WO2024184327A1 - Aqueous solutions of dantrolene - Google Patents

Abstract

The present invention relates to aqueous saline solutions comprising dantrolene and optionally also at least one additional active principle, such as botulinum neurotoxin, and their use for various therapeutic and/or aesthetic purposes. The solutions can be administered in any suitable way, but are advantageous in that they are particularly well adapted to intramuscular injection.

Description

Description

Title of Invention : Aqueous solutions of dantrolene j

Technical Field

[0001] The present invention relates to aqueous solutions comprising dantrolene and optionally also at least one additional active principle, such as botulinum neurotoxin, and their use for various therapeutic and/or aesthetic purposes. The solutions can be administered in any suitable way, but are advantageous in that they are particularly well adapted to intramuscular injection.

Background Art

[0002] Dantrolene is a drug approved as a myorelaxant and for the treatment of malignant hyperthermia. In indications associated with myorelaxant properties of Dantrolene but requiring local administration, it would be particularly useful to provide Dantrolene compositions optimized for intramuscular administration.

[0003] Solutions for intramuscular administration are typically saline aqueous homogenous solutions. However, dantrolene is poorly soluble in water and even less soluble in saline aqueous compositions.

[0004] At high concentrations of dantrolene, aqueous suspensions, rather than solutions, of dantrolene are typically used in the prior art, and the process of dantrolene resuspension is known to be lengthy and tedious. Suspensions are not well suited for intramuscular injection. There is therefore a need to improve the formulation of compositions comprising dantrolene, such as to make such compositions better optimized for intramuscular injection.

[0005] In view of intramuscular injection, it is even more desirable to provide the dantrolene formulation in the form of a ready-to-use injectable solution, which requires chemical stability of the formulation over a longer storage time, compared to the presently used compositions prepared by suspending Dantrolene in an aqueous solution shortly before use. Even though such ready- to-use compositions are more challenging to formulate, due to the need for longer shelf-life, these are particularly advantageous, as they reduce the burden on the health care professionals at the time of the treatment, are easier to use and reduce the risk of erroneous dosage, due to errors in the reconstitution process.

[0006] The pH of un-buffered solutions of dantrolene is mainly determined by the concentration of dantrolene, the pH increasing with the dantrolene concentration. In turn, in aqueous environments at basic and acidic pH, dantrolene suffers hydrolysis leading to the reduction of its activity. Aqueous solutions of dantrolene are thus poorly stable over time. Thus, for applications wherein dantrolene remains in solution for a prolonged time, such as ready-to-use compositions, it is sought to improve the chemical stability of dantrolene.

[0007] Dantrolene formulations have been intensively investigated in the prior art. The focus has been on compositions for systemic administration, in particular oral and intravenous administration route, as these are reflecting the approved routes of the main medical indications of dantrolene. However, compositions for oral or intravenous routes are often unsuitable for intramuscular administration. The prior art formulations of dantrolene are therefore not adapted to the administration of dantrolene by the intramuscular route.

[0008] Dantrolene has for example been formulated for intravenous injection as aqueous suspensions, as solutions in organic solvents, or as aqueous solutions with a high volume of solvent and low dantrolene concentration, or as aqueous suspensions having a basic pH.

[0009] Aqueous suspensions are unsuitable for the purpose of intramuscular injections, because, once injected, the particles from the suspension are confined to the fibrous or membranous tissues between muscle fibers forming agglomerates leading to poor absorption of the active principle.

[0010] Very dilute solutions are also unsuitable for intramuscular injections, because larger volumes are to be administered for a given dose, leading to greater/wider spreading of the injected composition. A larger volume is not a problem for intravenous injection, where the injected volume is diluted into the blood circulation. In contrast, it is an issue in case of intramuscular injection, because the spreading of a higher volume reduces the precision of the delivery of the active locally, causing diffusion into the surrounding tissue, in turn leading to diminution in duration and magnitude of effect in the targeted tissue and potential unwanted effect outside of the targeted zone. This is in particular critical for aesthetic applications and medical indications which require precise administration to specific muscles (such as inhibiting spasms in the upper limbs).

[0011 ] Formulations having an excessively basic pH (i.e. above 9) are also disadvantageous for intramuscular administration, because they cause discomfort or pain, and at very alkaline pH even necrosis, at the site of injection. See for example Clin Drug Investig. 2012 Jul 1 ; 32(7): 433-438.

[0012] In view of optimizing the formulation of compositions comprising dantrolene, there is therefore a need to identify compositions that are optimized for intramuscular injection, allow its solubilization and preferably ensure chemical stability over time.

[0013] Diverse adjuvants have been used in aqueous compositions of dantrolene, such as diverse carbohydrates. For example, W02017067980A1 disclosed that solubility of dantrolene could be improved in aqueous compositions comprising cyclodextrin or a derivative thereof at alkaline pH values. The compositions are for intravenous injection. The dantrolene to cyclodextrin molar ratio is of 1 :2 to 1 :30 and the pH is higher than 7, preferably 7.5 to 10.5, preferably 8.5 to 9.5. This document does not disclose any dantrolene saline solution. Saline compositions being preferred over non-saline solutions for intramuscular injection (in contrast to intravenous administration), the compositions of this document are disadvantageous for administration by this route.

[0014] Other prior art documents have disclosed compositions of dantrolene and cyclodextrin or derivatives thereof. For example, WO2018146187A1 discloses compositions of dantrolene with 2-hydroxypropyl-[3-cyclodextrin (HPBCD) for intravenous injection. Improvement of the stability and solubility of dantrolene is alleged. The dantrolene to HPBCD is 1 :1 to 1 :12. The pH is of 8.5 to 10.5. This document does not disclose any dantrolene saline solution. Saline compositions being preferred over non-saline solutions for intramuscular injection (in contrast to intravenous administration), the compositions of this document are disadvantageous for administration by this route.

[0015] More recently, W02020049670A1 aimed at improving the teaching of W02017067980A1 . It also relates to compositions for intravenous injection and discloses compositions having a lower dantrolene to cyclodextrin ratio of 1 :0.5 to 1 :2, which is alleged to reduce nephrotoxicity associated with cyclodextrin. The pH is also alkaline, in the range of 9.0 to 10.5. In combination with W02017067980A1 , this document suggests that decreasing the dantrolene to cyclodextrin ratio is advantageous. This document does not disclose any dantrolene saline solution. Saline compositions being preferred over non-saline solutions for intramuscular injection (in contrast to intravenous administration), the compositions of this document are disadvantageous for administration by this route.

[0016] Jansen A.C.A. et al, "Some physical-chemical properties of dantrolene and two of its analogues", International Journal of Pharmaceutics, vol. 75, no. 2-3 (1991 ), pages 193-199, discloses the solubility of dantrolene in aqueous saline solutions, at various pH and temperature. It also indicates that the solubility of dantrolene is increased in the presence of cyclodextrins, indicating complex formation. This publication focuses on the theoretical assessment of solubility, lipophilicity and complexation of dantrolene with different cyclodextrins (a-, [3- and y-cyclodextrin) and does not investigate the role of dantrolene to cyclodextrin ratio. The materials and methods do not provide the total concentration of dantrolene used, but only the total concentration of cyclodextrin and the concentration of dantrolene-cyclodextrin complexes based on the total cyclodextrin concentration (maximum 5%). The binding constant was calculated according to the formula

K = [complex] I [dantrolenef_ree] x [CDtotai]

The value of K does not provide information on the total concentration of dantrolene, in the absence of a description of the stoichiometric ratio of dantrolene and cyclodextrin in the complexes formed.

[0017] Jansen et al, 1991 , cited above, refers to another publication by the same author, related to a study of dantrolene bioavailability in rats (Jansen A.C.A. et al., "The influence of inclusion by cyclodextrins on absorption kinetics of dantrolene in the rat", Proceedings of the Fourth International Symposium on Cyclodextrins, pages 349-353 (1988), part of the Advances in Inclusion Science book series (AIS, volume 5)). In this publication, the dantrolene to cyclodextrin ratio can be calculated from the materials and methods section as being of 1 :15. Jensen et al, 1991 , aims at investigating physical-chemical properties of dantrolene, as part of such rat study. Therefore, without any indication to the contrary, there is no reason to believe that Jensen has used different total dantrolene concentrations in his second study, in particular knowing that he used total cyclodextrin concentrations closely matching those used in his 1988 study in rats.

[0018] It would further be desirable to optimize Dantrolene compositions to enable addition of one or more additional active principle(s) providing benefits in combination with dantrolene. In such case, an additional challenge is to provide a composition in which each of the different active principles is sufficiently soluble and preferably also chemically stable.

[0019] For example, compositions comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, preferably dantrolene, and Botulinum neurotoxin have been described in W02020/254690A1 . The combination of these active principles has been shown to result in an acceleration of the onset of action and/or an extension of the duration of action and/or an enhancement of the intensity of the effect of botulinum neurotoxin. It would therefore be advantageous to provide optimized solutions for co-administration of dantrolene and Botulinum neurotoxins.

