Liquid Neurotoxin Formulation Stabilized with Tryptophan or Tyrosine

Abstract

The invention relates to stable liquid neurotoxin formulations which are free of animal proteins, comprising a surfactant, an amino acid selected from tryptophan and tyrosine, a buffer comprising sodium, chloride and phosphate ions, which have a pH between 5.5 and 8, and which are stable for 2 months. These compositions are suitable for use in therapy and in particular for administration to a patient to achieve a desired therapeutic or aesthetic effect. The invention also relates to the use of an amino acid selected from tryptophan and tyrosine to protect a proteinaceous neurotoxin from degradation in a liquid composition which is free of animal derived proteins.

Claims

1. A liquid composition comprising: (i) a botulinum neurotoxin; (ii) a polysorbate; (iii) an amino acid selected from tryptophan and tyrosine; and (iv) a buffer comprising sodium, chloride, and phosphate ions; wherein: the liquid composition has a pH between 5.5 and 8 and is free of animal derived proteins; the amino acid is present at a concentration of from about 0.575 mg/ml to about 5 mg/ml and protects the botulinum neurotoxin from degradation, thereby providing a liquid composition that is stable for at least 2 months at 2 to 8 C.; the polysorbate is present at a concentration of 0.01% to 1% v/v; and the composition does not comprise albumin.

2. The liquid composition of claim 1, wherein the polysorbate is Polysorbate 20, Polysorbate 60 or Polysorbate 80.

3. The liquid composition of claim 1, wherein the amino acid is tryptophan.

4. The liquid composition of claim 1, wherein the buffer further comprises potassium ions.

5. The liquid composition of claim 1, wherein the composition has a pH between 6.0 and 7.5.

6. The liquid composition of claim 1, wherein no more than a 30% loss in extracellular proteolytic activity occurs over 2, 3, 6, 12, 18, 24 or 36 months at 5 C.

7. The liquid composition of claim 1, wherein the botulinum neurotoxin is a natural botulinum neurotoxin in complex form, a high purity natural botulinum neurotoxin, or a recombinant botulinum neurotoxin.

8. The liquid composition of claim 7, wherein the botulinum neurotoxin is a recombinant botulinum neurotoxin selected from a botulinum neurotoxin A, B, C, D, E, F or G, a modified botulinum neurotoxin, or a chimeric botulinum neurotoxin.

9. The liquid composition of claim 1, comprising: 4 to 10000 LD50 units of botulinum neurotoxin per mL; 0.001 to 1% v/v polysorbate; 0.575 mg/ml to 5 mg/ml tryptophan; 10 to 500 mM NaCl; 1 to 50 mM KCl; and 1 to 100 mM sodium phosphate; wherein the composition has a pH between 5.5 and 8 and is stable for 6 months at 5 C.

10. The liquid composition of claim 9, comprising: 10 to 2000 LD50 units of botulinum neurotoxin per mL; 0.05 to 0.2% v/v polysorbate 80; 0.575 mg/ml to 5 mg/ml tryptophan; 25 to 300 mM NaCl; 1 to 10 mM KCl; and 2 to 50 mM sodium phosphate; wherein the composition has a pH between 6.0 and 7.5 and is stable for 12 months at 5 C.

11. The liquid composition of claim 1, wherein the amino acid is L-tryptophan.

12. The liquid composition of claim 1, wherein the amino acid is present at a concentration of about 0.575 mg/ml to about 3 mg/ml.

13. The liquid composition of claim 1, wherein the amino acid is present at a concentration of about 0.74 mg/ml to about 5 mg/ml.

14. The liquid composition of claim 1, wherein the amino acid is present at a concentration of about 0.74 mg/ml to about 3 mg/ml.

15. A stabilized, botulinum neurotoxin ready-to-use (RTU) liquid composition comprising: 4 to 10000 LD50 units of botulinum neurotoxin per mL; 0.001 to 1% v/v polysorbate; 0.575 to 5 mg/mL amino acid selected from tryptophan and tyrosine; 10 to 500 mM NaCl; 1 to 50 mM KCl; and 1 to 100 mM sodium phosphate; wherein: the composition has a pH between 5.5 and 8; the composition does not comprise albumin; and the amino acid at a concentration of 0.575 to 5 mg/mL protects the botulinum neurotoxin from degradation, thereby providing a liquid composition that is stable for at least 2 months at 2 to 8 C. wherein no more than a 30% loss in extracellular proteolytic activity occurs over 2, 3, 6, 12, 18, 24 or 36 months at 5 C.

