ES3041383A2 - 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

Aqueous solutions of dantrolene

Technical field

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

Background

Dantrolene is an approved medication used as a muscle relaxant and for the treatment of malignant hyperthermia. For indications associated with the muscle relaxant properties of dantrolene but requiring local administration, it would be particularly useful to provide dantrolene formulations optimized for intramuscular administration.

Solutions for intramuscular administration are normally homogeneous aqueous saline solutions. However, dantrolene is poorly soluble in water and even less soluble in aqueous saline solutions.

At high concentrations of dantrolene, aqueous suspensions of dantrolene are typically used in the prior art, rather than solutions, and the dantrolene resuspension procedure is known to be lengthy and tedious. Suspensions are unsuitable for intramuscular injection. Therefore, there is a need to improve the formulation of dantrolene compositions so that they are better optimized for intramuscular injection.

Given the need for intramuscular injection, it is even more desirable to provide the dantrolene formulation as a ready-to-use injectable solution. This requires chemical stability of the formulation over a longer shelf life compared to currently used compositions prepared by suspending dantrolene in an aqueous solution shortly before use. Although such ready-to-use compositions are more difficult to formulate due to the need for a longer shelf life, they are particularly advantageous because they reduce the workload for healthcare professionals during treatment, are easier to use, and reduce the risk of incorrect dosage due to errors in the reconstitution procedure.

The pH of unbuffered dantrolene solutions is primarily determined by the dantrolene concentration, with pH increasing with increasing concentration. Furthermore, in aqueous environments with both basic and acidic pH, dantrolene undergoes hydrolysis, leading to a reduction in its activity. Consequently, aqueous dantrolene solutions are unstable over time. Therefore, for applications where dantrolene remains in solution for extended periods, such as ready-to-use compositions, efforts are made to improve the chemical stability of dantrolene.

Dantrolene formulations have been extensively researched in the prior art. Attention has been focused on compositions for systemic administration, particularly the oral and intravenous routes, as these reflect the approved routes for the main medical indications of dantrolene. However, oral or intravenous compositions are often unsuitable for intramuscular administration. Therefore, prior art dantrolene formulations are not adapted for intramuscular administration.

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

Aqueous suspensions are unsuitable for intramuscular injections because, once injected, the suspension particles become confined in the fibrous or membranous tissues between the muscle fibers, forming agglomerates that lead to poor absorption of the active ingredient.

Highly dilute solutions are also unsuitable for intramuscular injections because larger volumes must be administered for a given dose, leading to greater dispersion of the injected composition. A larger volume is not a problem for intravenous injection, where the injected volume is diluted in the bloodstream. However, it is a problem for intramuscular injection because the dispersion of a larger volume reduces the precision of local delivery of the active ingredient, causing diffusion into the surrounding tissue. This, in turn, leads to a decrease in the duration and magnitude of the effect in the target tissue and a potential for unwanted effects outside the target area. This is especially critical for aesthetic applications and medical indications that require precise delivery to specific muscles (such as the inhibition of spasms in the upper extremities).

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

In order to optimize the formulation of compositions comprising dantrolene, there is therefore a need to identify compositions that are optimized for intramuscular injection, that allow for solubilization and, preferably, that guarantee chemical stability over time.

Various adjuvants, such as various carbohydrates, have been used in aqueous dantrolene compositions. For example, WO2017067980A1 disclosed that the 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 molar ratio of dantrolene to cyclodextrin is 1:2 to 1:30, and the pH is above 7, preferably 7.5 to 10.5, and preferably 8.5 to 9.5. This document does not disclose any saline solution of dantrolene. Since saline compositions are preferred to non-saline solutions for intramuscular injection (as opposed to intravenous administration), the compositions in this document are disadvantageous for administration by this route.

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. Improved stability and solubility of dantrolene are claimed. The ratio of dantrolene to HPBCD is 1:1 to 1:12. The pH is 8.5 to 10.5. This document does not disclose any saline solution of dantrolene. Since saline compositions are preferred to non-saline solutions for intramuscular injection (as opposed to intravenous administration), the compositions in this document are disadvantageous for administration by this route.

More recently, WO2020049670A1 aimed to improve upon WO2017067980A1. It also addresses compositions for intravenous injection and discloses compositions with a lower dantrolene-to-cyclodextrin ratio of 1:0.5 to 1:2, purportedly reducing cyclodextrin-associated nephrotoxicity. The pH is also alkaline, in the range of 9.0 to 10.5. In conjunction with WO2017067980A1, this document suggests that decreasing the dantrolene-to-cyclodextrin ratio is advantageous. This document does not disclose any saline solutions of dantrolene. Since saline compositions are preferred to non-saline solutions for intramuscular injection (as opposed to intravenous administration), the compositions in this document are disadvantageous for administration by this route.

Jansen A.C.A. et al., “Some physical-chemical properties of dantrolene and two of its analogues”, International Journal of Pharmaceutics, vol. 75, no. 02-3 (1991), pages 193-199, reports the solubility of dantrolene in aqueous saline solutions at various pH levels and temperatures. It also indicates that the solubility of dantrolene increases in the presence of cyclodextrins, suggesting complex formation. This publication focuses on the theoretical evaluation of the solubility, lipophilicity, and complexation of dantrolene with different cyclodextrins (α-cyclodextrin, p-cycloderyrin, and γ-cyclodextrin) and does not investigate the role of the dantrolene-to-cyclodextrin ratio. The materials and methods section does not provide the total concentration of dantrolene used, but only the total concentration of cyclodextrin and the concentration of dantrolenecyclodextrin complexes based on the total cyclodextrin concentration (maximum 5%). The binding constant was calculated according to the formula

K = [complex] / [free dantrolene] x [total CD]

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

Jansen et al., 1991, cited above, refer to another publication by the same author, related to a study on the bioavailability of dantrolene 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 book series Advances in Inclusion Science (AIS, volume 5)). In this publication, the ratio of dantrolene to cyclodextrin can be calculated from the Materials and Methods section as 1:15. Jansen et al., 1991, aim to investigate the physicochemical properties of dantrolene as part of such a study in rats. Therefore, in the absence of any indication to the contrary, there is no reason to believe that Jensen used different total concentrations of dantrolene in his second study, particularly knowing that he used total concentrations of cyclodextrin that closely matched those used in his 1988 study in rats.

Furthermore, it would be desirable to optimize dantrolene formulations to allow the addition of one or more additional active ingredients that provide benefits in combination with dantrolene. In such a case, an additional challenge is to provide a formulation in which each of the different active ingredients is sufficiently soluble and, preferably, also chemically stable.

For example, WO2020/254690A1 describes compositions comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, preferably dantrolene, and botulinum neurotoxin. The combination of these active ingredients has been shown to accelerate the onset of action and/or prolong the duration of action and/or potentiate the intensity of the effect of botulinum neurotoxin. Therefore, it would be advantageous to provide optimized solutions for the co-administration of dantrolene and botulinum neurotoxins.

