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. A method for treating cancer in a patient comprising administering a reconstituted product of claim 4 to the patient, wherein the cancer being treated is breast cancer, esophageal cancer, gastric cancer, gall bladder cancer, bile duct cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, ovarian cancer, head and neck cancer, mesotheolioma cancer, stomach cancer, bowel cancer, lung cancer or colorectal cancer.

Description

EXAMPLES

(1) Exemplary Process Parameters of the Lyophilization.

(2) 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

(3) Online data recorded during a lyophilization is shown in FIG. 1.

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

(4) Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 3±2° 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 9.3±0.1. 11.65 g purified water were added. Overall 2.15 g sodium hydroxide 2M pH were needed to keep the pH at 9.3±0.1.

(5) 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.

(6) 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.

(7) When reconstituted solution is having an osmolality of 253 mosmol/kg.

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

(8) Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 3±2° 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 9.3±0.1. 12.74 g purified water were added. Overall 14.8 g sodium hydroxide 2M pH were needed to keep the pH at 9.3±0.1.

(9) 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.

(10) 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.

(11) When reconstituted solution is having an osmolality of 267 mosmol/kg.

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

(12) Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 3±2° 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.

(13) By the addition of sodium hydroxide 2M pH is held at 9.3±0.1. 17.05 g purified water were added. Overall 18.4 g sodium hydroxide 2M pH were needed to keep the pH at 9.3±0.1. The clear solution was kept for 2 hours at room temperature.

(14) 5.0 ml of the resulting clear solution were introduced per vial into 10 ml glass vials (7 vials). Vials were lyophilised.

(15) 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

(16) Under nitrogen 210 g purified water and 16.5 g sodium hydroxide 2M were cooled down to 3±2° 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.

(17) By the addition of sodium hydroxide 2M pH is held at 9.3±0.1. 17.66 g purified water were added. Overall 19.1 g sodium hydroxide 2M pH were needed to keep the pH at 9.3±0.1. The clear solution was kept for 2 hours under argon at room temperature.

(18) 5.0 ml of the resulting clear solution were introduced per vial into 10 ml glass vials (7 vials). Vials were lyophilised.

(19) 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)

(20) 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.

(21) 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

(22) 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)

(23) 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.

(24) 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

(25) 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.

(26) 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 μbar.

(27) 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

(28) 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.

(29) 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.

BRIEF DESCRIPTION OF THE DRAWING

(30) FIG. 1 shows data recorded for an example of the lyophilization method.