16. A liquid composition comprising: (i) a botulinum neurotoxin type A (BoNT/A); (ii) a polysorbate; (iii) an amino acid selected from tryptophan and tyrosine; and (iv) a buffer comprising sodium, chloride, and phosphate ions; wherein: the liquid composition has a pH between 5.5 and 8 and is free of animal derived proteins; the amino acid is present at a concentration of from about 0.575 mg/ml to about 5 mg/ml and protects the botulinum neurotoxin from degradation, thereby providing a liquid composition that is stable for at least 2 months at 2 to 8 C.; the polysorbate is present at a concentration of 0.01% to 1% v/v; and the composition does not comprise albumin.

Description

EXAMPLES

[0248] 1. Preparation of Stable Liquid Botulinum Toxin A Formulations

[0249] Liquid botulinum toxin preparations containing 15 ng/mL of highly purified BoNT/A, 15% v/v polysorbate 20, an amino acid selected from tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys) or a mixture of methionine (Met), tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys) (Sigma Aldrich), and Phosphate Buffer Saline (PBS from Calbiochem) (140 mM NaCl, 10 mM sodium phosphate and 3 mM KCl at pH 7.4 at 25 C.) were prepared, filtered using 0.22 m PVDF (polyvinylidenflourid) filters and stored in siliconized 2 mL glass syringes for 6 days at 40 C., after which a potency test was performed for each preparation.

[0250] For the potency test, the syringes containing the preparations were emptied in 2 mL glass vials (Chromacol, Gold) with lids containing PTFE treated rubber septa (Chromacol) or in 1.7 mL plastic micro centrifuge tubes (Axygen, Maximum Recovery) which both have low protein adsorption properties. The preparations were subsequently diluted using 0.9% NaCl solution with 3% human serum albumin (HSA). For each preparation, 50 L of sample was injected into the Gastrocnemius muscle of mice on the same day as the dilution was performed. The mice were monitored for 3 days and the degree of paralysis was recorded.

[0251] The results are shown in table 1.

TABLE-US-00002 TABLE 1 an accelerated storage test (6 days at 40 C.) of amino acid additions on BoNT/A stability. Potency test Formulation Dilution in 0.9 Inj. BoNT/A NaCl with 3% Dose Potency rating Buffer conc. Amino acid HSA (times) (ng) Day 1 Day 2 Day 3 PBS pH 7.4 15 ng/mL Trp 0.25 mg/mL + 2 0.25 WN 15% Cys 0.25 mg/mL + 20 0.025 WN polysorbate Met 0.25 mg/mL + 20 Tyr 0.25 mg/mL Cys 1 mg/mL 2 0.25 WN 20 0.025 WN Tyr 0.74 mg/mL 2 0.25 Sharp PA whole abdomen 20 0.025 WN Trp 1 mg/mL 2 0.25 20 0.025 WN 2 0.25 WN 20 0.025 WN Paralysis results in mice: = not analysed, WN = without note, PA = paralysis and = death.

[0252] Tyrosine and tryptophan were found to have a protective effect against BoNT/A degradation. Tryptophan was found to have the strongest protective effect. Cysteine, as well as the mixture containing all 4 amino acids did not have a protective effect.

[0253] 2. Preparation of a Stable Liquid Botulinum Toxin B Formulation

[0254] Liquid botulinum toxin preparations containing 350 ng/mL of highly purified BoNT/B, 15% v/v polysorbate 20, an amino acid selected from tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys) or a mixture of methionine (Met), tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys), and Phosphate Buffer Saline (PBS) at pH 7.4 were prepared, filtered using 0.22 m filters and stored in siliconized 2 mL glass syringes for two weeks at 40 C., after which a potency test was performed for each preparation as described above.

[0255] The results are shown in table 2.

TABLE-US-00003 TABLE 2 an accelerated storage test (two weeks at 40 C.) of amino acid additions on BoNT/B stability. Potency test Formulation Dilution in 0.9 Inj. BoNT/B NaCl with 3% Dose Potency rating Buffer conc. Amino acid HSA (times) (ng) Day 1 Day 2 Day 3 PBS pH 7.4 350 ng/mL Trp 0.25 mg/mL + 10 1.75 PA 15% Cys 0.25 mg/mL + polysorbate Met 0.25 mg/mL + 20 Tyr 0.25 mg/mL Cys 1 mg/mL 10 1.75 PA Tyr 0.575 mg/mL 10 1.75 Trp 1 mg/mL 10 1.75 35 0.5 PA Paralysis results in mice: = not analysed, WN = without note, PA = paralysis and = death.