Botulinum neurotoxins are toxins that disrupt neuronal transmission and act preferentially on cholinergic neurons; therefore, one of the main consequences of botulinum neurotoxin action is muscle relaxation due to the decreased release of neurotransmitters by cholinergic neurons. Botulinum neurotoxins are produced in complex form by the anaerobic, 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 have been identified, 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). Currently, eight, eight, twelve, and nine BoNT/A, BoNT/B, BoNT/E, and BoNT/F subtypes are known, respectively. Types A, B, E, and F are toxic to humans, while types C, D, and G more often cause toxicity in animals such as birds, horses, cattle, and primates. Type X was only recently described (Zhanget et al., Nature Communications, Volume 8, 14130 (2017)), following a report of infant botulism in Japan in 1995, and its toxicity appears to be limited in the species tested so far.

Botulinum neurotoxin complexes are present as high-molecular-weight protein complexes comprising two components: the enzymatically active neurotoxin component and an associated non-toxic bacterial protein component, which can be considered a coat protein containing both hemagglutinin and non-hemagglutinin proteins. The molecular weight of botulinum toxin complexes varies among different botulinum toxin serotypes, ranging from approximately 300 kDa to approximately 900 kDa; botulinum neurotoxins devoid of the non-toxic bacterial protein component have a molecular weight of approximately 150 kDa. Regarding aesthetic or therapeutic applications, 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 with a molecular weight of approximately 150 kDa for all known botulinum neurotoxin serotypes. This single-chain precursor is activated by proteolytic cleavage to generate a two-chain protein linked by disulfide bonds. The approximately 50 kDa light chain protein contains the catalytic domain and is a zinc-containing metalloprotease that acts as a zinc endopeptidase. The approximately 100 kDa heavy chain protein comprises a translocation domain and a receptor-binding domain. The heavy chain mediates binding to presynaptic cholinergic nerve terminals, particularly the presynaptic portion of the motor endplate or neuromuscular junction, and the internalization of the neurotoxin into the cell.

The natural property of botulinum neurotoxins to reversibly block the release of acetylcholine from presynaptic nerve endings in muscles and other effector tissues, such as the secretory glands of the sympathetic and parasympathetic nervous systems, makes them an important therapeutic option in numerous fields for controlling overstimulation of muscles (and other effector tissues) and the associated pain. For example, BoNT/A is currently used therapeutically in the following areas: movement disorders, especially for managing spasticity and dystonia, where BoNT/A has been well-documented as effectively relieving dystonia-related pain; urinary disorders, particularly overactive bladder; and secretory disorders, specifically hyperhidrosis and sialorrhea. In addition, botulinum neurotoxins are currently used in various cosmetic applications, such as smoothing facial lines or reducing glabellar, frown, and periorbital lines.

Despite sharing similar metalloprotease activity involved in the cleavage of proteins from the SNARE complex, botulinum neurotoxins can have different onsets and/or durations of action depending on their serotypes. For example, while BoNT/A and BoNT/F induce complete localized hind limb paralysis in mice within 2 days, BoNT/E produces the same effect within 24 hours. However, the duration of neuromuscular paralysis induced by BoNT/A 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 humans, although on a different timescale: BoNT-A paralysis is generally seen within a week and can last 3 to 4 months (Davletov et al., Trends in Neurosciences 28 (2005); pp. 446-452). In contrast, BoNT-E shows a faster onset (24 hours) but a much shorter duration (less than 1 month) in humans (Yoelin et al.,Plast Recontr Surg 142 (2018); pp. 847e-855e).

Due to its unique profile and activity in humans, most attention has focused on BoNT/A. Since 1991, several commercially available type A botulinum neurotoxins have been approved by the U.S. Food and Drug Administration (FDA). Available forms of BoNT/A include Botox®/Vistabel® (Allergan), Dysport®/Azzalure® (Ipsen Biopharm), and Xeomin®/Bocouture® (Merz Pharmaceuticals), among others. The only other commercially available BoNT serotype is BoNT/B Myobloc®/Neurobloc® (Solstice Neurosciences); however, its medical/cosmetic use is anecdotal. Improving the characteristics of BoNT remains an important goal to achieve in order to better meet medical needs.

In cases where dantrolene is combined with botulinum neurotoxin, there is a need to provide formulations comprising dissolved dantrolene and botulinum neurotoxin that allow the dantrolene to be dissolved at relatively high concentrations, thus enabling appropriate dosage of dantrolene in a small volume suitable for intramuscular injection. Ideally, these formulations should also have good stability for both dantrolene and botulinum neurotoxin to maintain high activity of both active ingredients. Furthermore, it is desirable that the effects of both dantrolene and botulinum neurotoxin be maintained, and preferably that synergy between them be achieved. It is also preferable to provide solutions ready for intramuscular injection.

Formulating compositions that combine dantrolene and botulinum neurotoxin is particularly challenging, as botulinum neurotoxin is only approved for intramuscular injection and several studies have reported that it is unstable at alkaline pH. Therefore, formulations with a pH above 7 are not optimal for botulinum neurotoxin administration. Consequently, the aim is to adapt prior art compositions to provide dantrolene and botulinum neurotoxin suitable for intramuscular injection, where the botulinum neurotoxin retains its activity.

The present invention aims to solve some or all of the problems mentioned above.

Summary of the invention

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, cyclodextrin or a derivative thereof, wherein the molar ratio of dantrolene or a pharmaceutically acceptable salt of dantrolene to cyclodextrin or a derivative thereof is 1:at least 40.

The composition of the first aspect is useful per se for administration in any indication where dantrolene is to be administered as an aqueous solution. This composition is advantageous due to the improved solubility of dantrolene resulting from the selection of optimal dantrolene-to-cyclodextrin ratios. The buffer is optional, as it does not affect solubility but only enhances the chemical stability of the dantrolene.

In a particular embodiment of this first aspect, the composition also comprises botulinum neurotoxin.

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 disease or disorder of skeletal muscle, a disease or disorder of smooth muscle and/or a disease or disorder of cardiac muscle cells.

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.

In a fourth aspect, the invention relates to a lyophilized product of an aqueous solution of the first or second aspect.

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

In a sixth aspect, the invention relates to a method for providing an aqueous solution according to the first or second aspect of the sixth aspect kit, comprising the steps of:

a) provide a fifth aspect kit

b) reconstitute the dantrolene composition if such composition is a lyophilized product, so as to obtain an aqueous solution of dantrolene in liquid form

c) thawing the aqueous dantrolene solution composition if such composition is supplied in frozen form, so as to obtain an aqueous dantrolene solution in liquid form

d) resuspend the lyophilized botulinum neurotoxin in the liquid aqueous composition of dantrolene provided in the kit or obtained at the end of steps b) or c), so that an aqueous solution is formed according to the first or second aspect;

e) Optionally, store the aqueous composition obtained at the end of step d) at a temperature of approximately 2 °C to approximately 8 °C for approximately 1 hour to approximately 7 days.

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, cyclodextrin, or a derivative thereof.

