Liquid botulinum toxin preparations and their use

Abstract

The present invention relates to a liquid formulation comprising (i) botulinum toxin, (ii) human serum albumin (HSA), (iii) hyaluronic acid, (iv) algitol, and optionally (v) an isotonic agent and / or (vi) a buffering agent. In a preferred embodiment, the HSA is used as a chelating agent. Furthermore, the present invention relates to the use of the liquid formulation in the treatment of therapeutic and cosmetic indications.

Description

[Technical Field]

[0001] The present invention relates to a liquid formulation comprising (i) botulinum toxin, (ii) human serum albumin (HSA), (iii) hyaluronic acid, and (iv) algitol. The liquid formulation is characterized by improved stability against light. The present invention further relates to a method for preparing the liquid formulation, and to the use of the liquid formulation in the treatment of therapeutic and cosmetic indications. [Background technology]

[0002] Botulinum neurotoxin (BoNT; or botulinum toxin (BT)) is a family of bacterial neurotoxins widely used to treat a growing variety of neurological, medical, and cosmetic conditions. There are seven widely recognized "classical" BoNT serotypes, known as BoNT / A to G. Currently, only two serotypes, A (BoNT / A) and B (BoNT / B), are used clinically. BoNT is produced by Clostridium species, particularly Clostridium botulinum, in the form of high molecular weight (up to approximately 900 kDa) complexes. These toxin complexes consist of an active 150 kDa neurotoxin and several complex-forming proteins (non-toxic neurotoxin-associated proteins, NAPs).

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

[0004] The formulation of BoNT is extremely difficult due to its structural complexity and the low concentrations of the products used. BoNT is highly sensitive to various conditions, including heat and alkaline pH. Therefore, since BoNT only functions when its structure is intact, the challenge in preparing BoNT for medical dosage forms is to formulate compositions that protect BoNT from inactivation or partial loss of its biological activity during the manufacture, storage, or use of the product.

[0005] Furthermore, BoNT has an extremely high potency, and the lethal dose for humans is only in the range of approximately 0.1 to 1 ng / kg. Therefore, BoNT pharmaceutical formulations contain only a very small amount of toxin, in the range of approximately 1 ng per vial. This exacerbates the known problem of loss of toxic activity due to surface denaturation. Consequently, one of the main challenges in formulating BoNT is minimizing activity loss during manufacturing and storage.

[0006] At the same time, BoNT formulations should be suitable for medical use in a wide range of different indications. This means that BoNT formulations should be suitable for injection into very different types of tissues, such as muscles, different layers of skin (dermis, subcutaneous tissue), or glands (e.g., salivary glands).

[0007] From the above perspective, the main BoNT products currently available are supplied as lyophilized powders, that is, in a form that remains stable for long periods even when stored at 2-8°C or room temperature. Lyophilized BoNT / A complexes were first introduced to the market in 1989 (Botox®, Allergan) and 1991 (Dysport®, Ipsen). In 2005, the first stable dosage form of pure 150kDa BoNT / A neurotoxin without complex-forming proteins was approved (Xeomin®, Merz Pharmaceuticals). However, these lyophilized products need to be reconstituted before use, and this process can lead to administration errors and sterility problems. Therefore, considerable effort has been made to develop liquid formulations of BoNT that are more convenient to use and easier to administer.

[0008] A commercially available liquid formulation of BoNT is available under the trade name Neurobloc® (Eisai). This 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-acetyltryptophan. Other commercially available liquid formulations are sold under the trade names Innotox® (Medytox) and Alluzience® (lpsen / Galderma). Both of these liquid formulations contain the BoNT / A toxin complex, as well as water, and in addition, sodium chloride, surfactants (Innotox®: polysorbate 20; Alluzience®: polysorbate 80), amino acids (Innotox®: methionine; Alluzience®: histidine), and additional excipients (Innotox®: sodium phosphate as a buffer; Alluzience®: sucrose).

