WO2025229073A1

WO2025229073A1 - Aqueous compositions of dantrolene

Info

Publication number: WO2025229073A1
Application number: PCT/EP2025/061858
Authority: WO
Inventors: Jean-Marc Le Doussal; Mickaël Machicoane; Paul ONILLON
Original assignee: Fastox Pharma SA
Current assignee: Fastox Pharma SA
Priority date: 2024-05-01
Filing date: 2025-04-30
Publication date: 2025-11-06
Anticipated expiration: 2026-11-01

Abstract

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

Description

Title of Invention: Aqueous compositions of dantrolene

Technical Field

[0001] The present invention relates to aqueous compositions comprising dantrolene and optionally also at least one additional active principle, such as botulinum neurotoxin, and their use for various therapeutic and/or aesthetic/cosmetic purposes. The compositions can be administered in any suitable way but are advantageous in that they are particularly well adapted to intramuscular injection, subcutaneous injection and/or intradermal 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] In cases wherein Dantrolene is administered via the intramuscular route into superficial muscles, the intramuscular administration often involves injection of part of the product into the dermis, so that the injected composition is at least partially administered intradermally and/or subcutaneously. The proportion of the injected product that is intradermally and/or subcutaneously injected depends on several parameters such as the injection technique the injected volume and the syringe/needle couple used by the practitioner. For example, when medical practitioners tend to inject larger volumes, or to withdraw the needle during injection of the product, the intradermally and/or subcutaneously administered portion can be significant. It would therefore be particularly advantageous to optimize Dantrolene composition for intramuscular administration, for subcutaneous administration and for intradermal administration at the same time.

[0004] Compositions for intramuscular administration are typically saline aqueous compositions, preferably homogeneous composition, more preferably solutions. However, dantrolene is poorly soluble in water and even less soluble in saline aqueous compositions. [0005] 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, subcutaneous or intradermal injection. There is therefore a need to improve the formulation of compositions comprising dantrolene, such as to make such compositions better optimized for intramuscular, subcutaneous and intradermal injection.

[0006] In view of intramuscular, subcutaneous and intradermal injection, it is even more desirable to provide the dantrolene formulation in the form of a ready-to-use injectable composition, 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 composition 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.

[0007] The pH of un-buffered compositions 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 compositions of dantrolene are thus poorly stable over time. Thus, for applications wherein dantrolene remains in the aqueous composition for a prolonged time, such as ready-to-use compositions, it is sought to improve the chemical stability of dantrolene.

[0008] 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 routes, 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, subcutaneous and intradermal administration. The prior art intravenous formulations of dantrolene are therefore not adapted to the administration of dantrolene by the intramuscular, subcutaneous or intradermal route. [0009] 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.

[0010] 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.

[0011] Very dilute solutions are also unsuitable for intramuscular, subcutaneous or intradermal 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, subcutaneous or intradermal 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).

[0012] 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.

[0013] In view of optimizing the formulation of compositions comprising dantrolene, there is therefore a need to identify compositions that are optimized for intramuscular, subcutaneous and intradermal injection, allow its solubilization and preferably ensure chemical stability over time.

[0014] Diverse excipients 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. This document does not disclose any dantrolene saline composition. Saline compositions being preferred over non-saline compositions for intramuscular, subcutaneous or intradermal injection (in contrast to intravenous administration), the compositions of this document are disadvantageous for administration by this route.

[0015] Other prior art documents have disclosed compositions of dantrolene and cyclodextrin or derivatives thereof. For example, WO2018146187A1 and W02020049670A1 discloses compositions of dantrolene with 2-hydroxypropyl-[3- cyclodextrin (HPBCD) for intravenous injection. These documents respectively allege improvement of the stability and solubility of dantrolene and reduced nephrotoxicity. Again, these documents do not disclose any dantrolene saline composition.

[0016] Cyclodextrin is however disadvantageous in that it tends to cause irritation of the dermis when the composition comprising cyclodextrin is administered intradermally (See EMA/CHMP/333892/2013 available from the website of the European Medicines Agency, which provides a background review for cyclodextrins used as excipients). This is also the case when the composition is administered subcutaneously. There is therefore a need to further improve the tolerance of dantrolene compositions in cases where the composition is administered wholly or partially intradermally and/or subcutaneously.

[0017] 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.

[0018] 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 compositions for co-administration of dantrolene and Botulinum neurotoxins.

[0019] 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.

[0020] 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 zinc- endopeptidase. 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.

[0021] 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.

[0022] 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).

[0023] 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.

[0024] 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 principles. 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 compositions for intramuscular injection.

[0025] 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.

[0026] The present invention aims at solving part or all of the above-mentioned problems.