Brief description of the drawings

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

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% NaCl (reference sample) at 4 °C (black circles) and 20 °C (black triangles), 0.5 mg/ml dantrolene formulation in a solution of “30% HPBCD L-histidine 10 mM” in 0.9% NaCl (final pH 7.4) at 4 °C (dark gray circles with dashed line) and 20 °C (dark gray triangles with dashed line), 0.5 mg/ml dantrolene formulation in a solution of “30% HPBCD L-histidine 10 mM EDTA 0.1 mM” in 0.9% NaCl (final pH of 7.4) at 4 °C (light gray triangles with dashed line) and at 20 °C (light gray triangles with dashed line).

Fig. 3. Overall paresis activity efficacy of onabotulinum toxin A (Ona) tested in 5 formulations in the DAS assay in rats: 0.9% NaCl (saline, reference sample), 5% Cremophor® EL, 5% Tween® 80, 30% HPBCD, 5% HPBCD. Overall efficacy is measured as the area under the paresis curve after 24 h (AUC24+).

Fig. 4. Overall paresis activity efficacy of onabotulinum toxin A (Ona) combined with dantrolene (FTP-501) tested in 5 formulations in the DAS assay in rats: 0.9% NaCl (saline, reference sample), 1.8% Cremophor® EL, 5% Tween® 20, and 2.2% HPBCD. Ona alone formulated in 0.9% NaCl or 5% Tween® 20 is provided as a reference. Overall efficacy is measured as the area under the paresis curve after 24 h (AUC24+).

Fig. 5. Overall paresis activity efficacy of onabotulinum toxin A (Ona) reconstituted in 0.9% NaCl (saline, reference sample), and incubated at room temperature before injection in rats for the DAS assay: extemporaneous, 1 h, 2 h, 4 h, 6 h, 24 h. Overall efficacy is measured as the area under the paresis curve, after 24 h (AUC24+).

Fig. 6. Overall paresis activity efficacy of onabotulinum toxin A (Ona) reconstituted with a 0.5 mg/ml dantrolene formulation in a solution of “HPBCD 30% L-histidine 10 mM EDTA 0.1 mM” in 0.9% NaCl (final pH 7.4), and incubated at room temperature before injection in rats for the DAS assay: extemporaneous, 1 h, 2 h, 4 h, 6 h, 24 h. Overall efficacy is measured as the area under the paresis curve, after 24 h (AUC24+).

Fig. 7. Overall paresis activity efficacy of onabotulinum toxin A (Ona) reconstituted with a 0.1 mg/ml dantrolene formulation in a solution of “HPBCD 6% L-histidine 2 mM EDTA 0.02 mM” in 0.9% NaCl (final pH 7.4), and incubated at room temperature prior to injection in rats for the DAS assay: extemporaneous, 1 h, 2 h, 4 h, 6 h, 24 h. Overall efficacy is measured as the area under the paresis curve, after 24 h (AUC24+).

Fig. 8. Overall paresis activity efficacy of onabotulinum toxin A (Ona) reconstituted with a 0.5 mg/ml dantrolene formulation in a solution of “HPBCD 30% L-histidine 10 mM EDTA 0.1 mM” in 0.9% NaCl (final pH 7.4), and incubated at 4 °C prior to 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 24 h (AUC24+).

Fig. 9. Dantrolene concentration in the solution obtained by reconstituting a lyophilized “0.5 mg/ml dantrolene in 30% HPBCD 10 mM L-histidine (final pH 7.4)” with 1.5 ml of 0.9% NaCl, and incubating said solution for 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours at 4 °C. The concentration is measured by UV dosing with (black) or without (gray) filter solutions.

Fig. 10. Dantrolene concentration in the solution obtained by reconstituting a lyophilized solution of “0.5 mg/ml dantrolene in 30% HPBCD 10 mM L-histidine (final pH 7.4)” with 1.5 ml of 0.9% NaCl, and incubating this solution for 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours at room temperature. The concentration is measured by UV dosing with (black) or without (gray) filter solutions.

Detailed description

Aqueous solutions

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 the dantrolene and, if present, the botulinum neurotoxin, exhibit satisfactory chemical stability, such solutions being particularly suitable for intramuscular injection.

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 molar ratio of dantrolene or a pharmaceutically acceptable salt of dantrolene to cyclodextrin or a derivative thereof is at least 1:40, and wherein the aqueous solution optionally comprises a buffer and/or at least one additional salt. Surprisingly, it was found that solutions of dantrolene or a pharmaceutically acceptable salt thereof comprising a high molar ratio of cyclodextrin or a derivative thereof to dantrolene exhibit significantly less dantrolene crystallization after prolonged storage in liquid form compared to solutions having a lower molar ratio of cyclodextrin or a derivative thereof to dantrolene.

More specifically, the ratio of dantrolene or a pharmaceutically acceptable salt thereof to cyclodextrin or a derivative thereof is approximately 1:40 to approximately 1:3000, preferably approximately 1:50 to approximately 1:700, more preferably approximately 1:55 to approximately 1:350, more preferably approximately 1:60 to approximately 1:185; most preferably approximately 1:60 to approximately 1:175.

It has been shown that the solubility of dantrolene decreases drastically in aqueous saline solutions compared to aqueous non-saline solutions, and particularly in aqueous NaCl solutions. Therefore, prior art compositions comprising dantrolene and salts in water are suspensions rather than solutions and are thus unsuitable for intramuscular injection. 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.

Dantrolene (IUPAC name 1-[(E)-[5-(4-nitrophenyl)furan-2-yl]methylidenamino]imidazolidin-2,4-dione) is an inhibitor of Ca2+ release through ryanodine receptor (RYR) channels and acts as a muscle relaxant. Dantrolene or any of its pharmaceutically acceptable salts are suitable for the purposes of the present invention. A dantrolene salt is defined as a deprotonated form of dantrolene (forming a dantrolene anion) with a cationic counterion. Such a 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 alkylammonium, polyalkylammonium, arylammonium, or ammonium cations from physiologically acceptable amino compounds, such as, for example, arginine, lysine, meglumine, tromethamine, choline, benzyltrimethylammonium, tetramethylammonium, N-methylpyridinium, tetrabutylammonium, 2-(2,3-dihydroxy-1-propylamino)quinolizinium, quinolizinium, 2-carbonyl-1-methylpyridinium, 2,3-dimethyl-1-phenyl-4-trimethylammonium-3-pyrazolin-5-one, dimethylammonium, or 1,3-dimethylimidazolium, and 2-(1-hydroxy-2-methyl)propyltrimethylammonium. 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 more preferred aspect, the pharmaceutically acceptable dantrolene salt is sodium dantrolene heptahydrate.

Dantrolene or a pharmaceutically acceptable salt thereof is preferably present in an amount of approximately 0.025 mg/ml to approximately 1.5 mg/ml, preferably from 0.1 mg/ml to approximately 1.1 mg/ml, more preferably from approximately 0.25 mg/ml to approximately 0.6 mg/ml, and most preferably from approximately 0.45 mg/ml to 0.5 mg/ml. Such concentrations of dantrolene are advantageous because they provide long-term physical stability to the composition.