[0009] U.S. Patent No. 8,372,645 discloses a method for stabilizing a botulinum neurotoxin of type A, B, C1, D, E, F or G, or a mixture of two or more botulinum neurotoxins, wherein the neurotoxin or mixture of neurotoxins does not contain a complex-forming protein that naturally forms a complex with the botulinum neurotoxin, and the method comprises mixing the neurotoxin in an aqueous solution with a non-protein stabilizer in an amount effective to retain the biological activity of the neurotoxin, wherein the aqueous solution does not contain mammalian-derived proteins. The non-protein stabilizer is selected from one or more of hyaluronic acid, polyvinylpyrrolidone, and polyethylene glycol. The composition may further comprise a polyalcohol selected from one or more of inositol, mannitol, and sorbitol. The aqueous solution contains a pH buffer. The composition may be lyophilized.

[0010] U.S. Patent No. 7,780,967 discloses a pharmaceutical composition consisting essentially of a high molecular weight polysaccharide and a botulinum toxin, wherein the botulinum toxin is stabilized by the high molecular weight polysaccharide and the pharmaceutical composition has reduced toxicity. The high molecular weight polysaccharide is selected from the group consisting of hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch, hydroxybutyl starch, and hydroxypentyl starch.

[0011] U.S. Patent Application Publication No. 2012 / 0141532 discloses a pharmaceutical composition comprising a botulinum neurotoxin and a viscous carrier for the botulinum neurotoxin, wherein the viscous carrier is selected from the group consisting of hyaluronic acid, carbomer, polyacrylic acid, cellulose polycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharide, polyacrylamide, polyvinyl alcohol, polyvinyl acetate, chitosans, alginates, and derivatives and mixtures thereof.

[0012] WO 2010 / 090677 discloses a solid form Clostridium toxin pharmaceutical composition free of animal proteins, comprising a Clostridium toxin active ingredient, an effective amount of a sugar excipient, and an effective amount of a surfactant excipient, wherein the sugar excipient is a monosaccharide, disaccharide or trisaccharide, and the surfactant excipient is a poloxamer, polysorbate, polyoxyethylene glycol dodecyl ether, or polyoxyethylene octyl phenyl ether. The composition is buffered to about pH 5.5 to about pH 6.5 using a citrate buffer, phosphate buffer, histidine buffer, or histidine phosphate buffer. The composition may further comprise an effective amount of sodium chloride. The non-protein polymer excipient is dextran, polyethylene glycol, polyethyleneimine, polyvinylpyrrolidone, polyvinyl acetate, inulin, starch, or a starch derivative. The composition further comprises an effective amount of a non-protein polymer excipient. The Clostridium toxin active ingredient is stable for at least one year when stored either at ambient temperature or below freezing temperature.

[0013] Finally, WO 2017 / 148915 discloses a botulinum toxin-containing composition comprising non-crosslinked hyaluronic acid and human serum albumin.

[0014] Despite these advances in the preparation of liquid botulinum toxin formulations, there remains a need for new options for developing a liquid formulation of botulinum toxin that is stable during transport and storage and ready for immediate use. Objectives of the Invention

[0015] An objective of the present invention is to provide a stable liquid formulation of botulinum toxin that is ready for immediate use in therapeutic and cosmetic treatments. Summary of the Invention

[0016] This invention is based on the unexpected discovery that using a pre-treated protein stabilizer based on human serum albumin (HSA) in the formulation of liquid botulinum toxin preparations reduces photosensitivity and consequently improves photostability. Furthermore, unexpectedly, even very small amounts of iron ions (Fe) are used. 3+ It was found that iron ions dramatically increased the photosensitivity of liquid botulinum toxin preparations containing human serum albumin (HSA). This is surprising because iron is relatively common and ubiquitous, and furthermore, other metal ions such as copper, cobalt, or nickel were found not to exhibit such destabilizing effects.

[0017] Furthermore, the present invention is based on the surprising discovery that tryptophan and N-acetyltryptophan reduce the photostability of liquid botulinum toxin preparations containing human serum albumin (HSA). This discovery is indeed surprising because commercially available HSA products typically contain sodium caprylate and N-acetyltryptophan to stabilize HSA at high temperatures, in addition to water and sodium chloride, which can lead to contamination of the botulinum toxin preparation. What is even more surprising is that tryptophan is listed as a stabilizing additive for botulinum toxin (see, for example, European Patent No. 3679946).