[0256] Tyrosine and tryptophan were found to have a protective effect against BoNT/B degradation. Cysteine, as well as the mixture containing all 4 amino acids also had a protective effect but to a weaker extent.

[0257] 3. Evaluation of Different Concentrations Of Tryptophan and Polysorbate 20

[0258] Liquid botulinum toxin preparations containing highly purified BoNT/A or BoNT/B and various concentrations of polysorbate 20 (PS 20) and tryptophan and Phosphate Buffer Saline (PBS) at pH 7.4 were prepared, filtered using 0.22 m filters and stored in siliconized 2 mL glass syringes. Hind limb paralysis potency tests were performed for each preparation as described above.

[0259] The results are shown in table 3.

TABLE-US-00004 TABLE 3 evaluation of Trp and polysorbate 20 (PS20) concentrations on BoNT/A or BoNT/B stability. POTENCY TESTING Formulation Potency rating (1-3 d) for samples stored at different temperatures and lengths BoNT Dilution in 0.9 Inj. 5 C. 25 C. conc. Trp PS20 NaCl with 3% Dose 6 months 5 weeks 4 months BoNT (ng/mL) (mg/mL) (%) HSA (times) (ng) 1 d 2 d 3 d 1 d 2 d 3 d 1 d 2 d 3 d BoNT/A 15 8 15 1 0.75 PA 5 0.15 WN 15 8 0.25 1 0.75 * 5 0.15 * WN.sup.2 15 1 15 1 0.75 5 0.15 PA.sup.3 BoNT/B 100 8 15 1 5 PA 10 0.5 PA.sup.1 PA PA 100 8 0.25 1 5 10 0.5 PA.sup.4 WN.sup.1 PA 100 1 15 1 5 10 0.5 PA.sup.4 PA PA Paralysis results in mice: = not analysed, WN = without note, PA = paralysis and = death. Paralysis degree (PA): .sup.1Toes affected; .sup.2Slightly numb in hind leg; .sup.3Both hind legs paralysed; .sup.4Hind leg paralysed; Elution buffer from purification (5): 50 mM sodium acetate pH 4.5 with 0.2% (v:v) polysorbate 20 and 400 mM sodium chloride. *a mix up of two BoNT/A dilutions of the 25 C. 8 mg/mL Trp 0.25% polysorbate 20 has probably occurred.

[0260] 4. Evaluation of Different Salt Concentrations in BoNT/B Preparations

[0261] Liquid botulinum toxin preparations containing 100 ng/mL of highly purified BoNT/B, polysorbate 20, tryptophan from various amino acid suppliers and a buffer selected from PBS pH 7.4 (Calbiochem), 12 nM phosphate buffer pH 7 (Apoteket) and 20 mM sodium acetate (NaAc) pH 5.5 (NaAc from Fluka and acetic acid from Merck) were prepared, filtered using 0.22 m filters and stored in siliconized 2 mL glass syringes. Hind limb paralysis potency tests were performed for each preparation as described above.

[0262] The results are shown in table 4.

TABLE-US-00005 TABLE 4 evaluation of different salt concentrations on stability of BoNT/B. Potency testing Potency rating (1-3 d) for samples stored at different length BoNT/B Trp Dilution in 0.9 Inj. 40 C. conc. manufacturer PS20 NaCl with 3% Dose 2 weeks 5 weeks (ng/mL) Buffer and conc. (%) HSA (times) (ng) 1 d 2 d 3 d 1 d 2 d 3 d 100 PBS Ajinomoto 15 1 5 pH 7.4 4 mg/mL 10 0.5 PA 100 PBS Sigma Aldrich 15 1 5 pH 7.4 4 mg/mL 10 0.5 PA 100 PBS Sigma Aldrich 15 1 5 pH 7.4 1 mg/mL 10 0.5 PA 100 PBS Sigma Aldrich 0.25 1 5 pH 7.4 4 mg/mL 10 0.5 PA PA.sup.2 100 12 mM Phosphate Sigma Aldrich 15 1 5 WN pH 7 4 mg/mL 10 0.5 WN.sup.1 100 20 mM NaAc Sigma Aldrich 15 1 5 PA pH 5.5 4 mg/mL 10 0.5 PA Paralysis results in mice: = not analysed, WN = without note, PA = paralysis and = death. .sup.1Some loss of function; .sup.2Weak paralysis

[0263] The results show that the preparations containing the PBS buffer (containing sodium, chloride, phosphate and potassium ions) appears to play a role in the stability of the botulinum toxin.