In a preferred aspect, the composition further comprises botulinum neurotoxin. “Botulinum neurotoxin” (BoNT) means at least one of the eight different naturally occurring botulinum neurotoxin serotypes, such as types A, B, C, D, E, F, G, and X, as well as modified, recombinant, hybrid, and chimeric botulinum neurotoxins. The terms botulinum neurotoxin and botulinum toxin are equivalent and may be used interchangeably. The term “botulinum toxin complex” or “toxin complex,” as used herein, refers to the approximately 150 kDa botulinum toxin protein molecule (belonging to any one of the botulinum toxin serotypes A–G, X), potentially together with associated endogenous non-toxic proteins (i.e., hemagglutinin protein and non-hemagglutinin protein produced by Clostridium botulinum bacteria). Note, however, that the botulinum toxin complex need not be derived from Clostridium botulinum bacteria as a unitary toxin complex. For example, botulinum toxin or modified botulinum toxin may first be prepared recombinantly and then combined with the non-toxic proteins. Recombinant botulinum toxin may also be purchased (e.g., from List Biological Laboratories, Campbell, CA).

A mutation in the coding sequence of a botulinum neurotoxin (BoNT) that introduces one or more amino acid substitutions is called a modified BoNT. A “modified” botulinum neurotoxin is a compound that has botulinum toxin activity but contains one or more chemical or functional alterations in any part or any amino acid chain compared to native, naturally occurring, or recombinant botulinum toxins. For example, botulinum toxin may be a modified neurotoxin, meaning a neurotoxin that has at least one of its amino acids deleted, modified, or substituted, compared to a native form. For instance, botulinum toxin may be modified to, for example, improve its properties or reduce unwanted side effects, while retaining the desired botulinum toxin activity. Botulinum toxin can also be a portion of the overall molecule that has been shown to have the necessary botulinum toxin activity, and in such a case, it can be used per se or as part of a combined or conjugated molecule, for example, a fusion protein. In such a case, the portion or fragment of botulinum neurotoxin might be, for example, a 50 kDa light chain (LC) of the toxin. Alternatively, botulinum toxin can be in the form of a botulinum toxin precursor, which may be non-toxic in itself, for example, a non-toxic zinc protease that becomes toxic upon proteolytic cleavage. “Hybrid and chimeric BoNT” refers to the mixture of heavy and light chain domains from different BoNT serotypes or subtypes. Hybrids and chimeric variants within BoNT serotypes and subtypes can be naturally occurring, such as BoNT/FA and BoNT/CD, or recombinant variants of BoNT. A recombinant botulinum neurotoxin may have its light chain and/or heavy chain produced recombinantly by a non-clostridial species.

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 chains of botulinum neurotoxin of type A, B, E. According to a more preferred embodiment, the botulinum neurotoxin used in the composition of the invention is of type A.

The botulinum neurotoxin is preferably present in a therapeutically effective amount, preferably in an amount from approximately 0.1 U/ml to approximately 4000 U/ml, preferably from 1 U/ml to approximately 400 U/ml, more preferably from approximately 5 U/ml to approximately 200 U/ml, most preferably from approximately 20 U/ml to approximately 100 U/ml.

Cyclodextrin and its derivatives are collectively referred to herein as “cyclodextrins.” Cyclodextrins are a family of cyclic oligosaccharides consisting of a macrocyclic ring of glucose subunits linked by α-1,4 glycosidic bonds. Cyclodextrins are produced from starch by enzymatic conversion. There are three different forms of cyclodextrins: α-cyclodextrins, β-cyclodextrins, and γ-cyclodextrins. A cyclodextrin derivative is defined herein 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)alkyl groups. In this document, cyclodextrins also refer to cyclodextrin salts or cyclodextrin derivatives, for example, sodium salt of cyclodextrin sulfate.

Examples of preferred cyclodextrins include P-cyclodextrin, 2,6-dimethyl-P-cyclodextrin, 2-hydroxyethyl-P-cyclodextrin, 2-hydroxyethyl-y-cyclodextrin, 2-hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-y-cyclodextrin, (2-carboxymethyl)propyl-P-cyclodextrin, sulfonylutrit-p-cyclodextrin, 2-hydroxypropyl-p-cyclodextrin, sulfonylutrit ether-p-cyclodextrin, and 2-hydroxyethyl-p-cyclodextrin. In a particularly preferred embodiment, the at least one cyclodextrin is selected from the group consisting of (3-cyclodextrin, 2,6-dimethyl-|3-cyclodextrin, 2-hydroxyethyl-|3-dodextrin, 2-hydroxyethyl-Y-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, (2-carboxymethoxy)propyl-|3-cyclodextrin, sulfobutyl ether-|3-cyclodextrin and, preferably, 2-hydroxypropyl-|3-cyclodextrin (HPBCD).

Cyclodextrin or a derivative thereof is preferably present in an amount of approximately 4% (w/v) to approximately 40% (w/v), preferably from approximately 10% (w/v) to approximately 35% (w/v), more preferably from approximately 10% (w/v) to approximately 30% (w/v), for example, from approximately 25% (w/v) to approximately 32% (w/v) or from approximately 28% (w/v) to approximately 32% (w/v). The inventors identified that such unexpectedly high concentrations of cyclodextrin or cyclodextrin derivatives were necessary to achieve physical stability when the salts are present in aqueous solution.

In a preferred embodiment, the aqueous composition of the present invention comprises a buffer. The buffer is not strictly necessary, as it does not affect the solubility of dantrolene or any of its salts. However, the buffer is particularly useful for reducing the alkalinity of the composition in the presence of high concentrations of dantrolene or its salts. Reducing alkalinity through the presence of a buffer is advantageous because it allows the use of high concentrations of dantrolene without compromising the chemical stability of this active ingredient. Indeed, dantrolene hydrolyzes under alkaline conditions. The presence of a buffer as described herein significantly reduces the hydrolysis of dantrolene. Therefore, compositions of the invention comprising a buffer as described herein can be stored for longer periods before administration, making such compositions much more practical for healthcare professionals. Furthermore, the reduction in pH improves the convenience and safety of intramuscular injection.

Any pharmaceutically acceptable buffer may be used. Preferably, the buffer is a buffer included in the pharmacopoeia. 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, preferably in the range of 6 to 8, and more preferably from 6.5 to 7.5.

The buffer is preferably present at a concentration of approximately 0.1 mM to approximately 20 mM, preferably from approximately 5 mM to approximately 15 mM, and more preferably approximately 10 mM. In a more preferred embodiment, the buffer is a histidine buffer and such buffer is present at a concentration of approximately 10 mM, or the buffer is phosphate and such buffer is present at a concentration of approximately 13 mM. Such relatively low buffer concentrations are advantageous because they allow for better solubility of dantrolene compared to higher buffer concentrations.

At least one salt, other than a dantrolene salt, cyclodextrin salt, or derivative thereof, is preferably present in an amount of approximately 0.45% to approximately 1.8%, more preferably 0.85% to approximately 0.95%, most preferably approximately 0.9% by weight, based on the total weight of the solution. This at least one salt, other than a dantrolene salt, cyclodextrin salt, or derivative thereof, is preferably an inorganic salt, preferably an alkali metal salt. 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 because they allow for proper solubilization, stability, and activity of BoNT.

