when stored under the same conditions until 9 months. The results are graphically illustrated in Example 10 (Table 6b) or Figure 8. In parallel, the content of the main degradation product, the pyrazino-s-triazine derivative of 4a-hydroxy-5-methyl-THF [MeFox], is only 0.06% when stored for 12 months at 25°C / 60%rh, whereas the MeFox content of the calcium salt of 5-methyl-(6S)-tetrahydrofolate has increased to more than 0.6% until 9 months. The results are graphically illustrated in Example 10 (Table 7) or Figure 9.

[0042] Thus, even under these conditions, the crystalline form of the present application shows an improved storage stability. These improved properties cannot be derived in view of the teaching of US patent 6,441,168 B1 either.

[0043] Another aspect of the present application is a process for obtaining a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, the process comprising the following steps:

[0044] i) preparing a mixture of 5-methyl-(6S)-tetrahydrofolate in water and adding L-isoleucine ethyl ester, optionally in a suitable solvent or solvent mixture;

[0045] ii) adding a base, optionally in a suitable solvent or solvent mixture, to achieve dissolution;

[0046] iii) heating the composition to at least 60°C;

[0047] iv) adding a suitable acid to adjust the pH to the range of 4 to 7;

[0048] v) crystallizing the mixture and cooling to a temperature of 1 °C to 30°C, optionally stirring the obtained suspension at the final temperature; and

[0049] vi) isolating the obtained solid material and optionally drying the product.

[0050] According to the disclosed process, the crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester can surprisingly be obtained in high efficiency and in high chemical, stereochemical and crystalline purity, even when prepared from 5-methyl-(6S)-tetrahydrofolic acid having an optical purity of 95.0%, which is formed in situ from (6S)-tetrahydrofolic acid benzenesulfonate having an optical purity of only 95.0%. Thus, when starting from (6S)-tetrahydrofolic acid benzenesulfonate having an optical purity of 95.0%, a crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester having an optical purity of more than 99% is finally obtained. Typically, the crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester prepared according to the disclosed process has an optical purity of more than 99%, preferably more than 99.5%, and a chemical purity of more than 98%, preferably more than 99%, even more preferably more than 99.5%.

[0051] Preferably, in step i), the mixture of 5-methyl-(6S)-tetrahydrofolic acid in water is prepared in situ from (6S)-tetrahydrofolic acid benzenesulfonic acid (prepared according to EP 0495204 B1 ), wherein (6S)-tetrahydrofolic acid benzenesulfonic acid is subjected to reductive methylation by using formaldehyde and sodium borohydride in an inert atmosphere.

[0052] Preferably, in step i), L-isoleucine ethyl ester is added in the form of its hydrochloride.

[0053] Preferably, the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-isoleucine ethyl ester hydrochloride in step i) is 1 : 1 to 1 :3.

[0054] Preferably, in step ii), the base used to enable the dissolution of 5-methyl-(6S)- tetrahydrofolic acid is an alkali hydroxide. More preferably, in step ii), the base is NaOH, KOH and / or a mixture thereof. The most preferred base in step ii) is aqueous sodium hydroxide.

[0055] Even more preferably, in step ii) or step iii), the pH after addition of the base is in the range of 6.5 to 9.0.

[0056] Most preferably, the solvent used in step i) and / or step ii) is water.

[0057] Optionally, charcoal is added in step iii) and the solution is then clarified by filtration.

[0058] Preferably, in step iv), aqueous hydrochloric acid is used as suitable acid.

[0059] More preferably, in step iv) the pH value obtained by the addition of the acid is set in the range of 4.4 to 6.4. Most preferably, in step iv) after the addition of the acid the pH is in the range of 5.4 to 6.

[0060] In step iii), iv) and / or v) seed crystals can be added, even more preferably seed crystals of the desired crystal modification.

[0061] Another aspect of the present application is a pharmaceutical composition, a food additive and / or a preparation comprising a salt of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester and optionally one or more acceptable excipients.

[0062] The salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester can be used as an ingredient for the production of a medicament and / or as a food additive.

[0063] The salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester can be used for the treatment of anemia, neural tube defects (NTD), cardiovascular diseases (CVD), depression, Alzheimer's disease and osteoporosis and / or for the dietary management of low plasma and / or low red blood cell folate (medical food).

[0064] The pharmaceutical composition according to the present application can be used for all modes of administration, preferably for oral, parenteral, intramuscular, intraspinal, intrathecal, periodontal, topical or rectal administration.