Summary of Invention

[0027] In a first aspect, the invention relates to an aqueous composition comprising dantrolene, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof and an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol. [0028] The composition of the first aspect is useful perse, for administration in any indication in which dantrolene is to be administered in the form of an aqueous composition, for example by the intramuscular, subcutaneous and/or intradermal route. This composition is advantageous, because of the high solubility of dantrolene and the very good tolerability of the composition when using the excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol. The buffer is only optional as it does not impact solubility, but only improves chemical stability of dantrolene.

[0029] In a particular embodiment of this first aspect, the composition further comprises Botulinum neurotoxin.

[0030] In a second aspect, the invention relates to an aqueous composition according to the first or second aspect, 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.

[0031] In a third aspect, the invention relates to the use of an aqueous composition according to the first or second aspect in an aesthetic and/or cosmetic (i.e. non- therapeutic) treatment.

[0032] In a fourth aspect, the invention relates to a lyophilizate of an aqueous composition of the first or second aspect.

[0033] In a fifth aspect, the invention relates to a kit comprising an aqueous, optionally frozen, composition of the first or second aspect, or a lyophilizate according to the fifth aspect, devoid of botulinum neurotoxin, and a lyophilized botulinum neurotoxin,

[0034] In a sixth aspect, the invention relates to a method of providing an aqueous composition 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 composition in liquid form; c) thawing the dantrolene aqueous composition if it is provided in frozen form, such as to obtain a dantrolene aqueous composition 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 composition 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.

Brief Description of Drawings

[0035] [Fig.1 ]: Graph representing the evolution of the erythema score over time after intradermal injection of 40pL of Compositions A to D to rats as described in Example 5.

[0036] [Fig. 2]: Graph representing the evolution of the mean erythema at day 2 after intradermal injection of increasing volumes of Compositions A to D to rats as described in Example 5.

[0037] [Fig. 3]: Graph representing the evolution of the mean DAS over time for Compositions E to G, as assessed in Example 6.

[0038] [Fig. 4]: Graph representing the mean AUC24+ of BoNT/A for each of Compositions E to G, as assessed in Example 6.

[0039] [Fig. 5] Graph representing the evolution of the mean AUC24+ of BoNT/A over time in a 0.9% NaCI solution (Saline, control), or in a Solutol HS15 + L- Histidine solution in 0.9% NaCI such as prepared in the first step of examples 1 and 2, or with the composition of the invention prepared in Example 2 as assessed in Example 7.

[0040] [Fig. 6] Graph representing the evolution of the concentration of Dantrolene in a lyophilizate of a composition according to the invention over time in the accelerated stability test at 40°C of Example 8.

[0041 ] [Fig. 7]: Graph representing the mean AUC24+ of BoNT/A administered in a 0.9% NaCI solution (Saline, control), 5% Cremophor EL, 5% Solutol HS15, 5% TWEEN 80, 30% 2-Hydroxypropyl-beta-cyclodextrin (CD) or 5% 2- Hydroxypropyl-beta-cyclodextrin (CD) (all in 0.9% NaCI) as assessed in Example 9.

Detailed description

Aqueous compositions

[0042] The present inventors have now developed aqueous compositions 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 and sustained activity, such compositions being particularly appropriate for intramuscular injection and well tolerated when administered subcutaneously and/or intradermally, without causing irritation of the skin.

[0043] The comparison of various excipients (see examples below) demonstrated that an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol provided an optimal balance between solubilization of dantrolene and tolerance upon subcutaneous and/or intradermal injection, while being compatible with Botulinum neurotoxin, which has previously been described as advantageous in combination with dantrolene.

[0044] In a first aspect, the aqueous compositions of the invention comprise dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof and an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol.

[0045] It was surprisingly found that compositions of dantrolene, of a pharmaceutically acceptable derivative thereof or of a pharmaceutically acceptable salt thereof comprising an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol make it possible to achieve high concentrations of Dantrolene.

[0046] In a preferred aspect, the composition of the invention is a clear (i.e. not turbid) composition, that preferably does not comprise any particle visible to the naked eye. Preferably the composition of the invention is a solution or a micellar composition. More preferably, the composition comprises dissolved dantrolene, micelles comprising dantrolene surrounded by and/or embedded in molecules of the excipient or a mixture of dissolved dantrolene and micelles. Dissolved dantrolene and micelles of dantrolene surrounded by molecules of the surfactant are advantageously particularly well tolerated for intramuscular, subcutaneous and/or intradermal administration. Micelles comprising dantrolene surrounded by and/or embedded in molecules of the excipient may also be referred to as “micelles of dantrolene” or “micellar dantrolene”.

[0047] In a preferred aspect, the composition further comprises at least one salt that is not a salt of Dantrolene, preferably such as defined below. The solubility of dantrolene has been shown to drastically drop in saline aqueous compositions compared to non-saline aqueous compositions, and in particular in aqueous compositions of NaCI. Surprisingly, hydroxy fatty acid esters of polyethylene glycol have proved to be excipients suitable to achieve high concentration of dissolved Dantrolene even in saline compositions, such as NaCI compositions.