In one particular aspect, the solution of the present invention comprises from 0.07 to 1.2 mg/ml, preferably from 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 at least one salt other than a dantrolene salt, cyclodextrin, or a derivative thereof, as defined above.

The solution of the present invention may optionally comprise one or more additional pharmaceutically acceptable excipients or adjuvants. 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 injection site. 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, hydroxyethyl starches, glycine, gelatin, calcium gluconeglucoheptonate, and mixtures thereof.

The present invention can have an osmolarity of between 350 and 700 mmol/kg, preferably between 400 and 650 mmol/kg.

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 fatty acids from castor oil (e.g., 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

d) organic solvents; and/or

e) particles that are larger than 5 nm after storage for one day at room temperature.

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

The aqueous solutions of the present invention are advantageously suitable for intramuscular injection and are therefore appropriate for this route of administration. This is advantageous because intramuscular administration is the primary route of administration for botulinum neurotoxin.

Therefore, in a preferred embodiment of the invention, the aqueous solution is free of organic solvents, because organic solvents are unsuitable for intramuscular injection. Furthermore, organic solvents are incompatible with the BoNT formulation, which is currently only approved for administration as an aqueous solution with 0.9% NaCl.

Given its use for intramuscular injection, it is also desirable to avoid the presence of undissolved particles, and in particular large undissolved particles, which agglomerate in tissues and are poorly bioavailable. It is also advantageous that the solution be stable and not precipitate after storage. Therefore, in another preferred aspect, the aqueous solution of the invention is free of particles larger than 5 nm after storage for one day at room temperature.

Particle size is defined herein as the equivalent mean diameter, as measured by dynamic light scattering. The mean is defined by the mass.

The aqueous solutions of the present invention are advantageously characterized by good stability after storage. Not only is physical stability achieved, but also chemical stability, since the degradation of dantrolene is limited to an acceptable level due to the adjustment of the composition components, as previously disclosed. Therefore, preferably, the concentration of dantrolene in the aqueous solution of the invention is maintained at acceptable levels after storage, as chemical degradation of the dantrolene is prevented. In a preferred embodiment, the concentration of (undegraded) dantrolene decreases by no more than approximately 12%, preferably no more than approximately 10%, more preferably no more than approximately 8%, and most preferably no more than approximately 6% after storage for at least 3 months at 4°C in liquid form.

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

The compositions of the present invention are advantageously suited for use in therapy or, in other words, for use as a medicament. Preferably, they are used in the treatment of muscular diseases or disorders, more preferably

a) at least one disease or disorder selected from:

i) a disease or disorder of skeletal muscle, such as movement disorder, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the hand, blepharospasm, eyelid disorder, strabismus, spasticity, cerebral palsy, focal spasticity, spasticity of the limbs, spasms, hemifacial spasm, tremors, tics, bruxism, apraxia and/or freezing of gait;

ii) smooth muscle disease or disorder, spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophagia, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, urinary dysfunction, fecal incontinence, constipation, anismus, anal fissure, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus muscle (priapism), other disorders of muscle tone and/or other disorders characterized by involuntary movements of muscle groups;

iii) a disease or disorder of the heart muscle cells, such as atrial fibrillation

(iv) a dermatological disease or disorder; and/or

b) pain associated with such disease or disorder.

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.

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

In 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 diseases or disorders, more preferably at least one disease or disorder such as those listed above, or pain associated with such a disease or disorder. Preferably, the medicament is for intramuscular injection.

In 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 of the diseases or disorders listed above, or pain associated with such a disease or disorder. More preferably, the treatment is administered by intramuscular injection.

Cosmetic use

The aqueous solutions of the present invention can be advantageously used in the cosmetic (i.e., non-therapeutic) treatment of any condition considered unsightly and involving muscle tension, activity, and/or volume. Therefore, the solutions of the invention are advantageously used to reduce 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.

Freeze-dried

The lyophilized products of the aqueous solutions described above are also the subject of the present invention. Such lyophilized products are obtained by lyophilizing 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 carried out by gently shaking the vial containing the diluent and the lyophilized product. After reconstitution, dissolution is preferably completed in less than 5 minutes.

In a preferred embodiment, the lyophilized powder is such that it provides a concentration of dantrolene or a pharmaceutically acceptable salt thereof of approximately 0.025 mg/mL to approximately 1.5 mg/mL, preferably from 0.1 mg/mL to approximately 1.1 mg/mL, more preferably from approximately 0.25 mg/mL to approximately 0.6 mg/mL, and most preferably from 0.45 mg/mL to 0.5 mg/mL after reconstitution with a suitable carrier for intramuscular injection. This concentration refers to the concentration of dissolved dantrolene or a pharmaceutically acceptable salt thereof. In a preferred embodiment, reconstitution is carried out in an aqueous saline solution as an acceptable carrier, with either 0.75 g of lyophilized powder and 1.5 mL of carrier, or 1.5 g of lyophilized powder and 3 mL of carrier.

Lyophilization can be carried out using any method known to a person skilled in the art, preferably by freeze-drying. Methods for reconstituting the lyophilized product are also well known to a person skilled in the art.

In a preferred aspect, the freeze-dried product has a moisture content of no more than approximately 0.05 to approximately 1% by weight, more preferably no more than approximately 0.6 to approximately 0.7% by weight.

In a preferred aspect, the freeze-dried product contains practically no sodium chloride. Preferably, the freeze-dried product is free of sodium chloride.

Kits

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 lyophilized botulinum neurotoxin as separate components. In such a case, the lyophilized botulinum toxin is resuspended in the aqueous solution of dantrolene before injection into a subject.

Therefore, in one embodiment, the present invention provides a kit comprising an aqueous solution, optionally frozen, according to the invention, or a freeze-dried version thereof according to the invention, devoid of botulinum neurotoxin, and a freeze-dried botulinum neurotoxin.

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

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, cyclodextrin or a derivative thereof, wherein the molar ratio of dantrolene or a pharmaceutically acceptable salt of dantrolene to cyclodextrin or a derivative thereof is 1:at least 40, and

b) a lyophilized botulinum neurotoxin.

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.

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 aqueous solution.

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

The kit of the invention can be advantageously used to prepare an aqueous solution according to the invention prior to injection into a subject. Such a method comprises the steps of:

a) provide a kit of the invention, as described above,

b) reconstitute the dantrolene composition if such composition is a lyophilized product, so as to obtain an aqueous solution of dantrolene in liquid form;

c) thawing the aqueous dantrolene solution composition if supplied in frozen form, so as to obtain a liquid aqueous dantrolene solution,

d) resuspend the lyophilized botulinum neurotoxin in the liquid aqueous composition of dantrolene provided in the kit or obtained at the end of steps b) or c), so as to form an aqueous solution according to one of the aspects of the invention described above;

e) Optionally, store the aqueous composition obtained at the end of step d) at a temperature of approximately 2 °C to approximately 8 °C for approximately 1 hour to approximately 7 days.

The methods of reconstitution, thawing, and resuspension are well known to those skilled in the technique. These steps can be performed using any method known to the practitioner.