[0065] In summary, the performance characteristics provided by the salt of 5-methyl-(6S)- tetrahydrofolate and L-isoleucine ethyl ester of the present application are advantageous for use in a medicament or as a food additive. In particular, the enhanced stability, the low change in water content in an environment of 20% to 75% relative humidity and the increased optical purity are unforeseeable for the person skilled in the art when crystallizing the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester. Examples

[0066] Powder X-ray diffraction

[0067] Stoe Stadi P with Mythen 1 K detector; Cu-Kal radiation; standard measurement conditions: transmission; tube power 40 kV and 40 mA; curved germanium monochromator; 0.02° 2 theta step size, 48 s step time, 1.5-50.5° 2 theta scan range; detector mode: step scan; 1° 2 theta detector step; standard sample preparation: 10 to 20 mg of sample placed between two pieces of acetate foil; sample holder: Stoe transmission sample holder; sample rotated during measurement. All sample preparation and measurements were performed in an ambient air atmosphere.

[0068] TG-FTIR

[0069] Thermogravimetric measurements were performed with a Netzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pan with pinhole, N2 atmosphere, heating rate 10 K / min).

[0070] DVS

[0071] DVS measurements were typically performed using an SPS 11-100 n "Sorptions Prüfsystem" from ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str. 23, 89079 Ulm (Germany).

[0072] Raman spectroscopy

[0073] FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman or a Bruker RFS 100 FT-Raman system using a near-infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. In the range from 3500 to -50 cm -1 -1, 64 scans with a resolution of 2 cm -1 -1 were accumulated; however, due to filter cutoff effects, only data above 100 cm -1 -1 were evaluated. The nominal laser power was typically 100 or 300 mW.

[0074] Example 1 : Preparation of salt crystalline form A of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester without seeding

[0075] A suspension of 2.00 g of 5-methyl-(6S)-tetrahydrofolic acid (assay: 95.6% w / w) in 20.0 ml of water was heated to 70°C and 2.13 g of L-isoleucine ethyl ester hydrochloride was added. The temperature in the suspension was 65°C and sodium hydroxide was added as a 30% (w / w) concentrated aqueous solution. A total of 0.95 g of 30% sodium hydroxide solution was added. The addition of sodium hydroxide resulted in a substantially clear solution which gradually turned into a concentrated suspension. The suspension was diluted with 20.0 ml of water and the heater was turned off to allow the mixture to cool to ambient temperature over about two hours. The reactor with the suspension was further cooled to about 10°C in an ice / water bath over half an hour and then filtered with a fritted glass filter and washed with 5 ml of cold water. The solid product was dried in a vacuum desiccator at 35°C for about 20 hours and checked by powder X-ray diffraction and identified as 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt crystalline Form A. The powder X-ray diffraction pattern of crystalline Form A is shown in Figure 1 and shows peaks at the 2 theta angles as shown in Table 1. HPLC analysis showed a purity of 98.65% area.

[0076] Table 1: 2 theta angles, d-spacings and qualitative intensities of crystalline Form A. Vs = very strong intensity, s = strong intensity, m = medium intensity, w = weak intensity, vw = very weak intensity. It should be noted that the intensity values can vary significantly due to preferred orientation effects.

[0077]

[0078]

[0079]

[0080] Example 2: Preparation of salt crystalline Form A of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester in the case of seeding

[0081] To 15 g of 5-methyl-(6S)-tetrahydrofolate (assay: 95.4% w / w, 6S-diastereomer: 97.74%) was added 225 g of water under nitrogen atmosphere. The pH was adjusted to 6.5 by the addition of aqueous sodium hydroxide solution (30% w / w). The mixture was heated to 66°C and further aqueous sodium hydroxide solution (30% w / w) was added to maintain the pH at 6.5. A solution of 15.2 g of L-isoleucine ethyl ester hydrochloride in 75 g of water was added at 66°C. After seeding with 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A at 66°C, the mixture was stirred at 66°C for 16 hours. The mixture was cooled to 1°C while the pH was adjusted to 5.7 by the addition of 1 molar aqueous hydrochloric acid solution. After stirring at 1°C for 1 hour, the crystalline material was isolated by suction filtration and washed with 44 g of water pre-cooled to 1°C. This material was dried under vacuum at 36°C for 22 hours to give 14.6 g of 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt, which corresponds to a theoretical yield of 75% (assay corrected). (Analytical data of the isolated product: purity: 98.9% area, assay: 73.95% w / w 5-methyl-(6S)-tetrahydrofolate corresponding to a 1 : 1 salt, loss on drying (residual water): 1.2% w / w, (6S)-diastereomer: 99.8%. TG-FTIR analysis indicated that the obtained solid product was substantially free of water as the mass loss at 150°C was no more than about 0.2%. H-NMR spectroscopic analysis indicated that the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester was about 1 : 1. The powder X-ray diffraction pattern shown in Figure 1 corresponds to Form A.

