Crystalline salts of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester

Abstract

The present invention refers to a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester is from 1:0.3 to 1:3.0 (in mol/mol) and/or hydrates and/or solvates thereof as well as to a process of obtaining the same.

Claims

1. A crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-valine ethyl ester is from 1:0.3 to 1:3.0 (in mol/mol) and/or hydrates and/or solvates thereof.

2. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-valine ethyl ester is from 1:0.5 to 1:2.5 (in mol/mol) and/or hydrates and/or solvates thereof.

3. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-valine ethyl ester is from 1:0.75 to 1:1.25 (in mol/mol) and/or hydrates and/or solvates thereof.

4. The crystalline salt of claim 1, wherein the ratio of 5-methyl-(6S)-tetrahydrofolic acid L-valine ethyl ester is approximately 1:1 (in mol/mol) and/or hydrates and/or solvates thereof.

5. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) selected from the following peaks located at 5.8, 6.9, 14.0, 19.0, 19.3, 22.2 and 25.9 (Form A).

6. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with characteristic peaks (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 5.8, 6.9, 14.0, 19.0, 19.3, 22.2 and 25.9 (Form A).

7. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) selected from the following peaks located at 5.8, 6.9, 12.6, 14.0, 14.9, 17.5, 18.0, 19.0, 19.3, 20.0, 22.2 and 25.9 (Form A).

8. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern substantially as shown in FIG. 1 (Form A).

9. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 4.9, 7.4, 8.5, 14.1, 15.8, 16.3, 17.2, 18.6, 22.2 and 24.5 (Form B).

10. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with peaks (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 4.9, 7.4, 8.5, 14.1, 15.8, 16.3, 17.2, 18.6, 22.2 and 24.5 (Form B).

11. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 4.9, 7.4, 8.5, 14.1, 15.8, 17.2, 18.4, 18.6, 20.3, 20.9, 21.1, 22.2, 24.5 and 25.7 (Form B).

12. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester and has a PXRD pattern substantially as shown in FIG. 2 (Form B).

13. The crystalline salt of claim 1 having at least 99 wt % or more chemical and/or stereoisomerical purity.

14. A process for obtaining the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester according to claim 1 comprising the steps of: i) providing a mixture of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester, optionally in a suitable solvent or a mixture of solvents ii) adding a base, optionally in a suitable solvent or a mixture of solvents, to dissolve the compounds; iii) heating the composition to at least 60° C. and optionally carrying out a clear filtration; iv) crystallizing and cooling the mixture to a temperature between 1° C. and 30° C., optionally adding more solvent or mixture of solvents; and v) isolating the obtained solid material and optionally drying the product.

15. The process of claim 14, characterized in that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester in step i) is in the range of from 1:1 to 1:3.

16. The process of claim 14, characterized in that the solvent is water.

17. The process of claim 14, characterized in that in step iii) and/or iv) seed crystals are added.

18. The process of claim 14, characterized in that L-valine ethyl ester is used as L-valine ethyl ester hydrochloride.

19. A pharmaceutical composition, food additive and/or preparation comprising the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester according to claim 1 and optionally one or more acceptable excipients.

20. The pharmaceutical composition according to claim 19 in the form of tablets, capsules, oral liquid preparations, powders, lyophilisates, granules, lozenges, reconstitutable powders, injectable or infusable solutions or suspensions or suppositories.

21. The pharmaceutical composition according to claim 19 further comprising at least one additional therapeutic agent.

22. The pharmaceutical composition according to claim 19, which is a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, peridontal, topical or rectal administration.

23. A method of producing a drug formulation or food additive formulation comprising: combining a drug or food additive with the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester according to claim 1.

24. A method for treating a subject for homocysteine-lowering, of anemia, neural tube defects, cardiovascular diseases, depression, cognitive impairment, Alzheimer's disease and osteoporosis and/or 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 comprising administering to the subject the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester according to claim 1.

