Liquid botulinum toxin preparations and uses thereof

Abstract

The present invention relates to a liquid botulinum toxin formulation comprising (i) a botulinum toxin, (ii) a stabilizing protein such as human serum albumin, (iii) a chelating agent (e.g., EDTA) or a phosphate salt, and optionally (iv) a calcium, magnesium, or zinc salt, (v) a tonicity agent, and / or (vi) a buffering agent. Additionally, the present invention relates to the use of the liquid botulinum toxin formulation for therapeutic and cosmetic treatments.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a liquid botulinum toxin formulation comprising (i) a botulinum toxin, (ii) a stabilizing protein such as human serum albumin, (iii) a chelating agent (e.g., EDTA) or a phosphate salt, and optionally (iv) a calcium, magnesium, or zinc salt, (v) a tonicity agent, and / or (vi) a buffering agent. Additionally, the present invention relates to the use of the liquid botulinum toxin formulation for therapeutic and cosmetic treatments. [Background technology]

[0002] 2. Background of the Invention Botulinum neurotoxins (BoNTs, or botulinum toxins (BTs)) are a family of bacterial neurotoxins that are widely used to treat an increasingly diverse range of neurological, medical, and cosmetic conditions. There are eight serotypes of BoNTs (BoNT / A-H). Two serotypes, A (BoNT / A) and B (BoNT / B), are currently in clinical use. BoNTs are produced in the form of high molecular weight (up to approximately 900 kDa) complexes by Clostridium species, particularly Clostridium botulinum. These toxin complexes consist of an active 150 kDa neurotoxin and several complex proteins (non-toxic neurotoxin-associated proteins, NAPs).

[0003] The 150 kDa neurotoxin is synthesized as an inactive single-chain polypeptide (~150 kDa) that is proteolytically cleaved to yield a light chain (LC, ~50 kDa) and a heavy chain (HC, ~100 kDa) linked by interchain disulfide bonds. The HC contains a C-terminal domain that mediates binding to receptors and an N-terminal domain that mediates translocation of LC across endosomal membranes. LC acts as a protease in neurons, cleaving neuronal SNARE proteins. This blocks fusion of synaptic vesicles with the plasma membrane, thus inhibiting neurotransmitter release from selected neurons.

[0004] Formulation of BoNT is very challenging due to the complexity of its structure and the low product concentrations used. BoNT is extremely sensitive to a variety of conditions, including heat and alkaline pH. Thus, since BoNT only functions when its structure is intact, the challenge in preparing a medical dosage form of BoNT is to formulate a composition that protects BoNT from inactivation or partial loss of biological activity during manufacture, storage, or use of the product. Furthermore, because the potency of BoNT is extremely high, with a lethal dose in humans ranging from only about 0.1 to 1 ng / kg, pharmaceutical formulations of BoNT contain extremely small amounts of toxin, ranging from only about 1 ng per vial. This exacerbates the known problem of loss of toxin activity due to surface denaturation. Thus, one of the main challenges in formulating BoNT is to minimize activity loss during manufacture and storage.

[0005] Due to these factors, the main BoNT products available today are provided as lyophilized powders, a form that is stable for long periods at 2-8°C or even at room temperature (Xeomin®). Lyophilized forms of the BoNT / A complex were first introduced to the market in 1989 (Botox®, Allergan) and 1991 (Dysport®, Ipsen). In 2005, the first stable dosage form of pure 150 kDa BoNT / A neurotoxin, free of protein complexes, was approved (Xeomin®, Merz Pharmaceuticals). However, these lyophilized products must be reconstituted before use, a process that can lead to dosing errors and sterility problems. Thus, there has been a great deal of effort to develop liquid formulations of BoNT that are more user-friendly and easier to administer.

[0006] The first liquid formulation of BoNT was approved in 2000 and launched in Europe in 2001 as Neurobloc®. Neurobloc® (Eisai) is a sterile solution of the BoNT / B complex formulated in a buffer containing disodium succinate, sodium chloride, human serum albumin (HSA), sodium caprylate, and sodium N-acetyltryptophanate. However, the product has an acidic pH, which means it can be painful when injected. For BoNT / A, only two liquid formulations have received marketing authorization to date: Innotox® (Medytox), which is limited to domestic Asian markets (e.g., South Korea, approved in 2013), and Alluzience® (lpsen / Galderma), which was approved for use in Europe in 2021. Both of these liquid formulations contain the BoNT / A toxin complex and, in addition to water, sodium chloride, detergents (Innotox®: polysorbate 20, Alluzience®: polysorbate 80), amino acids (Innotox®: methionine, Alluzience®: histidine), and additional excipients (Innotox®: sodium phosphate as a buffering agent, Alluzience®: sucrose). Summary of the Invention [Problem to be solved by the invention]

[0007] Despite these advances in the preparation of liquid botulinum toxin formulations, there remains a need for new options for developing ready-to-use liquid formulations of botulinum toxin that are stable during shipping and storage.

[0008] Objective of the invention It is an object of the present invention to provide a stable, ready-to-use liquid formulation of botulinum toxin for therapeutic and cosmetic treatments. [Means for solving the problem]

[0009] Summary of the Invention The present invention is based on the surprising discovery that the addition of a chelating agent (e.g., EDTA) or phosphate to a liquid formulation of botulinum toxin containing human serum albumin (HSA) results in an improved formulation that is both heat and light stable.

[0010] Thus, in one embodiment, the present invention provides a liquid formulation comprising: (i) botulinum toxin, (ii) a stabilizing protein, preferably human serum albumin (HSA), and (iii) Chelating agents or phosphates.

[0011] Optionally, the liquid formulation of the invention further comprises (iv) a salt of calcium, magnesium or zinc, or a mixture thereof; or (v) an isotonicity agent (e.g., sodium chloride); or (vi) a buffering agent (e.g., a phosphate buffer or a histidine buffer, or both); or two of components (iv)-(vi) (e.g., (iv) and (v), (iv) and (vi), or (v) and (vi)), or all three of components (iv)-(vi).

[0012] In another aspect, the invention relates to the liquid formulations of the invention for use in therapy, in particular for use in the treatment of neuromuscular disorders, pain, sialorrhea, hyperhidrosis, urological disorders, and neurological disorders.

[0013] In a further aspect, the present invention relates to the cosmetic (aesthetic) use of the liquid formulation of the present invention for the treatment of a cosmetic condition, preferably a skin condition, in particular a skin line or crease.

[0014] In yet another aspect, the present invention relates to a method for treating a disease or condition comprising administering to a person in need thereof an effective amount of the liquid formulation of the present invention.

[0015] Preferred embodiments of the liquid botulinum toxin formulations, their uses and methods of use according to the invention are set out in the accompanying claims.

[0016] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The present invention is based on the unexpected discovery that the presence of a chelating agent (e.g., EDTA) or a phosphate salt in a liquid formulation of botulinum toxin containing human serum albumin (HSA) results in an improved formulation that is stable to light as well as heat. Additionally, it has been unexpectedly found that the addition of calcium, magnesium, or zinc salts reduces the pain of injection.