[0020] Botulinum neurotoxins are toxins impairing neuronal transmission that are preferentially active on cholinergic neurons; one of the main consequences of botulinum neurotoxin action is thus muscle relaxation due to decreased release of neurotransmitters by the cholinergic neuron. Botulinum neurotoxins are produced as complexes by an anaerobic and spore-forming bacterium Clostridium botulinum and to a lesser extent by other Clostridium species, such as C. butyricum, C. barati, C. sporogenes and C. argentinense. Eight different serotypes of botulinum neurotoxins known as type A (BoNT/A), B (BoNT/B), C (BoNT/C), D (BoNT/D), E (BoNT/E), F (BoNT/F), G (BoNT/G), and X (BoNT/X) were identified. There are currently 8, 8, 12 and 9 known BoNT/A, BoNT/B, BoNT/E and BoNT/F subtypes respectively. Types A, B, E and F are toxic for human beings while types C, D and G more often cause toxicity for example in birds, horses, cattle and primates. Type X has only been described recently (Zhang et al., Nature Communications, volume 8, 14130 (2017)), after a case report of infant botulism in Japan in 1995, and its toxicity appears limited in tested species so far.

[0021] Botulinum neurotoxin complexes are present in the form of high-molecular weight protein complexes comprising two components; namely the enzymatically active neurotoxin component and an associated non-toxic bacterial protein component which can be regarded as a coat protein including hemagglutinin and non-hemagglutinin proteins. The molecular weight of the botulin toxin complexes varies among the distinct botulinum toxin serotypes from about 300 kDa to about 900 kDa; botulinum neurotoxins devoid of the non-toxic bacterial protein component has a molecular weight of about 150 kDa. Regarding the aesthetic or therapeutic application, the coat protein is reported to have no significant function and does not contribute to the pharmacological properties. The neurotoxin component is expressed as an inactive single-chain precursor having a molecular weight for all of the known botulinum neurotoxin serotypes of about 150 kDa. This single chain precursor is activated by proteolytic cleavage to generate a disulfide- linked two-chain protein. The about 50 kDa light chain protein contains the catalytic domain and is a zinc-containing metalloprotease and acts as a zincendopeptidase. The about 100 kDa heavy chain protein comprises a translocation domain and a receptor- binding domain. The heavy chain mediates binding to the presynaptic cholinergic nerve terminals, in particular to the presynaptic part of the motor end plate or neuromuscular junction, and internalization of the neurotoxin into the cell.

[0022] The natural property of botulinum neurotoxins to reversibly block the release of acetylcholine from the presynaptic nerve terminals in muscles and other effector tissues such as the secretory glands in the orthosym pathetic and parasympathetic systems makes them an important therapeutic option in numerous fields to control muscle (and other effector tissues) overstimulation, and associated pain. For example, BoNT/A is currently used therapeutically in the fields of: movement disorders, especially for managing spasticity and dystonia, where BoNT/A is well-described to efficiently relief dystonia-related pain; urinary disorders, notably overreactive bladder; secretory disorders, namely hyperhidrosis and sialorrhea. Moreover, botulinum neurotoxins are now currently used in a variety of aesthetic indications, such as smoothing facial lines or reducing glabellar, frown and periorbital lines.

[0023] Despite sharing a similar metalloprotease activity involved in the cleavage of proteins of the SNARE complex, botulinum neurotoxins may have different onset and/or duration of action depending on their serotypes. For example, whereas BoNT/A and BoNT/F induces a complete localized paralysis in the hind leg of mice within 2 days, BoNT/E produces the same effect within 24 hours. Nevertheless, the duration of BoNT/A- induced neuromuscular paralysis is 28 days in mice, compared to only 5 and 8 days for toxins E and F respectively. A similar pattern has been observed in human, although at a different timescale: BoNT-A paralysis is generally observed within a week and can last for 3 to 4 months (Davletov et al., TRENDS in Neurosciences 28 (2005); pp. 446-452). On the contrary, BoNT-E shows faster onset (24 hours) but much shorter duration (less than 1 month) in human (Yoelin et al., Plast Recontr Surg 142 (2018); pp. 847e-855e).

[0024] Because of its unique profile, and activity in human, most attention has been focused on BoNT/A. Since 1991 , several commercial botulinum neurotoxins of type A were approved by the U.S. Food and Drug Administration (FDA). The available forms of BoNT/A are Botox®/Vistabel® (Allergan), Dysport®/Azzalure® (Ipsen Biopharm) and Xeomin®/Bocouture® (Merz Pharmaceuticals) among others. The only other BoNT serotype that is currently commercially available is BoNT/B Myobloc®/Neurobloc® (Solstice Neurosciences); its medical/aesthetic use is anecdotal, though. Today, improving BoNT characteristics is still an important goal to reach, in order to better answer medical needs.

[0025] In cases where dantrolene is combined with Botulinum neurotoxin, there is a need to provide formulations comprising dissolved dantrolene and botulinum neurotoxin enabling dantrolene dissolution at relatively high concentrations, thus enabling appropriate dosage of dantrolene in a small volume suitable for intramuscular injection, and preferably having good stability of both dantrolene and botulinum neurotoxin, to maintain high activity of both active principle. It is further desirable that the effects of the dantrolene and botulinum neurotoxin be maintained and preferably that a synergy between dantrolene and botulinum neurotoxin be achieved. Preferably, it is also desirable to provide ready-to use solutions for intramuscular injection.

[0026] The formulation of compositions combining dantrolene and Botulinum neurotoxin is particularly challenging, as Botulinum neurotoxin is only approved for intramuscular injection and because Botulinum neurotoxin has been reported in several studies to be unstable at alkaline pH. Formulations with a pH above 7 are therefore suboptimal for administration of botulinum neurotoxin. It is therefore sought to adapt the compositions of the prior art to provide dantrolene and Botulinum neurotoxin suitable for intramuscular injection, wherein Botulinum neurotoxin retains its activity.

[0027] The present invention aims at solving part or all of the above-mentioned problems.

Summary of Invention

[0028] In a first aspect, the invention relates to an aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof and cyclodextrin or a derivative thereof, optionally a buffer, and optionally at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof, wherein the dantrolene or dantrolene pharmaceutically acceptable salt to cyclodextrin or derivative thereof molar ratio is of 1 :at least 40.

[0029] The composition on the first aspect is useful per se, for administration in any indication in which dantrolene is to be administered in the form of an aqueous solution. This composition is advantageous, because of the improved solubility of dantrolene due to the selection of optimal dantrolene to cyclodextrin ratios. The buffer is only optional as it does not impact solubility, but only improves chemical stability of dantrolene.

[0030] In a particular embodiment of this first aspect, the composition further comprises Botulinum neurotoxin.

[0031] In a second aspect, the invention relates to an aqueous solution according to the first or second aspect, for use in therapy (or in other words for use as a medicament), preferably for use in the treatment of a skeletal muscle disease or disorder, a smooth muscle disease or disorder and/or a cardiac muscle cell disease or disorder. [0032] In a third aspect, the invention relates to the use of an aqueous solution according to the first or second aspect in a cosmetic treatment.

[0033] In a fourth aspect, the invention relates to a lyophilizate of an aqueous solution of the first or second aspect.

[0034] In a fifth aspect, the invention relates to a kit comprising an aqueous, optionally frozen, solution of the first or second aspect, or a lyophilizate according to the fifth aspect, devoid of botulinum neurotoxin, and a lyophilized botulinum neurotoxin,

[0035] In a sixth aspect, the invention relates to a method of providing an aqueous solution according to the first or second aspect from the kit of the sixth aspect comprising the steps of: a) providing a kit of the fifth aspect, b) reconstituting the dantrolene composition if such composition is a lyophilizate, such as to obtain a dantrolene aqueous solution in liquid form; c) thawing the dantrolene aqueous solution composition if it is provided in frozen form, such as to obtain a dantrolene aqueous solution in liquid form, d) resuspending the lyophilized botulinum neurotoxin in the liquid aqueous composition of dantrolene provided in the kit or obtained in the end of steps b) or c), such as to form an aqueous solution according to the first or second aspect; e) optionally storing the aqueous composition obtained in the end of step d) at a temperature of about 2°C to about 8°C for about 1 hour to about 7 days.

[0036] In a seventh aspect, the invention relates to an aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof, botulinum neurotoxin and at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof salt.

Brief Description of Drawings

[0037] [Fig.1 ] Dantrolene concentration measured by HPLC in 1 mg/mL Dantrolene formulation in a 30% HPBCD solution in 0.9% NaCI after 1 day, 1 week, 2 weeks, 1 month and 3.5 months and 6 months of storage at 4°C (circles), 20°C (triangles) and 40°C (up to 1 month only, squares).