Description

EXAMPLES

[0044] Powder X-ray diffraction

[0045] Stoe Stadi P equipped with a Mythen1K Detector; Cu-Kα1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; curved Ge monochromator; 0.02°2θ step size, 48 s step time, 1.5-50.5°2θ scanning range;

[0046] detector mode: step scan; 1°2θ detector step; standard sample preparation: 10 to 20 mg sample was placed between two acetate foils; sample holder: Stoe transmission sample holder; the sample was rotated during the measurement. All sample preparation and measurement was done in an ambient air atmosphere.

[0047] TG-FTIR

[0048] Thermogravimetric measurements were carried out with a Netzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with a pinhole, N.sub.2 atmosphere, heating rate 10 K/min).

[0049] DVS

[0050] DVS measurements are typically performed with an SPS11-100n “Sorptions Prüfsystem” from ProUmid (formerly “Projekt Messtechnik”), August-Nagel-Str. 23, 89079 Ulm (Germany).

[0051] DVS measurements were conducted as follows: The sample was placed on an aluminum holder on top of a microbalance and allowed to equilibrate at 50% RH before starting the pre-defined humidity program:

[0052] (1) two hours kept at 50% constant relative humidity (RH) then

[0053] (2) raised RH to 95% at a rate of 5% per hour

[0054] (3) maintained RH at 95% for five hours

[0055] (4) reduced to 0% RH at a rate of 5% per hour

[0056] (5) maintained RH at 0% for five hours

[0057] (6) raised RH to 95% at a rate of 5% per hour

[0058] (7) maintained RH at 95% for five hours

[0059] (8) reduced to 0% RH at a rate of 5% per hour

[0060] (9) maintained RH at 0% for five hours

[0061] (10) raised to 50% RH at a rate of 5% per hour

[0062] (11) maintained RH at 50% for about one hour

Example 1: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine-ethyl ester without Seeding

[0063] 478 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate was dissolved at room temperature in 2.00 mL of a 1.00 molar aqueous sodium hydroxide solution and 546 mg of L-valine ethyl ester hydrochloride was added. The solution was stirred at room temperature for about 20 minutes and 0.300 mL of a 1.00 molar aqueous hydrochloric acid solution was added. While stirring, the solution gradually changed into a concentrated suspension. 1.00 mL of water was added and the suspension was sonicated. An additional 0.300 mL of a 1 molar aqueous hydrochloric acid solution was added and the suspension again sonicated. The suspension was stirred at room temperature for about 70 minutes and the solid material was separated by centrifugal filtration under ambient conditions. 0.5 mL of water was added to the wet solid material in the filter centrifuge device and centrifugation was repeated. This wash step was repeated twice more using 0.50 mL and 1.00 mL of water, respectively. The wet filter cake was transferred into a fritted glass filter and air dried by drawing ambient air (about 22° C./about 34% r.h.) through the glass filter for about 20 minutes. The dried material was examined by .sup.1H-NMR and identified as a 5-methyl-(6S)-tetrahydrofolic acid L-valine ethyl ester 1:1 salt. Analysis of the sample by TG-FTIR showed a water content of about 0.5%. Powder X-ray diffraction was carried out and the PXRD pattern of the L-valine ethyl ester salt Form A, substantially as depicted in FIG. 1 was obtained.

Example 2: Vacuum Drying of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester

[0064] About 112 mg of the crystalline material according to Example 1 was vacuum dried at room temperature and about 10 mbar for about one hour, followed by ventilation using ambient air (about 22° C./about 23% r.h.). Analysis of the sample by TG-FTIR showed a water content of about 0.3%.

[0065] An aliquot of the sample is prepared between two acetate foils for PXRD at room temperature and about 25% relative humidity. Powder X-ray diffraction was carried out and the PXRD pattern of the L-valine ethyl ester salt Form A, as depicted in FIG. 1 was obtained which exhibits peaks at 2-theta angles as listed in Table 1.