[0018] The liquid formulations of the present invention are ready to use without the need for reconstitution prior to injection, and therefore offer the advantage of improved safety and accuracy of administration compared to powdered, lyophilized botulinum toxin formulations. Furthermore, the liquid formulations exhibit superior stability, which facilitates transportation and storage, as well as handling by the physician.

[0019] Without wishing to be bound by theory, it is believed that the observed light sensitivity is due to the involvement of stabilizing proteins, such as, for example, HSA. Furthermore, the surprising finding that the liquid formulations of the present invention have improved photostability, i.e., reduced loss of toxin activity due to light (reduced light sensitivity), is believed to be due to the chelating agent or phosphate interacting with or affecting components contained in the HSA or the HSA material used, thereby avoiding or minimizing the adverse effects of light on the ability of HSA to stabilize botulinum toxin.

[0020] The increased photostability of the liquid botulinum toxin formulations of the invention simplifies the manufacturing process, allows the product to be filled and packaged without extensive photoprotection, and reduces the potential for loss of activity due to storage exposed to light at the physician's location until use. Overall, the reduced sensitivity of the liquid formulations of the invention improves the overall ease of handling and use of the botulinum toxin during injection procedures.

[0021] In one embodiment, the present invention provides a method for producing a method for treating a pulmonary arthritis, comprising: (i) botulinum toxin, (ii) a stabilizing protein, and (iii) liquid formulations containing a chelating agent or a phosphate salt.

[0022] As used herein, the term "comprising", including and containing, as well as variations thereof, such as "comprises", "includes" and "contains", are intended to refer to a non-exclusive inclusion, such that a process, method, product-by-process, composition or formulation that comprises, includes or contains an element or list of elements may include other elements not expressly listed for such process, method, product-by-process, composition or formulation, rather than only including those elements. Furthermore, within the framework of the present invention, each of the terms "comprises", "comprising", "includes", "including", "contains", "containing" and variations thereof are intended to be replaceable with the terms "consists" or "consisting" or any variations thereof (e.g., "consists essentially of"), which are understood to refer to an exclusive inclusion of the indicated elements.

[0023] As used in the context of this specification, the terms "a," "an," "the," and similar references are to be construed to cover both the singular and the plural, and thus may also refer to "at least one" or "two or more," unless otherwise specified herein or clearly contradicted by context.

[0024] The term "liquid formulation" as used herein generally refers to an aqueous formulation, typically an aqueous solution. The term "liquid formulation" as used herein can be used interchangeably with "liquid composition". Preferably, the liquid formulation is a pharma- ceutically acceptable liquid formulation. The term "pharma-ceutically acceptable" as used herein means that the liquid formulation does not cause unacceptable side effects when administered to a human patient or subject. That is, the liquid formulation is suitable for human use. The aqueous solution may be a buffer, with or without saline, or may be saline, such as buffered (e.g., phosphate and / or histidine buffered) saline.

[0025] The liquid formulation of the present invention can be stored in any suitable container system. A suitable container system for storing the liquid formulation of the present invention is any device that is sealable or sealed and has a partially or completely sealed space that can be used to contain, store, and / or transport the liquid formulation. The container system is preferably a sealed (or sealed) container made of glass or plastic (e.g., organic polymer), or partially or mainly made of glass or plastic, including, for example, a container in the form of (i) a syringe, (ii) a vial, (iii) a carpule, or (iv) an ampoule. In a preferred embodiment of the present invention, the liquid formulation is stored in a syringe in the form of a pre-filled syringe, as known in the art.

[0026] With regard to component (i), the botulinum toxin is not particularly limited and includes botulinum toxins of any serotype (BoNT / A to G). For example, the botulinum toxin may be of serotype A or B (BoNT / A, BoNT / B). Preferably, the botulinum toxin is of serotype A, more preferably of serotype A1 (BoNT / A1), and most preferably of BoNT / A1 produced by the Clostridium botulinum Hall strain. Furthermore, the botulinum toxin may be other botulinum toxins, such as naturally occurring neurotoxins obtained from Clostridium botulinum, or botulinum toxins obtained from alternative sources, including recombinant techniques, genetic or chemical modifications.

[0027] Furthermore, as used herein, the term "botulinum toxin" ("BT"), and the synonymously used term "botulinum neurotoxin" ("BoNT"), are intended to refer to pure botulinum neurotoxin and / or any complex thereof, i.e., any complex of pure botulinum neurotoxin and a complex protein (referred to as a "toxin complex").

[0028] The term "pure botulinum neurotoxin" as used herein means a botulinum neurotoxin that does not contain complex proteins (sometimes referred to as "neurotoxic components"), or more precisely, a botulinum neurotoxin that does not contain neurotoxin-associated complex proteins (NAPs). A pure botulinum neurotoxin is an (active) neurotoxic polypeptide that ultimately inhibits the release of acetylcholine. It is a two-chain protein consisting of a light chain (LC, about 50 kDa) and a heavy chain (HC, about 100 kDa) linked by a disulfide bond. Thus, the active neurotoxic polypeptide is also referred to herein as "150 kDa neurotoxin," "botulinum neurotoxin (150 kD)," or "neurotoxic component."

[0029] The term "toxin complex" as used herein refers to a macromolecular complex of a neurotoxic component and a series of complex proteins (NAPs). In particular, the term "toxin complex" includes the 900 kDa, 500 kDa, and 300 kDa Clostridium botulinum type A toxin complexes. The complex proteins are non-toxic non-hemagglutinins (NTNHA) and, in strains of serotypes A-D, different hemagglutinins (HA). For example, the 900 kDa complex is contained in onabotulinumtoxinA (Botox® / Vistabel®, Allergan, Inc., Irvine, CA, USA). Toxin complexes as active agents are also contained in Dysport® (Azzalure®, psen, Paris, France), Alluzience® (lpsen / Galderma), and Innotox® (Medytox).

[0030] The concentration of botulinum toxin in the liquid preparation of the present invention is in the range of 1 to 1000 U / ml, preferably in the range of 10 to 200 U / ml, more preferably in the range of 20 to 150 U / ml, for example, 50 U / ml or 100 U / ml.

[0031] As used herein, the term "unit" or "U" refers to the biological activity (biological potency) of a toxin and is the dose that is lethal to 50% of the mice tested (LD 50 ) More specifically, in the context of the present invention, unless otherwise stated, LD 50 is measured using the Mouse Bioassay (MBA). The MBA is the mean lethal dose (LD) of a toxin / neurotoxin following intraperitoneal injection in mice. 50 ), i.e., the amount of toxin / neurotoxin that can kill 50% of a group of mice. On this basis, one unit (U) of toxin / neurotoxin as used herein is defined as one mouse LD 50 (1.0LD 50 = 1.0U). 50The mouse bioassay is the gold standard of various biological, chemical, or immunological detection methods for botulinum toxins and is known to those of skill in the art (see, e.g., Pearce, LB; Borodic, GE; First, ER; MacCallum, RD Measurement of botulinum toxin activity: Evaluation of the lethality assay. Toxicol. Appl. Pharmacol. 1994, 128:69-77). Those of skill in the art can determine the appropriate botulinum toxin concentration depending on the serotype and intended use.

