Resin toxin compositions

Abstract

The present invention provides non-aqueous solid and liquid compositions comprising resiniferatoxin and a surfactant. The non-aqueous solid and liquid compositions can be used to prepare aqueous compositions for the treatment of pain, particularly osteoarthritis-related joint pain. In addition, the present invention provides kits containing the non-aqueous solid and liquid compositions and a diluent and methods of making the compositions. The non-aqueous solid and liquid compositions allow for longer storage and recovery of resiniferatoxin compared to resiniferatoxin alone prior to reconstitution with a diluent.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of pharmacy and to compositions of resiniferatoxin, their manufacture and use. BACKGROUND

[0002] Resiniferatoxin (RTX) is a naturally occurring compound produced by the Moroccan cactus Euphorbia resinifera and is a potent analog of capsaicin, the active ingredient of chili peppers. At very small doses, resiniferatoxin is known as a transient receptor potential cation channel subfamily V member 1 (TRPV1) agonist and has been investigated for its therapeutic efficacy in a variety of diseases or afflictions, such as pain relief, overactive bladder, interstitial cystitis, and rhinitis.

[0003] Resiniferatoxin is known to readily bind to a variety of substances and surfaces, making it challenging to handle the very small amounts required for low doses of the compound. In addition, it breaks down into fragments upon thawing and, therefore, is thawed immediately prior to use and diluted in a solvent, such as ethanol or dimethyl sulfoxide, to prevent breakdown.

[0004] Accordingly, it is an object of the present invention to provide resiniferatoxin compositions that are storable for a prolonged period of time without significant breakdown, particularly non-aqueous solid and liquid compositions of resiniferatoxin and a surfactant with optional additional components. The non-aqueous compositions can be diluted with a diluent, such as an aqueous diluent, for therapeutic applications. Kits, methods of manufacture, and methods of use of the resiniferatoxin compositions are also provided. Additional objects of the invention will become apparent from the following description of the invention, examples, and claims. SUMMARY

[0005] In a first aspect, the present invention relates to a non-aqueous solid or non-aqueous liquid composition of resiniferatoxin comprising (a) resiniferatoxin; and (b) a surfactant. In certain embodiments, the non-aqueous solid composition is a lyophilized solid. The invention also relates to a kit comprising (a) a first kit component comprising a non-aqueous solid or non-aqueous liquid composition; and (b) a second kit component comprising a diluent. Methods of using the non-aqueous solid and non-aqueous liquid compositions to manufacture a medicament for treating pain are also contemplated. In specific embodiments, the pain is osteoarthritis-related joint pain.

[0006] In another aspect, the present application provides an aqueous composition of resiniferatoxin comprising (a) about 0.005 mM to about 10 mM resiniferatoxin; (b) about 0.01% to about 10% by volume of a surfactant; and (c) at least one buffering agent; wherein the pH of the composition is about 7.7 to about 8.3. The aqueous composition can be used to treat pain. In some embodiments, the present application provides a method of treating pain comprising administering to a subject a therapeutically effective amount of the aqueous composition disclosed herein. In some embodiments, the aqueous composition is suitable for intra-articular administration. In specific embodiments, the pain is osteoarthritis-associated joint pain. DETAILED DESCRIPTION

[0007] DEFINITIONS

[0008] The term "about," and like terms, in connection with a property or value, includes the exact property or precise value, as well as any property or value that would be regarded, as a person of ordinary skill in the art would regard, as being within normal or acceptable variability of the property or value, in connection with the art and the method by which the property or value is measured or determined. The term "about" generally refers to a range of values ±10% of the specified value. For example, the phrase "about 200" includes ±10% of 200, or 180 to 220.

[0009] The term "kit," as used herein, means that the components included in the kit are physically separable and distinguishable from one another, but are provided or sold together for the purpose of administration or use together.

[0010] The terms "lyophilized," "lyophilization," and "lyophilize," as used herein, refer to the process of freeze-drying, in which a sample is first frozen and dried (typically under vacuum) to remove water and / or other solvents.

[0011] The term "non-aqueous," as used herein, refers to a solid or liquid composition that is substantially free of water (water of hydration excepted), preferably less than 2% water by weight as measured by standard techniques such as, for example, thermogravimetric measurements. The non-aqueous compositions described herein can contain small amounts of water absorbed from ambient moisture.

[0012] The terms "resiniferatoxin" and "RTX," as used herein, refer to a naturally occurring chemical produced by the Moroccan cactus resin, Euphorbia resinifera, or synthetically produced, and has the following structure and a molar mass of 628.71:

[0013]

[0014] The term "D-a-tocopheryl polyethylene glycol succinate" is used herein synonymously with the terms "D-a-tocopheryl polyethylene glycol 1000 succinate" and the abbreviation "TPGS". D-a-tocopheryl polyethylene glycol succinate is a non-ionic surfactant.

[0015] The term "solid composition" as used herein refers to a solid in crystalline, powder, and / or amorphous form or any mixture thereof. It is in the solid state at normal temperature and pressure (NTP) as defined by the U.S. National Institute of Standards and Technology (NIST). The solid compositions described herein can contain trace amounts of water or other solvents (in addition to water of hydration) preferably less than 2% by weight of water or other solvents. The solid compositions can be stored at room temperature or lower, preferably about 4°C, about 10°C, or about 25°C, prior to reconstitution with a liquid solvent.

[0016] The term "buffer" or "buffer solution" means a combination of two or more components that buffers the pH (i.e., minimizes changes in pH) upon the addition of an acid or a base. A buffer or buffer solution can also include auxiliary compounds such as neutral salts (e.g., sodium chloride) that contribute little to the buffering effect but are incorporated for physiological reasons, e.g., to adjust the osmotic pressure of the buffer solution.