[0038] [Fig. 2] Long-term stability test of three formulations at two different temperatures: 0.5 mg/mL Dantrolene formulation in a 30% HPBCD solution in 0.9% NaCI (reference sample) at 4°C (black circles) and at 20°C (black triangles), 0.5 mg/mL Dantrolene formulation in a “30% HPBCD + 10 mM L-Histidine” solution in 0.9% NaCI (pH7.4 final) at 4°C (dashed dark grey circles) and at 20°C (dashed dark grey squares), 0.5 mg/mL Dantrolene formulation in a “30% HPBCD + 10 mM L-Histidine + 0.1 mM EDTA” solution in 0.9% NaCI (pH7.4 final) at 4°C (dashed light grey squares) and at 20°C (dashed light grey triangles).

[0039] [Fig. 3] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) tested in 5 formulations in the rat DAS assay: 0.9% NaCI (saline, reference sample), Cremophor® EL 5%, Tween®80 5%, HPBCD 30%, HPBCD 5%. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+).

[0040] [Fig. 4] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) combined with dantrolene (FTP-501 ) tested in 5 formulations in the rat DAS assay: 0.9% NaCI (saline, reference sample), Cremophor® EL 1.8%, Tween®20 5%, HPBCD 2.2%. Ona alone formulated in 0.9% NaCI or Tween®20 5% are provided as reference. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+).

[0041 ] [Fig. 5] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) reconstituted in 0.9% NaCI (saline, reference sample), and incubated at room temperature before injection in rats for the DAS assay: extemporaneous, 1 h, 2h, 4h, 6h, 24h. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+).

[0042] [Fig. 6] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) reconstituted with a 0.5 mg/mL dantrolene formulation in a “30% HPBCD + 10 mM L-Histidine + 0.1 mM EDTA” solution in 0.9% NaCI (pH7.4 final), and incubated at room temperature before injection in rats for the DAS assay: extemporaneous, 1 h, 2h, 4h, 6h, 24h. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+). [0043] [Fig. 7] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) reconstituted with a 0.1 mg/mL dantrolene formulation in a “6% HPBCD + 2 mM L-Histidine + 0.02 mM EDTA” solution in 0.9% NaCI (pH7.4 final), and incubated at room temperature before injection in rats for the DAS assay: extemporaneous, 1 h, 2h, 4h, 6h, 24h. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+).

[0044] [Fig. 8] Overall efficacy of the paresis activity of Onabotulinum toxin A (Ona) reconstituted with a 0.5 mg/mL dantrolene formulation in a “30% HPBCD + 10 mM L-Histidine + 0.1 mM EDTA” solution in 0.9% NaCI (pH7.4 final), and incubated at 4°C before injection in rats for the DAS assay: extemporaneous, 7 days, 14 days, 28 days. Overall efficacy is measured as the Area Under the paresis Curve, after 24h (AUC24+).

[0045] [Fig. 9] Concentration of dantrolene in the solution obtained by reconstituting a “0.5 mg/mL dantrolene in 30% HPBCD + 10 mM L-Histidine (pH7.4 final)” lyophilizate with 1 .5 mL of 0.9% NaCI, and incubating said solution 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours at 4°C. Concentration is measured by UV-dosing with (black) or without (gray) filtering solutions.

[0046] [Fig. 10] Concentration of dantrolene in the solution obtained by reconstituting a “0.5 mg/mL dantrolene in 30% HPBCD + 10 mM L-Histidine (pH7.4 final)” lyophilizate with 1 .5 mL of 0.9% NaCI, and incubating said solution 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours at room temperature. Concentration is measured by UV-dosing with (black) or without (gray) filtering solutions.

Detailed Description

Aqueous solutions

[0047] The present inventors have now developed aqueous solutions of dantrolene and optionally botulinum neurotoxin having a therapeutically effective dose in a reasonably small volume, having good physical stability, wherein dantrolene and, if present, botulinum neurotoxin, exhibit satisfying chemical stability, such solutions being particularly appropriate for intramuscular injection.

[0048] In a first aspect, the aqueous solutions of the invention comprise dantrolene or a pharmaceutically acceptable salt thereof and cyclodextrin or a derivative thereof, wherein the dantrolene or dantrolene pharmaceutically acceptable salt to cyclodextrin or derivative thereof molar ratio is of 1 : at least 40, and wherein the aqueous solution optionally comprises a buffer and/or at least one additional salt. It was surprisingly found that solutions of dantrolene or a pharmaceutically acceptable salt thereof comprising a high molar ratio of cyclodextrin or derivative to dantrolene exhibit a significantly lower crystallization of dantrolene upon extended storage in liquid form compared to solutions having a lower molar ratio of cyclodextrin or derivative to dantrolene.

[0049] More specifically, the ratio of dantrolene or pharmaceutically acceptable salt thereof to cyclodextrin or derivative thereof is about 1 :40 to about 1 :3000, preferably about 1 :50 to about 1700, more preferably about 1 :55 to about 1 :350, more preferably about 1 :60 to about 1 :185; most preferably about 1 :60 to about 1 :175.

[0050] The solubility of dantrolene has been shown to drastically drop in saline aqueous solutions compared to non-saline aqueous solutions, and in particular in aqueous solutions of NaCI. Therefore, the prior art compositions comprising dantrolene and salts in water are suspensions rather than solutions and are therefore unsuitable for intramuscular injections. Now, the present inventors have developed advantageous aqueous saline solutions comprising dantrolene or a pharmaceutically acceptable salt thereof, , at least one salt (in addition to any dantrolene salt, cyclodextrin salt and/or buffer salt). Preferably, the salt is as defined below.

[0051] Dantrolene (IIIPAC name 1-[(E)-[5-(4-nitrophenyl)furan-2- yl]methylideneamino]imidazolidine-2, 4-dione) is an inhibitor of Ca²⁺ release through ryanodine receptor (RYR) channels and acts a muscle relaxant. Dantrolene or any of its pharmaceutically acceptable salts are suitable for the purpose of the present invention. A dantrolene salt is defined as deprotonated form of dantrolene (forming a dantrolene anion) with a cationic counterion. Such cationic counterion is preferably selected from alkali metals, such as sodium or potassium, alkaline earth metals, such as calcium or magnesium, ammonium, pyridinium, substituted pyridinium, quinolidinium, or substituted ammonium cations such as alkyl ammonium, polyalkyl ammonium, aryl ammonium or ammonium cations of physiologically acceptable amino compounds, such as for example arginine, lysine, meglumine, tromethamine, choline, benzyltrimethylammonium, tetramethylammonium, N-methylpyridinium, tetrabutylammonium, 2-(2,3-dihydroxy-1 -proylamino)-quinolizinium, quinolizinium, 2-carbonyl-1 -methylpyridinium, 2,3- dimethyl-1 -phenyl-4-trimethyl-ammonium-3- pyrazolin-5-one, dimethylammonium or 1 ,3-dimethylimidazolium and 2-(1 - hydroxy-2-methyl)propyltri-methylammonium. Preferably the dantrolene salt is a salt of the dantrolene anion with an alkali metal counterion, preferably with a sodium or potassium counterion, more preferably a sodium counterion. In a most preferred aspect, the pharmaceutically acceptable salt of dantrolene is dantrolene sodium heptahydrate.

[0052] The dantrolene or pharmaceutically acceptable salt thereof is preferably present in an amount of about 0,025 mg/ml to about 1 ,5 mg/ml, preferably 0,1 mg/ml to about 1 ,1 mg/ml, more preferably about 0,25 mg/ml to about 0,6 mg/ml, most preferably about 0.45 mg/ml to 0,5 mg/ml. Such concentrations of dantrolene are advantageous in that it provides long-term physical stability to the composition.

[0053] In a preferred aspect, the composition further comprises botulinum neurotoxin. By "botulinum neurotoxin" (BoNT) is meant naturally occurring at least one of the eight different serotypes of botulinum neurotoxins known as type A, B, C, D, E, F, G and X as well as modified, recombinant, hybrid and chimeric botulinum neurotoxins. The expression botulinum neurotoxin and botulinum toxin are equivalent and can be used interchangeably. The term "botulinum toxin complex" or "toxin complex" as used herein refers to the approximately 150 kD botulinum toxin protein molecule (belonging to any one of botulinum toxin serotypes A-G, X), potentially along with associated endogenous non-toxin proteins (i.e., hemagglutinin protein and non-toxin non-hemagglutinin protein produced by Clostridium botulinum bacteria). Note, however, that the botulinum toxin complex does not need to be derived from Clostridium botulinum bacteria as one unitary toxin complex. For example, botulinum toxin or modified botulinum toxin may be recombinantly prepared first and then subsequently combined with the non-toxin proteins. Recombinant botulinum toxin can also be purchased (e.g., from List Biological Laboratories, Campbell, CA).