TABLE-US-00001 TABLE 1 2-theta angles, d-spacings and qualitative intensities for 5- methyl-(6S)-tetrahydrofolic acid L-valine ethyl ester salt Form A according to example 2. Vs = very strong, s = strong, m = medium, w = weak, and vw = very weak in intensity. It should be noted that intensity values can vary substantially due to preferred orientation effects. angle °2Θ d-spacing [Å] qualitative intensity 5.8 15.3 s 6.9 12.8 vs 8.5 10.3 w 10.7 8.2 w 11.6 7.6 w 11.9 7.5 w 12.6 7.0 m 12.9 6.9 w 14.0 6.3 vs 14.9 5.93 m 15.3 5.78 w 15.6 5.69 w 15.9 5.59 w 16.2 5.45 w 16.8 5.26 w 17.5 5.06 m 18.0 4.92 m 18.6 4.78 w 19.0 4.67 s 19.3 4.60 s 19.6 4.52 w 20.0 4.43 m 21.1 4.21 w 22.2 3.99 s 22.8 3.90 w 23.3 3.81 w 24.1 3.70 w 24.4 3.64 w 24.9 3.58 m 25.1 3.54 w 25.4 3.50 m 25.9 3.44 s 27.0 3.30 w 27.8 3.21 w 28.2 3.16 w 28.6 3.12 w 29.4 3.04 w

Example 3: Hydrate Formation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester

[0066] About 100 mg of the crystalline material produced according to Example 1 was stored at room temperature and 75% relative humidity for 14 days. An aliquot of the sample was prepared for PXRD between two Kapton foils at room temperature and at about 55% relative humidity. Powder X-ray diffraction was carried out and the PXRD pattern of the L-valine ethyl ester salt Form B, as depicted in FIG. 2 was obtained which exhibits peaks at 2-theta angles as listed in Table 2.

TABLE-US-00002 TABLE 2 2-theta angles, d-spacings and qualitative intensities for 5- methyl-(6S)-tetrahydrofolic acid L-valine ethyl ester salt Form B according to Example 3. Vs = very strong, s = strong, m = medium, w = weak, and vw = very weak in intensity. It should be noted that intensity values can vary substantially due to preferred orientation effects. angle °2Θ d-spacing [Å] qualitative intensity 4.9 18.2 m 5.6 15.9 w 7.4 12.0 vs 8.5 10.4 s 9.7 9.1 w 10.1 8.7 w 11.8 7.5 w 12.0 7.4 w 12.3 7.2 w 14.1 6.3 s 14.8 5.97 w 15.5 5.70 w 15.8 5.60 m 16.3 5.45 m 17.2 5.15 s 18.4 4.82 m 18.6 4.76 s 19.0 4.66 w 19.6 4.53 w 19.9 4.46 w 20.3 4.37 m 20.9 4.25 m 21.1 4.21 m 21.6 4.10 w 22.2 4.01 s 23.3 3.82 w 23.8 3.74 w 24.2 3.68 w 24.5 3.63 s 24.8 3.58 w 25.4 3.50 w 25.7 3.46 m 26.2 3.39 w 27.2 3.27 w 28.6 3.12 w 29.1 3.07 w 29.4 3.03 w 29.9 2.98 vw 30.4 2.94 w 30.7 2.91 w 31.0 2.88 w

Example 4: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine-ethyl ester

[0067] 4.78 g of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 94.6%, [6S]-content 97.6%) were dissolved at room temperature in 20.0 mL of a 1.0 molar aqueous sodium hydroxide solution. 5.47 g of L-valine ethyl ester hydrochloride were added. To the solution then 3.0 mL of a 1.0 molar aqueous hydrochloric acid solution was added. While stirring, the solution gradually changed into a light suspension. Another 2.0 mL of a 1.0 molar aqueous hydrochloric acid solution was added in portions of 0.5 mL. The light suspension the was seeded with crystalline as obtained in Example 1. Another 1.0 mL of a 1.0 molar aqueous hydrochloric acid solution was added. The solid material was separated by filtration and washed with 3.0 mL water. The received solid was dried at 36° C./0-10 mbar. The dried material (2.61 g, corresponding to 43% assay corrected yield) was examined by PXRD and HPLC. The assay for 5-methyl-(6S)-tetrahydrofolic acid was 74.8%w/w. Powder X-ray diffraction pattern substantially as depicted in FIG. 1 (Form A) was obtained. HPLC disclosed a [6S]-content of 99.9%.