[0048] 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, any of its pharmaceutically acceptable derivative or any of its pharmaceutically acceptable salts are suitable for the purpose of the present invention.

[0049] 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.

[0050] A pharmaceutically acceptable derivative of dantrolene is defined as any chemical derivative of dantrolene that retains the therapeutic activity of dantrolene as described herein. Examples of suitable pharmaceutically acceptable derivatives of dantrolene include azumolene, pCI-dantrolene and o- nitrodantrolene. In the present disclosure, a reference to dantrolene may generally include its suitable pharmaceutically acceptable derivatives. The dantrolene, pharmaceutically acceptable derivative thereof or pharmaceutically acceptable salt thereof is preferably present in the composition in an amount of about 0.025 mg/mL to about 0.16 mg/mL, preferably about 0.05 mg/mL to about 0.16 mg/mL, preferably about 0.05 mg/mL to about 0.15 mg/mL, more preferably about 0.1 mg/mL to about 0.15 mg/mL, based on the total volume of the composition. Such concentrations of dantrolene are advantageous in that they provide long-term physical stability to the composition.

[0051] 'About" in the context of amount values refers to an average deviation of maximum +/-20 %, preferably +/-10 %, most preferably +/-5 % based on the indicated value. For example, an amount of about 20 mg/m² refers to 20 mg/m² +/-6 mg/m², preferably 20 mg/m² +/-4 mg/m², most preferably 20 mg/m² +/-2 mg/m². This includes also the value itself with any deviation.

[0052] 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).

[0053] Mutation in the coding sequence of a BoNT that introduces 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 non-toxic, 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.

[0054] 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. [0055] The botulinum neurotoxin is preferably present in the composition in a therapeutically effective amount, preferably in an amount of from about 0,1 ll/rnl to about 4000 U/mL, preferably 1 ll/rnl to about 400 U/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, based on the total volume of the composition.

[0056] Hydroxy fatty acid esters of polyethylene glycol are esters of fatty acids and polyethylene glycol, wherein the fatty acid has one or more hydroxyl group(s) attached to its principal chain known for various uses, for example as emulsifiers or surfactants. The present inventors have discovered that such compounds could also be used as excipients for increasing the concentration of dissolved or micellar Dantrolene in aqueous and aqueous saline compositions. In a preferred aspect, the hydroxy fatty acid ester of polyethylene glycol is a hydroxy fatty acid ester of a long-chain fatty acid, i.e. a hydroxy fatty acid ester of a fatty acid consisting of an aliphatic chain of 13 to 21 carbon atoms. Preferably, the fatty acid is a saturated fatty acid, such as myristic acid, palmitic acid, stearic acid and arachidic acid. More preferably, the fatty acid is selected from palmitic acid, stearic acid and oleic acid, most preferably it is stearic acid. In a more preferred aspect, the hydroxy fatty acid is selected from 3-hydroxystearic acid, 12- hydroxystaric acid and 17 hydroxystearic acid, most preferably 12-hydroxystearic acid. 12-hydroxystearic acid ester of polyethylene glycol is advantageously known as an excipient in the pharmaceutical industry. In a specific example of the hydroxy fatty acid esters of polyethylene glycol are esters of fatty acids and polyethylene glycol is commercially available as Solutol® HS-15, CAS No 61909- 81-7 or Kolliphor® HS 15.

[0057] In a preferred aspect the hydroxy fatty acid ester is not an ester of an unsaturated fatty acid, a cis isomer of a fatty acid, or specifically ricinoleic acid.

[0058] In a preferred aspect, the polyethylene glycol in the hydroxy fatty acid ester has a molecular weight of between 200 and 1000 Da, preferably between 400 and 800 Da, most preferably 660 Da.

[0059] The excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol is preferably present in the composition in an amount of at least about 10 mg/ml, preferably at least about 20 mg/ml, more preferably at least about 40 mg/ml, such as about 10 mg/ml to about 60 mg/ml, preferably about 20 mg/ml to about 60 mg/ml, more preferably about 40 mg/ml to about 60 mg/ml, most preferably 40 mg/ml to about 55 mg/ml, based on the total volume of the composition.

[0060] The term “free polyethylene glycol” as used herein refers to unesterified polyethylene glycol.

[0061] 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.

[0062] 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 composition close to neutral pH, preferably in the range of 6 to 8, preferably 6.5 to 7.5.