[0032] Alternatively, cell-based assays can be used to determine botulinum toxin activity, such as those described in WO2009 / 114748, WO2013 / 049508, or WO2014 / 207109. 50 Calibration using reference standards allowed the conversion of botulinum toxin activity results obtained in cell-based assays to mouse LD 50 It can be correlated with the results obtained in the assay.

[0033] LD used by manufacturers of commercial botulinum toxin preparations 50Because of differences in testing, the unit strengths they present for commercially available botulinum toxin preparations are proprietary and cannot be easily compared. Thus, within the framework of the present invention, the conversion rates shown below are used to determine the efficacy of botulinum toxin A ("INCO", Xeomin®, Bocouture®; botulinum toxin serotype A, free of complex proteins; Merz Pharmaceuticals GmbH), onabotulinum toxin A ("ONA", Botox®, Vistabel®; botulinum toxin complex of serotype A; Allergan Inc.), abobotulinum toxin A ("ABO", Dysport®, Azzalure®; botulinum toxin complex of serotype A; Medicis Pharmaceutica lCorp., Galderma Lab.), rimabotulinum toxin B ("RIM", Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences The relative potencies of ONA, INCO, botulinum toxin serotype A, and PurTox® ("TBD"; botulinum toxin serotype A; Mentor Worldwide LLC) are established. For purposes of use herein, the conversion ratio of ONA to INCO is 1:1. The conversion ratio of ONA / INCO:ABO is 1:2.5. The conversion ratio of ONA / INCO:RIM is 1:50 and the conversion ratio of ONA / INCO:TBD is 1:1.5.

[0034] With respect to component (ii) of the liquid formulation of the present invention, the term "stabilizing protein" generally refers to a polypeptide that increases the stability of a botulinum toxin. As used herein, the term "polypeptide" is used interchangeably with the term "protein" and refers to a peptide consisting of more than 10 amino acids, preferably more than 50 amino acids, more preferably more than 100 amino acids, most preferably more than 200 amino acids, and preferably up to 1000 amino acids linked by peptide bonds. Particularly preferred herein are polypeptides containing 150-1000 amino acids, or 250-800 amino acids, particularly 300-1000 amino acids or 350-700 amino acids.

[0035] In the present invention, the stabilizing protein can be, for example, human serum albumin (HSA), ovalbumin, casein, or a mixture thereof. Particularly preferred for use herein are human serum albumin, ovalbumin, or a mixture thereof, and most preferred is HSA.

[0036] The stabilizing protein or mixture of stabilizing proteins may be present in the liquid formulation in an amount of 0.001-2.0% w / v, preferably 0.01-1.0% w / v, more preferably 0.01-0.5% w / v, and most preferably 0.02-0.3% w / v or 0.03-0.10% w / v. The most preferred stabilizing protein, human serum albumin (HSA), is preferably present in the liquid formulation in an amount of 0.001-1.00% w / v, more preferably 0.01-0.3% w / v, and most preferably 0.02-0.15% w / v. As used herein, the term "human serum albumin" or "HSA" refers to donor HSA (HSA derived from human blood, more precisely HSA derived from human plasma), recombinant HSA, and / or HSA derived from any other source. Preferably, the human serum albumin is recombinant HSA or HSA derived from human blood.

[0037] Without wishing to be bound by theory, it was surprisingly found that photostability decreased with increasing HSA concentration and therefore it is believed that the light sensitivity of the formulations of the present invention is at least in part related to components contained in the HSA or the HSA material used.

[0038] Regarding component (iii), the chelating agent is not particularly limited as long as it can bind metal ions. The term "chelating agent" as used herein is also referred to as "chelating agent" or "sequestering agent". The chelating agent as used herein is generally a metal ion-binding organic compound. The metal ion generally forms multiple coordinate bonds with the organic chelating agent, which acts as a multidentate ligand.

[0039] Chelators suitable for use in the present invention include, but are not limited to, aminopolycarboxylic acids (e.g., aminopolycarboxylic acids having 3-6, preferably 4, carboxylic acid functional groups) and other compounds such as citric acid, porphyrins, TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), TETA (triethylenetetramine), and mixtures thereof. Representative aminopolycarboxylic acids include NTA (nitrilotriacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TED (ethylenediaminotriacetic acid), EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), and TTHA (triethylenetetraminehexaacetic acid).

[0040] Particularly preferred chelating agents for use in the present invention include those having the general formula (I): (HO2CCH2)2N-RN(CH2CO2H)2(I) The R group is not particularly limited and may contain zero, one or two carboxylic acid functional groups. Preferably, the R group contains zero or one carboxylic acid functional group, and most preferably, no carboxylic acid functional group.

[0041] Examples of compounds of general formula (I) include, for example, EDTA, EGTA, BAPTA, DTPA, and TTHA. Particularly preferred for use in the present invention are EDTA, EGTA, and DTPA, more preferably EDTA and DTPA, and most preferably EDTA. Any mixture of the above chelating agents can also be used in the present invention.

[0042] The chelating agent may be present at a concentration of at least 0.01 mM, or at least 0.1 mM, or at least 1 mM. Preferably, the chelating agent is present at a concentration of 0.01 to 100 mM, or at a concentration of 0.1 to 50 mM, more preferably at a concentration of 0.05 to 20 mM, and most preferably at a concentration of 1 to 10 mM.

[0043] Further, with respect to component (iii), it is contemplated herein that in the liquid formulation of the present invention, instead of or in addition to the chelating agent, a phosphate may be used to reduce the photosensitivity of the formulation. Indeed, it has been unexpectedly found that the presence of a phosphate also significantly improves photostability. The phosphate may be present in the liquid formulation at a concentration of at least 0.1 mM, or at least 1 mM, or at least 5 mM. Preferably, the phosphate is present in the liquid formulation at a concentration of 0.1-100 mM, preferably 1-50 mM, more preferably 5-30 mM.

[0044] Preferably, the liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) human serum albumin, and (iii) a chelating agent, preferably EDTA. This preferred liquid formulation, as well as other liquid formulations described herein above, may further comprise one or more of optional components (iv), (v), and (vi) described below, i.e., (iv), or (v), or (vi), or (iv) and (v), or (iv) and (vi), or (v) and (vi), or (vi), (v) and (vi).

[0045] In a preferred embodiment of the invention, the liquid formulation may further comprise a salt of an alkaline earth metal or a transition metal, preferably further comprising: (iv) Salts of calcium, magnesium, or zinc, or mixtures thereof.

[0046] Preferred salts are calcium, magnesium and zinc salts, more preferably calcium and magnesium salts, and most preferably calcium salts. In the presence of EDTA, the addition of these salts forms EDTA complexes, i.e., EDTA with the added metal cation complexed as the central atom. The nature of the anion of the salt is not critical, the only requirement being that the salt is soluble in the aqueous formulation of the present invention. For example, the anion may be the anion of pharma-ceutically acceptable inorganic and organic acids. Alternatively, the chelating agent itself may form the anion of the metal salt, and components (iii) and (iv) are added to the liquid formulation, for example as Na2CaEDTA.

[0047] Suitable counter anions include salts, such as acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, glycolylarsanilate, hexanoate, hydrabamine, hydroxynaphthoate, iodide, lactobionate, nitrate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, acetate, succinate, sulfate, tartrate, and teoclate.