[0017] Solid compositions

[0018] In one aspect, the present application provides a non-aqueous solid composition of resinotoxin comprising resinotoxin and a surfactant. Optionally, the composition comprises one or more additional additives. In some preferred embodiments, the solid composition is a lyophilized composition, also known as a lyophilizate or freeze-dried product. In some embodiments, the surfactant can be a non-ionic surfactant. Exemplary non-ionic surfactants include D-a-tocopheryl polyethylene glycol succinate, PEG-20 hydrogenated castor oil, PEG-35 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-100 hydrogenated castor oil, PEG-1000 hydrogenated castor oil, PEG-2000 hydrogenated castor oil, PEG-4000 hydrogenated castor oil, PEG-6000 hydrogenated castor oil, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polysorbate 100. In some embodiments, the surfactant is selected from the group consisting of D-a-tocopheryl polyethylene glycol succinate, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-35 hydrogenated castor oil, and polysorbate 80. In particular embodiments, the surfactant is D-a-tocopheryl polyethylene glycol succinate or polysorbate 80. In more particular embodiments, the surfactant is D-a-tocopheryl polyethylene glycol succinate.

[0019] The composition can also include a polyethylene glycol and / or an additional surfactant, where the additional surfactant is optionally a poloxamer. Exemplary polyethylene glycols include PEG 1000, PEG 1450, PEG 1600, PEG 3000, PEG 3350, PEG 4000, PEG 4500, PEG 6000, PEG 8000, and PEG 20000. In some embodiments, the polyethylene glycol is PEG 6000. The poloxamer can be, for example, poloxamer 101, 182, 188, 237, 331, 338, or 407. In some embodiments, the poloxamer is poloxamer 188, 237, or 407. In another embodiment, the poloxamer is poloxamer 188.

[0020] Other optional additives include solid components of the buffer solution. Components can include, for example, NaCl, CaCl2x2H2O, KCl, tris(hydroxymethyl)aminomethane, and combinations thereof. In particular embodiments, the non-aqueous solid composition comprises resin toxin, D-a-tocopheryl polyethylene glycol succinate, NaCl, CaCl2x2H2O, KCl, and tris(hydroxymethyl)aminomethane, and is preferably lyophilized. In some embodiments, the non-aqueous solid compositions disclosed herein do not comprise a monosaccharide or sugar alcohol.

[0021] Non-aqueous solid compositions of resinotoxin can be obtained by combining resinotoxin, a surfactant, and optional additives, if any, in a solvent to form a solution or dispersion, and then drying the solution or dispersion. For example, a stock solution of resinotoxin in an organic solvent such as absolute ethanol can be prepared at a concentration of about 0.1 pg / mL to about 1000 pg / mL, about 0.2 pg / mL to about 200 pg / mL, or about 20 pg / mL to about 50 pg / mL. Other organic solvents that can be used include glycerol or DMSO. The stock solution (e.g., 50 pL) can be diluted with a diluent (e.g., 950 pL) containing a surfactant and optionally polyethylene glycol or an additional surfactant (e.g., poloxamer), followed by drying. In some embodiments, the diluent is an aqueous diluent. In some embodiments, the aqueous diluent comprises at least one buffering agent and optionally at least one isotonicity agent. In some embodiments, the at least one buffering agent is suitable for maintaining a pH of about 7.7 to about 8.3. In other embodiments, the aqueous diluent comprises about 140 mM-150 mM NaCl, about 3 mM-5 mM CaCl2x2H2O, about 3 mM-5 mM KCl, about 8 mM-12 mM tris(hydroxymethyl)aminomethane, and a pH of about 7.7 to about 8.3. In particular embodiments, the aqueous diluent comprises about 147 mM NaCl, about 4 mM CaCl2x2H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane, and a pH of about 8.0.

[0022] Drying or solvent removal can be performed by a variety of techniques, including vacuum evaporation, spray drying, microencapsulation, lyophilization, other drying techniques, and combinations thereof. In some embodiments, the solution is frozen and dried by lyophilization.

[0023] Non-aqueous solid compositions are generally more stable at or below room temperature than either solid resinotoxin alone or aqueous liquid compositions of resinotoxin. The solids can be stored at or below room temperature prior to reconstitution and use. Exemplary storage temperatures include about 0 °C, about 4 °C, or about 25 °C, or in the range of about -5 °C to about 25 °C, about 0 °C to about 25 °C, about 0 °C to about 15 °C, or about 0 °C to about 10 °C, or about 0 °C to about 5 °C.

[0024] In some additional embodiments, the non-aqueous solid compositions of the present application are characterized in that they readily dissolve in an aqueous carrier or diluent. In these embodiments, the solid composition can be combined with an aqueous diluent, preferably sterile, such that upon shaking a liquid solution containing the resin toxin in dissolved form is obtained. In some embodiments, at least 90% of the resin toxin is recovered after the solid is dissolved in an aqueous diluent. In other embodiments, at least 95% of the resin toxin is recovered after the solid is dissolved in an aqueous diluent. In some embodiments, at least 90% of the resin toxin is recovered after storage at about -5 °C to about 25 °C, about 0 °C to about 25 °C, about 0 °C to about 15 °C, or about 0 °C to about 10 °C, or about 0 °C to about 5 °C for up to about 1 month to about 24 months, about 6 months to about 24 months, or about 12 months to about 24 months. In one particular embodiment, at least 90% of the resin toxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 24 months. In another particular embodiment, at least 95% of the resin toxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 12 months.

[0025] Liquid compositions

[0026] In another aspect, the present application provides non-aqueous liquid compositions of resin toxin comprising resin toxin and a surfactant. Optionally, the composition comprises one or more additional additives. As with the non-aqueous solid compositions described herein, the non-aqueous liquid compositions provide stable formulations of resin toxin that can readily be converted to liquid aqueous compositions that can be used, for example, as injection solutions for delivering effective amounts of resin toxin to patients in need thereof. The non-aqueous liquid compositions of the present application can also be referred to as concentrates or pre-concentrates, respectively.