[0018] Furthermore, it was found that the presence of non-protein stabilizers, including hyaluronic acid, and algitol contribute to improving the photostability of the liquid formulation.

[0019] Therefore, in the first aspect, the present invention relates to water and the following components: (i) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Algitol A liquid formulation containing, (I) The liquid formulation contains a chelating agent, (II) The liquid formulation contains a chelating agent and a metal salt selected from salts of calcium, magnesium, zinc, and mixtures thereof, (III) The human serum albumin is used in the following steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. Including, or subjected to a process, (IV) The liquid formulation is produced in the following steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. The present invention provides a liquid formulation that has been subjected to a process including [the specified element].

[0020] In a second aspect, the present invention relates to water and the following components: (i) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Algitol, which is sorbitol The present invention provides a liquid formulation containing the following:

[0021] In one embodiment of the first and second aspects, the liquid formulation comprises components (v) and (vi): (v) tonicity agent; (vi) buffer It further includes one or both of the above.

[0022] In one embodiment of the first or second aspect, the botulinum toxin is a botulinum neurotoxin complex, or Botulinum toxin is a botulinum neurotoxin that does not contain complex-forming proteins, or, The botulinum toxin is of serotype A, or Botulinum toxin is a serotype A botulinum neurotoxin that does not contain complex-forming proteins, or Botulinum toxin is of serotype A and is present at concentrations of 1 to 1000 U / ml, or Botulinum toxin is a serotype A botulinum neurotoxin that does not contain complex-forming proteins and is present at concentrations of 1 to 1000 U / ml.

[0023] In one embodiment of the first or second aspect, human serum albumin is present in the liquid formulation at a concentration of 0.001 to 1.00% w / v.

[0024] In one embodiment of the first aspect and embodiments referenced thereto, the chelating agent is selected from the group consisting of aminopolycarboxylic acids having 3 to 6 carboxylic acid functional groups, citrates, porphyrins, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof. Preferably, the chelating agent is of general formula (I): (HO2CCH2)2N-RN(CH2CO2H)2(I) An aminopolycarboxylic acid [wherein R is either free of carboxylic acid groups or contains one or two carboxylic acid groups], preferably an aminopolycarboxylic acid selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(α-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, or The chelating agent in question is 2,2',2''-nitrilotriacetic acid.

[0025] In one embodiment of the first aspect and embodiments relating thereto, the metal salt is present in the liquid formulation in an amount of 0.01 to 100 mM, or the metal salt is calcium chloride and is present in the liquid formulation in an amount of 0.01 to 100 mM.

[0026] In one embodiment of the first and second embodiments or embodiments referenced thereto, the liquid formulation is Fe 3+ The ions are contained at a concentration of less than 1 μM, preferably less than 500 nM, and more preferably less than 250 nM.

[0027] In one embodiment of the first and second embodiments or embodiments relating thereto, the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0028] In one embodiment of the first and second aspects or embodiments referenced thereto, the hyaluronic acid is not crosslinked, or Hyaluronic acid is present at a concentration of 2-10 mg / ml, and this hyaluronic acid is not cross-linked.

[0029] In a further embodiment, hyaluronic acid is present at a concentration of 2 to 10 mg / ml and is cross-linked.

[0030] In one embodiment of the first aspect and embodiments referenced thereto, the algitol is selected from glycerol, mannitol, isomalt, lactitol, sorbitol, xylitol, treitol, erythritol, and arabitol, preferably mannitol and sorbitol.

[0031] In one embodiment of the first and second aspects or embodiments referenced thereto, algitol is present at a concentration of 20 to 100 mg / ml.

[0032] In one embodiment of the first and second aspects, and in embodiments relating thereto, (v) The isotonic agent is present in the liquid formulation in an amount of 0.01 to 2.0% w / v, or the isotonic agent is sodium chloride, or the isotonic agent is sodium chloride and is present in the liquid formulation in an amount of 0.01 to 2.0% w / v; (vi) The buffer is present in the liquid formulation at a concentration of 1 to 100 mM, or the buffer is citric acid, an amino acid, a phosphate, or a mixture thereof, or the buffer is histidine, or the buffer is histidine, a phosphate, or a mixture thereof, and the histidine and phosphate are present in the liquid formulation at a concentration of 1 to 100 mM.