[0048] Preferably, the salt is a chloride. Particularly preferred salts include calcium chloride (i.e., CaCl2 and its hydrates), magnesium chloride (i.e., MgCl2 and its hydrates), and zinc chloride (i.e., ZnCl2 and its hydrates). In the presence of EDTA, the addition of these salts results in the formation of EDTA complexes, i.e., EDTA with the added metal cation complexed as the central atom.

[0049] In general, it is expected that the concentration of the metal ion of the salt is approximately equimolar to the concentration of the chelating agent. The salt can be present in the liquid formulation at a concentration of at least 0.01 mM, or at least 0.1 mM, or at least 1 mM. Preferably, the salt is present in the liquid formulation at a concentration of 0.01 to 100 mM, preferably 0.1 to 50 mM, more preferably 0.05 to 20 mM, and most preferably 1 to 10 mM.

[0050] Surprisingly, the inventors of the present invention have discovered that the addition of an alkaline earth metal or transition metal salt, particularly a calcium, magnesium, or zinc salt, significantly reduces the pain of injection of the liquid formulations of the present invention while maintaining storage and photostability. This discovery was unexpected because (i) the liquid botulinum toxin formulations described herein were found to cause unpleasant injection pain that was neither foreseen nor predicted, and (ii) the addition of an alkaline earth metal or transition metal salt, such as calcium chloride, was surprisingly found to dramatically reduce the injection pain while (iii) maintaining the storage and photostability of the liquid formulations, even though it was believed that a free (uncomplexed) chelating agent was required to achieve the photostabilizing effect. That is, the central metal atom (e.g., Ca 2+ It has been surprisingly found that chelating agents, including EDTA, can protect botulinum toxins from light.

[0051] According to the present invention, the liquid formulation may optionally further comprise: (v) Tonicity agent.

[0052] The term "isotonicity agent" as used herein refers to an agent that is added to an injection formulation to make the osmotic properties of the formulation similar to physiological fluid. The isotonicity agent is also called an "osmotic pressure regulator". The isotonicity agent is not particularly limited and can be selected from the group consisting of, for example, sugars, salts, polymers, and mixtures thereof.

[0053] Exemplary tonicity agents include sucrose, glucose, sodium carbonate, amino acids, polyethylene glycol (PEG), dextran, cyclodextrin, and colloids (e.g., colloidal polysaccharides).Typically, the concentration of the tonicity agent is in the range of 0-2.0% w / v, particularly 0.01-2.0% w / v, or 0.1-1.5% w / v, more particularly 0.6-1.2% w / v.

[0054] Preferably, the isotonicity agent is sodium chloride (NaCl), which may be present in the liquid formulation of the present invention in an amount of 0.01-2.0% w / v, preferably 0.1-1.5% w / v, more preferably 0.5-1.2% w / v, or 0.8-1.0% w / v, and most preferably 0.9% w / v.

[0055] According to the present invention, the liquid formulation may optionally further comprise: (vi) Buffering agents.

[0056] The term "buffering agent" as used herein refers to an agent that maintains the pH of a liquid formulation within an acceptable range, i.e., an agent that can control the pH of the formulation. A suitable buffering agent is one that does not chemically react with other ingredients and is present in an amount sufficient to provide the desired degree of pH buffering. Such buffering agents include, for example, amino acids, acetate, malic acid, ascorbate, citrate, tartrate, fumarate, succinate, phosphate, bicarbonate, Tris, Bis-Tris, ACES, MES, BES, MOPS, HEPES, TES, PIPES, tricine, and imidazole.

[0057] Preferably, the buffer is a phosphate, an amino acid, or a mixture thereof. It should be noted that phosphate can play a dual role in the present invention, i.e., it can function as a phosphate pH buffer and exert a stabilizing effect as component (iii) of the present invention. The amino acid can be selected from aspartic acid, glycine, glutamic acid, histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof, and is preferably selected from histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof. Most preferably, the amino acid is histidine. The most preferred buffer for use herein is histidine, a phosphate, or a mixture thereof.

[0058] The concentration of the buffer in the liquid formulation of the present invention is preferably 1 to 100 mM, more preferably 2 to 50 mM, and most preferably 5 to 20 mM. When the buffer is an amino acid (e.g., histidine), the buffer may be present in the liquid formulation at a concentration of 1 to 100 mM, preferably 2 to 50 mM, more preferably 5 to 20 mM, and most preferably 10 mM. When the buffer is a phosphate, the buffer may be present in the liquid formulation at a concentration of 1 to 100 mM, preferably 2 to 50 mM, more preferably 5 to 20 mM, and most preferably 10 mM.

[0059] The pH of the liquid formulation of the present invention is typically in the range of 5.0 to 8.0, particularly in the range of 5.5 to 7.5, and preferably in the range of 5.5 to 7.0 or 6.0 to 7.5, more preferably in the range of 6.0 to 7.0, and most preferably in the range of 6.0 to 6.5.

[0060] In addition, the liquid formulation of the present invention may further comprise one or more additional pharma- ceutically acceptable excipients, unless otherwise specified or intended. For example, the liquid formulation may comprise one or more of glycerol, sucrose, lactose, mannitol, hyaluronic acid, lactic acid, citric acid, amino acids, benzyl alcohol, lidocaine, gelatin, hydroxyethyl starch (HES), polyethylene oxide, and polysorbates (e.g., polysorbate 20, polysorbate 80). Other suitable pharma-ceutically acceptable excipients include those well known in the art (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania).

[0061] On the other hand, it is also contemplated that the liquid formulations of the present invention are specifically free of certain components (i.e., compounds, materials, or substances), such as detergents, polysaccharides, amino acids, stabilizing peptides, and the like (including any combination thereof). As used herein, the term "detergent" is used synonymously with "surfactant" and is intended to include non-ionic detergents and ionic detergents. As used herein, the term "stabilizing peptide" generally refers to a peptide of 5-50 amino acids, e.g., 10-40 amino acids, or 15-30 amino acids. Thus, the term "stabilizing peptide" excludes HSA.

[0062] In one embodiment, the liquid formulation of the present invention does not contain a detergent, in particular does not contain a polysorbate, more particularly does not contain polysorbate 20 and / or polysorbate 80. In another embodiment, the liquid formulation of the present invention does not contain an alginate. In another embodiment, the liquid formulation of the present invention does not contain a succinate. In another embodiment, the liquid formulation of the present invention does not contain one or more (e.g., 2, 3, 4, or 5) amino acids selected from the group consisting of arginine, glutamic acid, methionine, tryptophan, and serine. In another embodiment, the liquid formulation of the present invention does not contain sugars, such as monosaccharides, oligosaccharides, polysaccharides, or mixtures thereof. In particular, the liquid formulation of the present invention may contain one or more (e.g., 2, 3, or 4) of sucrose, lactose, maltose, and trehalose. It is also contemplated in the present invention that the liquid formulation lacks one or more, or all, of the above compounds.