[0063] The buffer is preferably present at a concentration in the composition of about 0,1 mM to about 40 mM, preferably about 5 mM to about 30 mM, most preferably about 10 mM to about 20 mM. In a most preferred embodiment, the buffer is histidine buffer and such buffer is present at a concentration of about 10 mM to about 20 mM. Such relatively low buffer concentrations are advantageous in that it allows better dantrolene solubility, compared to higher buffer concentrations. [0064] The at least one salt that is not a salt of dantrolene, or of a derivative thereof is preferably present in the composition 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 volume of the composition. Such at least one salt that is not a salt of dantrolene, 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 high solubilization, stability and activity of BoNT.

[0065] In a particular aspect, the composition of the present invention comprises about 0.025 mg/mL to about 0.16 mg/mL, preferably about 0.05 mg/mL to about 0.16 mg/mL, more preferably about 0.05 mg/mL to about 0.15 mg/mL, preferably about 0.1 mg/mL to about 0.15 mg/mL, of dissolved dantrolene or micellar dantrolene, based on the total volume of the composition. For the purpose of the present invention, micellar dantrolene refers to dantrolene present in the composition in the form of micelles, i.e. at least partially surrounded by molecules of the excipient. 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, or of a derivative thereof, such as defined above.

[0066] The composition of the present invention can optionally comprise one or more further pharmaceutically acceptable excipient or adjuvant. Preferably, the composition 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.

[0067] The composition of the present invention preferably has an osmolarity of between about 250 to 600 mmol/kg. preferably 330 to 450 mmol/kg. [0068] In a preferred aspect, the aqueous composition does not comprise polyvinylpyrrolidones, such as povidone (synthetic polymer comprising linear chains of 1-vinyl-2-pyrrolidone).

[0069] In another preferred aspect, the aqueous composition does not comprise ethoxylated glycerol esters, such as castor oil fatty acids (for example the commercially available Cremophor® EL).

[0070] In still another preferred aspect, the aqueous composition does not comprise synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers (for example those commercially available as Lutrol®)

[0071 ] More preferably the aqueous composition does not comprise any polyvinylpyrrolidones, such as povidone (synthetic polymer comprising linear chains of 1-vinyl-2-pyrrolidone), ethoxylated glycerol esters, such as castor oil fatty acids (for example the commercially available Cremophor® EL) and synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers (for example those commercially available as Lutrol®).

[0072] The excipient as used in the present invention is advantageously capable of dissolving dantrolene, such as to obtain a composition of the invention having sufficiently high dantrolene concentration, even without the use of any ionic surfactant. Also, the excipient allows to retain activity of botulinum neurotoxin.

[0073] The aqueous compositions 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.

[0074] Thus, in a preferred aspect of the invention, the aqueous composition does not comprise any organic solvent, 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 compositions with 0.9% NaCI.

[0075] 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 composition be stable and does not precipitate upon storage.

[0076] The aqueous compositions of the present invention are advantageously characterized by a good stability upon storage. Not only 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 composition 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.

[0077] In one aspect of the invention, the aqueous composition according to the present invention is in frozen form. Indeed, freezing the composition increases its chemical and physical stability during storage.

Compositions for use as a medicament

[0078] The compositions of the present invention are advantageously 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.

[0079] In a preferred aspect of the invention the composition of the invention is for use in the treatment of a disease or disorder, wherein the composition of the invention is administered by intramuscular injection.

[0080] In other words, the present invention relates to a method of treatment comprising administering an aqueous composition 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 composition according to the present invention, preferably administering a composition according to the invention by the intramuscular injection.

[0081] In still other words, the present invention relates to the use of a composition 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.

[0082] In still other words, the present invention relates to the use of a composition 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. Aesthetic and/or cosmetic use

[0083] The aqueous compositions of the present invention can advantageously be used in an aesthetic and/or cosmetic (i.e. non-therapeutic) treatment of any condition considered as un-aesthetic and involving muscles tension, activity and/or volume. Thus, the compositions 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.

[0084] Preferably the aqueous composition is used in a in an aesthetic and/or cosmetic treatment by intramuscular, subcutaneous and/or intradermal injection.

Lyophilizates

[0085] Lyophilizates of the above-described aqueous compositions 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 composition 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.

[0086] 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 0.16 mg/mL, preferably from about 0.05 mg/mL to about 0.16 mg/mL, preferably from about 0.05 mg/mL to about 0.15 mg/mL, more preferably about 0.1 mg/mL to about 0.15 mg/mL, upon reconstitution with a carrier suitable for intramuscular injection, based on the total weight of the reconstituted composition. 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 composition 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. [0087] 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.

[0088] 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%.

[0089] In a preferred aspect, the lyophilizate comprises substantially no sodium chloride. Preferably, the lyophilizate is free from sodium chloride.

Kits

[0090] As an alternative to the aqueous composition of the present invention comprising dantrolene and Botulinum neurotoxin, the present invention also provides a kit comprising an aqueous composition of dantrolene and a botulinum neurotoxin in lyophilized form as separate components. In such instance, the lyophilized botulinum is resuspended in the aqueous composition of dantrolene before being injected to a subject.