[0264] 5. Evaluation of Different Stabilizers

[0265] Liquid botulinum toxin preparations containing 15 ng/mL of highly purified BoNT/A, a polysorbate 20 (PS20) or polysorbate 80 (PS80) or HSA, tryptophan and PBS were prepared, filtered using 0.22 m filters and stored in siliconized 2 mL glass syringes. Hind limb paralysis potency tests were performed for each preparation as described above.

[0266] The results are shown in table 5.

TABLE-US-00006 TABLE 5 evaluation of different surfactants on stability of BoNT/A. Potency testing Potency rating (1-3 d) for samples stored at different length BoNT/A Dilution in 0.9 Inj. 40 C. conc. NaCl with 3% Dose 6 days 4 weeks 3 months (ng/ml) Buffer Trp Stabiliser HSA (times) (ng) 1 d 2 d 3 d 1 d 2 d 3 d 1 d 2 d 3 d 15 PBS 1 mg/mL PS 80 1.7 0.45 PA.sup.3 pH 7.4 0.25% 15 0.05 WA 15 PBS 1 mg/mL PS 20 1.7 0.45 PA.sup.2 pH 7.4 0.25% 15 0.05 PA WN 15 PBS HSA 1.7 0.45 pH 7.4 1 mg/mL 15 0.05 PA.sup.1 Paralysis results in mice: = not analysed, WN = without note, PA = paralysis and = death. .sup.1Severe paralysis both hind legs; .sup.2Angles paws; .sup.3Severe paralysis

[0267] 6. Evaluation of Different Formulations

[0268] Liquid botulinum toxin preparations containing 10 ng/mL of highly purified BoNT/A, 0.25% PS80, 1 mg/mL tryptophan and PBS were prepared as described above. The pH was adjusted to 6.6 and 7.0 by adding HCl. Each preparation was stored 5 weeks at 40 C.

[0269] Each preparation was then diluted 10 times and hind limb paralysis potency tests were performed as described above (0.05 ng per injection). In both cases, hind limb paralysis was observed at day 3. The paralysis was stronger with the pH 6.6 preparation.

[0270] 7. Evaluation of Different Formulations

[0271] Liquid botulinum toxin preparations containing 0.3 ng/mL of highly purified BoNT/A, a polysorbate selected from PS20 and PS80, 1 mg/mL tryptophan and 12 mM PBS at pH 7.4 were prepared as described above. The pH of each preparation was adjusted to pH 6.6 or 6.9 by adding 1.2 M HCl.

[0272] Polysorbate 20 was tested at one concentration, 0.2% w/v, corresponding to about 20 times its CMC (critical micellar concentration, about 0.01% w/v at 21 C.). Polysorbate 80 was tested 0.04% and 0.2% w/v, corresponding respectively to about 20 and 100 times its CMC (about 0.002% w/v at 21 C.).

TABLE-US-00007 TABLE 6 Choice of polysorbate and pH Composition PS20% w/v PS20% w/v pH PS20-1 0.2 6.6 PS20-2 0.2 6.9 PS80-1 0.04 6.6 PS80-2 0.04 6.9 PS80-3 0.2 6.6

[0273] For each preparation, a volume of 0.5 mL was filled in 1 mL long glass syringes (BD) and sealed with a fluorocarbon coated plunger.

[0274] The potency was measured by hind limb paralysis test on mice as described above.

[0275] No decrease in potency was observed in any formulation after 6 months storage at 5 C. and after 25 C.

[0276] 8. Evaluation of Different Formulations

[0277] Nineteen different formulations containing highly purified botulinum neurotoxin type A were prepared with varying concentrations of polysorbate 80, tryptophan, sodium phosphate, sodium chloride, potassium chloride and varying pH. Each formulation had a target nominal potency of 500 U/mL. Each formulation was degassed, filtered through 0.2 m filter and filled into vials. Nitrogen gas was used as a protective atmosphere in the vials. The filling was performed in an anaerobic chamber. Each formulation was filled in 1 mL aliquots in a nitrogen atmosphere in 2 mL glass vials capped with FluroTec stoppers sealed with aluminium flip off seals and stored upright.