[0054] Mutation in the coding sequence of a BoNT that introduce one or more amino acid substitution is named modified BoNT. By "modified" botulinum neurotoxin is meant a compound that has botulinum toxin activity but contains one or more chemical or functional alterations on any part or on any amino acid chain relative to naturally occurring or recombinant native botulinum toxins. For instance, the botulinum toxin may be a modified neurotoxin that is a neurotoxin which has at least one of its amino acids deleted, modified or replaced, as compared to a native form. For instance, the botulinum toxin may be one that has been modified in a way that, for instance, enhances its properties or decreases undesirable side effects, but that still retains the desired botulinum toxin activity. The botulinum toxin may also be a portion of the overall molecule that has been shown to possess the necessary botulinum toxin activity, and in such case may be used per se or as part of a combination or conjugate molecule, for instance a fusion protein. In such case, the portion or fragment of the botulinum neurotoxin may be, for example, a 50 kDa light chain (LC) of the toxin. Alternatively, the botulinum toxin may be in the form of a botulinum toxin precursor, which may itself be nontoxic, for instance a non-toxic zinc protease that becomes toxic on proteolytic cleavage. "Hybrid and chimeric" BoNT refers to the mixing of heavy and light chain domains of different serotypes or different subtypes of BoNT. The hybrid and chimeric within BoNT serotypes and subtypes may be natural, such as BoNT/FA and BoNT/CD, or recombinant variants of BoNT. A recombinant botulinum neurotoxin can have the light chain and/or the heavy chain thereof made recombinantly by a non-Clostridial species.

[0055] According to a preferred embodiment, the botulinum neurotoxin used in the composition of the invention is of type A, B, E or a combination of heavy and light chain of type A, B, E botulinum neurotoxin. According to a more preferred embodiment, the botulinum neurotoxin used in the composition of the invention is of type A.

[0056] The botulinum neurotoxin is preferably present in a therapeutically effective amount, preferably in an amount of from about 0,1 ll/rnl to about 4000 LI/mL, preferably 1 ll/rnl to about 400 LI/mL, more preferably from about 5 ll/rnl to about 200 ll/rnl, most preferably from about 20 ll/rnl to about 100 ll/rnl.

[0057] Cyclodextrin and derivatives thereof are herein collectively designated as "cyclodextrins". Cyclodextrins are a family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1 ,4 glycosidic bonds. Cyclodextrins are produced from starch by enzymatic conversion. Three different forms of cyclodextrins exist: a-cyclodextrins, [3-cyclodextrins, and y-cyclodextrins. A cyclodextrin derivative is herein defined as a cyclodextrin comprising at least one substituent, preferably selected from the group consisting of linear or branched alkyl, hydroxyalkyl, carboxyalkyl, alkylcarbonyl, carboxyalkoxyalkyl, sulfoalkyl, alkylcarbonyloxyalkyl, alkoxycarbonylalkyl, sulfate and hydroxy-(mono or poly)alky I groups. Cyclodextrins herein also refers to salts of cyclodextrins or cyclodextrin derivatives, for example cyclodextrin sulfate sodium salt.

[0058] Examples of preferred cyclodextrins include (3-cyclodextrin, 2,6-dimethyl-|3- cyclodextrin, 2-hydroxyethyl-[3-cyclodextrin, 2-hydroxyethyl-y-cyclodextrin, 2- hydroxypropyl-[3-cyclodextrin, 2-hydroxypropyl-y-cyclodextrin, (2- carboxymethoxy)propyl-[3-cyclodextrin, sulfonylbutyl-[3-cyclodextrin, 2- hydroxypropyl-[3-cyclodextrin, sulfobutylether-[3-cyclodextrin and 2-hydroxyethyl- [3-cyclodextrin. In an especially preferred embodiment, the at least one cyclodextrin is selected from the group consisting of [3-cyclodextrin, 2,6-dimethyl- [3-cyclodextrin, 2-hydroxyethyl-[3-cyclodextrin, 2-hydroxyethyl-y-cyclodextrin, 2- hydroxypropyl-y-cyclodextrin, (2-carboxymethoxy)propyl-[3-cyclodextnn, sulfobutylether-[3-cyclodextrin and preferably 2-hydroxypropyl-[3-cyclodextrin (HPBCD).

[0059] The cyclodextrin or derivative thereof is preferably present in an amount of about 4% (m/v) to about 40% (m/v), preferably about 10% (m/v) to about 35% (m/v), more preferably about 10% (m/v) to about 30% (m/v), for example about 25% (m/v) to about 32% (m/v) or about 28% (m/v) to about 32% (m/v). The inventors identified that such unexpectedly high concentrations of cyclodextrin or cyclodextrin derivatives was required to obtain physical stability when salts are present in the aqueous solution.

[0060] In a preferred aspect, the aqueous composition of the present invention comprises a buffer. The buffer is not required as it does not impact the solubility of dantrolene or any salt thereof. The buffer is, however, particularly useful to reduce the alkalinity of the composition in the presence of high concentrations of dantrolene or salts thereof. Reducing the alkalinity through the presence of a buffer is favorable as it enables using high concentrations of dantrolene without compromising the chemical stability of this active principle. Indeed, dantrolene is hydrolyzed under alkaline conditions. The presence of a buffer as described herein significantly reduces dantrolene hydrolysis. Thus, the compositions of the invention comprising a buffer such as described herein can be stored for a longer time before administration, which makes such compositions much more practical for health practitioners. Furthermore, reducing the pH improves the comfort and the safety of intramuscular injection.

[0061 ] Any pharmaceutically acceptable buffer can be used. Preferably, the buffer is a pharmacopeia-listed buffer. Preferably, the buffer is selected from the group consisting of histidine buffer, phosphate buffer, citrate buffer, carbonate buffer, arginine buffer, lysine buffer, meglumine buffer, tromethamine buffer, acetate buffer, phosphate-citrate buffer, succinate buffer and mixtures thereof. In one embodiment, the buffer maintains the pH of the solution close to neutral pH, preferably in the range of 6 to 8, preferably 6.5 to 7.5.

[0062] The buffer is preferably present at a concentration of about 0,1 mM to about 20 mM, preferably about 5 mM to about 15 mM, most preferably about 10 mM. In a most preferred embodiment, the buffer is histidine buffer and such buffer is present at a concentration of about 10 mM or the buffer is phosphate and such buffer is present at a concentration of about 13 mM. Such relatively low buffer concentrations are advantageous in that it allows better dantrolene solubility, compared to higher buffer concentrations.

[0063] The at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof is preferably present in an amount of about 0,45% to about 1 ,8%, more preferably 0,85 to about 0,95 %, most preferably about 0,9% by weight, based on the total weight of the solution. Such at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof is preferably an inorganic salt, preferably a salt of an alkali metal. In a preferred aspect, the salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, ammonium chloride and combinations thereof. Such preferred salts are advantageous in that they allow correct solubilization, stability and activity of BoNT.

[0064] In a particular aspect, the solution of the present invention comprises from 0.07 to 1.2 mg/mL, preferably 0.15 to 0.5 mg/mL, of dissolved dantrolene. In a more preferred aspect such amounts of dissolved dantrolene are achieved even in the presence of least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof, such as defined above.

[0065] The solution of the present invention can optionally comprise one or more further pharmaceutically acceptable excipient or adjuvant. Preferably, the solution of the present invention comprises a tonicity modifier. A tonicity modifier is an excipient designed to reduce local irritation by preventing osmotic shock at the site of injection. Preferably the tonicity modifier is a pharmaceutically acceptable osmolality agent, more preferably selected from the group consisting of mannitol, fructose, glucose, gluconolactone, gluconate, sucrose, lactose, trehalose, dextrose, dextran, hydroxyl ethyl starches, glycine, gelatin, calcium gluconoglucoheptonate, and mixtures thereof.

[0066] The present invention may have an osmolarity of between about 350 to 700 mmol/kg. preferably 400 to 650 mmol/kg.

[0067] In a preferred aspect, the aqueous solution is free from any one or more or even more preferably free from all of the following: a) polyvinylpyrrolidones, such as povidone (synthetic polymer comprising linear chains of 1 -vinyl-2-pyrrolidone); b) ethoxylated glycerol esters, such as castor oil fatty acids (for example the commercially available Cremophor® EL); c) synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers (for example those commercially available as Lutrol®); and/or

[0068] The cyclodextrin or derivative thereof of the present invention is advantageously capable of dissolving dantrolene, such as to obtain a solution of the invention having sufficiently high dantrolene concentration, even without the use of any surfactant. As shown in the examples below, the inventors have identified that a cyclodextrin derivative (HPBCD) allowed to retain the best activity of botulinum neurotoxin among the tested excipients.