[0091] Therefore, in an embodiment, the present invention provides a kit comprising an aqueous, optionally frozen, composition according to the invention, or a lyophilizate thereof according to the invention, devoid of botulinum neurotoxin, and a lyophilized botulinum neurotoxin.

[0092] Preferably, the aqueous composition and the lyophilized botulinum neurotoxin of the kit are each provided in a suitable container.

[0093] In a specific embodiment, the kit comprises: a) an aqueous composition comprising dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof, an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol, optionally a buffer and optionally at least one salt that is not a salt of dantrolene, and b) a lyophilized botulinum neurotoxin. [0094] In another specific embodiment, the kit comprises: a) an aqueous saline composition comprising dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof, and b) a lyophilized botulinum neurotoxin.

[0095] Dantrolene and salts thereof, the excipient, botulinum neurotoxin, buffers and salts are as defined in any of the embodiments described above with respect to the aqueous composition.

[0096] The aqueous composition can optionally be provided in frozen form.

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

[0097] The kit of the invention can advantageously be used to prepare an aqueous composition 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 composition in liquid form; c) thawing the dantrolene aqueous composition if it is provided in frozen form, such as to obtain a dantrolene aqueous composition 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 composition 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.

[0098] 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

[0099] A first composition according to the present invention was prepared, comprising the ingredients provided in [Table 1]:

[0100] [Table 1]

1 ) Polyethylene glycol 12-hydoxystearate (Kolliphor® HS 15)

[0101] A Solutol HS15 and L-Histidine solution was prepared according to the following procedure (for 100mL):

- weighting 4.5g of Solutol (Kolliphor HS 15);

- weighting 310.4mg of L-Histidine;

- dissolving the Solutol HS15 and the L-Histidine in 90 mL of NaCI 0.9% solution under agitation in a rotating wheel until complete dissolution (approx. 2 hours);

- adjusting the volume to 100mL with NaCI 0.9%; and

- filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm).

[0102] The obtained formulation was then used to dissolve dantrolene sodium according to the following procedure:

- weighing 15mg of dantrolene sodium powder in glass vial;

- adding 100mL of the Solutol HS15 and L-Histidine solution (previously prepared as described above) to form a suspension;

- vortexing the suspension for 30 seconds;

- agitating on a rotating wheel for 1 hour; - adjusting the pH to 7.4 with HCI 10%;

- filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm); and

- checking that the obtained solution was clear.

[0103] Part of the resulting solution was then used to reconstitute BoNT/A lyophilizate. An amount of 1 .25 mL of the solution was added to a 50U vial of OnabotulinumtoxinA (Allergan) to obtain a final composition comprising 40U/mL of BoNT/A. Both the dantrolene composition and the dantrolene + BoNT compositions are ready for injection for any appropriate indication.

Example 2: compositions according to the invention

[0104] A second composition according to the present invention was prepared, comprising the ingredients provided in [Table 2]:

[0105] [Table 2]

2) Polyethylene glycol 12-hydoxystearate (Kolliphor® HS 15)

[0106] A Solutol HS15 and L-Histidine solution was prepared according to the following procedure (for 100mL):

- weighting 4.5g of Solutol (Kolliphor HS 15);

- weighting 310.4 mg of L-Histidine;

- adjusting the volume to 100mL with NaCI 0.9%; and

- filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm). [0107] The obtained formulation was then used to dissolve dantrolene sodium according to the following procedure:

- weighing 15mg of dantrolene sodium powder in glass vial;

- vortexing the suspension for 30 seconds;

- agitating on a rotating wheel for 1 hour;

- adjusting the pH to 7.4 with HCI 10%;

- filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm); and

- checking that the obtained solution is clear.

Example 3: compositions according to the invention

[0108] A third composition according to the present invention was prepared, comprising the ingredients provided in [Table 3]:

[0109] [Table 3]

1 ) Polyethylene glycol 12-hydoxystearate (Kolliphor® HS 15)

[0110] A Solutol HS15 and L-Histidine solution was prepared according to the following procedure (for 100mL):

- weighting 4.5g of Solutol (Kolliphor HS 15);

- weighting 310.4mg of L-Histidine;

- dissolving the Solutol HS15 and the L-Histidine in 90 mL of water under agitation in a rotating wheel until complete dissolution (approx. 2 hours);

- adjusting the volume to 100m L with water; and - filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm).

[0111] The obtained formulation was then used to dissolve dantrolene sodium according to the following procedure:

- weighing 15mg of dantrolene sodium powder in glass vial;

- adding 100mL of the Solutol HS15 and L-Histidine solution (previously prepared as described above) to the dantrolene to form a suspension;

- vortexing the suspension for 30 seconds;

- agitating on a rotating wheel for 1 hour;

- adjusting the pH to 7.4 with HCI 10%;

- filtering the solution on 0.22pM filter (Millex-GV PVDF 033mm); and

- checking that the obtained solution was clear.