[0027] The non-aqueous liquid composition of resinotoxin can be obtained by combining resinotoxin, a surfactant, and optional additional additives in a solvent to form a non-aqueous solution. In some embodiments, the solvent is anhydrous ethanol. In some embodiments, the surfactant can be a non-ionic surfactant. In some embodiments, the non-aqueous liquid composition comprises (a) about 0.005 mM to about 10 mM of resinotoxin, preferably about 0.01 mM to about 1 mM of resinotoxin, more preferably about 0.1 mM to about 1 mM of resinotoxin, even more preferably about 0.1 mM to about 0.2 mM of resinotoxin. Exemplary non-ionic surfactants include D-alpha-tocopheryl polyethylene glycol succinate, PEG-20 hydrogenated castor oil, PEG-35 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-100 hydrogenated castor oil, PEG-1000 hydrogenated castor oil, PEG-2000 hydrogenated castor oil, PEG-4000 hydrogenated castor oil, PEG-6000 hydrogenated castor oil, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polysorbate 100. In some embodiments, the surfactant is selected from the group consisting of D-alpha-tocopheryl polyethylene glycol succinate, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-35 hydrogenated castor oil, and polysorbate 80. In certain embodiments, the surfactant is D-alpha-tocopheryl polyethylene glycol succinate. In other embodiments, the surfactant is polysorbate 80. Optional additives can include polyethylene glycol and / or additional surfactants, wherein the additional surfactants are optionally poloxamer. Exemplary polyethylene glycols include PEG 1000, PEG 1450, PEG 1600, PEG 3000, PEG 3350, PEG 4000, PEG 4500, PEG 6000, PEG 8000, and PEG 20000. In some embodiments, the polyethylene glycol is PEG 6000. The poloxamer can be, for example, poloxamer 101, 182, 188, 237, 331, 338, or 407. In some embodiments, the poloxamer is poloxamer 188, 237, or 407. In another embodiment, the poloxamer is poloxamer 188.

[0028] In one particular embodiment, the non-aqueous liquid composition comprises resinotoxin and polysorbate 80. In another particular embodiment, the non-aqueous liquid composition comprises resinotoxin and D-alpha-tocopheryl polyethylene glycol succinate. In some embodiments, the non-aqueous liquid compositions disclosed herein do not comprise a monosaccharide or a sugar alcohol.

[0029] The non-aqueous liquid composition of resinotoxin can be obtained by combining resinotoxin, a surfactant, and optional additives in a solvent to form the non-aqueous liquid composition, for example, preparing a sterile solution of RTX and the corresponding surfactant and / or poloxamer in EtOH abs. with the desired concentration, and dispensing aliquots of this solution into vials and sealing with a stopper and crimping.

[0030] The inventors have discovered that the non-aqueous liquid composition is more stable at or below room temperature than the solid resinotoxin alone or the aqueous liquid composition of resinotoxin. Exemplary storage temperatures include about 0 °C, about 4 °C, or about 25 °C, or in the range of about -5 °C to about 25 °C, about 0 °C to about 25 °C, about 0 °C to about 15 °C, or about 0 °C to about 10 °C, or about 0 °C to about 5 °C. In some embodiments, at least 90% of the resinotoxin is recovered after storage at about -5 °C to about 25 °C, about 0 °C to about 25 °C, about 0 °C to about 15 °C, or about 0 °C to about 10 °C, or about 0 °C to about 5 °C for up to about 1 month to about 24 months, about 6 months to about 24 months, or about 12 months to about 24 months. In one particular embodiment, at least 90% of the resinotoxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 24 months. In another particular embodiment, at least 95% of the resinotoxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 12 months.

[0031] Kit

[0032] In another aspect, the present application provides a kit comprising (a) a first kit component comprising a non-aqueous solid composition or a non-aqueous liquid composition as described herein and (b) a second kit component comprising a diluent.

[0033] In some embodiments, the diluent can be an aqueous solution or an organic solvent. Exemplary organic solvents include absolute ethanol, glycerol, and dimethyl sulfoxide (DMSO). A particular diluent is an aqueous diluent. The aqueous diluent can comprise only water or can comprise at least one buffer and optionally at least one isotonic agent. The buffer can be suitable for maintaining the pH at about 7.7 to about 8.3 upon combining the first and second kit components. In some embodiments, the aqueous diluent comprises about 140 mM-150 mM NaCl, about 3 mM-5 mM CaCl2x2H2O, about 3 mM-5 mM KCl, about 8 mM-12 mM tris(hydroxymethyl)aminomethane, and a pH of about 7.7 to about 8.3. In a particular embodiment, the aqueous diluent comprises about 147 mM NaCl, about 4 mM CaCl2x2H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane, and a pH of about 8.0.

[0034] In some embodiments, the first kit component is a non-aqueous solid composition comprising a resin toxin, a surfactant, and optionally at least one buffer and optionally at least one isotonicity agent, and the second kit component is water. In certain embodiments, the non-aqueous solid composition is lyophilized. In some embodiments, the buffer and optional isotonicity agent comprises NaCl, CaCl2x2H2O, KCl, and tris(hydroxymethyl)aminomethane. In certain embodiments, when the first kit component (non-aqueous solid composition) is combined with the second kit component (water), the resulting aqueous solution has a pH of about 7.7 to about 8.3.

[0035] In other embodiments, the first kit component is a non-aqueous liquid composition comprising a resin toxin and a surfactant, and the second kit component is an aqueous diluent comprising at least one buffer and optionally at least one isotonicity agent. In some embodiments, when the first kit component (non-aqueous liquid composition) is combined with the second kit component (aqueous diluent containing at least one buffer and optionally at least one isotonicity agent), the resulting aqueous solution has a pH of about 7.7 to about 8.3.