Examples

Example 1: compositions according to the invention

A first composition was prepared according to the present invention, comprising the components provided in Table 1:

Table 1

The molar ratio of dantrolene to HPBCD in this composition is 172. The pH is 7.4.

A second composition was prepared according to the present invention, comprising the components provided in Table 2:

Table 2

The molar ratio of dantrolene to HPBCD in this composition is 64. The pH is 7.4.

Example 2: Effect of various excipients on the solubility of sodium dantrolene

Various excipients (surfactants or cosolvents) were examined to determine their ability to solubilize dantrolene and thus increase the soluble concentration of dantrolene in the final reconstituted formulation comprising dantrolene and botulinum neurotoxin.

Each vehicle was prepared by solubilizing the corresponding excipient in a 0.9% sodium chloride solution at various concentrations. Table 3 shows, for each excipient, the mass of excipient 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.

Table 3

Next, sodium dantrolene (source: Abcam; designated as “dantrolene” in Examples 2 to 7 below) was added to each vehicle. Saturation was achieved by adding an excess of active ingredient to a given volume of the test medium. The suspension was then stirred by magnetic stirring for 24 hours at 20 °C, away from light.

Next, the supernatant was isolated by centrifugation (double centrifugation for 20 minutes at 15000 rpm at 20 °C), diluted in a mixture of solvents allowing its injection into the chromatographic system.

The concentration of dantrolene in solution for each medium was then determined by HPLC with external standardization. The HPLC method used in this study is presented in Table 4. Given the UV spectrum of dantrolene, the chromatograms were extracted at 386 nm, which provides a satisfactory compromise between the response factor and initial stability.

In an initial series of tests, the solubility of dantrolene was determined in different vehicles prepared in 0.9% sodium chloride solution: Cremophor® EL 5%, Lutrol® F68 5%, Tween® 80 5%, and HPBCD 30%. The solubility of dantrolene in 0.9% sodium chloride solution was also determined for reference purposes. The solubility levels of dantrolene at 20 °C (HPLC results) in these six vehicles are reported in Table 5.

The solubility of dantrolene in 0.9% NaCl (0.017 mg/ml) was as expected. Except for Lutrol® F68, the different surfactants tested (5% Cremophor® EL and 5% Tween® 80, both in 0.9% NaCl) improved the solubility of dantrolene, with 0.13 mg/ml, 0.14 mg/ml, and 0.18 mg/ml solubilized, respectively. The 30% HPBCD solution in 0.9% NaCl was the vehicle that most significantly increased the solubility of dantrolene, reaching approximately 1.8 mg/ml of soluble dantrolene concentration.

Table 4

Table 5

In a second series of experiments, the solubility of dantrolene was determined in different vehicles by mixing a 0.9% NaCl solution with a water-miscible cosolvent. The four cosolvents tested and their percentages in 0.9% NaCl are as follows: 10% propylene glycol, 10% PEG400, 10% glycerol, and 10% ethanol. The resulting dantrolene solubility levels at 20 °C (HPLC results) in these four vehicles are presented in Table 6.

Table 6

In all these vehicles tested, the solubility of dantrolene did not even reach 0.05 mg/ml, indicating that none of these co-solvents is a suitable excipient to better solubilize dantrolene.

In a third series of experiments, the suitability of the polymer polyvinylpyrrolidone (PVP K17) as an excipient was tested. The equilibrium solubility of sodium dantrolene was measured at 20 °C in a vehicle comprising 10% PVP K17 in 0.9% NaCl. The results are provided in Table 7. PVP K17 did not increase the solubility of dantrolene.

Table 7

In conclusion, these results demonstrated that the highest solubility of dantrolene was obtained in the 30% HPBCD solution, with a solubilized dantrolene concentration of approximately 1.8 mg/ml. In the surfactant solutions (5% Cremophor® EL and 5% Tween® 80, both in 0.9% NaCl), the solubility of dantrolene only increased slightly. The other excipients did not increase the solubility of dantrolene.

Example 3: Evaluation of the HPBCD concentration needed to solubilize dantrolene at a concentration of 1 mg/ml

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

Table 8

These results demonstrate that a minimum of 20% HPBCD in 200 mM NaCl is required to solubilize dantrolene to the target concentration of 1 mg/ml during 24 hours of stirring at 20 °C.

Example 4: Evaluation of the stability of the dantrolene formulation in a 30% HPBCD solution in 0.9% NaCl.

Based on these results, longer storage tests were performed. 0.5 mg/ml and 1 mg/ml dantrolene formulations were prepared in a 30% HPBCD solution in 0.9% NaCl and stabilized at 4 °C. 1.5 g and 3 g of HPBCD, respectively, were weighed out. A few milliliters of 0.9% NaCl solution were added, and the solution was stirred until completely dissolved. The volume was then brought up to 10 ml with 0.9% aqueous NaCl solution. The solution was then magnetically stirred to ensure complete homogenization.

After manufacturing, both formulated samples were analyzed by HPLC for dantrolene content (after filtration of a small sample volume), and aliquots were then taken into antibiotic bottles for a stability study. These aliquots were subsequently analyzed by HPLC after 1, 3, and 7 days at 4 °C, protected from light, for the soluble fraction of dantrolene. The results are presented in Table 9.

Table 9

Both samples were shown to be chemically stable (no decrease in dantrolene content was observed) and physically stable (no dantrolene crystallization was observed) for at least 7 days of storage at 4 °C, protected from light.

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

Table 10

The equilibrium solubility of dantrolene after 24 hours at 4 °C in the same vehicle (30% HPBCD solution in 0.9% NaCl) was 1.5 mg/mL. Based on this result, a formulation of 1 mg/mL dantrolene in 30% HPBCD in 0.9% NaCl was expected to remain stable long-term at 4 °C. These results prompted a more comprehensive evaluation of the formulation's stability in a 30% HPBCD solution in 0.9% NaCl. Therefore, additional experiments were performed to confirm long-term stability.

A 1 mg/ml dantrolene formulation was prepared in a 30% HPBCD solution in 0.9% NaCl as previously disclosed. After complete solubilization, the solution was filtered through a sterile filter unit (Millex® PVDF 0.22 µm), analyzed by HPLC at T0 for dantrolene content determination, and then aliquots were taken into antibiotic bottles for stability testing at different temperatures (4 °C, 20 °C, and 40 °C) for 6 months, as well as at -20 °C for a freeze/thaw cycle test.

The samples were then analyzed by HPLC (after filtration, Millex® PVDF 0.45 µm) 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 (only up to 1 month), protected from light, to determine the dantrolene concentration. A stability sample was also analyzed after a freeze-thaw cycle.

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

Table 11

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

Regarding physical stability, no crystallization was observed in the different stability samples studied stored at 4 °C, 20 °C and 40 °C.

Regarding chemical stability, marked chemical degradation (a significant decrease in dantrolene content, as well as an increase in impurity content) was observed after just one day of storage at 40 °C. After one month, the degradation reached approximately 60%. After one week of storage at 20 °C, slight degradation also began to be observed, reaching approximately 10% after one month, 30% after 3.5 months, and 45% after six months. At 4 °C, degradation was also evident, with a decrease in dantrolene content of approximately 6% after 3.5 months and 13% after six months.

