Stable lyophilisates comprising 5,10-methylene-(6R)-tetrahydrofolic acid

Abstract

The present invention is directed to stable lyophilisates comprising 5,10-methylene-(6R)-tetrahydrofolic acid or a pharmaceutically acceptable salt thereof and a dicarboxylic acid, or a salt thereof, as well as, a processes of obtaining the same, and the use of such products.

Claims

1. A stable lyophilisate comprising 5,10-methylen-(6R)-tetrahydrofolic acid, or a pharmaceutically acceptable salt thereof, and a dicarboxylic acid, or a salt thereof.

2. A stable lyophilisate according to claim 1, wherein the dicarboxylic acid is succinic, maleic, malic, tartaric, fumaric or oxalic acid.

3. A stable lyophilisate according to claim 1, having a chemical purity of greater than 98% and a stereoisomeric purity of greater than 99%.

4. A reconstituted product obtained by dissolving the lyophilisate of claim 1 in water or a liquid pharmaceutically acceptable vehicle.

5. A reconstituted product according to claim 4, wherein the water is sterile water for injection.

6. A reconstituted product according to claim 4, further comprising a pharmaceutically acceptable carrier.

7. A reconstituted product according to claim 4, further comprising an additional pharmaceutically acceptable active ingredient.

8. A reconstituted product according to claim 4, further comprising a buffer.

9. A process for preparing a lyophilisate according to claim 1 comprising the steps of (i) dissolving 5,10-methylene-(6R)-tetrahydrofolic acid, or a pharmaceutically acceptable salt thereof, in water containing a dicarboxylic acid, or a salt thereof; (ii) freezing the water; and (iii) thereafter removing the frozen water under vacuum.

10. A lyophilisate according to claim 9, wherein NaOH is added in step (i).

11. (canceled)

12. A method for treating cancer in a patient comprising administering a reconstituted product of claim 4 to the patient.

Description

EXAMPLES

Exemplary Process Parameters of the Lyophilization.

[0066]

TABLE-US-00002 Ice Shelf condenser temper- temper- Time Cumulative Step ature ature Pressure step time 1 Loading 4 atm 00:01 0:01 2 Freezing, 45 atm 01:30 1:31 ramp 3 Freezing 45 atm 02:00 3:31 4 Vacuum 45 70 0.05 00:30 4:01 adjustment 5 Primary drying, 0 70 0.05 01:45 5:46 ramp 6 Primary 0 70 0.05 41:00 46:46 drying 7 Secondary 25 70 0.05 12:00 58:46 drying, ramp 8 Secondary 25 70 0.05 06:00 64:46 drying

[0067] Online data recorded during a lyophilization is shown in FIG. 1.

Example 1: Lyophilisate Containing 5,10-methylen-(6R)-tetrahydrofolic acid and malic acid

[0068] Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 32 C. (resulting pH 14.0). 6.96 g malic acid and then 5.70 g 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate salt were added (pH is decreasing to 13.0) and rinsed with 2.5 g purified water. By the addition of sodium hydroxide 2M pH is held at 9.30.1. 11.65 g purified water were added. Overall 2.15 g sodium hydroxide 2M pH were needed to keep the pH at 9.30.1.

[0069] 5.0 ml of the resulting clear solution (being 5.181 g to 5.184 g) were introduced per vial into 10 ml glass vials (36 vials). Vials were immediately frozen with liquid nitrogen and lyophilised at <10.sup.1 mbar.

[0070] Vials obtained were containing a lyophilisate of 102 mg 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as free acid) with malic acid. Cake weight was 258 mg. 5,10-methylene-(6R)-tetrahydrofolic acid is showing a purity of 97.42% area measured by HPLC. Water content was 2.7% w/w resp. 7.0 mg per vial.

[0071] When reconstituted solution is having an osmolality of 253 mosmol/kg.

Example 2: Lyophilisate Containing 5,10-methylen-(6R)-tetrahydrofolic acid and succinic acid

[0072] Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 32 C. (resulting pH 14.0). 10.56 g sodium succinate hexahydrate and then 5.70 g 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate salt were added (pH is decreasing to 13.8) and rinsed with 2.5 g purified water. By the addition of sodium hydroxide 2M pH is held at 9.30.1. 12.74 g purified water were added. Overall 14.8 g sodium hydroxide 2M pH were needed to keep the pH at 9.30.1.

[0073] 5.0 ml of the resulting clear solution (being 5.0959 g and 5.1079 g) were introduced per vial into 10 ml glass vials (36 vials). Vials were immediately frozen with liquid nitrogen and lyophilised at <10.sup.1 mbar.