[0036] In some embodiments, the kit can be supplied in the following forms:

[0037] (a) separate compartments of one primary package (such as a bag divided into two or more "pouches" by a lamination seam, or one kit component is contained in a glass vial and the other kit component is held in the screw cap of the glass vial);

[0038] (b) separate primary packages packaged together in one secondary package (such as separate sets of bags or glass vials for two or more kit components, two or more sets of bags or glass vials are sold in the same carton);

[0039] (c) separate primary packages packaged in two or more separate secondary packages held together in turn by paper or plastic overwrap, ribbons, sleeves, etc. (such as separate sets of bags or glass vials for two or more kit components, two or more sets of bags or glass vials are sold in two or more cartons, which are in turn wrapped with shrink foil);

[0040] (d) combinations thereof (such as the first kit component is provided in a multi-dose carton of paperboard optionally with a dosing scoop, the carton of paperboard is sold in a carton with multiple glass vials, each containing the second kit component).

[0041] Optionally, the kits of the present application can further comprise written instructions on how best or preferably to combine and use the two or more kit components.

[0042] Aqueous compositions for injection and use

[0043] In another aspect, the present application provides an aqueous composition of resiniferatoxin comprising (a) resiniferatoxin (b) a surfactant (c) and at least one buffering agent, wherein the pH of the composition is about 7.7 to about 8.3. The aqueous composition can be prepared by combining the first and second kit components as described above. In some embodiments, the aqueous composition comprises (a) about 0.005 mM to about 10 mM resiniferatoxin, preferably about 0.01 mM to about 1 mM resiniferatoxin, more preferably about 0.1 mM to about 1 mM resiniferatoxin, even more preferably about 0.1 mM to about 0.2 mM resiniferatoxin; (b) about 0.01% to about 10% by volume of a surfactant, preferably about 0.01% to about 1% by volume of a surfactant, more preferably about 0.1% to about 1% by volume of a surfactant, even more preferably about 0.2% to about 0.5% by volume of a surfactant, especially 0.35% by volume of a surfactant; and (c) at least one buffering agent; wherein the pH of the composition is about 7.7 to about 8.3. In other embodiments, the aqueous composition comprises (a) about 0.005 mM to about 10 mM resiniferatoxin, preferably about 0.01 mM to about 1 mM resiniferatoxin, more preferably about 0.1 mM to about 1 mM resiniferatoxin, even more preferably about 0.1 mM to about 0.2 mM resiniferatoxin; (b) about 0.01% to about 10% by volume of a surfactant, preferably about 0.01% to about 1% by volume of a surfactant, more preferably about 0.1% to about 1% by volume of a surfactant, even more preferably about 0.2% to about 0.5% by volume of a surfactant, especially 0.35% by volume of a surfactant; (c) about 140 mM - 150 mM NaCl; (d) about 3 mM - 5 mM CaCl2 x 2 H2O; (e) about 3 mM - 5 mM KCl; (f) and about 8 mM - 12 mM tris(hydroxymethyl)aminomethane; wherein the pH of the composition is about 7.7 to about 8.3. In particular embodiments, the aqueous composition comprises (a) about 0.01 mM to about 1 mM resiniferatoxin, preferably about 0.1 mM to about 1 mM resiniferatoxin, more preferably about 0.1 mM to about 0.2 mM resiniferatoxin; (b) about 0.01% to about 1% by volume of a surfactant, more preferably about 0.1% to about 1% by volume of a surfactant, even more preferably about 0.2% to about 0.5% by volume of a surfactant, especially 0.35% by volume of a surfactant; (c) about 147 mM NaCl; (d) about 4 mM CaCl2 x 2 H2O; (e) about 4 mM KCl; (f) and about 10 mM tris(hydroxymethyl)aminomethane; wherein the pH of the composition is about 8.0. In particular embodiments, the aqueous composition does not comprise a monosaccharide or a sugar alcohol.

[0044] Methods of use

[0045] The non-aqueous solid compositions, non-aqueous liquid compositions, and / or aqueous compositions of resinotoxins disclosed herein can be used in the manufacture of a medicament for treating pain. Accordingly, the present application provides the non-aqueous solid compositions, non-aqueous liquid compositions, and / or aqueous compositions of resinotoxins disclosed herein for use as a medicament. In some embodiments, the compositions disclosed herein are used in the treatment of pain. Accordingly, the present application provides the non-aqueous solid compositions, non-aqueous liquid compositions, and / or aqueous compositions of resinotoxins disclosed herein for use in a method for treating pain. Also provided are the non-aqueous solid compositions, non-aqueous liquid compositions, and / or aqueous compositions of resinotoxins disclosed herein for use in the manufacture of a medicament for treating pain. In other embodiments, the present disclosure provides a method for treating pain comprising administering to a subject a therapeutically effective amount of any of the non-aqueous solid compositions, non-aqueous liquid compositions, and / or aqueous compositions described herein. In particular embodiments, the aqueous compositions are suitable for intra-articular administration. In particular embodiments, the pain is osteoarthritis-related joint pain.

[0046] The following examples are intended to illustrate the application and should not be construed as limiting the scope of the application.