[0112] The resulting solution was dispensed in glass vials (1 .5 mL per vial) and frozen at -20°C for 24h. Lyophilization was then performed for 4 days at -20°C under P = 1 .03 mbar. Finally, vials were stored at 4°C for further use.

Example 4: Effect of various excipients on dantrolene sodium solubility

[0113] Diverse excipients (surfactants or co-solvents) were screened for their ability to solubilize dantrolene and therefore to increase the dantrolene soluble concentration in a final reconstituted formulation comprising dantrolene and botulinum neurotoxin.

[0114] Each vehicle was prepared by solubilizing the corresponding excipient into 0.9% sodium chloride solution at various concentrations. [Table 4] 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. [0115] [Table 4]

[0116] Dantrolene sodium (origin: Abeam; designated as "Dantrolene" in the following examples 3 to 8) 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.

[0117] 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.

[0118] 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 5], 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. [0119] [Table 5]

[0120] 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 5% Solutol HS15. 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 6], [0121] [Table 6]

[0122] The Dantrolene solubility in 0.9% NaCI (0.017 mg/mL) was consistent with the expected one. Lutrol® F68 in 0.9% NaCL did not improve the Dantrolene solubility. In contrast, 5% Solutol HS15, 5% Cremophor® EL and 5% Tween®80, all in 0.9% NaCI improved the Dantrolene solubility, with 0.16 mg/mL, 0.14 mg/mL and 0.18 mg/mL solubilized respectively.

[0123] 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 were 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 7],

[0124] [Table 7]

[0125] In all these tested vehicles, the Dantrolene solubility did not even reach 0.05 mg/mL, indicating that none of these co-solvents is a suitable excipient for better solubilizing Dantrolene.

[0126] In conclusion, these results demonstrate that addition of 5% Solutol HS15 to 0.9% NaCI solution increased Dantrolene solubility, with an almost 10-fold increase of the solubility, compared to the control NaCI solution.

Example 5: Tolerance of dantrolene formulations after intradermal injection

[0127] For tolerance assessment, groups of 3 rats (2 cages) were randomly assigned to each product before injection. Rats were injected intradermally with 5-40 pL of injection solution in the right flank (previously shaved) using a 33-gauge needle (Ref. TSK 33G 0.26x13mm Low Dead Space Al 33013) attached to a Hamilton glass Syringe 100 pL (Ref. 74344). For a given injection volume group, the different products were injected in the same animal, at distance (1 cm) from each other; each injected area for these subgroups was circled with a black marker to facilitate further monitoring. The left flank was not injected. During the injection procedure, the animals were held by one operator and injected by another. No anesthesia was required.

[0128] The rats were observed just after injection, at 0.5, 1 , 2 hours, at day 1 and daily until day 5. Specific attention was paid to any change of skin morphology and signs of inflammation or tissue damage near the point of injection. In this regard, erythema at injection sites were observed and scored as follows:0 = no erythema, 1 = slight erythema (barely perceptible), 2 = erythema (well defined), 3 = necrosis.

[0129] The following compositions were injected: a) Composition A (control): NaCL 0.9% (designated as "saline" in [Fig. 1] and [Fig. 2]), b) Composition B (control): Solutol HS15 and L-Histidine solution in NaCI 0.9% (prepared in the first step of the process described in the first step of Examples 1 and 2) (designated as "F92" in [Fig. 1] and [Fig. 2]); c) Composition C (invention): 15 mg/mL of the dantrolene composition obtained in Example 2 (designated as "0.15mg/mL dantrolene in F92" in [Fig. 1] and [Fig. 2]); and d) Composition D (comparative): 0.15 mg/mL dantrolene formulated in 2- Hydroxypropyl-beta-cyclodextrin (HPBCD) 9% (designated as "0.15mg/mL dantrolene in 9% HPBCD" in [Fig. 1] and [Fig. 2]).

[0130] The plot in [Fig. 1] shows erythema score evolution over time after injection of each of Compositions A to D.

[0131] No erythema could be detected for any of Compositions A, B and C (controls and invention). By contrast, in rats treated with Composition D (comparative), erythema appeared at 2h, and were persistent for up to 5 days. This indicates that Solutol HS15 was well tolerated after intradermal injection, whereas HPBCD was not, at this concentration.

[0132] The plot in [Fig. 2] shows erythema score at day 2, with respect to injected volume, for each of Compositions A to D.