[0082] Example 3: Preparation of a salt of 5-methyl-(6S)-tetrahydrofolate and L isoleucine ethyl ester salt Form A

[0083] Example 3: Preparation of a salt of 5-methyl-(6S)-tetrahydrofolate and L isoleucine ethyl ester salt Form A Example 3: Preparation of a salt of 5-methyl-(6S)-tetrahydrofolate and L isoleucine ethyl ester salt Form A

[0084] Example 4: Preparation of salt Form B of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester

[0085] To a mixture of 6.0 ml water and 452 mg of 5-methyl-(6S)-tetrahydrofolate L- isoleucine ethyl ester salt Form A was added 6.0 ml of a 0.5 M solution of L- isoleucine ethyl ester hydrochloride in water and 2.0 ml of ethanol at room temperature. The pH was adjusted to pH ~ 6-7 by the addition of 0.250 ml of 1 N NaOH and the mixture was then heated to 75 °C. The temperature of the light yellow suspension was maintained at 75 °C for 30 minutes. The mixture was then allowed to cool to 25 °C over approximately three hours. Stirring at 25 °C was continued over approximately 4 hours and a solid sample, designated PP555-P40a, was recovered by filtration by centrifugation and examined by PXRD without drying. The solid product was identified as 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form B.

[0086] Table 2: 2 theta angles, d-spacings and qualitative intensities for Form B. Vs = very strong intensity, s = strong intensity, m = medium intensity, w = weak intensity, vw = very weak intensity. It should be noted that the intensity values can vary significantly due to preferred orientation effects.

[0087]

[0088]

[0089]

[0090] Example 5: Preparation of salt Form C of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester

[0091] To 81 mg of amorphous 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt (produced by freeze-drying a solution in 4: 1 water-dioxane) was added 1.0 ml of 2: 1 (by volume) ethanol-water and the mixture was stirred at room temperature for two days. The suspension was then filtered and the solid was subjected to powder X-ray diffraction. The powder X-ray diffraction showed that a crystalline form, designated Form C, was obtained. 1 H-NMR spectroscopic analysis showed a molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester of about 2.8: 1.

[0092] Table 3: 2 theta angles, d-spacings and qualitative intensities for Form C. Vs = very strong intensity, s = strong intensity, m = medium intensity, w = weak intensity, vw = very weak intensity. It should be noted that the intensity values can vary significantly due to preferred orientation effects.

[0093]

[0094]

[0095]

[0096] Example 6: Preparation of a salt of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester from 5-methyl-(6S)-tetrahydrofolate formed in situ from (6S)- tetrahydrofolate benzenesulfonate

[0097] To a mixture of 20 g [6S]-tetrahydrofolate benzenesulfonate (prepared according to EP 0 495 204, tetrahydrofolate assay: 72.2% w / w, (6S)-diastereomer: 95.9%) and 50 g water, 9.9 mL of an aqueous sodium hydroxide solution (30% w / w sodium hydroxide) were added at 20-30 °C under stirring. Then 0.83 mL of an aqueous sodium hydroxide solution of 1 molar were added and the mixture was cooled to 0-5 °C. At 0-5 °C, 3.13 mL of an aqueous formaldehyde solution (concentration: 36.9% w / w) were added and after 1 hour of stirring, 0.31 mL of an aqueous sodium hydroxide solution (30% w / w sodium hydroxide) were added, followed by a mixture of 14 g water, 1.7 g of an aqueous sodium hydroxide solution (30% w / w sodium hydroxide) and 3.07 g of sodium borohydride. The mixture was heated to about 63 °C and stirred for 90 minutes. After cooling to ambient temperature, 7.9 mL of an aqueous hydrochloric acid solution (37% w / w) were added, followed by 4.1 mL of an aqueous sodium hydroxide solution (30% w / w sodium hydroxide). A small amount of sodium tetraborate was added and the mixture was cooled to 0-5 °C and stirred for about 22 hours. The solids were removed by filtration (suction) and washed with 5 g water. To the filtrate, a mixture of 15.86 grams of L-isoleucine ethyl ester hydrochloride and 30 grams of water was added at ambient temperature under stirring. The mixture was heated to about 66 °C and a small amount of crystalline 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A was added for seeding. At about 66 °C, 27.1 mL of an aqueous 1 molar hydrochloric acid solution were added. A suspension was formed. The mixture was cooled to about 20 °C within about 90 minutes while further adding 33.2 mL of an aqueous 1 molar hydrochloric acid solution. The crystalline product was isolated by filtration (suction) and washed with 84 mL of water pre-cooled in an ice bath. The product was dried at room temperature under vacuum (10 mbar) for about 60 hours to give 17.2 grams of crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester Form A (5-methyl-(6S)-tetrahydrofolate assay: 74.0% w / w, purity: 98.6% area, (6S)-diastereomer: 99.5%), which corresponds to an assay corrected chemical yield of 88.1%.