[0069] The aqueous solutions of the present invention are advantageously for intramuscular injection, and thus are suitable for this route of administration. This is advantageous, as intramuscular administration is the main route of administration of botulinum neurotoxin.

[0070] Thus, in a preferred aspect of the invention, the aqueous solution is free of organic solvents, because organic solvents are not appropriate for intramuscular injection. Also, organic solvents are not compatible with BoNT formulation, which is currently only approved for administration in the form of an aqueous solutions with 0.9% NaCI.

[0071 ] In view of the use for intramuscular injection, it is further desired to avoid the presence of undissolved particles, and in particular of large undissolved particles, which agglomerate in the tissues, and are poorly bioavailable. It is further advantageous that the solution be stable and does not precipitate upon storage. Thus, in another preferred aspect, the aqueous solution of the invention is free from particles having a size over 5 nm, upon storage for one day at room temperature. Particle size is herein defined as the equivalent mean diameter, as measured by Dynamic Light Scattering. The mean is defined by mass.

[0072] The aqueous solutions of the present invention are advantageously characterized by a good stability upon storage. Not only the physical stability, but also chemical stability is achieved, as degradation of dantrolene is limited to an acceptable limit, due to the finetuning of the components of the composition, as disclosed above. Thus, preferably, the concentration of dantrolene in the aqueous solution of the invention remains at acceptable levels upon storage, as dantrolene chemical degradation is avoided. In a preferred aspect, the concentration of (un-degraded) dantrolene decreases by not more than about 12%, preferably not more than about 10%, more preferably not more than about 8%, most preferably not more than about 6% upon storage for at least 3 months at 4°C in liquid from.

[0073] In one aspect of the invention, the aqueous solution according to the present invention is in frozen form. Indeed, freezing the solution increases its chemical and physical stability during storage. Solutions for use in therapy

[0074] The compositions of the present invention are advantageously for use in therapy or in other words for use as a medicament. Preferably they are for use in the treatment of muscular diseases or disorders, more preferably a) at least one disease or disorder selected from: i) a skeletal muscle disease or disorder, such as movement disorder, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal hand dystonia, blepharospasm, eyelid disorder, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasms, hemifacial spasm, tremors, tics, bruxism, apraxia and/or freezing of gait; ii) a smooth muscle disease or disorder, spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophagia, gastroparesis, spasmodic colitis, neurogenic bladder, overreactive bladder, interstitial cystitis, benign prostatic hyperplasia, urinary dysfunction, fecal incontinence, constipation, anismus, anal fissure, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernous muscle (priapism), other muscle tone disorders and/or other disorders characterized by involuntary movements of muscle groups; iii) a cardiac muscle cell disease or disorder such as atrial fibrillation. iv) a dermatological disease or disorder; and/or b) pain associated with such disease or disorder.

[0075] In a preferred aspect of the invention the solution of the invention is for use in the treatment of a disease or disorder, wherein the solution of the invention is administered by intramuscular injection.

[0076] In other words, the present invention relates to a method of treatment comprising administering an aqueous solution according to the invention and more particularly to a method for the treatment of muscular diseases or disorders, more preferably at least one disease or disorder such as listed above, or pain associated with such disease or disorder, wherein the method comprises administering a solution according to the present invention, preferably administering a solution according to the invention by the intramuscular injection.

[0077] In still other words, the present invention relates to the use of a solution according to the invention for the manufacture of a medicament, preferably a medicament for the treatment of muscular disease or disorders, more preferably at least one disease or disorder such as listed above, or pain associated with such disease or disorder. Preferably the medicament is a medicament for intramuscular injection.

[0078] In still other words, the present invention relates to the use of a solution according to the invention for the treatment of a disease or disorder, preferably a muscular disease or disorder, more preferably at least one disease or disorder such as listed above, or pain associated with such disease or disorder. More preferably, the treatment is administered by intramuscular injection.

Cosmetic use

[0079] The aqueous solutions of the present invention can advantageously be used in a cosmetic (i.e. non-therapeutic) treatment of any condition considered as un- aesthetic and involving muscles tension, activity and/or volume. Thus, the solutions of the invention are advantageously used for reducing wrinkles, lines, such as glabellar lines, furrows, crow’s feet, muscle volume, such as masseter or calf volume, hypertrophic scars and/or other dermatological conditions.

Lyophilizates

[0080] Lyophilizates of the above-described aqueous solutions are also an object of the present invention. Such lyophilizates are obtained by lyophilization of a composition of the invention as described above and can be reconstituted using a pharmaceutically acceptable liquid diluent prior to injection, preferably intramuscular injection. Such liquid diluent is preferably water for injection, or an aqueous saline solution for injection. Reconstitution is preferably performed by gently swirling the vial with the diluent and the lyophilizate. Upon reconstitution, the dissolution is preferably completed in less than 5 minutes.

[0081] In a preferred aspect, the lyophilizate is such that it provides a concentration of dantrolene or pharmaceutically acceptable salt thereof of about 0,025 mg/ml to about 1 ,5 mg/ml, preferably 0,1 mg/ml to about 1 ,1 mg/ml, more preferably about 0,25 mg/ml to about 0,6 mg/ml, most preferably 0,45 mg/ml to 0,5 mg/ml upon reconstitution with a carrier suitable for intramuscular injection. Such concentration refers to the concentration of dissolved dantrolene or pharmaceutically acceptable salt thereof. In a preferred aspect, the reconstitution is performed in a saline aqueous solution as acceptable carrier, with either 0.75 g of lyophilizate and 1 .5 mL of carrier, or 1 .5 g of lyophilizate and 3 mL of carrier.

[0082] Lyophilization can be performed using any method known to the person skilled in the art, preferably by freeze-drying. Methods for reconstitution of the lyophilizate are well-known to the person skilled in the art.

[0083] In a preferred aspect, the lyophilizate has a moisture content of no more than about 0.05 to about 1 wt%, more preferably no more than about 0.6 to about 0.7 wt%.

[0084] In a preferred aspect, the lyophilizate comprises substantially no sodium chloride. Preferably, the lyophilizate is free from sodium chloride.

Kits

[0085] As an alternative to the aqueous solution of the present invention comprising dantrolene and Botulinum neurotoxin, the present invention also provides a kit comprising an aqueous solution of dantrolene and a botulinum neurotoxin in lyophilized form as separate components. In such instance, the lyophilized botulinum is resuspended in the aqueous solution of dantrolene before being injected to a subject.

[0086] Therefore, in an embodiment, the present invention provides a kit comprising an aqueous, optionally frozen, solution according to the invention, or a lyophilizate thereof according to the invention, devoid of botulinum neurotoxin, and a lyophilized botulinum neurotoxin.

[0087] Preferably, the aqueous solution and the lyophilized botulinum neurotoxin of the kit are each provided in a suitable container.

[0088] In a specific embodiment, the kit comprises: a) an aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof, cyclodextrin or a derivative thereof, optionally a buffer and optionally at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof, wherein the dantrolene or dantrolene pharmaceutically acceptable salt to cyclodextrin or derivative thereof molar ratio is of 1 :at least 40, and b) a lyophilized botulinum neurotoxin.

[0089] In another specific embodiment, the kit comprises: a) an aqueous saline solution comprising dantrolene or a pharmaceutically acceptable salt thereof, and b) a lyophilized botulinum neurotoxin.

[0090] Dantrolene and salts thereof, cyclodextrin or derivatives thereof, botulinum neurotoxin, buffers and salts are as defined in any of the embodiments described above with respect to the aqueous solution.

[0091] The aqueous solution can optionally be provided in frozen form.

Methods for providing an aqueous solution of the invention using the kit of the invention

[0092] The kit of the invention can advantageously be used to prepare an aqueous solution according to the invention before injection to a subject. Such method comprises the steps of: a) providing a kit of the invention, such as described above, b) reconstituting the dantrolene composition if such composition is a lyophilizate, such as to obtain a dantrolene aqueous solution in liquid form; c) thawing the dantrolene aqueous solution composition if it is provided in frozen form, such as to obtain a dantrolene aqueous solution in liquid form, d) resuspending the lyophilized botulinum neurotoxin in the liquid aqueous composition of dantrolene provided in the kit or obtained in the end of steps b) or c), such as to form an aqueous solution according to of the abovedescribed aspects of the invention; e) optionally storing the aqueous composition obtained in the end of step d) at a temperature of about 2°C to about 8°C for about 1 hour to about 7 days. [0093] Methods for reconstitution, thawing and resuspension are well known to the person skilled in that art. Such steps can be performed using any method known in the art.

Examples

Example 1 : compositions according to the invention

[0094] A first composition according to the present invention was prepared, comprising the ingredients provided in [Table 1]:

[0095] [Table 1]

[0096] The dantrolene to HPBCD molar ratio in such composition is of 172. The pH is 7.4.