[0047] Examples

[0048] Materials

[0049] The following materials were used to prepare the exemplary non-aqueous solid and liquid compositions described herein: Acetonitrile (ACN®; Batch: STBH6791, Sigma-Aldrich, Austria); Ammonium formate (Formic acid ammonium salt; Batch: BCBD3147V, Sigma-Aldrich, Austria); Calcium chloride dihydrate (CaCI2 2H2O; Batch: S28238-156, Sigma-Aldrich, Austria); Cremophor EL (Cremophor® EL; Batch: 86741436W0, BASF; Germany); Cremophor RH60 (Cremophor® RH60; Batch: 91191656P0, BASF; Germany); Formic acid (Formic acid; Batch: 396236476, Carl Roth, Karlsruhe, Germany); Hydrochloric acid (HCl; Batch: H2116K1, Carl Roth, Germany); Mannitol (Mannitol; Batch: 1560453, Thermo Scientific, Austria); Orthophosphoric acid 85% for HPLC (Orthophosphoric acid; Batch: Z0440928, Merck, Germany); PEG 6000 (Polyethylene glycol 6000; Cremophor® PEG 6000; Batch: 2005TS1586, Croda; Germany); Potassium chloride (KCI; Batch: 351173748, Carl Roth, Germany); RTX solution 10 mg mL-1 provided by Mestex (RTX solution; Batch: LIMS200949145, Carbogen Amcis, Switzerland); Sodium chloride (NaCI; Batch: 177255242, Carl Roth, Germany); Sodium phosphate monobasic dihydrate (NaH2P04 H2O; Batch: BCBG8983V, Sigma-Aldrich, Austria); TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate; Vitamin E TPGS; Batch: BCX8795, Sigma-Aldrich, Austria); Tris (Tris(hydroxymethyl)aminomethane, Trizma Ph. Eur, (Trizma®; Batch: S28238-156, Sigma-Aldrich, Vienna, Austria); and Tween 80 (Polysorbate 80, Tween® 80; Batch: K47176961 602, Merck Millipore, Vienna, Austria). -1 (ACN®; Batch: STBH6791, Sigma-Aldrich, Austria); Ammonium formate (Formic acid ammonium salt; Batch: BCBD3147V, Sigma-Aldrich, Austria); Calcium chloride dihydrate (CaCI2 2H2O; Batch: S28238-156, Sigma-Aldrich, Austria); Cremophor EL (Cremophor® EL; Batch: 86741436W0, BASF; Germany); Cremophor RH60 (Cremophor® RH60; Batch: 91191656P0, BASF; Germany); Formic acid (Formic acid; Batch: 396236476, Carl Roth, Karlsruhe, Germany); Hydrochloric acid (HCl; Batch: H2116K1, Carl Roth, Germany); Mannitol (Mannitol; Batch: 1560453, Thermo Scientific, Austria); Orthophosphoric acid 85% for HPLC (Orthophosphoric acid; Batch: Z0440928, Merck, Germany); PEG 6000 (Polyethylene glycol 6000; Cremophor® PEG 6000; Batch: 2005TS1586, Croda; Germany); Potassium chloride (KCI; Batch: 351173748, Carl Roth, Germany); RTX solution 10 mg mL-1 provided by Mestex (RTX solution; Batch: LIMS200949145, Carbogen Amcis, Switzerland); Sodium chloride (NaCI; Batch: 177255242, Carl Roth, Germany); Sodium phosphate monobasic dihydrate (NaH2P04 H2O; Batch: BCBG8983V, Sigma-Aldrich, Austria); TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate; Vitamin E TPGS; Batch: BCX8795, Sigma-Aldrich, Austria); Tris (Tris(hydroxymethyl)aminomethane, Trizma Ph. Eur, (Trizma®; Batch: S28238-156, Sigma-Aldrich, Vienna, Austria); and Tween 80 (Polysorbate 80, Tween® 80; Batch: K47176961 602, Merck Millipore, Vienna, Austria).

[0050] Example 1

[0051] Preparation of non-aqueous solid RTX compositions

[0052] A stock solution of 1 mg / ml RTX in EtOH abs. was prepared. From this stock solution a working solution containing the desired RTX concentration in EtOH was prepared. Test vials were prepared by combining 50 μΐ of the RTX working solution with 950 μΐ of the buffer containing the corresponding surfactant and / or poloxamer. All prepared vials were homogenized, covered with parafilm, frozen and subsequently lyophilized to yield the non-aqueous solid RTX compositions.

[0053] Exemplary compositions

[0054] Exemplary non-aqueous solid compositions that have been prepared are summarized in Table 1 below.

[0055] Table 1

[0056]

[0057]

[0058] Table 1 shows the non-aqueous solid compositions that have been prepared. "Mestex buffer" (MB) contains 147 mM NaCl, 4 mM CaCl2x2H2O, 4 mM KCl, 10 mM tris(hydroxymethyl)aminomethane and has a pH of about 8.0.

[0059] Stability testing

[0060] For certain exemplary non-aqueous compositions described herein, the % values of the amount of resin toxin detected by HPLC after lyophilization and reconstitution are provided in Table 2 below. After the lyophilization process, all vials were sealed and stored under the corresponding conditions as indicated in Table 1. At each time point (months (m)), the required amount of vial was reconstituted with 1 mL of water for injection by gently shaking the vial until complete dissolution of the lyophilized product was achieved. After 5 minutes, a sample was taken, diluted 1 : 1 with ethanol and analyzed by HPLC. In addition, a calibration curve was established at each time point using a dilution of RTX made from the stock solution to ensure comparability of the data obtained.

[0061] Table 2

[0062]

[0063] Table 2 shows the stability data of the non-aqueous solids over 12 months. All reconstituted solutions were clear and colorless. The pH was 8.0 ± 0.2.

[0064] Example 2

[0065] Preparation of non-aqueous liquid RTX compositions

[0066] A sterile solution of RTX and corresponding surfactant and / or poloxamer in EtOH abs. with the desired concentration was prepared. 0.5 ml of this solution was dispensed into 10R vials and sealed with a stopper and crimped. Such formulations were stored at 5°C.

[0067] The exemplary non-aqueous liquid compositions that have been prepared are summarized in Table 3 below.

[0068] In addition to the non-aqueous solid compositions, RTX was also formulated with different surfactants in EtOH (abs) as liquid pre-concentrates. At each time point the contents of the vials were diluted 1 to 20 with Mestex buffer without surfactant. After 5 minutes, samples were taken, diluted 1 : 1 with ethanol and analyzed by HPLC. In addition, a calibration curve was established at each time point using RTX dilutions made from the stock solution to ensure comparability of the data obtained.