[0098] Example 7: Preparation of the salt of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester

[0099] To 1670 g of water, 250 g of 5-methyl-(6S)-tetrahydrofolate (assay: 96.2% w / w) and 256.1 g of L-isoleucine ethyl ester hydrochloride were added at ambient temperature under stirring. 500 g of water were added and the pH was adjusted to pH = 7.3 by adding 134.6 mL of an aqueous sodium hydroxide solution (30% w / w sodium hydroxide). The mixture was heated to about 66°C under stirring and a small amount of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester Form A was added for seeding. At 66°C, 385 mL of a 1 molar aqueous hydrochloric acid solution were added within about 30 minutes. The mixture was cooled to about 20°C within about 90 minutes while further 365 mL of a 1 molar aqueous hydrochloric acid solution were added. The crystalline product was isolated by filtration (suction) and washed with 733 g of water pre-cooled in an ice bath. The product was dried at about 40°C under vacuum (10 mbar) for about 60 hours to yield 300.8 grams of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester Form A (5-methyl-(6S)-tetrahydrofolate assay: 74.3% w / w, purity: 98.7% area), which corresponds to a 92.9% assay corrected chemical yield.

[0100] Example 8: Preparation of the salt of 5-methyl-(6S)-tetrahydrofolate and L- isoleucine ethyl ester

[0101] To 250.5 kg water in a reaction vessel at ambient temperature was added under stirring 31.0 kg 5-methyl-(6S)-tetrahydrofolate (assay: 96.8% w / w, 6S-diastereomer: 98.3%) and 32.0 kg L-isoleucine ethyl ester hydrochloride. 16.5 kg water was added and the pH was adjusted to pH = 7.3 by addition of 18.6 kg aqueous sodium hydroxide solution (30% w / w sodium hydroxide). The mixture was heated to about 66°C under stirring and the solids were removed by filtration. The filter was washed via the reaction vessel with 20.3 kg water. The filtrate and the washing were combined in a crystallization vessel and heated to about 65°C. 37.5 grams of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester Form A were added for seeding. At about 65°C 64.5 kg of 1 M aqueous hydrochloric acid solution were added within 30 minutes. The mixture was cooled to 20°C within 90 minutes and the pH was adjusted to pH = 5.4 by addition of 1.5 kg of 1 M aqueous hydrochloric acid solution. The mixture was stirred at about 20°C for 1 hour and the crystalline product was isolated by centrifugation. The product was washed with 45 kg water pre-cooled to about 3°C. The product was then dried at 50°C under vacuum for 17 hours to give 23.63 kg 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester Form A (5-methyltetrahydrofolate assay 77.2% w / w, 6S-diastereomer: 99.5%), which corresponds to an assay corrected yield of 60.8%. The yield of this example is not representative because part of the crystalline product was lost during centrifugation due to a technical fault.

[0102] Example 9: Hygroscopicity and water content (DVS experiment)

[0103] The water content of a sample of 5-methyl-(6S)-tetrahydrofolate calcium salt was measured and found to be 12.4%. TG-FTIR analysis of a sample of 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A prepared according to Example 1 or 2 showed that the sample was essentially free of water.