[0074] Vials obtained were containing a lyophilisate of 102 mg 5,10-methylene-(6R)-tetrahydrofolic acid (calculated as free acid) with succinic acid. Cake weight was 242 mg. 5,10-methylene-(6R)-tetrahydrofolic acid is showing a purity of 97.35% area measured by HPLC. Water content was 1.1% w/w resp. 2.6 mg per vial.

[0075] When reconstituted solution is having an osmolality of 267 mosmol/kg.

Example 3: Lyophilisate Containing 5,10-methylen-(6R)-tetrahydrofolic acid and maleic acid

[0076] Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 32 C. 13.92 g di-sodium maleate hydrate and then 5.70 g 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate salt were added (pH is decreasing to 13.8) and rinsed with 2.5 g purified water. Cooling was withdrawn and temperature was let to increase to room temperature.

[0077] By the addition of sodium hydroxide 2M pH is held at 9.30.1. 17.05 g purified water were added. Overall 18.4 g sodium hydroxide 2M pH were needed to keep the pH at 9.30.1. The clear solution was kept for 2 hours at room temperature.

[0078] 5.0 ml of the resulting clear solution were introduced per vial into 10 ml glass vials (7 vials). Vials were lyophilised.

[0079] Vials obtained were containing a lyophilisate of 5,10-methylene-(6R)-tetrahydrofolic acid with maleic acid. 5,10-methylene-(6R)-tetrahydrofolic acid is showing a purity of 97.54% area measured by HPLC.

Example 4: Lyophilisate Containing 5,10-methylen-(6R)-tetrahydrofolic acid and fumaric acid

[0080] Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 32 C. 6.26 g di-sodium fumarate and then 5.70 g 5,10-methylene-(6R)-tetrahydrofolic acid hemisulfate salt were added (pH is decreasing to 13.8) and rinsed with 2.5 g purified water. Cooling was withdrawn and temperature was let to increase to room temperature.

[0081] By the addition of sodium hydroxide 2M pH is held at 9.30.1. 17.66 g purified water were added. Overall 19.1 g sodium hydroxide 2M pH were needed to keep the pH at 9.30.1. The clear solution was kept for 2 hours under argon at room temperature.

[0082] 5.0 ml of the resulting clear solution were introduced per vial into 10 ml glass vials (7 vials). Vials were lyophilised.

[0083] Vials obtained were containing a lyophilisate of 5,10-methylene-(6R)-tetrahydrofolic acid with fumaric acid. 5,10-methylene-(6R)-tetrahydrofolic acid is showing a purity of 96.80% area measured by HPLC.

Example 5: Long Term Stability of Lyophilisates Containing 5,10-methylen-(6R)-tetrahydrofolic acid and a dicarboxylic acid (Content of (6R)-5,10-CH.SUB.2.-THF)

[0084] In order to determine the long-term stabilities of lyophilisates of 5,10-CH.sub.2-(6R)-THF prepared according to Examples 1-4, lyophilisates were stored in air at +5 C., +25 C./60% relative humidity and +40 C./75% relative humidity. The content of 5,10-CH.sub.2-(6R)-THF remaining was measured by HPLC at periodic intervals and is given by comparison with the initial value (% rel.). The results are shown in Tables 1 and 2.

TABLE-US-00003 TABLE 1 Long-term stability of a lyophilisate containing 5,10- methylen-(6R)-tetrahydrofolic acid and malic acid Relative content of (6R)-5,10-CH.sub.2-THF over storage time [months] 0 1 2 3 6 12 +5 C. 100.0 101.2 100.1 100.0 101.5 +25 C./60% rh 100.0 99.3 101.0 99.8 98.6 100.9 +40 C./75% rh 100.0 99.5 100.6 100.1 99.4

TABLE-US-00004 TABLE 2 Long-term stability of a lyophilisate containing 5,10- methylen-(6R)-tetrahydrofolic acid and succinic acid Relative content of (6R)-5,10-CH.sub.2-THF over storage time [months] 0 1 2 3 6 12 +5 C. 100.0 98.8 99.0 (75.8).sup.1 100.5 +25 C./60% rh 100.0 98.0 99.4 98.8 98.9 98.8 +40 C./75% rh 100.0 98.1 98.7 98.4 96.7 .sup.1Most likely lab error
Tables 1 and 2 clearly show that lyophilsates of 5,10-CH.sub.2-(6R)-THF are highly stable over a long period of time even at elevated temperatures.