[0133] No erythema could be detected for the Compositions A, B and C (controls and invention), irrespective of the injected volume. By contrast, for Composition D (comparative), injected volumes superior to 5 pL led to erythema. These results comfort the previous conclusion, showing that Solutol HS15 is well tolerated after intradermal injection, much better than HPBCD, which was used in the prior art. Example 6: Compatibility of the compositions of the invention with BoNT/A

[0134] Paresis activity was assessed relying on the Digit Abduction Score assay in rats, as described below.

Tested compositions

[0135] Three compositions were compared: a) Composition E (control): solution of Botulinum neurotoxin (BoNT/A) in 0.9% NaCI (designated as Ona (saline) in [Fig. 3] and [Fig. 4], b) Composition F: solution of Botulinum neurotoxin (BoNT/A) in 0.9% NaCI Solutol HS15 and L-Histidine solution as prepared in the first step of Examples 1 and 2 (designated as "Ona (F92)" in [Fig. 3] and [Fig. 4], c) Composition G (invention): solution of Botulinum neurotoxin (BoNT/A) in the Dantrolene composition of Example 2 (designated as "Ona + Dantrolene (F92)" in [Fig. 3] and [Fig. 4],

Animals

[0136] 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.

[0137] 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

[0138] Animals were injected (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)

[0139] 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.

[0140] 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

[0141] 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.

[0142] 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.

[0143] 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+).

[0144] [Fig. 3] shows that when Botulinum Neurotoxin A (Ona) was reconstituted in a 0.9% NaCI Solutol HS15 and L-histidine solution (Composition F), and further diluted for injection in rat, its activity was similar to when reconstituted in saline (Composition E). Furthermore, the combination of dantrolene with Ona in this formulation (Composition G) was able to enhance Ona activity. This enhancement could be quantified in [Fig. 4] (DAS AUC after 24h, ** = p<0.01 ). Example 7: In-use stability of BoNT/A in the compositions of the invention

[0145] [Fig. 5] shows the in-use stability of Botulinum toxin A (Ona) reconstituted with either a 0.9% NaCI solution (Saline, control), or in a Solutol HS15 + L-Histidine solution in 0.9% NaCI such as prepared in the first step of examples 1 and 2, or with the composition of the invention prepared in Example 2. These results demonstrate that Botulinum neurotoxin activity was stable during the first 4 hours, and then decreased, in both the reference and Solutol HS15 formulations.

[0146] This stability of 4 hours post reconstitution is sufficient to ensure appropriate time for injection to a subject after reconstitution of the lyophilizate in at the practitioner's facilities.

Example 8: Stability of Solutol-based lyophilizate

[0147] Lyophilizates were produced as described in Example 3. To estimate their stability over time, the lyophilizates were stored at 40°C for 4 weeks. At 7, 14, 21 and 28 days, lyophilizates were reconstituted by adding 1 .5 mL of saline solution in the glass vial, and by gently swirling the vial. Rapid dissolution of the lyophilizate cake was observed. Dantrolene concentration was dosed by HPLC. Two replicates were used for each time point experiment. The concentration of Dantrolene in the solution used for the preparation of the lyophilizate (see Example 3) was used as a reference.

[0148] The maximal dantrolene loss observed in reconstituted lyophilizate, as compared to dantrolene concentration in solution before lyophilization, was 6.3% (see [Fig. 6]). As a conclusion, Solutol HS15-based lyophilizates containing dantrolene are stable for 4 weeks at 40°C.

Example 9: Compatibility of the diverse excipients with BoNT/A

[0149] To test the compatibility of different excipients with BoNT/A, each vehicle was prepared by solubilizing the corresponding excipient into 0.9% sodium chloride solution at various concentrations. Table 8 shows for each excipient the excipient mass used for a final vehicle volume of 10 mL and the excipient concentration. The following vehicles were prepared: [0150] Table 8:

[0151 ] In accordance with Example 1 , an amount of 1 .25 mL of the vehicle was added to a 50U vial of OnabotulinumtoxinA (Allergan) to obtain a final composition comprising 40U/mL of BoNT/A.

[0152] DAS of BoNT/A in the different vehicles was assessed as described in Example 6. The results are provided in [Fig. 7], 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 Solutol HS15 had low impact on Ona activity.

Claims

[Claim 1] An aqueous composition comprising dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof and an excipient comprising at least one hydroxy fatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol, and wherein the aqueous composition optionally comprises a buffer and/or at least one additional salt that is not a salt of dantrolene.

[Claim 2] The aqueous composition according to [claim 1], further comprising Botulinum neurotoxin.

[Claim 3] The aqueous composition according to any one of [Claim 1] or [Claim 2], 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 pharmaceutically acceptable derivative of dantrolene is defined as a chemical derivative of dantrolene which retains dantrolene activity, preferably wherein the pharmaceutically acceptable derivative of dantrolene is selected from azumolene, pCI-dantrolene and o-nitrodantrolene. 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 the hydroxy fatty acid ester of polyethylene glycol is a hydroxy fatty acid ester of polyethylene glycol wherein the fatty acid ester consists of an aliphatic chain of 13 to 21 carbon atoms, such as myristate, palmitate, stearate and arachidate acid, preferably stearate.