[0104] A sample of 5-methyl-(6S)-tetrahydrofolate calcium salt and a sample of 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A prepared according to Example 1 or 2 (each about 20 mg) were tested by dynamic vapor sorption analysis (DVS) over a relative humidity range from 0 to 75% r.h. The DVS measurement was performed using an SPS 11-100 n "Sorptions Prüfsystem" from ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str. 23, 89079 Ulm (Germany). The measurement was performed as follows: The sample was placed on an aluminum or platinum holder on top of a microbalance and allowed to equilibrate at 50% RH, after which the predefined humidity program was started:

[0105] (1) five hours at 50% relative humidity (RH), then

[0106] (2) scanning to 50→0% RH at a rate of 5% per hour

[0107] (3) maintaining a constant RH at 0% for 5 hours

[0108] (4) increasing the RH to 75% at a rate of 5% per hour

[0109] (5) maintaining a constant RH at 75% for 5 hours

[0110] (6) scanning to 50% RH at a rate of 5% per hour

[0111] The results of 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A were compared to the results of the calcium salt, showing that the water content of 5-methyl-(6S)-tetrahydrofolate L-isoleucine ethyl ester salt Form A varied by less than 0.8% over the tested range, while the water content of the calcium salt of 5-methyl-(6S)-tetrahydrofolate varied by more than 6%. The results are illustrated graphically in Figure 5.

[0112] Example 10: Stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester

[0113] In order to compare the long-term stability of the compound of the present application, the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, with the long-term stability of the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolate prepared according to EP 1 044 975 B1, respective stability data have been generated at various temperatures and humidities.

[0114] (a) Stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 25°C / 60% rh

[0115] The crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolate and the salt form A of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester prepared according to the literature procedure (EP 1 044 975 B1 ) were stored at 25 °C / 60 % rh. The content of residual 5-methyl-(6S)-tetrahydrofolate in the samples was measured by HPLC at periodic intervals. The results are shown in Table 4a and Figure 6. The remaining content of 5-methyl-(6S)-tetrahydrofolate was also compared to the initial value at the time of preparation (relative %). The results are shown in Table 4b. In addition, the content of the main degradation product, the pyrazino-s-triazine derivative of 4a-hydroxy-5-methyl-THF (MeFox), was measured by HPLC at periodic intervals and is disclosed as absolute value (% w / w). The results are shown in Table 5 and Figure 7.

[0116] Table 4a: Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 25 °C / 60 % rh (% w / w)

[0117]

[0118]

[0119] Table 4b: Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 25 °C / 60 % rh (relative %).

[0120]

[0121] Table 5: Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 25 °C / 60 % rh (main degradation product [MeFox])

[0122]

[0123] (b) Stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 40 °C / 75 % rh

[0124] The crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolate and the salt form A of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester prepared according to the literature procedure (EP 1 044 975 B1 ) were stored at 40°C / 75%rh. The content of residual 5-methyl-(6S)-tetrahydrofolate in the samples was measured by HPLC at periodic intervals. The results are shown in Table 6a and Figure 8. The remaining content of 5-methyl-(6S)-tetrahydrofolate was also compared to the initial value at the time of preparation (relative %). The results are shown in Table 6b. In addition, the content of the main degradation product, the pyrazino-s-triazine derivative of 4a-hydroxy-5-methyl-THF (MeFox), was measured by HPLC at periodic intervals and disclosed as absolute values (% w / w). The results are shown in Table 7 and Figure 9.

[0125] Table 6a: Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 40°C / 75%rh (% w / w)

[0126]

[0127] Table 6b: Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester at 40°C / 75%rh (relative %)

[0128]

[0129]

[0130] Table 7: Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)- tetrahydrofolate at 40°C / 75%rh (main degradation product [MeFox])

[0131]

[0132] The Tables 4 to 7 with the stability data of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester disclosed in the present invention clearly show that:

[0133] i) the stability of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester differs significantly compared to the crystalline calcium salt of 5-methyl-(6S)- tetrahydrofolate, and

[0134] ii) the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester is much more stable than the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolate for a very long time.

[0135] Example 11: Isomeric enrichment of the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester

[0136] When preparing the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester starting from 5-methyl-(6S)-tetrahydrofolate, the following isomeric enrichment (measured by HPLC) can be obtained

[0137]

[0138] When preparing the salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester starting from 5-methyl-(6S)-tetrahydrofolate benzenesulfonate prepared in situ, the following isomeric enrichment (measured by HPLC) can be obtained

[0139]

Claims

1. A crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester is from 1:0.75 to 1:1.25, in mol / mol. The salt is characterized as a salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, and has a PXRD pattern containing characteristic peaks selected from those at 5.8, 6.9, 14.0, 17.5 and 22.2, crystal form A, the characteristic peaks being represented by 2θ ± 0.2°2θ CuKα radiation.