[0063] In one embodiment, the liquid formulation of the invention lacks: (i) detergent and monosaccharides, oligosaccharides, and polysaccharides; (ii) detergent and any amino acid, or detergent and all amino acids except histidine; (iii) detergent and stabilizing peptides; (iv) monosaccharides, oligosaccharides, and polysaccharides and any amino acid, or monosaccharides, oligosaccharides, and polysaccharides and all amino acids except histidine; (v) monosaccharides, oligosaccharides, and polysaccharides and stabilizing peptides; (vi) any amino acid and stabilizing peptide, or all amino acids except histidine and stabilizing peptides; (vii) detergent, monosaccharides, oligosaccharides, and polysaccharides and any amino acid, or detergent; (viii) detergents, monosaccharides, oligosaccharides, and polysaccharides and stabilizing peptides; (ix) detergents, any amino acids, and stabilizing peptides, or detergents, all amino acids other than histidine, and stabilizing peptides; (x) monosaccharides, oligosaccharides, and polysaccharides, any amino acids and stabilizing peptides, or monosaccharides, oligosaccharides, and polysaccharides, all amino acids other than histidine, and stabilizing peptides; (xi) detergents, monosaccharides, oligosaccharides, and polysaccharides, any amino acids and stabilizing peptides, or detergents, monosaccharides, oligosaccharides, and polysaccharides, all amino acids other than histidine, and stabilizing peptides.

[0064] In another embodiment, the liquid formulation of the present invention does not contain any amino acids other than histidine. In another embodiment, the liquid formulation of the present invention does not contain any monosaccharides, disaccharides, or trisaccharides. In another embodiment, the liquid formulation of the present invention does not contain any stabilizing peptides or proteins other than HSA. In another embodiment, the liquid formulation of the present invention does not contain any phosphate, for example in the form of a phosphate buffer.

[0065] In a preferred embodiment of the invention, the liquid formulation is deficient in: (i) succinate and a detergent (e.g., polysorbate), (ii) succinate and methionine, (iii) succinate and sucrose, (iv) a detergent (e.g., polysorbate) and methionine, (v) a detergent (e.g., polysorbate) and sucrose, (vi) methionine and sucrose, (vii) succinate, a detergent (e.g., polysorbate), and methionine, (xii) a mixture of succinate, a detergent (e.g., polysorbate), and methionine, (xiii) a mixture of succinate, a detergent (e.g., polysorbate), and sucrose, (xiv) a mixture of succinate, a detergent (e.g., polysorbate), and methionine, (xv) a mixture of succinate, a detergent (e.g., polysorbate), and methionine, (xvi) a mixture of succinate, a detergent (e.g., polysorbate), and methionine, (xv) a mixture of succinate, a detergent (e.g., polysorbate), and sucrose, (xv ... (i) succinate, a detergent (e.g., polysorbate), and sucrose, (ix) succinate, methionine, and sucrose, (x) a detergent (e.g., polysorbate), methionine, and sucrose, and (xi) succinate, a detergent (e.g., polysorbate), methionine, and sucrose, (xii) a detergent (e.g., polysorbate) and histidine, (xiii) a detergent (e.g., polysorbate), histidine, and sucrose.

[0066] A preferred liquid formulation of the invention comprises (i) a botulinum toxin, (ii) HSA, and (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2, where R contains zero or one or two carboxylic acid groups. A preferred liquid formulation of the invention comprises (i) a botulinum toxin in a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, and (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2, where R contains zero or one or two carboxylic acid groups, at a concentration of 0.05-20 mM.

[0067] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, and (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA. A preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, and (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA, at a concentration of 0.05-20 mM.

[0068] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, and (iii) EDTA. A preferred liquid formulation of the present invention comprises (i) a botulinum toxin in a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, and (iii) EDTA in a concentration of 0.05-20 mM.

[0069] A preferred liquid formulation of the invention comprises (i) a botulinum toxin, (ii) HSA, (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2, where R does not contain a carboxylic acid group or contains one or two carboxylic acid groups, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride. A preferred liquid formulation of the invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2, where R does not contain a carboxylic acid group or contains one or two carboxylic acid groups, at a concentration of 0.05-20 mM, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, at a concentration of 0.05-20 mM.

[0070] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, and (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride. A preferred liquid formulation of the invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA at a concentration of 0.05-20 mM, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, at a concentration of 0.05-20 mM.

[0071] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, (iii) EDTA, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride. A preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, (iii) EDTA at a concentration of 0.05-20 mM, and (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, at a concentration of 0.05-20 mM.

[0072] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2 (wherein R contains zero, one or two carboxylic acid groups), (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, and (v) a phosphate buffer and / or histidine. A preferred liquid formulation of the invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, (iii) a chelating agent of the general formula (HO2CCH2)2N-RN(CH2CO2H)2 (wherein R contains zero, one or two carboxylic acid groups) at a concentration of 0.05-20 mM, (iv) a calcium, magnesium or zinc salt or mixtures thereof, preferably calcium chloride, at a concentration of 0.05-20 mM, and (v) phosphate buffer and / or histidine at a concentration of 2-50 mM.

[0073] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA, (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, and (v) a phosphate buffer and / or histidine. A preferred liquid formulation of the present invention comprises: (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-0.5% w / v; (iii) a chelating agent selected from the group consisting of DOTA, TED, EDTA, EGTA, BAPTA, DTPA, and TTHA, or a chelating agent selected from the group consisting of EDTA, EGTA, BAPTA, DTPA, and TTHA, at a concentration of 0.05-20 mM; (iv) a calcium, magnesium, or zinc salt, or a mixture thereof, preferably calcium chloride, at a concentration of 0.05-20 mM; and (v) a phosphate buffer and / or histidine at a concentration of 2-50 mM.

[0074] A preferred liquid formulation of the present invention comprises (i) a botulinum toxin, (ii) HSA, (iii) EDTA, (iv) a calcium, magnesium, or zinc salt or a mixture thereof, preferably calcium chloride, and (v) a phosphate buffer and / or histidine. A preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml, (ii) HSA in an amount of 0.01-0.5% w / v, (iii) EDTA at a concentration of 0.05-20 mM, (iv) a calcium, magnesium, or zinc salt or a mixture thereof, preferably calcium chloride, and (v) a phosphate buffer and / or histidine at a concentration of 2-50 mM.

[0075] Particularly preferably, the above preferred liquid formulation further comprises sodium chloride, particularly sodium chloride at a concentration of about 0.9% w / v. Furthermore, the pH is preferably in the range of 6.0 to 7.5. Furthermore, the botulinum toxin is preferably serotype A, particularly the neurotoxic component of serotype A.

[0076] Exemplary preferred liquid formulations of the present invention include: Formulation 1 : 50U / ml BoNT / A 0.9% NaCl (9 mg / mL) 0.05% HSA (0.5 mg / mL) 0.1%Na2-EDTA (1mg / ml; approx. 2.7mM), pH 5.5 to 8.0 (e.g., 6.0), and optionally pH buffer (e.g., 10 mM phosphate or 10 mM histidine) Formulation 2 : 50U / ml BoNT / A 0.9% NaCl (9 mg / mL) 0.085% HSA (0.85 mg / mL) 0.155% histidine (1.55 mg / ml; approximately 10 mM) 0.13%Na2-EDTA (1.3mg / mL; approx. 3.5mM) 0.039% CaCl2 (390μg / mL; approx. 3.5mM) pH 5.5 to 8.0 (e.g., 6.0), and optionally pH buffer (e.g., 10 mM phosphate)

[0077] The present invention also relates to a liquid formulation comprising a botulinum toxin, wherein the toxin activity does not decrease by more than 35% compared to the initial toxin activity when the liquid formulation is stored at an elevated temperature of 40° C. for 4 weeks. In another embodiment, the present invention relates to a liquid formulation comprising a botulinum toxin, wherein the liquid formulation is stored at an elevated temperature of 250 W / m 2 Even after 7 hours of exposure to a light source, the toxin activity does not decrease by more than 35% compared to the initial toxin activity.