[Claim 4] The aqueous composition according to any one of [Claim 1] to [Claim 3], comprising:

Dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof in an amount of about 0.025 mg/mL to about 0.16 mg/mL, preferably about 0.05 mg/mL to about 0.16 mg/mL, preferably about 0.05 mg/mL to about 0.15 mg/mL, more preferably about 0.1 mg/mL to about 0.15 mg/mL, based on the total volume of the composition; botulinum neurotoxin in a therapeutically effective amount, preferably in an amount of from about 0,1 U/ml to about 4000 U/mL, preferably 1 U/ml to about 400 U/mL, more preferably from about 5 U/ml to about 200 U/ml, most preferably from about 20 U/ml to about 100 U/ml, based on the total volume of the composition; and/or the excipient comprising the hydroxyfatty acid ester of polyethylene glycol, optionally in admixture with free polyethylene glycol, in an amount of at least about 10 mg/ml, preferably at least 20 mg/ml, more preferably at least 40 mg/ml, such as about 10 mg/ml to about 60 mg/ml, preferably about 20 mg/ml to about 60 mg/ml, more preferably about 40 mg/ml to about 60 mg/ml, most preferably 40 mg/ml to about 55 mg/ml based on the total volume of the composition.

[Claim 5] The aqueous composition according to any one of [Claim 1] or [Claim 4], wherein the hydroxy fatty acid ester of polyethylene glycol is selected from 3-hydroxystearate of polyethylene glycol, 12-hydroxystearate of polyethylene glycol and 17-hydroxystearate of polyethylene glycol.

[Claim 6] The aqueous composition 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, phosphate-citrate buffer, succinate buffer and mixtures thereof.

[Claim 7] The aqueous composition according to any one of [Claim 1] to [Claim 6], wherein the at least one salt that is not a salt of dantrolene is preferably present in the composition 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 composition 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] The aqueous composition according to any one of [Claim 1] to [Claim 7], wherein the composition 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] The aqueous composition according to any one of [Claim 1] to [Claim 8], wherein the composition : a) does not comprise any polyvinylpyrrolidones, such as povidone (synthetic polymer comprising linear chains of 1-vinyl-2-pyrrolidone); b) does not comprise any ethoxylated glycerol esters, such as castor oil fatty acids; c) does not comprise any synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers; and/or d) does not comprise any organic solvent.

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

[Claim 11] The aqueous composition for use according to [Claim 10], for use in the treatment of muscular diseases or disorders.

[Claim 12] The aqueous composition 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 13] Non-therapeutic use of an aqueous composition according to any one of [Claim 1 ] to [Claim 9] in an aesthetic and/or cosmetic 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 14] A lyophilizate of an aqueous composition according to any one of [Claim 1 ] to [Claim 9],

[Claim 15] A kit comprising a) A dantrolene composition selected from i. An aqueous composition comprising dantrolene, a pharmaceutically acceptable derivative thereof or a pharmaceutically acceptable salt thereof, at least one hydroxy fatty acid ester of polyethylene glycol, optionally a buffer and optionally at least one salt that is not a salt of dantrolene, wherein such aqueous composition is either in liquid or in frozen form; and ii. a lyophilizate of an aqueous composition of i.; and b) a lyophilized botulinum neurotoxin.

[Claim 16] A method of providing an aqueous composition according to any one of [Claim 1] to [Claim 9] from the kit of [Claim 15], comprising the steps of: a) providing a kit of [Claim 15], b) reconstituting the dantrolene composition if such composition is a lyophilizate, such as to obtain a dantrolene aqueous composition in liquid form; c) thawing the dantrolene aqueous composition if it is provided in frozen form, such as to obtain a dantrolene aqueous composition 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 composition according to any one of [Claim 1] to [Claim 9]; e) optionally storing the aqueous composition obtained at 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 17] A method for treating a disease or disorder in a subject comprising administering an aqueous composition according to any one of [Claim 1] to [Claim 9] to the subject.

[Claim 18] The method according to [claim 17], wherein the disease or disorder is a muscular disease or disorder.

[Claim 19] The method according to [Claim 17] for treating a 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; and/or iv) a dermatological disease or disorder; and/or for treating pain associated with such disease or disorder.

[Claim 20] Use of an aqueous composition according to any one of [Claim 1] to [Claim 9] for the manufacture of a medicament.

[Claim 21] The use according to [Claim 20], wherein the medicament is for the treatment of a muscular disease or disorder.

[Claim 22] The use according to [Claim 20] wherein the medicament is for the treatment of a 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; and/or iv) a dermatological disease or disorder; and/or for treating pain associated with such disease or disorder.