[0033] In one embodiment of the first and second aspects or embodiments referenced thereto, the pH of the liquid formulation is in the range of 5.0 to 8, preferably 5.5 to 6.5.

[0034] In a third embodiment, the present invention relates to a method for preparing a liquid formulation as described in the first embodiment or any embodiment referenced therefrom, wherein the method is Water and, (I) Chelating agent or, (II) Chelating agents, and metal salts selected from salts of calcium, magnesium, zinc, and mixtures thereof. This includes mixing components (i) to (iv) and optionally (v) and / or (vi) in a liquid containing, (III) The method is steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; (b) A step of contacting the human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. including, or (IV) The method is steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. Includes, Preferably, the contact in step (a) or step (b) is carried out by mixing a composition containing human serum albumin with a chelating agent or a chelating agent and a metal salt, or the contact in steps (d) and (e) is carried out by mixing a liquid formulation containing components (i) to (iv) with a chelating agent or a chelating agent and a metal salt to adjust the concentration of the chelating agent in the liquid formulation to 1 mM to 500 mM. This concerns the method, Alternatively, the present invention relates to a method for preparing a liquid formulation as described in the second embodiment or any embodiment referenced thereto, The present invention relates to a method comprising mixing components (i) to (iv), and optionally (v) and / or (vi), in a liquid containing water.

[0035] In a fourth aspect, the present invention relates to liquid formulations according to any of the first and second embodiments or embodiments referenced thereto, for use in the treatment of neuromuscular diseases, pain, salivation, hyperhidrosis, urinary tract disorders, and neurological disorders.

[0036] In a fifth embodiment, the present invention relates to the use of liquid formulations described in the first or second embodiment or any embodiment referenced thereto in cosmetic / non-therapeutic applications.

[0037] In a sixth aspect, the present invention relates to a method for treating a disease or condition, comprising administering an effective amount of a liquid formulation described in any of the first or second embodiments or embodiments referenced herein to a body in need. [Modes for carrying out the invention]

[0038] definition

[0039] As used herein, the terms “comprising,” as well as “including” and “containing,” and any variation thereof such as “comprise,” “include,” and “contain,” are intended to refer to non-exclusive inclusion, and a process, method, product-by-process, composition, or formulation that comprises, includes, or contains some element or list of elements may include not only those elements but also other elements not expressly enumerated for such process, method, product-by-process, composition, or formulation. In addition, within the framework of the present invention, the terms “comprise,” “comprising,” “include,” “including,” “contain,” and any variation thereof, and any variation thereof, are intended to be replaced with “consist,” or “consisting,” or any variation thereof (e.g., “consist essentially of”), which are understood to refer to the exclusive inclusion of the elements shown.

[0040] The terms “a,” “an,” and “the,” as used in the context of this invention, and similar references, are to be interpreted as encompassing both singular and plural forms, and therefore, unless otherwise specifically indicated herein or unless clearly inconsistent with the context, may also relate to “at least one” or “two or more.”

[0041] As used herein, the terms “liquid formulation” or “liquid botulinum toxin formulation” generally refer to aqueous formulations, typically aqueous solutions. In this specification, the term “liquid formulation” may be used interchangeably with “liquid composition.” Preferably, a liquid formulation is a pharmaceutically acceptable liquid formulation, i.e., a liquid pharmaceutical formulation. As used herein, “pharmaceutically acceptable” means that, when administered to a human patient or subject, the liquid formulation does not cause unacceptable adverse side effects; that is, it means the liquid formulation is suitable for use in humans. The aqueous solution may be a buffer solution with or without saline, and may be a physiological saline such as a buffered (e.g., phosphate and / or histidine-buffered) saline.