Example 6: Long Term Stability of Lyophilisates Containing 5,10-methylen-(6R)-tetrahydrofolic acid and a dicarboxylic acid (Content of Stability Indicator 10-formyl-(6R)-tetrahydrofolic acid)

[0085] In order to determine the long-term stabilities of lyophilisates of (6R)-5,10-CH.sub.2-THF prepared according to Examples 1-4, lyophilisates were stored in air at +5 C., +25 C./60% relative humidity and +40 C./75% relative humidity. The content of the main degradation product 10-formyl-(6R)-tetrahydrofolic acid was measured by HPLC at periodic intervals. The results are shown in Tables 3 and 4.

TABLE-US-00005 TABLE 3 Long-term stability of a lyophilisate containing 5,10- methylen-(6R)-tetrahydrofolic acid and malic acid Content of 10-formyl-(6R)-tetrahydrofolic acid over storage time [months] 0 1 2 3 6 12 +5 C. 0.16 0.18 0.18 0.16 0.17 +25 C./60% rh 0.16 0.18 0.18 0.19 0.17 0.17 +40 C./75% rh 0.16 0.18 0.18 0.19 0.18

TABLE-US-00006 TABLE 4 Long-term stability of a lyophilisate containing 5,10- methylen-(6R)-tetrahydrofolic acid and succinic acid Content of 10-formyl-(6R)-tetrahydrofolic acid over storage time [months] 0 1 2 3 6 12 +5 C. 0.14 0.15 0.17 0.15 0.15 +25 C./60% rh 0.14 0.17 0.17 0.19 0.18 0.20 +40 C./75% rh 0.14 0.19 0.21 0.22 0.22
Tables 3 and 4 confirm that lyophilsates of 5,10-CH.sub.2-(6R)-THF are highly stable over a long period of time even at elevated temperatures.

[0086] No melting or collapse occurs during the lyophilization process of 5,10-CH.sub.2-(6R)-THF according to the present invention. Process is running at shelf temperature of 30 C. and pressure of 200 bar.

[0087] Extensive cake shrinkage is reduced, but a lid showing a dense structure is formed on top of the lyophilisates of the present invention. Optionally, annealing for 1 to 2 hours at temperatures around 5 C. to 2 C. is performed.

Example 7: Stability of Reconstituted Lyophilisates Containing 5,10-methylen-(6R)-tetrahydrofolic acid and a dicarboxylic acid

[0088] In order to determine the stabilities of reconstituted lyophilisates of (6R)-5,10-CH.sub.2-THF prepared according to Examples 1-4, lyophilisates containing 5,10-methylen-(6R)-tetrahydrofolic acid per vial were re-dissolved in 10 mL water (dissolving time). After further 50 min the content of 5,10-methylen-(6R)-tetrahydrofolic acid was measured. Then the solution was cooled to 18 C. and stored for 12 days at 18 C. After that the content of 5,10-methylen-(6R)-tetrahydrofolic acid was again measured. The vials were then warmed up to room temperature and stored for another 5 days. Then the content of 5,10-methylen-(6R)-tetrahydrofolic acid was again measured. Corresponding reference vials containing 5,10-methylen-(6R)-tetrahydrofolic acid and tri-sodium citrate, 5,10-methylen-(6R)-tetrahydrofolic acid and sodium acetate, and just 5,10-methylen-(6R)-tetrahydrofolic acid (without a dicarboxylic acid) were treated and measured equivalently. All results (measured by HPLC) were calculated relative to the initial value. The results are shown in Table 5.

TABLE-US-00007 TABLE 5 Stability of reconstituted lyophilisates containing 5,10- methylen-(6R)-tetrahydrofolic acid and a dicarboxylic acid Content of 10-formyl-(6R)-tetrahydrofolic acid over storage time after further 5,10-methylen-(6R)- Dissolving further further 5 days at tetrahydrofolic time [min] 0-value 50 min at 12 days at room acid and . . . room temperature 18 C. temperature tri-sodium citrate 55 100.0 99.52 99.10 74.75 (Reference) di-sodium fumarate 74 100.0 99.42 97.54 68.39 di-sodium maleate 69 100.0 99.53 97.80 70.75 sodium acetate 73 100.0 99.46 97.68 65.92 (Reference) - (Reference) 66 100.0 99.26 98.70 63.48
Table 5 confirms that reconstituted lyophilsates of 5,10-CH.sub.2-(6R)-THF are acceptably stable over time when stored at low temperatures.