[0097] A second composition according to the present invention was prepared, comprising the ingredients provided in [Table 2]:

[0098] [Table 2]

[0099] The dantrolene to HPBCD molar ratio in such composition is of 64. The pH is 7.4.

Example 2: Effect of various excipients on dantrolene sodium solubility

[0100] Diverse excipients (surfactants or co-solvents) were screened for their ability to solubilize dantrolene and therefore to increase the dantrolene soluble concentration in the final reconstituted formulation comprising dantrolene and botulinum neurotoxin.

[0101] Each vehicle was prepared by solubilizing the corresponding excipient into 0.9% sodium chloride solution at various concentrations. [Table 3] shows for each excipient the excipient mass used for a final vehicle volume of 10 mL and the excipient concentration. In the second part of the study, other vehicles were also prepared in the same way.

[0102] [Table 3]

[0103] Dantrolene sodium (origin: Abeam; designated as "Dantrolene" in the following examples 2 to 7) was then added to each vehicle. The saturation was obtained by adding an excess of active ingredient to a given volume of test medium. The suspension was then stirred by magnetic stirring over 24 hours at 20°C, away from light.

[0104] The supernatant was then isolated by centrifugation (double centrifugation during 20 minutes at 15000 rpm at 20°C), diluted in a solvent mixture allowing its injection into the chromatographic system.

[0105] The Dantrolene concentration in solution for each medium was then determined by HPLC with external standardization. The HPLC method used in this study is disclosed in [Table 4], Given the UV spectrum of Dantrolene, the extraction of chromatograms was performed at 386 nm which gives a satisfactory compromise between the response factor and the baseline stability.

[0106] In a first series of trials, the solubility of Dantrolene was determined in different vehicles, prepared in 0.9% sodium chloride solution: 5% Cremophor® EL, 5% Lutrol® F68, 5% Tween®80 and 30% HPBCD. The Dantrolene solubility in 0.9% sodium chloride solution was also determined to serve as a reference data. The Dantrolene solubility levels at 20°C (HPLC results) in these six vehicles are reported in [Table 5],

[0107] The Dantrolene solubility in 0.9% NaCI (0.017 mg/mL) was consistent with the expected one. Except with Lutrol® F68, the different tested surfactants (5% Cremophor® EL and 5% Tween®80, all in 0.9% NaCI) improved the Dantrolene solubility, with 0.13 mg/mL, 0.14 mg/mL and 0.18 mg/mL solubilized respectively. The 30% HPBCD solution in 0.9% NaCI was the vehicle that most significantly increased the Dantrolene solubility, reaching about 1.8 mg/mL soluble concentration of Dantrolene.

[0108] [Table 4]

[0109] [Table 5]

[0110] In a second series of experiments, the solubility of Dantrolene was determined in different vehicles wherein a 0.9% NaCI solution was admixed with a water miscible co-solvent. The four tested co-solvents and their percentages in 0.9% NaCI are the following: 10% propylene glycol, 10% PEG400, 10% glycerol and 10% ethanol. The resulting Dantrolene solubility levels at 20°C (HPLC results) in these four tested vehicles are presented in [Table 6],

[0111] [Table 6]

[0112] In all these tested vehicles, the Dantrolene solubility did not even reach 0.05 mg/mL, indicating that a none of these co-solvents is a suitable excipient for better solubilizing Dantrolene.

[0113] In a third series of experiment, the suitability as an excipient of one polymer, polyvinylpyrrolidone PVP K17, was tested. Dantrolene sodium equilibrium solubility was measured at 20°C in a vehicle comprising 10% PVP K17 in 0.9% NaCI. The results are provided in [Table 7], PVP K17 did not increase Dantrolene solubility.

[0114] [Table 7]

[0115] In conclusion, these results demonstrated that the highest Dantrolene solubility was obtained in the 30% HPBCD solution, with a solubilized concentration of Dantrolene of about 1.8 mg/mL. In surfactant solutions (5% Cremophor® EL and 5% Tween®80, all in 0.9% NaCI), the Dantrolene solubility was only slightly increased. The other excipients were not successful in increasing Dantrolene solubility.

Example 3: Assessment of HPBCD concentration required to solubilize Dantrolene at a concentration of 1 mg/mL

[0116] The equilibrium solubility of Dantrolene was measured in HPBCD solutions at 18%, 20% and 25% in 200 mM NaCI. The obtained Dantrolene solubility levels at 20°C (HPLC results) in these three HPBCD solutions are presented in [Table 8],

[0117] [Table 8]

[0118] These results demonstrate that a minimum of 20% HPBCD in 200 mM NaCI is required for solubilizing the Dantrolene at the target concentration of 1 mg/mL over 24 hours of stirring at 20°C.

Example 4: stability assessment of Dantrolene formulation in a 30% HPBCD solution in 0.9% NaCI

[0119] Based on these results, longer storage tests were performed. A 0.5 mg/mL and a 1 mg/mL Dantrolene formulations in a 30% HPBCD solution in 0.9% NaCI were prepared to be put in stability at 4°C. An amount of 1 .5 g, respectively an amount of 3 g of HPBCD was weighed. A few milliliters of 0.9% NaCI solution were added, and the solution was stirred until complete solubilization. The volume was completed to 10mL with 0.9% NaCI aqueous solution. The solution was then magnetically stirred for complete homogenization.

[0120] After manufacturing, both formulated samples were analyzed by HPLC for Dantrolene content determination (after filtration of a small sample volume) and then aliquoted in antibiotic bottles for a stability study. The aliquots were then analyzed by HPLC after 1 day, 3 days and 7 days at 4°C, protected from light, for Dantrolene soluble fraction determination. The results are provided in [Table 9],

[0121] [Table 9]

[0122] Both formulated samples were shown to be chemically (no Dantrolene content decrease evidenced) and physically (no Dantrolene crystallization observed) stable for at least 7 days of storage at 4°C, protected from light.

[0123] Both formulated samples were also characterized for their osmolarity and pH. The results are provided in [Table 10] below. Even if quite high, the osmolarity of both formulated samples remained acceptable for an intramuscular or a subcutaneous injection (see Wei Wang, “Tolerability of hypertonic injectables”, Int J Pharm. 2015 Jul 25;490(1 -2):308-15).

[0124] [Table 10]

[0125] The equilibrium Dantrolene solubility after 24 hours at 4°C in the same vehicle (30% HPBCD solution in 0.9% NaCI) was of 1.5 mg/mL. Based on this result, a 1 mg/mL Dantrolene formulation in 30% HPBCD in 0.9% NaCI is expected to remain stable over the long-term at 4°C. These results triggered a more complete stability assessment of the formulation in a 30% HPBCD solution in 0.9% NaCI. Thus additional experiments were performed to confirm stability over the longer term.

[0126] A 1 mg/mL Dantrolene formulation in a 30% HPBCD solution in 0.9% NaCI was prepared as disclosed above. After complete solubilization, the solution was filtered on a sterile filter unit (Millex® PVDF 0.22 pm), analyzed by HPLC at TO for Dantrolene content determination, and then aliquoted in antibiotic bottles to be put in stability at different temperatures (4°C, 20°C and 40°C) for 6 months, as well as at -20°C for testing a freeze/thaw cycle.

[0127] The samples were then analyzed by HPLC (after filtration, Millex® PVDF 0.45 pm) after 1 day, 1 week, 2 weeks, 1 month, 3.5 months and 6 months of storage at 4°C, 20°C and 40°C (up to 1 month only), protected from light, for Dantrolene concentration determination. A stability sample was also analyzed after a freeze/thaw cycle.

[0128] The stability results are illustrated in [Fig. 1 ] (for Dantrolene concentration results), and reported in [Table 11 ] (for chromatographic purity results). These results demonstrate that compositions of the invention are sufficiently stable, even in the absence of a buffer. [0129] [Table 11]

[0130] In summary, sufficient stability was demonstrated for a duration of about three months. As it will be shown below in Example 5, the shelf life is further increased when a buffer is added to the dantrolene composition.

[0131] With respect to physical stability, no crystallization was observed in the different studied stability samples stored at 4°C, 20°C and 40°C.

[0132] With respect to chemical stability, a marked chemical degradation (significant Dantrolene content decrease, as well as impurity content increase) was observed from 1 day of storage at 40°C. After 1 month, the degradation reached about 60%. After 1 week of storage at 20°C, a slight degradation also started to be observed, for reaching about 10% after 1 month, 30% after 3.5 months, and 45% after 6 months. At 4°C, a degradation was also evidenced, with a Dantrolene content decrease of about 6% after 3.5 months and 13% after 6 months.