[0042] Botulinum toxin

[0043] With respect to component (i), the botulinum toxin is not particularly limited and includes any serotype of botulinum toxin (e.g., 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 serotype A, more preferably serotype A1 (BoNT / A1), and most preferably BoNT / A1 produced by the Clostridium botulinum Hall strain. Furthermore, the botulinum toxin may be any other botulinum toxin, such as a natural neurotoxin obtained from the bacterium Clostridium botulinum, or botulinum toxin obtained from alternative sources including recombinant technology and genetic or chemical modification.

[0044] Furthermore, as used herein, the terms “botulinum toxin” (“BT”) and the synonymous 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 with complex-forming proteins (referred to as “toxin complexes”).

[0045] As used herein, the term “pure botulinum neurotoxin” means botulinum neurotoxin that does not contain the complex-forming protein (sometimes called the “neurotoxic component”), more precisely, botulinum neurotoxin that does not contain the neurotoxin-associated complex-forming protein (NAP). Pure botulinum neurotoxin is an (active) neurotoxic polypeptide that ultimately inhibits the release of acetylcholine. It is a double-stranded protein consisting of a light chain (LC; approximately 50 kDa) and a heavy chain (HC; approximately 100 kDa) linked together by a disulfide bond. Thus, the active neurotoxic polypeptide is also referred herein as “150 kDa neurotoxin,” “Botulinum neurotoxin (150 kD),” or “neurotoxic component.”

[0046] As used herein, the term “toxin complex” refers to a macromolecular complex of a neurotoxic component with a set of complex-forming proteins (NAPs). In particular, the term “toxin complex” includes 900 kDa, 500 kDa, and 300 kDa botulinum toxin type A complexes. The complex-forming proteins are non-toxic non-hemagglutinins (NTNHAs), and in strains of serotypes A-D, various hemagglutinins (HAs). For example, the 900 kDa complex is found in onabotulinum toxin A (Botox® / Vistabel®, Allergan, Inc., Irvine, CA, USA). Toxin complexes as activators are found in Dysport® (Azzalure®, Ipsen, Paris, France), Alluzience® (Ipsen / Galderma), and Innotox® (Medytox).

[0047] The concentration of botulinum toxin in the liquid formulation of the present invention may be 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.

[0048] As used herein, the terms “unit” or “U” refer to the biological activity (biological potency) of a toxin and relate to the lethal dose (LD50) in 50% of the mice tested. More specifically, in the context of this invention, unless otherwise specified, LD50 refers to the biological activity (biological potency) of a toxin. 50 This is measured using a mouse bioassay (MBA). The MBA determines the mean lethal dose (LD50) of the toxin / neurotoxin after intraperitoneal injection into mice, i.e., the amount of toxin / neurotoxin that can kill 50% of a group of mice. Based on this, one unit (U) of toxin / neurotoxin as used herein is defined as 1 mouse LD50 (1.0 LD50 = 1.0 U). The LD50 mouse bioassay is the gold standard among various biological, chemical, or immunological detection methods for botulinum toxin and is known to those skilled in the art (see, for example, Pearce, LB; Borrodic, GE; First, ER; MacCallum, RD: Measurement of botulinum toxin activity: Evaluation of the lethality assay. Toxicol. Appl. Pharmacol. 1994, 128:69-77). Those skilled in the art can determine a suitable botulinum toxin concentration depending on the serotype and intended use.

[0049] Alternatively, botulinum toxin activity can be determined using cell-based assays, as described in International Publication Nos. 2009 / 114748, 2013 / 049508, or 2014 / 207109. Those skilled in the art will be able to correlate the botulinum toxin activity obtained from cell-based assays with the results obtained from mouse LD50 assays by calibration using an LD50 reference standard.

[0050] LD used by manufacturers of commercially available botulinum toxin preparations 50Because the tests differ, the unit potency indicated by manufacturers of commercially available botulinum toxin preparations is unique to each product and cannot be easily compared. Therefore, within the framework of the present invention, incobotulinum toxin A ("INCO"; Xeomin®, Bocouture®; botulinum toxin serotype A without complex-forming protein; 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 Pharmaceutical Corp., Galderma Lab.), limabotulinum toxin B ("RIM"; Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences Inc.), and PurTox® ("TBD"; botulinum toxin serotype A; Mentor Worldwide To establish the comparative titers of (LLC), the conversion ratios provided below are used. As used herein, the conversion ratio between ONA and INCO is 1:1. The conversion ratio between ONA / INCO:ABO is 1:2.5. The conversion ratio between ONA / INCO:RIM is 1:50, and the conversion ratio between ONA / INCO:TBD is 1:1.5.