[0069] In Table 3 below, the results of the stability of RTX in such compositions are shown as a percentage of the To value measured after initial filtration and centrifugation.

[0070] Table 3

[0071]

[0072] 1 = TWEEN 14% 2 = Tween 0.08%

[0073] Table 3 shows the stability data for the non-aqueous liquid compositions. All reconstituted solutions were clear and colorless.

[0074] The use of non-aqueous liquid formulations is less preferred compared to the non-aqueous solid compositions described herein, due to the fact that all non-aqueous liquid compositions still contain 5% solvent as well as the fact that for such formulations additional vials with the corresponding buffer have to be prepared and delivered.

[0075] Example 3

[0076] Reconstitution and short-term stability of non-aqueous solid RTX compositions

[0077] An appropriate amount of water for injection (WFI) was added to vials containing lyophilized RTX compositions as prepared in Example 1 comprising TPGS / Mestex buffer (“TPGS / MB” as shown in Table 2 above, at three different concentrations 0.1%, 0.35% and 0.7%) to prepare ready-to-use medicaments for intra-articular injection into a joint. As can be seen from Table 4 below, the reconstituted lyophilized RTX compositions were stable over the 4 hours measured, independent of the TPGS concentration used.

[0078] Table 4

[0079]

[0080] Table 4 shows the results of the stability study of the formulations RTX / EtOH / 0.10%, 0.35% or 0.70% TPGS / Mestex buffer. The samples were diluted 1 : 1 with EtOH prior to HPLC analysis. The indicated values are the mean (n > 3) ± SD.

[0081] List of items

[0082] The present application relates in particular to the following specific embodiments:

[0083] 1. A non-aqueous composition of resin toxin, the composition comprising:

[0084] (a) resin toxin; and

[0085] (b) a surfactant.

[0086] 2. The composition of item 1, wherein the composition is a solid composition.

[0087] 3. The composition of item 2, wherein the solid composition is a lyophilized composition.

[0088] 4. The composition of any one of items 2 to 3, wherein the surfactant is a non-ionic surfactant.

[0089] 5. The composition of item 4, wherein the surfactant is selected from the group consisting of D-a-tocopheryl polyethylene glycol succinate, PEG-20 hydrogenated castor oil, PEG-35 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-100 hydrogenated castor oil, PEG-1000 hydrogenated castor oil, PEG-2000 hydrogenated castor oil, PEG-4000 hydrogenated castor oil, PEG-6000 hydrogenated castor oil, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polysorbate 100.

[0090] 6. The composition of item 5, wherein the surfactant is selected from the group consisting of D-a-tocopheryl polyethylene glycol succinate, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-35 hydrogenated castor oil, and polysorbate 80.

[0091] 7. The composition of item 6, wherein the surfactant is D-a-tocopheryl polyethylene glycol succinate.

[0092] 8. The composition of item 6, wherein the surfactant is PEG-60 hydrogenated castor oil.

[0093] 9. The composition of item 6, wherein the surfactant is PEG-40 hydrogenated castor oil.

[0094] 10. The composition of item 6, wherein the surfactant is PEG-35 hydrogenated castor oil.

[0095] 11. The composition of item 6, wherein the surfactant is polysorbate 80.

[0096] 12. The composition of any one of items 2 to 11, further comprising:

[0097] (d) polyethylene glycol and / or

[0098] (e) an additional surfactant,

[0099] wherein the additional surfactant is optionally a poloxamer.

[0100] 13. The composition of item 12, wherein the polyethylene glycol is PEG 1000, PEG 1450, PEG 1600, PEG 3000, PEG 3350, PEG 4000, PEG 4500, PEG 6000, PEG 8000, or PEG 20000.

[0101] 14. The composition of item 13, wherein the polyethylene glycol is PEG 6000.

[0102] 15. The composition of any one of items 2 to 14, wherein at least 90% of the resin toxin is recovered after the solid is dissolved in an aqueous diluent.

[0103] 16. The composition of item 15, wherein at least 95% of the resin toxin is recovered after the solid is dissolved in an aqueous diluent.

[0104] 17. The composition of any one of items 2 to 16, wherein at least 90% of the resin toxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 24 months.

[0105] 18. The composition of item 17, wherein at least 95% of the resin toxin is recovered after storage at 0 °C to 25 °C for up to about 3 months to about 12 months.

[0106] 19. The composition of any one of items 2 to 18, wherein the composition further comprises NaCl.

[0107] 20. The composition of any one of items 2 to 19, wherein the composition further comprises CaCl2 x 2 H2O.

[0108] 21. The composition of any one of clauses 2-20, wherein the composition further comprises KCl.

[0109] 22. The composition of any one of clauses 2-21, wherein the composition further comprises tris(hydroxymethyl)aminomethane.

[0110] 23. The composition of any one of clauses 2-7 and 12-22, wherein the composition comprises resin toxin, D-a-tocopheryl polyethylene glycol succinate, NaCl, CaCl2x2H2O, KCl, and tris(hydroxymethyl)aminomethane.

[0111] 24. The composition of any one of clauses 2-23 for use in the manufacture of a medicament for the treatment of pain.

[0112] 25. The composition of clause 24, wherein the pain is osteoarthritis-related joint pain.

[0113] 26. A kit comprising:

[0114] (a) a first kit component comprising the composition of any one of clauses 2-25; and

[0115] (b) a second kit component comprising a diluent.

[0116] 27. The kit of clause 26, wherein the diluent is an aqueous diluent.

[0117] 28. The kit of clause 27, wherein the aqueous diluent comprises at least one buffering agent and optionally at least one isotonicity agent.

[0118] 29. The kit of clause 28, wherein the at least one buffering agent is suitable for maintaining a pH of about 7.7 to about 8.3 upon combination of the first and second kit components.