[0133] The stability was also assessed in a frozen sample. Samples were prepared as described above and were frozen after complete solubilization. The sample was kept at -20°C for 1 , 2 and 3 months. The Dantrolene concentration was assessed by HPLC. The results are provided in [Table 12], [0134] [Table 12]

Example 5: Effect of histidine buffer on physical and chemical stability of Dantrolene formulation

[0135] The long-term stability of three formulations was assessed at 4°C and 20°C, as well as after successive freeze/thaw cycles (followed by a short storage period at 4°C). The three formulations were the following: a) a 0.5 mg/mL Dantrolene formulation in a 30% HPBCD solution in 0.9% NaCI (reference sample) b) a 0.5 mg/mL Dantrolene formulation in a “30% HPBCD + 10 mM L- Histidine” solution in 0.9% NaCI (pH7.4 final) c) a 0.5 mg/mL Dantrolene formulation in a “30% HPBCD + 10 mM L- Histidine + 0.1 mM EDTA” solution in 0.9% NaCI (pH7.4 final)

[0136] After complete solubilization (the samples were kept under magnetic stirring for one hour at 20°C, protected from light), the pH of each solution was measured and adjusted to 7.4 (by adding small volumes of 1 N HCI) for both formulations with L-H istidine. After one more hour of magnetic stirring, each solution was filtered under sterile conditions (laminar flow hood, sterile filter unit (Millex® PVDF 0.22 pm), sterile antibiotic bottle) and then aliquoted in 2 mL sterile amber antibiotic vials to be put in stability at 4°C and 20°C for 12 months. Some vials were also stored at -20°C for studying the effect of successive freeze/thaw cycles, all protected from light.

[0137] The manufactured solutions were analyzed at TO for Dantrolene content determination and pH measurement. The stability samples were then analyzed by HPLC (after filtration, Millex® PVDF 0.45 pm) after 1 month, 3 months and 6 months of storage at 4° and 20°C, protected from light, for Dantrolene soluble fraction determination. The aspect (macroscopic observation) as well as the pH of the stability samples were also reported. Storage is still ongoing at the filing date and further analysis is planned at later timepoints.

[0138] Regarding the stability samples submitted to successive freeze/thaw cycles, the samples were thawed at 20°C for 1 .5 hours before being frozen for 1 .5 hours, etc. In practice, and for each studied solution, the stability samples subjected to 1 freeze/thaw cycle were first kept at -20°C for 2 or 3 weeks, before being thawed at 20°C for 1 .5 hours, and finally stored at 4°C for 1 week or 2 weeks, respectively, before being analyzed. In the same way, the stability samples subjected to 3 or 5 freeze/thaw cycles are thawed/frozen/thawed by 1 ,5-hour periods (except a frozen time of one night before the fourth cycle), and then stored at 4°C for 1 week or 2 weeks before to be analyzed.

[0139] No physical degradation (no observed crystallization) and no significant pH change was evidenced in the different stability samples stored over 6 months at 4°C and 20°C.

[0140] With respect to chemical stability, the presence of 10 mM L-Histidine (with or without 0.1 mM EDTA) significantly improved the chemical stability of Dantrolene in the tested formulated samples stored both at 4°C and at 20°C. Indeed, the formulations with the L-histidine buffer exhibited about twice less degradation compared to the reference sample, as shown on [Fig. 2],

[0141 ] Regarding the samples submitted to successive freeze/thaw cycles, no significant degradation is observed other than that due to the storage at 4°C for 1 or 2 weeks.

Example 6: Compatibility of diverse excipients with botulinum neurotoxin

[0142] Paresis activity was assessed relying on the Digit Abduction Score assay in rats, as described below.

Animals

[0143] Experiments were carried out with female adult Sprague-Dawley rats (200g), obtained from Janvier Labs/France. Rats were acclimatized for at least one week prior to use with access to food and water ad libitum in animal facility. Unless otherwise stated, 6 rats for each condition were employed. [0144] The body weight of animals was recorded all along the study on day 0, 2, 4, 7 and once a week until the end of experiment.

Treatment

[0145] Animals were injected (25 pL or 40 pL per rat) using a 33-gauge needle attached to a 100pL Hamilton Syringe. On the initial day of the experiment, before the injections, rodents were pre-screened for a “zero” DAS response. Then, rats received intramuscular (IM) injections into the right tibialis anterior (TA) muscle. The left TA muscle was left non-injected as control.

Digit abduction score assay (DAS)

[0146] The muscle paralysis was measured using the digit score abduction (DAS) assay as reported by Broide et al.(TOXICON, 2013, 71 , pp. 18-24). Animals were scored for DAS response at different time points following the compound injections by eliciting digit abduction: 30 minutes; 1 hour; 2 hours; 6 hours; 12 hours; 24 hours; 2 days, 4 days, 5 days, 6 days, 7 days, 8 days and once a day until two consecutive measurements on two consecutive days were equal to zero (DAS = 0). The varying degrees of digit abduction were scored on a five-point scale (0 = normal to 4 = maximum reduction in digit abduction, i.e. none of the toes can abduct) by two separate observers who were blinded to the treatment.

[0147] As typically performed for rat DAS studies, the DAS response were induced by grasping the rat around the torso, lifting into the air and simulating a drop to a flat surface. The rat reflexively usually braces for impact by spreading the digits in its hind paws and the DAS response was immediately scored with the animal facing up in a reclining position.

Data analysis

[0148] As described above, a DAS score of T was assigned when loss of abduction was observed with the 1st digit. A DAS score of ‘2’ was given when three digits are coupled together. A score of ‘3’ was given when four digits are conjoined, and rats received a score of ‘4’ when all five digits of the right paw were grouped together during abduction. [0149] The DAS responses at each time point were measured and kinetics (onset of muscle weakening and duration of efficacy) were assessed and compared in the different groups of rats.

[0150] For each rat, areas under the DAS curve (in DAS*days units) were calculated using series of Riemann sums between T=24 hours to the end of the experiment (AUC24+).

[0151] [Fig. 3] shows that when Botulinum Neurotoxin A (Ona) was reconstituted in a 0.9% NaCI solution containing 5% Tween®80, its activity was impaired. On the contrary, a similar solution containing HPBCD (up to 30%) had low impact on Ona activity.

[0152] [Fig. 4] shows that when Botulinum Neurotoxin A (Ona) was reconstituted in a 0.9% NaCI solution containing 4.5% Tween®20, its activity was impaired. Likewise, the combination of dantrolene (FTP-501) with Ona in this formulation was not able to enhance Ona activity; on the contrary, in 0.9% NaCI solution, the same combination (Ona + FTP-501 ) showed a better activity than Ona alone. Of note, dantrolene enhancement was similar in a formulation containing HPBCD.

[0153] [Fig. 5], [Fig. 6] and [Fig. 7] show the in-use stability of Botulinum toxin A (Ona) reconstituted with either a 0.9% NaCI solution (reference), or formulations containing “HPBCD + L-Histidine + EDTA” in 0.9% NaCI, at room temperature. These results demonstrate that Ona activity was stable during the first 4-6 hours, and then decreased, in both the reference and HPBCD formulations.

[0154] [Fig. 8] shows the in-use stability of Botulinum toxin A (Ona) reconstituted with either a 0.9% NaCI solution (reference), or formulations containing “HPBCD + L- Histidine + EDTA” in 0.9% NaCI, at 4°C. These results demonstrate that Ona activity was stable during the first 14 days, and then decreased, in both the reference and HPBCD formulations.

Example 7: Properties of HPBCD-based lyophilizates

[0155] A HPBCD-based formulation containing dantrolene in lyophilizate form to be reconstituted in 0.9% NaCI, was prepared as follows. A 300 mg/mL HPBCD + 10 mM L-Histidine solution was first prepared in water; Dantrolene was then added to obtain a final concentration of 500 pg/mL. The pH was then adjusted to 7.4 by adding HCI 10%. The obtained composition was filtered on a 0.2 pm PVDF membrane and 1 ,5mL aliquots were dispensed in glass vials and frozen at -20°C for 24 hours. Lyophilization was then performed by applying a pressure of 1 ,03mbar at -20°C for 4 days. Lyophilizates were then stored at 4°C until analysis.

[0156] In a first experiment, the lyophilizates were reconstituted with 1 .5 mL of NaCI 0.9%. Instant reconstitution was observed, and UV-dosing of dantrolene showed that the desired concentration of 500 pg/mL dantrolene was obtained, as shown in [Fig. 9] and [Fig. 10], first point on the left. Reconstituted vials were then incubated at 4°C ([Fig. 9]) or room temperature ([Fig. 10]) for 24 hours. From these curves, an in-use stability of at least 24 hours at 4°C and room temperature could be concluded.

[0157] In a second experiment, the lyophilizates were stored at 4°C for 20 months, then were reconstituted with 1 .5 mL of NaCI 0.9%, and dantrolene content was dosed by UV-dosing. A concentration of 472 ± 6 pg/mL (±SD, n=2) could be retrieved, demonstrating the stability of the lyophilizates during at least 20 months at 4°C.