[0078] Moreover, the present invention further relates to a method for preparing a liquid botulinum toxin formulation as described herein, comprising combining the components as described herein, particularly components (i)-(iii), and optionally one or more of components (iv)-(vi). The preparation of the liquid formulations of the present invention is not particularly limited, and the respective techniques are known to those skilled in the art. As mentioned above, liquid formulations of botulinum toxin are generally aqueous solutions, preferably saline, more preferably saline, and most preferably buffered (e.g., phosphate-buffered or histidine-buffered) saline.

[0079] Preferably, the salt is dissolved first, then the HSA is added, the pH is adjusted if necessary, and finally the botulinum toxin is added. This order is not essential, but is believed to safely maintain maximum specific activity of the botulinum toxin. Preferably, the method for preparing a liquid botulinum toxin formulation does not include reconstitution of a lyophilized botulinum toxin formulation in powder form.

[0080] The present invention further relates to a method for stabilizing a liquid botulinum toxin formulation comprising mixing (i) a botulinum toxin, (ii) a stabilizing protein, preferably human serum albumin (HSA), (iii) a chelating agent or a phosphate salt, and optionally one or more of components (iv)-(vi), where components (i)-(v) are as defined herein.

[0081] The invention further relates to the use of a chelating agent as defined herein to improve the photostability of an aqueous botulinum toxin formulation comprising (i) a botulinum toxin, and (ii) a stabilizing protein, preferably human serum albumin, and optionally one or more of components (iv)-(vi), where components (i), (ii), and (iv)-(vi) are as defined herein. "Improved photostability" in this context refers to an aqueous botulinum toxin formulation comprising a chelating agent, the formulation being capable of withstanding a UV-induced ... 2 The biological activity of botulinum toxin after exposure to 250 W / m2 light for 7 hours at the same temperature (e.g., 20°C) is 2 The botulinum toxin has a biological activity that is at least 20% greater, preferably at least 40% greater, more preferably at least 60% greater, and most preferably at least 80% greater than the botulinum toxin activity of an aqueous botulinum toxin formulation that does not contain a chelating agent after exposure to light for 7 hours.

[0082] The present invention further relates to the use of a phosphate salt to improve the photostability of an aqueous botulinum toxin formulation comprising (i) a botulinum toxin, and (ii) a stabilizing protein, preferably human serum albumin, and optionally one or more of components (iv)-(vi), where components (i), (ii), and (iv)-(vi) are as defined herein. "Improved photostability" in this context refers to the photostability of an aqueous botulinum toxin formulation comprising a phosphate salt at a temperature (e.g., 20° C.) of 250 W / m 2 The biological activity of botulinum toxin after 7 hours of exposure to light of 250 W / m at the same temperature (e.g., 20°C) 2The biological botulinum toxin activity of the phosphate-free aqueous botulinum toxin formulation after exposure to 7 hours of light is at least 10% greater, preferably at least 20% greater, more preferably at least 30% less, and most preferably at least 40% greater than the biological botulinum toxin activity of the phosphate-free aqueous botulinum toxin formulation after exposure to 7 hours of light.

[0083] Furthermore, the present invention relates to the use of a calcium, magnesium, or zinc salt, or a mixture thereof, to reduce the injection pain of a botulinum toxin formulation comprising (i) a botulinum toxin, (ii) a stabilizing protein, preferably human serum albumin (HSA), (iii) a chelating agent or a phosphate salt, and optionally one or more of components (iv)-(vi), wherein components (i)-(vi) are as defined herein.

[0084] In another aspect, the present invention relates to a liquid formulation of the present invention for use in therapy.

[0085] In particular, the liquid formulation of the present invention can be used to treat neuromuscular disorders, pain, sialorrhea, hyperhidrosis, urological disorders, and neurological disorders. Examples of neuromuscular disorders include dystonia, cervical dystonia, spasms, post-stroke spasticity, blepharospasm, tremors, hyperkinetic movement disorders, and cerebral palsy. Urological disorders include, in particular, the treatment of symptoms characterized by detrusor overactivity, overactive bladder, neurogenic bladder, and interstitial cystitis, vulvodynia and chronic pelvic pain, benign prostatic hyperplasia (BPH), and detrusor-sphincter dyssynergia (DSD). Examples of neurological disorders include chronic migraine, trigeminal neuralgia, peripheral neuropathic pain, diabetic neuropathic pain, and depression.

[0086] In yet another aspect, the present invention relates to the cosmetic use of the liquid formulation of the present invention for the treatment of a cosmetic condition.

[0087] This aspect of the invention relates to purely aesthetic uses of the liquid formulations of the invention.Preferred cosmetic conditions to be treated include skin conditions, especially the treatment of skin, especially facial wrinkles.

[0088] The term "wrinkle" as used herein is broadly interpreted to include not only fine lines, but also lines, rhytids, creases, furrows, and folds. The terms "line", "fine lines", "wrinkles", "furrows", and "folds" are often used interchangeably since they share similar definitions. In the present invention, "line" is generally interchangeable with "fine lines", but may preferably refer to shallower depressions in the skin than "fine lines". "Folds" are interchangeable with wrinkles and lines, and are preferably linear depressions. "Wrinkles" are interchangeable with fine lines, lines, and folds. It preferably refers to mild fine lines, and may describe specific fine lines in specific locations. "Rhytid" as used herein is essentially the same as fine lines. However, "rhytid" preferably refers to a skin structure formed by a collection of irregular lines. "Deep wrinkles" are deep creases or deep lines in the skin.

[0089] Preferably, the wrinkles treated in accordance with the present invention are facial wrinkles including horizontal forehead wrinkles, glabellar wrinkles, periorbital wrinkles, crow's feet, bunny lines (i.e., lines radiating downward on the sides of the nose), nasolabial folds, upper lip radial lines, lower lip radial lines, corners of the mouth lines, marionette lines, perioral lines, commissure lines at the corners of the mouth, whisker lines, and cobble chin lines.

[0090] To treat the facial wrinkles mentioned above, botulinum toxin is typically administered by intramuscular injection into the following muscles: frontalis (horizontal line of the forehead), proximal and corrugator supercilii (glabellar lines), lateral orbicularis oculi (crow's feet / periorbital wrinkles), nasal, proximal and transverse nasal muscles (bunny lines), levator labii superioris nasolabial folds (nasolabial folds), orbicularis oris (upper and lower radial lip lines), depressor anguli oris (angular line of the mouth, marionette lines, commissure of the mouth, labial and mental lines), and mentalis (perioral line, cobblestone chin).