[0278] The stability of the 19 formulations was assessed at 5 C., 25 C. and 37 C. using BoTest to measure potency.

TABLE-US-00008 TABLE 7 Excipient compositions Sodium Poly- Exp phosphate Tryptophan sorbate 80 NaCl KCl Name pH (mM) (mg/mL) (v %) (mM) (mM) N1 6.3 2 0.25 0.01 25 0 N2 7.2 2 0.25 0.01 255 0 N3 6.3 50 0.25 0.01 255 10 N4 7.2 50 0.25 0.01 25 10 N5 6.3 2 3 0.01 255 10 N6 7.2 2 3 0.01 25 10 N7 6.3 50 3 0.01 25 0 N8 7.2 50 3 0.01 255 0 N9 6.3 2 0.25 1 25 10 N10 7.2 2 0.25 1 255 10 N11 6.3 50 0.25 1 255 0 N12 7.2 50 0.25 1 25 0 N13 6.3 2 3 1 255 0 N14 7.2 2 3 1 25 0 N15 6.3 50 3 1 25 10 N16 7.2 50 3 1 255 10 N17 6.75 10 1.625 0.1 140 3

TABLE-US-00009 TABLE 8 Packaging components Article Article number, Supplier Clear glass vial of boro 1097221, Schott silicate Type I plus, 2 mL Grey Flurotec coated bromobutyl 1356 4023/50, West stopper Westar RS, 13 mm Aluminium flip off seals, 13 mm 5920-6623, West

[0279] For all formulations the solution remained clear and for most parts colourless.

[0280] The excipients concentrations tested in this study seem not to affect the pH of the formulations during the time interval tested. The potency results are presented in table 9.

TABLE-US-00010 TABLE 9 Botest potency results Remaining potency compared to base line (BoTest) Baseline potency 2 months 6 months 6 months (U/mL) (Botest) 37 C. 25 C. 5 C. pH 0 month U/mL % U/mL % U/mL % N1 6.3 86 0 0 0 0 39 45 N2 7.2 474 210 44 389 82 485 102 N3 6.3 449 288 64 406 90 512 114 N4 7.2 299 62 21 368 123 406 136 N5 6.3 378 263 70 385 102 422 112 N6 7.2 93 0 0 0 0 52 56 N7 6.3 238 0 0 0 0 239 100 N8 7.2 375 305 81 402 107 450 120 N9 6.3 196 0 0 100 51 294 150 N10 7.2 354 201 57 408 115 411 116 N11 6.3 438 197 45 372 85 492 112 N12 7.2 411 96 23 295 72 417 101 N13 6.3 304 185 61 403 133 416 137 N14 7.2 206 0 0 0 0 183 89 N15 6.3 197 155 79 231 117 214 109 N16 7.2 476 227 48 390 82 685 144 N17 6.75 402 250 62 286 71 488 121

[0281] For several compositions there was no more than 30% loss in potency over 6 months at 5 C. and/or no more than about 40% loss in potency over 3 months at 25 C. and/or no more than about 50% loss in potency over 2 months at 37 C.

[0282] 9. Evaluation of PS 60

[0283] A formulation containing highly purified botulinum neurotoxin type A was prepared with 0.1% (v/v) PS60, 1 mg/mL L-Tryptophan, 10 mM sodium phosphate, 140 mM sodium chloride, 3 mM potassium chloride and water for injection. The pH was adjusted to 6.75 with HCl. The formulation had a target nominal potency of 100 U/mL. The formulation was degassed, filtered through 0.2 um filters and filled into 2 mL vials aseptically in an anaerobic chamber with nitrogen atmosphere with a fill volume of 1 mL. Nitrogen gas was used as protective atmosphere in the vials. The vials were capped with FluroTec stoppers sealed with aluminium flip off seals.

TABLE-US-00011 TABLE 10 Packaging components Article Article number, Supplier Clear glass vial of boro 1097221, Schott silicate Type I plus, 2 mL 13 mm Inj stopper coated INJ13TB3WRS, Nordic Pack bromobutyl 4023-50 grey Blue aluminium flip off seals, 13 mm 5920-1164, Nordic Pack

[0284] The potency over time at 37 C. and 25 C. was measured by the MLD50 test as described herein.

[0285] At 37 C., the remaining potency after 9 weeks was around 50-55% of the initial potency.

[0286] At 25 C., the remaining potency after 3 months was about 80% of the initial potency.