Stability was also evaluated in a frozen sample. Samples were prepared as described above and frozen after complete solubilization. The sample was stored at -20 °C for 1, 2, and 3 months. Dantrolene concentration was determined by HPLC. The results are presented in Table 12.

Table 12

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

The long-term stability of three formulations was evaluated 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 as follows: a) a formulation of 0.5 mg/ml dantrolene in a 30% HPBCD solution in 0.9% NaCl (reference sample)

b) a formulation of 0.5 mg/ml of dantrolene in a solution of “HPBCD 30% L-histidine 10 mM” in 0.9% NaCl (final pH 7.4)

c) a formulation of 0.5 mg/ml of dantrolene in a solution of “HPBCD 30% L-histidine 10 mM EDTA 0.1 mM” in 0.9% NaCl (final pH 7.4).

After complete solubilization (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 HCl) for both L-histidine formulations. After a further 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 into sterile 2 mL amber antibiotic vials for stabilization at 4 °C and 20 °C for 12 months. Some vials were also stored at -20 °C to study the effect of successive freeze/thaw cycles, all protected from light.

The prepared solutions were analyzed at T0 for dantrolene content determination and pH measurement. Subsequently, stability samples were analyzed by HPLC (after filtration, Millex® PVDF 0.45 pm) after 1, 3, and 6 months of storage at 4°C and 20°C, protected from light, for the determination of the soluble fraction of dantrolene. The appearance (macroscopic observation) and pH of the stability samples were also reported. Storage is ongoing at the time of submission, and further analyses are planned at subsequent time points.

Regarding stability samples subjected to successive freeze-thaw cycles, the samples were thawed at 20 °C for 1.5 hours before being refrozen for 1.5 hours, and so on. In practice, for each solution studied, stability samples subjected to one freeze-thaw cycle were first stored at -20 °C for two or three weeks, before being thawed at 20 °C for 1.5 hours, and finally stored at 4 °C for one or two weeks, respectively, before analysis. Similarly, stability samples subjected to three or five freeze-thaw cycles were thawed/frozen/thawed in 1.5-hour intervals (except for an overnight freezing period before the fourth cycle), and then stored at 4 °C for one or two weeks before analysis.

No physical degradation (no crystallization was observed) nor any significant change in pH was evident in the different stability samples stored for 6 months at 4 °C and 20 °C.

Regarding 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 at both 4 °C and 20 °C. In fact, the L-histidine buffered formulations showed approximately half the degradation of the reference sample, as shown in [Fig. 2].

Regarding samples subjected to successive freezing/thawing cycles, no significant degradation was observed other than that due to storage at 4 °C for 1 or 2 weeks.

Example 6: Compatibility of various excipients with botulinum neurotoxin

Paresis activity was assessed based on the rat finger abduction scoring assay, as described below.

Animals

The experiments were conducted using adult female Sprague-Dawley rats (200 g), obtained from Janvier Labs/France. The rats were acclimated for at least one week prior to use, with access to food and water ad libitum in the animal facility. Unless otherwise indicated, 6 rats were used for each condition.

The body weight of the animals was recorded throughout the study on days 0, 2, 4, 7 and once a week until the end of the experiment.

Treatment

The animals were injected (25 pl or 40 pl per rat) using a 33-gauge needle attached to a 100 pl Hamilton syringe. On the first day of the experiment, prior to the injections, the rodents underwent a pre-test to ensure that the DAS response was “zero.” The rats then received intramuscular (i.m.) injections into the right tibialis anterior (TA) muscle. The left TA muscle was left uninjected as a control.

Finger Abduction Scoring Trial (DAS)

Muscle paralysis was measured using the finger abduction scoring (DAS) assay as reported by Broide et al. (TOXICON, 2013, 71, pp. 18-24). The animals' DAS response was scored at different time points following injections of the compound, inducing finger 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 daily until two consecutive measurements on two consecutive days were equal to zero (DAS = 0). The different degrees of finger abduction were scored on a five-point scale (from 0 = normal to 4 = maximum reduction in finger abduction, i.e., neither finger can be abducted) by two separate observers blinded to the treatment.

As is standard practice for DAS studies with rats, the DAS response was induced by grasping the rat by the torso, lifting it into the air, and simulating a fall onto a flat surface. The rat reflexively prepares for impact by spreading the toes of its hind feet, and the DAS response was immediately scored with the animal lying supine in a recumbent position.

Data analysis

As described above, a DAS score of “1” was assigned when loss of abduction was observed with the 1st toe. A DAS score of “2” was assigned when all three toes were brought together. A score of “3” was assigned when four toes were brought together, and rats received a score of “A” when all five toes of the right paw were grouped together during abduction.

DAS responses were measured at each time point and the kinetics (onset of muscle weakening and duration of effectiveness) were evaluated and compared in the different groups of rats.

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

Figure 3 shows that when botulinum neurotoxin A (Ona) was reconstituted in a 0.9% NaCl solution containing 5% Tween® 80, its activity was altered. In contrast, a similar solution containing HPBCD (up to 30%) had little impact on the activity of Ona.

Figure 4 shows that when botulinum neurotoxin A (Ona) was reconstituted in a 0.9% NaCl solution containing 4.5% Tween® 20, its activity was altered. Similarly, the combination of dantrolene (FTP-501) with Ona in this formulation was unable to potentiate the activity of Ona; conversely, in a 0.9% NaCl solution, the same combination (Ona FTP-501) showed better activity than Ona alone. It is worth noting that the enhancement of dantrolene was similar in a formulation containing HPBCD.

Figures 5, 6, and 7 show the in-use stability of botulinum toxin A (Ona) reconstituted with a 0.9% NaCl solution (reference) or formulations containing HPBCD L-histidine EDTA in 0.9% NaCl, at room temperature. These results demonstrate that the activity of Ona was stable for the first 4–6 hours and then decreased in both the reference and HPBCD formulations.

Figure 8 shows the in-use stability of botulinum toxin A (Ona) reconstituted with a 0.9% NaCl solution (reference), or with formulations containing “HPBCD L-histidine EDTA” in 0.9% NaCl, at 40°C. These results demonstrate that the activity of Ona was stable for the first 14 days, and then decreased, in both the reference and HPBCD formulations.

Example 7: Properties of HPBCD-based lyophilized products

A lyophilized HPBCD-based formulation containing dantrolene was prepared for reconstitution in 0.9% NaCl as follows. First, a 300 mg/ml solution of 10 mM HPBCD L-histidine was prepared in water; then, dantrolene was added to obtain a final concentration of 500 pg/ml. Next, the pH was adjusted to 7.4 by adding 10% HCl. The resulting composition was filtered through a 0.2 µm PVDF membrane, and 1.5 ml aliquots were dispensed into glass vials and frozen at -20 °C for 24 hours. Lyophilization was then carried out at a pressure of 1.03 mbar at -20 °C for 4 days. The lyophilized samples were then stored at 4 °C until analysis.