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

3. The crystalline salt of claim 1 or 2, characterized in that... The salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, and has a PXRD pattern containing at least one other characteristic peak selected from the following at 8.5, 12.5, 12.9, 14.9, 16.2, 17.9, 18.9, 19.2, 24.4, 25.4, 25.8, 25.9 and 34.6, crystal form A, the characteristic peak being represented by 2θ ± 0.2°2θ, CuKα radiation.

4. The crystalline salt of claim 1 or 2, characterized in that... The salts of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester have the PXRD pattern shown in Figure 1, crystal form A.

5. The crystalline salt of claim 1 or 2, characterized in that... The salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, and has a Raman spectrum containing at least one characteristic peak selected from the following: 1607, 1571, 1506, 1250, and 650 nm. Crystal form A, the characteristic peak is expressed in wavenumber cm⁻¹. -1 ±2 cm -1 express.

6. The crystalline salt of claim 1 or 2, characterized in that... The salt is a salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester, and has a Raman spectrum containing at least one characteristic peak selected from the following: 2961, 2938, 1607, 1571, 1506, 1468, 1332, 1250, 1153, 922, 860, and 650 nm. Crystal form A, the characteristic peak is expressed in wavenumber cm⁻¹. -1 ±2cm -1 express.

7. The crystalline salt of claim 1 or 2, characterized in that... The salts of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester have the Raman spectra shown in Figure 4, crystal form A.

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

9. The crystalline salt of claim 8, having a stereoisomeric purity of at least 99%.

10. A method for obtaining a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9, the method comprising the steps of: i) Prepare a mixture of 5-methyl-(6S)-tetrahydrofolate in water and add L-isoleucine ethyl ester, optionally in a suitable solvent or solvent mixture. ii) Add a base, optionally in a suitable solvent or mixture of solvents, to achieve dissolution; iii) Heat the composition to at least 60°C; iv) Add a suitable acid to adjust the pH to the range of 4 to 7; v) Crystallize the mixture and cool it to a temperature of 1°C to 30°C, optionally stirring the resulting suspension at the final temperature; and vi) Separate the obtained solid material and optionally dry the product.

11. The method of claim 10, characterized in that... In step i), the molar ratio of 5-methyl-(6S)-tetrahydrofolate to L-isoleucine ethyl ester is 1:1 to 1:

3.

12. The method of claim 10 or 11, characterized in that... In step i), a mixture of 5-methyl-(6S)-tetrahydrofolate in water is prepared in situ.

13. The method of claim 10 or 11, characterized in that... In step i), L-isoleucine ethyl ester is used in its hydrochloride form.

14. The method of claim 10 or 11, characterized in that... In step ii), an aqueous solution of sodium hydroxide is used to dissolve the compound, and in step iv), an aqueous solution of hydrochloric acid is used for pH adjustment.

15. The method of claim 10 or 11, characterized in that... In step ii), after the addition of alkali, the pH is in the range of 6.5 to 9.

0.

16. The method of claim 10 or 11, characterized in that... Add carbon in step iii), and then filter the solution to clarify it.

17. The method of claim 10 or 11, characterized in that... The solvent is water.

18. The method of claim 10 or 11, characterized in that... In step iv), the pH value obtained by adding acid is set to be in the range of 5.4 to 6.

4.

19. The method of claim 10 or 11, characterized in that... Seed crystals are added in steps iii), iv) and / or v).

20. A pharmaceutical composition comprising a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9, and optionally one or more acceptable excipients.

21. The pharmaceutical composition according to claim 20, prepared from the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9, and in the form of tablets, capsules, oral liquid preparations, powders, lyophilized preparations, granules, lozenges, reconfigurable powders, injectable or infusionable solutions or suspensions or suppositories.

22. The pharmaceutical composition according to claim 20 or 21, further comprising at least one additional therapeutic agent.

23. The pharmaceutical composition according to claim 20 or 21, wherein it is a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, periodontal, or rectal administration.

24. The pharmaceutical composition according to claim 20 or 21, wherein it is a pharmaceutical composition for topical application.

25. A food additive comprising a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9, and optionally one or more acceptable excipients.

26. A formulation comprising a crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9, and optionally one or more acceptable excipients.

27. Use of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9 in the preparation of pharmaceutical ingredients.

28. Use of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9 for the preparation of a medicament for the treatment of 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 erythrocyte folate and / or low cerebrospinal fluid folate and / or low peripheral or central nervous system folate.

29. Use of the crystalline salt of 5-methyl-(6S)-tetrahydrofolate and L-isoleucine ethyl ester according to any one of claims 1 to 9 as a food additive.

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