[0119] 30. The kit of clause 29, wherein the aqueous diluent comprises about 140 mM-150 mM NaCl, about 3 mM-5 mM CaCl2x2H2O, about 3 mM-5 mM KCl, about 8 mM-12 mM tris(hydroxymethyl)aminomethane, and a pH of about 7.7 to about 8.3.

[0120] 31. The kit of clause 30, wherein the aqueous diluent comprises about 147 mM NaCl, about 4 mM CaCl2x2H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane, and a pH of about 8.0.

[0121] 32. A method of making a non-aqueous solid composition of resinotoxin, the method comprising:

[0122] (a) mixing resinotoxin and a surfactant in a solvent;

[0123] (b) removing the solvent.

[0124] 33. The method of item 32, wherein in step (a) a buffer is combined with the resinotoxin and surfactant in a solvent.

[0125] 34. The method of any one of items 32 to 33, wherein the solvent is removed by lyophilization.

[0126] 35. A non-aqueous solid composition of resinotoxin made according to the method of any one of items 32 to 34.

[0127] 36. The composition of item 1, wherein the composition is a liquid composition.

[0128] 37. The composition of item 36, wherein the concentration of resinotoxin is about 0.005 mM to about 10 mM resinotoxin, preferably about 0.01 mM to about 1 mM resinotoxin, even more preferably about 0.1 mM to about 0.2 mM resinotoxin; (b) about 0.01% to about 10% by volume of a surfactant, preferably about 0.01% to about 1% by volume of a surfactant, even more preferably about 0.1% to about 0.5% by volume of a surfactant, in particular 0.35% by volume of a surfactant.

[0129] 38. The composition of item 36 or 37, wherein the surfactant is a non-ionic surfactant.

[0130] 39. The composition of item 38, wherein the surfactant is selected from the group consisting of D-a-tocopheryl polyethylene glycol succinate, PEG-20 hydrogenated castor oil, PEG-35 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-100 hydrogenated castor oil, PEG-1000 hydrogenated castor oil, PEG-2000 hydrogenated castor oil, PEG-4000 hydrogenated castor oil, PEG-6000 hydrogenated castor oil, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polysorbate 100.

[0131] 40. The composition of item 39, wherein the surfactant is selected from the group consisting of D-a-tocopheryl polyethylene glycol succinate, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-35 hydrogenated castor oil, and polysorbate 80.

[0132] 41. The composition of item 40, wherein the surfactant is D-alpha-tocopheryl polyethylene glycol succinate.

[0133] 42. The composition of item 40, wherein the surfactant is PEG-60 hydrogenated castor oil.

[0134] 43. The composition of item 40, wherein the surfactant is PEG-40 hydrogenated castor oil.

[0135] 44. The composition of item 40, wherein the surfactant is PEG-35 hydrogenated castor oil.

[0136] 45. The composition of item 40, wherein the surfactant is polysorbate 80.

[0137] 46. The composition of any one of items 36-45, further comprising:

[0138] (d) polyethylene glycol; and / or

[0139] (e) an additional surfactant,

[0140] wherein the additional surfactant is optionally a poloxamer.

[0141] 47. The composition of item 46, wherein the polyethylene glycol is PEG 1000, PEG 1450, PEG 1600, PEG 3000, PEG 3350, PEG 4000, PEG 4500, PEG 6000, PEG 8000, and PEG 20000.

[0142] 48. The composition of item 47, wherein the polyethylene glycol is PEG 6000.

[0143] 49. The composition of any one of items 36-48, wherein at least 90% of the resinotoxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 24 months.

[0144] 50. The composition of item 49, wherein at least 95% of the resinotoxin is recovered after storage at about 0 °C to about 25 °C for up to about 3 months to about 12 months.

[0145] 51. The composition of any one of items 36-50, for use in the manufacture of a medicament for the treatment of pain.

[0146] 52. The composition of item 51, wherein the pain is osteoarthritis-related joint pain.

[0147] 53. A kit comprising:

[0148] (a) a first kit component comprising the composition of any one of items 36 to 52; and

[0149] (b) a second kit component comprising a diluent.

[0150] 54. The kit of item 53, wherein the diluent is an aqueous diluent.

[0151] 55. The kit of item 54, wherein the aqueous diluent comprises at least one buffering agent and optionally at least one isotonic agent.

[0152] 56. The kit of item 55, wherein the at least one buffering agent is suitable for maintaining a pH of about 7.7 to about 8.3 upon combination of the first and the second kit components.

[0153] 57. The kit of item 56, wherein the aqueous diluent comprises about 140 mM - 150 mM NaCl, about 3 mM - 5 mM CaCl2x2 H2O, about 3 mM - 5 mM KCl, about 8 mM - 12 mM tris(hydroxymethyl)aminomethane, and a pH of about 7.7 to about 8.3.

[0154] 58. The kit of item 57, wherein the aqueous buffer diluent comprises about 147 mM NaCl, about 4 mM CaCl2x2 H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane, and a pH of about 8.0.

[0155] 59. A method of making a non-aqueous liquid composition of resin toxin, the method comprising combining resin toxin, a surfactant, and a non-aqueous solvent.

[0156] 60. A non-aqueous liquid composition of resin toxin made according to the method of item 59.

[0157] 61. An aqueous composition of resin toxin, the composition comprising:

[0158] (a) about 0.005 mM to about 10 mM resin toxin, preferably about 0.01 mM to about 1 mM resin toxin, more preferably about 0.1 mM to about 1 mM resin toxin, even more preferably about 0.1 mM to about 0.2 mM resin toxin;

[0159] (b) about 0.01% to about 10% by volume of a surfactant, preferably about 0.01% to about 1% by volume of a surfactant, more preferably about 0.1% to about 1% by volume of a surfactant, even more preferably about 0.1% to about 0.5% by volume of a surfactant, especially 0.35% by volume of a surfactant; and

[0160] (c) at least one buffering agent;

[0161] wherein the pH of the composition is about 7.7 to about 8.3.