In the first experiment, the lyophilized samples were reconstituted with 1.5 mL of 0.9% NaCl. Instantaneous reconstitution was observed, and UV dosing of dantrolene showed that the desired concentration of 500 pg/mL was achieved, as shown in Figures 9 and 10, first point on the left. The reconstituted vials were then incubated at 4 °C (Figure 9) or at room temperature (Figure 10) for 24 hours. From these curves, in-use stability of at least 24 hours at 4 °C and at room temperature could be concluded.

In a second experiment, the lyophilized samples were stored at 4 °C for 20 months, then reconstituted with 1.5 mL of 0.9% NaCl, and the dantrolene content was measured by UV dosing. A concentration of 472 ± 6 µg/mL (±SD, n=2) could be recovered, demonstrating the stability of the lyophilized samples for at least 20 months at 4 °C.

Claims (16)

1. An aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof and cyclodextrin or a derivative thereof, wherein the molar ratio of dantrolene or a pharmaceutically acceptable salt of dantrolene to cyclodextrin or a derivative thereof is at least 1:40, and wherein the aqueous solution optionally comprises a buffer and/or at least one additional salt that is not a salt of dantrolene, cyclodextrin, or a derivative thereof. 2. Aqueous solution according to claim 1, further comprising botulinum neurotoxin. 3. An aqueous solution 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 alkylammonium, polyalkylammonium, arylammonium, or ammonium cations from physiologically acceptable amino compounds, such as, for example, arginine, lysine, meglumine, tromethamine, choline, benzyltrimethylammonium, tetramethylammonium, N-methylpyridinium, tetrabutylammonium, 2-(2,3-dihydroxy-1-propylamino)-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)propyltrimethylammonium; where the botulinum neurotoxin is selected from the group consisting of serotypes A (BoNT/A), B (BoNT/B), E (BoNT/E) and combinations of heavy and light chains thereof; and/or wherein the cyclodextrin or a derivative thereof is selected from the group consisting of p-cyclodextrin, 2,6-dimethyl-p-cyclodextrin, 2-hydroxyethyl-p-cyclodextrin, 2-hydroxyethyl-Y-cyclodextrin, 2-hydroxypropyl-p-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, (2-carboxymethyl)propyl-p-cyclodextrin, sulfonylutrit-p-cyclodextrin, 2-hydroxypropyl-p-cyclodextrin, sulfonylutrit ether-p-cyclodextrin and 2-hydroxyethyl-p-cyclodextrin, preferably from the group consisting of p-cyclodextrin, 2,6-dimethyl-p-cyclodextrin, 2-hydroxyethyl-p-cyclodextrin, 2-hydroxyethyl-Y-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, (2-carboxymethoxy)propyl-P-cyclodextrin, sulfobutyl ether-P-cyclodextrin and preferably 2-hydroxypropyl-P-cyclodextrin (HPBCD). 4. Aqueous solution according to any one of claim 1 to claim 3, comprising: dantrolene or a pharmaceutically acceptable salt thereof in an amount of approximately 0.025 mg/ml to approximately 1.5 mg/ml, preferably from 0.1 mg/ml to approximately 1.1 mg/ml, more preferably from approximately 0.25 mg/ml to approximately 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 from approximately 0.1 U/ml to approximately 4000 U/ml, preferably from 1 U/ml to approximately 400 U/ml, more preferably from approximately 5 U/ml to approximately 200 U/ml, most preferably from approximately 20 U/ml to approximately 100 U/ml; and/or cyclodextrin or a derivative thereof in an amount from approximately 4% (w/v) to approximately 40% (w/v), preferably from approximately 10% (w/v) to approximately 35% (w/v), more preferably from approximately 10% (w/v) to approximately 30% (w/v), for example, from approximately 25% (w/v) to approximately 32% (w/v), or from approximately 28% (w/v) to approximately 32% (w/v). 5. Aqueous solution according to any one of claim 1 to claim 4, wherein the ratio of dantrolene or a pharmaceutically acceptable salt thereof to cyclodextrin or a derivative thereof is from approximately 1:40 to approximately 1:3000, preferably from approximately 1:50 to approximately 1:700, more preferably from approximately 1:55 to approximately 1:350, more preferably from approximately 1:60 to approximately 1:185; most preferably from approximately 1:60 to approximately 1:175. 6. 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, phosphate-citrate buffer, succinate buffer and mixtures thereof. 7. An aqueous solution according to any one of claim 1 to claim 6, wherein the at least one salt, which is not a dantrolene salt, cyclodextrin salt, or a derivative thereof, is preferably present in an amount of approximately 0.45% to approximately 1.8%, more preferably 0.85% to approximately 0.95%, most preferably approximately 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. 8. 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, hydroxyethyl starches, glycine, gelatin, calcium gluconoglucoheptonate, and mixtures thereof. 9. 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 fatty acids from castor oil; c) synthetic copolymers of ethylene oxide and propylene oxide, such as poloxamers; d) organic solvents; and/or e) particles that are larger than 5 nm after storage for one day at room temperature. 10. Aqueous solution according to any one of claim 1 to claim 9 for use as a medicament. 11. 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 disease or disorder of skeletal muscle, such as movement disorder, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the hand, blepharospasm, eyelid disorder, strabismus, spasticity, cerebral palsy, focal spasticity, spasticity of the limbs, 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, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, urinary dysfunction, fecal incontinence, constipation, anismus, anal fissure, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus muscle (priapism), other disorders of muscle tone and/or other disorders characterized by involuntary movements of muscle groups; iii) a disease or disorder of the cardiac muscle cells, such as atrial fibrillation; (iv) a dermatological disease or disorder; and/or b) pain associated with such disease or disorder. 12. Use of an aqueous solution according to any one of claim 1 to claim 9 in a non-therapeutic 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. 13. Lyophilized product of an aqueous solution according to any one of claim 1 to claim 9. 14. 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, cyclodextrin or a derivative thereof, wherein the molar ratio of dantrolene or a pharmaceutically acceptable salt of dantrolene to cyclodextrin or a derivative thereof is at least 1:40, and wherein such aqueous solution is in either liquid or frozen form; and ii. a lyophilized form of an aqueous solution of i.; and b) a lyophilized botulinum neurotoxin. 15. Method for providing an aqueous solution according to any one of claim 1 to claim 9 of the kit according to claim 14, comprising the steps of: a) provide a kit according to claim 14, b) reconstitute the dantrolene composition if such composition is a lyophilized product, so as to obtain an aqueous solution of dantrolene in liquid form; c) thawing the aqueous dantrolene solution composition if such composition is provided in frozen form, so as to obtain an aqueous dantrolene solution in liquid form, d) resuspending the lyophilized botulinum neurotoxin in the liquid aqueous dantrolene composition provided in the kit or obtained at the end of steps b) or c), so as to form an aqueous solution according to any one of claim 1 to claim 9; e) Optionally, store the aqueous composition obtained at the end of step d) at a temperature of approximately 2 °C to approximately 8 °C for approximately 1 hour to approximately 7 days. 16. Aqueous solution comprising dantrolene or a pharmaceutically acceptable salt thereof, botulinum neurotoxin and at least one salt that is not a salt of dantrolene, cyclodextrin or a derivative thereof.