[0091] A further preferred cosmetic application of the liquid formulation of the present invention relates to its use in cosmetic applications including rejuvenation and / or improvement of the quality of the skin of the face and / or body.

[0092] In yet another aspect, the present invention relates to a method of treating a disease or condition comprising administering to a person in need thereof an effective amount of the liquid formulation of the present invention.

[0093] The disease or condition may be any one of the diseases and conditions mentioned above, regardless of whether it is a therapeutic or cosmetic indication.Accordingly, in one embodiment, the present invention relates to a (non-therapeutic) method of treating a cosmetic condition, preferably a skin condition, comprising administering an effective amount of the liquid formulation of the present invention to a person in need thereof.In another embodiment, the present invention relates to a (non-therapeutic) method of treating a cosmetic condition, preferably a skin condition, comprising injecting an effective amount of the liquid formulation of the present invention to a person in need thereof.

[0094] A further preferred method of the present invention relates to a method for rejuvenating and / or improving the quality of facial and / or body skin, comprising administering to a person in need thereof an effective amount of a liquid formulation of the present invention.

[0095] The person to be treated is not particularly limited other than having a disease or condition that can be treated according to the present invention. Those skilled in the art can determine the appropriate dosing regimen for treating a given therapeutic or cosmetic indication. In particular, injections may be intradermal, subdermal (subcutaneous), or intramuscular, depending on the disease or condition to be treated. EXAMPLES

[0096] The following examples illustrate liquid botulinum toxin formulations according to the invention. Percentages are weight / volume (w / v) unless otherwise stated.

[0097] Example 1 thermal stability The thermal stability of the botulinum toxins was determined by measuring the change in biological activity of the botulinum toxins over time at an elevated temperature of 40° C. Biological activity was determined using a cell-based potency assay (CBA) as described in WO2013 / 049508 and WO2014 / 207109.

[0098] Briefly, neuronal cells were incubated with a sample containing a neurotoxin and a reference standard of known potency. After the incubation period, the cells were lysed and the amount of cleaved SNAP25 protein was determined by immunoassay. The biological activity of the sample was then calculated by comparing the cleavage rate of cells treated with the sample with that of cells treated with the reference standard.

[0099] An HSA-containing composition ("HSA composition"; 50 U / ml BoNT / A, 0.1% HSA, 0.47% sucrose, 0.9% NaCl) was compared to Comparative Composition 1 (50 U / ml BoNT / A, 0.015% polysorbate 20, 0.02% methionine, 0.9% NaCl, 0.078% NaH2PO4; pH 6.5) and Comparative Composition 2 (50 U / ml BoNT / A, 0.01% polysorbate 80, 0.155% histidine, 0.4% sucrose, 0.9% NaCl; pH 6.5). A portion of each composition was stored in the dark at 2-8°C and analyzed by CBA within 7 days ("T0"). The remaining portions of the compositions were stored at elevated temperature (40°C) for 2 weeks and then biological activity was measured. The botulinum toxin used in all compositions was the 150 kDa BoNT / A neurotoxin without complexing proteins. The results are shown in Table 1. [Table 1]

[0100] As can be seen from Table 1, the HSA composition containing human serum albumin (HSA) is significantly more stable than comparative compositions 1 and 2, which lack HSA as a stabilizing protein. Thus, the presence of a stabilizing protein such as HSA is important in achieving sufficiently high toxin thermal stability.

[0101] The effect of HSA on botulinum toxin stability was further tested by measuring the biological activity of BoNT / A (150 kDa) in liquid compositions with different HSA concentrations (75 U / ml BoNT / A, 0.03-1.0 mg / ml HSA, 10 mM phosphate, 10 mM histidine, 0.9% NaCl, pH 6.0) immediately after the manufacturing process (T0 time point above; 0 was the same for all tested samples). The results are shown in Table 2. [Table 2]

[0102] As can be seen from Table 2, the toxin activity increases with increasing HSA concentration.

[0103] Example 2 light sensitivity When the stability of various liquid formulations as mentioned above was investigated, it was surprisingly found that light significantly affected the stability of the toxin. This finding was further investigated by photostability testing using a SUNTEST CPS+ instrument (ATLAS Material Testing Technology LLC). The instrument is equipped with a filter set providing a spectral distribution in the wavelength range of 320-800 nm, which corresponds to ID65 (indirect daylight standard for indoors) of ISO 10977, and also equipped with a window glass filter according to ICH Q1B. The photostability testing was performed at 250 W / m 2 The test took place for seven hours.

[0104] Specifically, BoNT / A (150 kDa) compositions containing buffered 0.9% NaCl (pH 6.0) and different concentrations of human serum albumin (0.03 to 1.0 mg / ml HSA) were prepared and then irradiated with 250 W / m 2The compositions were exposed to light for 7 hours. For comparison, identical BoNT / A (150 kDa) compositions containing different concentrations of HSA (0.03 mg / mL, 0.1 mg / mL, 0.3 mg / mL, and 1.0 mg / mL) were stored in the dark for the same time (controls). The biological activity of the light-exposed compositions and each of the dark-stored control compositions was then measured. The relative BoNT / A activity, expressed as a percentage of the respective control, was calculated as a measure of photostability. The results are shown in Table 3. [Table 3]

[0105] The results in Table 3 show that the photostability of BoNT / A in HSA-containing compositions decreases with increasing HSA concentration. Thus, although thermal stability increases with increasing HSA concentration (see Example 1), unexpectedly, HSA was found to have a detrimental effect on photostability. Further testing (results not shown) revealed that the use of an alternative HSA product (recombinant HSA instead of donor HSA) or extensive dialysis of HSA to remove potential small molecule impurities (<10 kDa) did not alter these findings, i.e., did not affect photosensitivity.

[0106] Example 3 photostability Further tests were carried out to find HSA-containing compositions with low light sensitivity, i.e., high photostability. These tests included the preparation of a liquid composition (composition "1") containing BoNT / A (150 kDa) without complexing proteins, 0.1% HSA, 0.47% saccharose, and 0.9% NaCl. In addition, two liquid compositions corresponding to composition 1 were prepared, but each of them further contained 2.7 mM EDTA (composition "1+EDTA") and 20 mM phosphate (composition "1+phosphate"; pH 6.0). In addition, a DTPA-containing composition (composition "2(DTPA)") was prepared, containing BoNT / A (150 kDa), 0.01% HSA, 0.015% polysorbate 20, 10 mM phosphate, 0.155% histidine, 2.7 mM DTPA, 0.9% NaCl, pH 6.0.

[0107] Compositions 1, 1+EDTA, 1+phosphate, and 2(DTPA) were heated at 250 W / m as described above. 2 The compositions were exposed to light for 7 hours. In parallel, identical compositions were stored in the dark for the same period of time (controls). The biological BoNT / A activity was then measured in comparison to the respective control compositions stored in the dark. The results are shown in Table 4. [Table 4]

[0108] As can be seen from Table 4, the addition of the chelating agents EDTA and DTPA significantly improves the photostability of HSA-containing BoNT / A compositions. Phosphate was also found to exert a similar, but weaker, positive effect.