[0162] 62. The composition of item 61, comprising about 140 mM - 150 mM NaCl, about 3 mM - 5 mM CaCl2x2 H2O, about 3 mM - 5 mM KCl, about 8 mM - 12 mM tris(hydroxymethyl)aminomethane.

[0163] 63. The composition of item 62, comprising about 147 mM NaCl, about 4 mM CaCl2x2 H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane.

[0164] 64. The composition of any one of items 61 to 63, wherein the composition is suitable for intra-articular administration.

[0165] 65. The composition of any one of items 61 to 64, wherein the composition is obtainable by combining the first and the second kit components as defined in any one of items 26 to 31 or 53 to 58.

[0166] 66. The composition of any one of items 61 to 65, for use in the treatment of pain.

[0167] 67. The composition of item 66, wherein the pain is osteoarthritis-related joint pain.

[0168] 68. A method of treating pain, comprising administering to a subject a therapeutically effective amount of the composition of any one of items 61 to 65.

[0169] 69. The method of item 68, wherein the pain is osteoarthritis-related joint pain.

[0170] 70. The composition of any one of items 1 to 25, 35 to 52, and 60 to 67, wherein the composition does not comprise a monosaccharide or a sugar alcohol.

[0171] 71. A dilution of the composition of any one of items 1 to 6, comprising:

[0172] (a) about 0.005 mM to about 10 mM resin toxin, preferably about 0.01 mM to about 1 mM resin toxin, more preferably about 0.1 mM to about 1 mM resin toxin, even more preferably about 0.1 mM to about 0.2 mM resin toxin;

[0173] (b) about 0.01% to about 10% by volume of a surfactant, preferably about 0.01% to about 1% by volume of a surfactant, more preferably about 0.1% to about 1% by volume of a surfactant, even more preferably about 0.1% to about 0.5% by volume of a surfactant, in particular 0.35% by volume of a surfactant; and

[0174] (c) at least one buffering agent;

[0175] wherein the pH of the composition is about 7.7 to about 8.3.

[0176] 72. The dilution of the composition of item 71 comprising about 140 mM - 150 mM NaCl, about 3 mM - 5 mM CaCl2 x 2 H2O, about 3 mM - 5 mM KCl, about 8 mM - 12 mM tris(hydroxymethyl)aminomethane.

[0177] 73. The dilution of the composition of item 72 comprising about 147 mM NaCl, about 4 mM CaCl2 x 2 H2O, about 4 mM KCl, about 10 mM tris(hydroxymethyl)aminomethane.

[0178] 74. The dilution of the composition of any one of items 71 to 73, wherein the composition is suitable for intra-articular administration.

[0179] 75. The dilution of the composition of any one of items 71 to 74, wherein the composition is obtainable by combining the first and the second kit components as defined in any one of items 26 to 31 or 53 to 58.

[0180] 76. The dilution of the composition of any one of items 71 to 75 for use in the treatment of pain.

[0181] 77. The dilution of the composition of item 76, wherein the pain is osteoarthritis- related joint pain.

[0182] 78. A method of treating pain comprising administering to a subject a therapeutically effective amount of the dilution of the composition of any one of items 71 to 75.

[0183] 79. The method of item 78, wherein the pain is osteoarthritis-related joint pain.

[0184] 80. A diluent for the composition of any one of items 1 to 25, 35 to 52, and 71 to 77, wherein the composition does not comprise a monosaccharide or sugar alcohol.

Claims

1. A solid lyophilized, non-aqueous composition of a resin toxin, said composition comprising: (a) resin toxins; and (b) Surfactants, The surfactant mentioned therein is D-α-tocopherol polyethylene glycol succinate.

2. The composition of claim 1, wherein at least 90% of the resin toxin is recovered after the solid is dissolved in an aqueous diluent.

3. The composition of claim 1, wherein at least 90% of the resin toxin is recovered after storage at 0°C to 25°C for 3 to 24 months.

4. The composition according to any one of claims 1 to 3, wherein the composition comprises resin toxin, D-α-tocopherol polyethylene glycol succinate, NaCl, CaCl2 x 2 H2O, KCl, and three (hydroxymethyl)aminomethane.

5. The composition according to any one of claims 1 to 3, wherein the composition does not contain monosaccharides or sugar alcohols.

6. A reagent kit comprising: (a) a first reagent kit component comprising the composition as described in any one of claims 1 to 5; and (b) Second kit component, which contains diluent.

7. The kit of claim 6, wherein the diluent is an aqueous solution.

8. The kit of claim 6, wherein when the first kit component is combined with the second kit component, the resulting aqueous solution has a pH of 7.7 to 8.

3.

9. The kit according to any one of claims 6 to 8, wherein the diluent is an aqueous solution and comprises at least one buffer and optionally at least one isotonic agent.

10. The kit of claim 9, wherein the buffer is adapted to maintain pH between 7.7 and 8.3 when combining the first kit component and the second kit component.

11. The kit according to any one of claims 6 to 8, wherein the diluent is aqueous and comprises 140-150 mM NaCl, 3-5 mM CaCl2 x 2 H2O, 3-5 mM KCl, 8-12 mM tris(hydroxymethyl)aminomethane, and has a pH of 7.7 to 8.

3.

12. The kit according to any one of claims 6 to 8, wherein the kit is supplied in the following form: (a) A separate compartment for primary packaging; (b) Separate primary packages that are packaged together in a secondary package; (c) A separate primary package contained in two or more separate secondary packages, the secondary packages being held together in sequence by paper or plastic wrapping paper, ribbon, sleeve, etc.; or (d) Their combination.

13. The composition of any one of claims 1 to 5 or the kit of any one of claims 6 to 12, for use in a method for treating pain.

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