Example 5: Hygroscopicity and Water Content (DVS Experiments)

[0068] 21 mg of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester according to Example 2 was weighed into an aluminum sample pan for DVS measurement. A DVS measurement was performed with an SPS11-100n “Sorptions Prüfsystem” from ProUmid, August-Nagel-Str. 23, 89079 Ulm (Germany). For relative humidity (RH) scans, change rates of 5% per hour were used. The sample pan was placed into the instrument and a defined relative humidity change program was started according to the following steps: [0069] (1) Maintained RH for 2 hours at 50%, then [0070] (2) scanned RH from 50.fwdarw.0% at a rate of 5% per hour and maintained RH at 0% for 5 hours, then [0071] (3) scanned RH from 0.fwdarw.75% at a rate of 5% per hour and maintained RH at 75% for 5 hours, then [0072] (4) scanned RH from 75.fwdarw.0% RH at a rate of 5% per hour and maintained RH at 0% for 5 hours. [0073] (5) scanned RH from 0.fwdarw.75% at a rate of 5% per hour and maintained RH at 75% for 5 hours, then [0074] (6) scanned RH from 75.fwdarw.50% RH at a rate of 5% per hour and maintained RH at 50% for 2 hours.

[0075] In parallel, the very same protocol was applied to a sample of the calcium salt as a reference and the result is displayed in FIG. 3. Within the relative humidity range from 0 to 55% the relative sample mass for the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester (solid line) changes less than 1% while for the calcium salt (dashed line) the relative sample mass changes by about 6%.

Example 6: Kinetic Solubility of the Crystalline Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-valine ethyl ester

[0076] 42.5 mg of the anhydrous form of the crystalline 5-methyl-(6S)-tetrahydrofolic acid L-valine ethyl ester salt (Form A) according to Example 2 was weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified/de-ionized water (for instance water for chromatography) was added to the solid using an adjustable volumetric pipette. The mixture was vigorously agitated at room temperature for one minute. After one minute a dilute suspension was observed suggesting that most of the sample was dissolved. The dilute suspension was filtered by centrifugal filtration and 1.50 mL of the aqueous solution was transferred into a tared glass vial (about 10 mL volume).

[0077] The water was evaporated in an air dryer at 40° C. for about 15 hours, then at 50° C. for about eight hours, subsequently drying was completed at 50° C. under vacuum (10 to 20 mbar) for about 13 hours. The solubility was determined by gravimetric evaluation of the solid residue. The solubility was 12.5 mg of 5-methyl-(6S)-tetrahydrofolic acid per mL.

[0078] Reference example 1: Kinetic solubility of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid

[0079] 42.5 mg of the anhydrous form of the crystalline 5-methyl-(6S)-tetrahydrofolic acid calcium salt was weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified/de-ionized water (for instance water for chromatography) was added to the solid using an adjustable volumetric pipette. The mixture was vigorously agitated at room temperature for one minute. After one minute a suspension was observed. The suspension was filtered by centrifugal filtration and 1.50 mL of the aqueous solution was transferred into a tared glass vial (about 10 mL volume). The water was evaporated in an air dryer at 40° C. for about 15 hours, then at 50° C. for about eight hours, subsequently drying was completed at 50° C. under vacuum (10 to 20 mbar) for about 13 hours. The solubility was determined by gravimetric evaluation of the solid residue. The solubility was 9.0 mg of 5-methyl-(6S)-tetrahydrofolic acid per mL.