[0109] Example 4 Photostability and storage stability in the presence of chelate complexes In the following experiments, it was unexpectedly found that the subcutaneous injection of the liquid formulation of the present invention (0.9% NaCl, 3.5 mM EDTA, 10 mM histidine, 0.085% HSA, pH 6.0) caused a relatively strong injection pain. Even a very small amount of EDTA (100 μl at a concentration of only 2.7 mM s.c.) caused a very unpleasant pain. Various controls were used to exclude the possibility that the pain was caused by a placebo effect or pH. Histidine could also be excluded as a cause. The burning pain was found to be due to the complexing agent EDTA (results not shown). This finding was unexpected in view of the fact that disodium EDTA is used in the treatment of heavy metal poisoning, in chelation therapy, and in the formulation of vaccines.

[0110] However, it was surprisingly found that the addition of calcium or magnesium ions to liquid botulinum toxin formulations significantly reduced the pain of injection while maintaining the photoprotective effect. Also, a positive effect on storage stability at 40°C was obtained not only by the addition of EDTA, but also by the addition of EDTA complexes (EDTA with magnesium, calcium, or zinc ions).

[0111] More specifically, the experiments carried out compared five different liquid compositions of BoNT / A 150 kDa (without complexing proteins): Composition 1: 0.103% HSA, 20 mM histidine, 0.9% NaCl, pH 6.0 Composition 2: 0.085% HSA, 10 mM histidine, 0.9% NaCl, 3.5 mM EDTA, pH 6.0 Composition 3: 0.085% HSA, 10 mM histidine, 0.9% NaCl, 3.5 mM EDTA, 3.5 mM MgCl2, pH 6.0 Composition 4: 0.085% HSA, 10 mM histidine, 0.9% NaCl, 3.5 mM EDTA, 3.5 mM ZnCl2, pH 6.0. Composition 5: 0.085% HSA, 10 mM histidine, 0.9% NaCl, 3.5 mM EDTA, 3.5 mM CaCl2, pH 6.0.

[0112] Relative BoNT / A activity was measured by exposure to light (250 W / m 2 The results are shown in Table 5. [Table 5]

[0113] As can be seen, the compositions containing EDTA are significantly more stable to light than the compositions without EDTA. 2+ ), Calcium (Ca 2+ ), and zinc (Zn 2+ ) complexing metal ions has only a minor effect on the stabilizing properties.

[0114] These results are surprising because the complexing properties of free EDTA were thought to be essential to protect BoNT / A from the destabilizing effects of light exposure, yet in the experiments described above, the concentrations of free EDTA were very low (e.g., in the range of 100 pM for zinc). However, contrary to expectations, the addition of metal ions such as magnesium and zinc did not abolish the photoprotective effect of EDTA.

[0115] Furthermore, the relative BoNT / A activity was measured after storage at 40° C. for 2 and 4 weeks, compared with the control sample at TO (immediately after preparation of the composition). The results are shown in Table 6. [Table 6]

[0116] The results show that the formulations containing EDTA are more stable than those without EDTA when stored at 40° C. Unexpectedly, the results further show that magnesium and zinc ions as the central metal ions of the EDTA complex have little effect on the stabilizing properties.

Claims

1. A liquid formulation comprising: (i) botulinum toxin, (ii) a stabilizing protein, and (iii) Chelating agents or phosphates.

2. 2. The liquid formulation of claim 1, wherein the stabilizing protein is selected from human serum albumin, ovalbumin, casein, and mixtures thereof, or the stabilizing protein is human serum albumin.

3. 3. The liquid formulation of claim 1, wherein the stabilizing protein is human serum albumin, and the human serum albumin is present in the liquid formulation at a concentration of 0.001 to 1.00% w / v.

4. 3. The liquid formulation of claim 1 or 2, wherein the chelating agent is selected from the group consisting of aminopolycarboxylic acids having 3 to 6 carboxylic acid functional groups, citrate salts, porphyrins, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof.

5. 5. The liquid formulation of claim 4, wherein the chelating agent is represented by the general formula (I): (HO ２ CCH ２ ) ２ N-R-N(CH ２ CO ２ H) ２ (I) is an aminopolycarboxylic acid of the formula wherein R contains zero, one or two carboxylic acid groups, and the aminopolycarboxylic acid is preferably selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), and mixtures thereof.

6. 3. The liquid formulation of claim 1, wherein the chelating agent is present in the liquid formulation at a concentration of 0.01 to 100 mM.

7. 3. The liquid formulation of claim 1 or 2, wherein the botulinum toxin is a botulinum neurotoxin that does not contain a complex protein, or wherein the botulinum toxin is serotype A; or wherein the botulinum toxin is a botulinum neurotoxin of serotype A that does not contain complex proteins; or wherein the botulinum toxin is serotype A and is present at a concentration of 1 to 1000 U / ml; Or a liquid formulation wherein the botulinum toxin is a botulinum neurotoxin of serotype A that does not contain complex proteins and is present at a concentration of 1 to 1000 U / ml.

8. 3. The liquid formulation of claim 1 or 2, comprising: (i) botulinum toxin, (ii) human serum albumin, and (iii) EDTA.

9. 3. The liquid formulation of claim 1 or 2, wherein the liquid formulation is (iv) further comprising a salt of calcium, magnesium, or zinc, or a mixture thereof; or wherein the liquid formulation is (iv) A liquid formulation further comprising a salt of calcium, magnesium, or zinc, or a mixture thereof, wherein said salt is present in said liquid formulation in an amount of 0.01 to 100 mM, and wherein said salt is calcium chloride and is present in said liquid formulation in an amount of 0.01 to 100 mM.

10. 3. The liquid formulation of claim 1 or 2, wherein the liquid formulation is (v) further comprising a tonicity agent; or wherein the liquid formulation is (v) A liquid formulation further comprising a tonicity agent, wherein said tonicity agent is present in said liquid formulation in an amount of 0.01-2.0% w / v, or wherein said tonicity agent is sodium chloride, or wherein said tonicity agent is sodium chloride and is present in said liquid formulation in an amount of 0.01-2.0% w / v.

11. 3. The liquid formulation of claim 1 or 2, wherein the liquid formulation is (vi) further comprising a buffer; or wherein the liquid formulation is (vi) A liquid formulation further comprising a buffering agent, wherein said buffering agent is present in said liquid formulation in an amount of 1 to 100 mM, or wherein said buffering agent is an amino acid, a phosphate, or a mixture thereof, or wherein said buffering agent is histidine, or wherein said buffering agent is histidine, a phosphate, or a mixture thereof, and said histidine and phosphate are present in said liquid formulation in an amount of 1 to 100 mM.

12. 3. The liquid formulation according to claim 1 or 2, wherein the pH is in the range of 5.0 to 8.

0.

13. 3. A liquid formulation according to claim 1 or 2 for use in therapy, in particular for use in the treatment of neuromuscular disorders, pain, salivation, hyperhidrosis, urinary disorders and neurological disorders.

14. 10. Cosmetic use of a liquid formulation according to claim 1 or 2 for the treatment of a cosmetic condition.

15. 10. A method of treating a disease or condition comprising administering an effective amount of the liquid formulation of claim 1 or 2 to a person in need thereof.