Claims

Claims

[Claim 1 ] [An aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof and cyclodextrin or a derivative thereof, wherein the dantrolene or dantrolene pharmaceutically acceptable salt to cyclodextrin or derivative thereof molar ratio is of 1 :at least 40, and wherein the aqueous solution optionally comprises a buffer and/or at least one additional salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof.

[Claim 2] An aqueous solution according to [claim 1 ], further comprising Botulinum neurotoxin.

[Claim 3] An aqueous solution according to any one of [Claim 1 ] or [Claim 16], wherein the pharmaceutically acceptable dantrolene salt is defined as a salt of a deprotonated form of dantrolene with a cationic counterion selected from the group consisting of alkali metals, such as sodium or potassium, alkaline earth metals, such as calcium or magnesium, ammonium, pyridinium, substituted pyridinium, quinolidinium, and substituted ammonium cations such as alkyl ammonium, polyalkyl ammonium, aryl ammonium or ammonium cations of physiologically acceptable amino compounds, such as for example arginine, lysine, meglumine, tromethamine, choline, benzyltrimethylammonium, tetramethylammonium, N-methylpyridinium, tetrabutylammonium, 2-(2,3- dihydroxy- l-proylamino)-quinolizinium, quinolizinium, 2-carbonyl-l-methylpyridinium, 2,3- dimethyl- l-phenyl-4- trimethyl-ammonium-3-pyrazolin-5-one, dimethylammonium or 1 ,3- dimethylimidazolium and 2-(l-hydroxy-2-methyl)propyltri-methylammonium; wherein the botulinum neurotoxin is selected from the group consisting of the serotypes A (BoNT/A), B (BoNT/B), E (BoNT/E) and combinations of heavy and light chains thereof; and /or wherein the cyclodextrin or derivative thereof is selected from the group consisting of [3-cyclodextrin, 2,6-dimethyl-[3-cyclodextrin, 2-hydroxyethyl-[3- cyclodextrin, 2-hydroxyethyl-y-cyclodextrin, 2-hydroxypropyl-[3-cyclodextrin, 2-hydroxypropyl-y-cyclodextrin, (2-carboxymethoxy)propyl-[3-cyclodextrin, sulfonylbutyl-[3-cyclodextrin, 2-hydroxypropyl-[3-cyclodextrin, sulfobutylether- [3-cyclodextrin and 2-hydroxyethyl-[3-cyclodextrin, preferably from the group consisting of [3-cyclodextrin, 2,6-dimethyl-[3-cyclodextrin, 2-hydroxyethyl-[3- cyclodextrin, 2-hydroxyethyl-y-cyclodextrin, 2-hydroxypropyl-y-cyclodextrin, (2-carboxymethoxy)propyl-[3-cyclodextrin, sulfobutylether-[3-cyclodextrin and preferably 2-hydroxypropyl-[3-cyclodextnn (HPBCD).

[Claim 4] An aqueous solution according to any one of [Claim 1 ] to [Claim 3], comprising: dantrolene or a pharmaceutically acceptable salt thereof in an amount of about 0,025 mg/ml to about 1 ,5 mg/ml, preferably 0,1 mg/ml to about 1 ,1 mg/ml, more preferably about 0,25 mg/ml to about 0,6 mg/ml, most preferably from 0.45 mg/ml to 0,5 mg/ml; botulinum neurotoxin in a therapeutically effective amount, preferably in an amount of from about 0,1 ll/rnl to about 4000 LI/mL, preferably 1 ll/rnl to about 400 LI/mL, more preferably from about 5 ll/rnl to about 200 ll/rnl, most preferably from about 20 ll/rnl to about 100 ll/rnl; and/or cyclodextrin or a derivative thereof in an amount of about 4% (m/v) to about 40% (m/v), preferably about 10% (m/v) to about 35% (m/v), more preferably about 10% (m/v) to about 30% (m/v), for example about 25% (m/v) to about 32% (m/v), or about 28% (m/v) to about 32% (m/v).

[Claim 5] An aqueous solution according to any one of [Claim 1 ] to [Claim 4], wherein the ratio of dantrolene or pharmaceutically acceptable salt thereof to cyclodextrin or derivative thereof is about 1 :40 to about 1 :3000, preferably about 1 :50 to about 1700, more preferably about 1 :55 to about 1 :350, more preferably about 1 :60 to about 1 : 185; most preferably about 1 :60 to about 1 :175.

[Claim 6] An aqueous solution according to any one of [Claim 1 ] to [Claim 5], wherein the buffer is selected from the group consisting of histidine buffer, phosphate buffer, citrate buffer, carbonate buffer, arginine buffer, lysine buffer, meglumine buffer, tromethamine buffer, acetate buffer, phosphatecitrate buffer, succinate buffer and mixtures thereof.

[Claim 7] An aqueous solution according to any one of [Claim 1 ] to [Claim 6], wherein the at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof is preferably present in an amount of about 0,45% to about 1 ,8%, more preferably 0,85 to about 0,95 %, most preferably about 0,9% by weight, based on the total weight of the solution and/or wherein such salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, ammonium chloride and combinations thereof.

[Claim 8] An aqueous solution according to any one of [Claim 1] to [Claim 7], wherein the solution further comprises a tonicity modifier, preferably a pharmaceutically acceptable osmolality agent, more preferably selected from the group consisting of mannitol, fructose, glucose, gluconolactone, gluconate, sucrose, lactose, trehalose, dextrose, dextran, hydroxyl ethyl starches, glycine, gelatin, calcium gluconoglucoheptonate, and mixtures thereof.

[Claim 9] An aqueous solution according to any one of [Claim 1] to [Claim 8], wherein the solution is free from any one or more or all of, preferably free from all of the following: a) polyvinylpyrrolidones, such as povidone (synthetic polymer comprising linear chains of 1-vinyl-2-pyrrolidone); b) ethoxylated glycerol esters, such as castor oil fatty acids; c) synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers; d) organic solvents; and/or e) particles having a size over 5 nm upon storage for one day at room temperature.

[Claim 10] An aqueous solution according to any one of [Claim 1] to [Claim 9] for use as a medicament,

[Claim 11] An aqueous solution for use according to [Claim 10], for use in the treatment of a) at least one disease or disorder selected from: i) a skeletal muscle disease or disorder, such as movement disorder, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal hand dystonia, blepharospasm, eyelid disorder, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasms, hemifacial spasm, tremors, tics, bruxism, apraxia and/or freezing of gait; ii) a smooth muscle disease or disorder, spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophagia, gastroparesis, spasmodic colitis, neurogenic bladder, overreactive bladder, interstitial cystitis, benign prostatic hyperplasia, urinary dysfunction, fecal incontinence, constipation, anismus, anal fissure, , uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernous muscle (priapism), other muscle tone disorders and/or other disorders characterized by involuntary movements of muscle groups; iii) a cardiac muscle cell disease or disorder such as atrial fibrillation; iv) a dermatological disease or disorder; and/or b) pain associated with such disease or disorder.

[Claim 12] Use of an aqueous solution according to any one of [Claim 1 ] to [Claim 9] in a cosmetic non-therapeutic treatment, preferably for reducing wrinkles, lines, such as glabellar lines, furrows, crow’s feet, muscle volume, such as masseter or calf volume, hypertrophic scars and/or other dermatological conditions.

[Claim 13] A lyophilizate of an aqueous solution according to any one of [Claim 1 ] to [Claim 9],

[Claim 14] A kit comprising a) A dantrolene composition selected from i. An aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof, cyclodextrin or a derivative thereof, optionally a buffer and optionally at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof, wherein the dantrolene or dantrolene pharmaceutically acceptable salt to cyclodextrin or derivative thereof molar ratio is of at least 1 :40, and wherein such aqueous solution is either in liquid or in frozen form; and ii. a lyophilizate of an aqueous solution of i.; and b) a lyophilized botulinum neurotoxin.

[Claim 15] A method of providing an aqueous solution according to any one of [Claim 1] to [Claim 9] from the kit of [Claim 14], comprising the steps of: a) providing a kit of [Claim 14], b) reconstituting the dantrolene composition if such composition is a lyophilizate, such as to obtain a dantrolene aqueous solution in liquid form; c) thawing the dantrolene aqueous solution composition if it is provided in frozen form, such as to obtain a dantrolene aqueous solution in liquid form, d) resuspending the lyophilized botulinum neurotoxin in the liquid aqueous composition of dantrolene provided in the kit or obtained in the end of steps b) or c), such as to form an aqueous solution according to any one of [Claim 1] to [Claim 9]; e) optionally storing the aqueous composition obtained in the end of step d) at a temperature of about 2°C to about 8°C for about 1 hour to about 7 days.

[Claim 16] An aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof, botulinum neurotoxin and at least one salt that is not a salt of dantrolene, of cyclodextrin or of a derivative thereof. ]