[0051] Human serum albumin (HSA)

[0052] With respect to component (ii) of the liquid formulation of the present invention, human serum albumin (HSA) acts as a stabilizing protein. The term “stabilizing protein” generally refers to polypeptides that contribute to improved stability of botulinum toxin. HSA can be present in the liquid formulation in an amount of 0.001 to 2.0% w / v, preferably 0.001 to 1.00% w / v, more preferably 0.01 to 0.5% w / v, even more preferably 0.02 to 0.3% w / v, and most preferably 0.03 to 0.15% w / v.

[0053] In the context of this invention, the terms “human serum albumin” or “HSA” are intended to refer to donor HSA (HSA derived from human blood, more precisely from human plasma) and recombinant HSA. In one preferred embodiment, human serum albumin is donor HSA. In another preferred embodiment, human serum albumin is recombinant HSA.

[0054] In the liquid formulation according to the first embodiment, the HSA or the preliminary formulation containing the HSA is free from metal cations that may adversely affect stability, particularly photostability, especially Fe 3+ It is used as a chelating agent to bind or remove ions. This is disclosed in more detail below.

[0055] Hyaluronic acid

[0056] With respect to component (iii), the term "hyaluronic acid" as used in the context of the present invention is also known as HA, hyaluronic acid salt, or hyaluronan, and has the following structural formula: [ka] It is an anionic, non-sulfated glycosaminoglycan that can be represented by [this].

[0057] Therefore, hyaluronic acid can be considered a polymer of disaccharides, which are composed of two glucose derivatives, namely D-glucuronic acid and DN-acetylglucosamine. In this disaccharide, glucuronic acid is β(1→3) glycosidically linked to N-acetylglucosamine, which in turn is β(1→4) glycosidically linked to the next glucuronic acid molecule.

[0058] It should be noted that in this invention, the term "hyaluronic acid" includes all pharmaceutically acceptable salts, hydrates, and / or solvates thereof. Preferably, hyaluronic acid exists in the form of a sodium salt.

[0059] When used in the context of the present invention, the term "hyaluronic acid" can be understood in its broadest sense as any part of hyaluronic acid known in the art.

[0060] Hyaluronic acid can exist in both an uncrosslinked, linear form and a crosslinked form.

[0061] According to a preferred embodiment of the present invention, the hyaluronic acid used in the liquid formulation according to the present invention is not crosslinked, or Hyaluronic acid is present at a concentration of 2-10 mg / ml, and this hyaluronic acid is not cross-linked.

[0062] According to another embodiment, hyaluronic acid is present at a concentration of 2 to 10 mg / ml, and the hyaluronic acid is cross-linked.

[0063] In one embodiment, the hyaluronic acid used in the liquid formulation according to the present invention may also be subjected to chelating, similar to the purification of HSA, in order to remove metal ions that may adversely affect photostability.

[0064] In a preferred embodiment, the hyaluronic acid used in the liquid formulation according to the present invention has an average molecular weight of 100 kDa to 8 MDa, preferably 500 kDa to 7 MDa, more preferably 1 MDa to 6 MDa, more preferably 2 MDa to 5 MDa, more preferably 2 MDa to 4.5 MDa, more preferably 2.5 to 4.5 MDa, more preferably 2.5 MDa to 4.0 MDa, and particularly 3.0 to 3.8 MDa.

[0065] In a more preferred embodiment, the hyaluronic acid used in the liquid formulation according to the present invention is not crosslinked and has an average molecular weight of 100 kDa to 8 MDa, preferably 500 kDa to 7 MDa, more preferably 1 MDa to 6 MDa, more preferably 2 MDa to 5 MDa, more preferably 2 MDa to 4.5 MDa, more preferably 2.5 to 4.5 MDa, more preferably 2.5 MDa to 4.0 MDa, and particularly 3.0 to 3.8 MDa.

[0066] Algitol

[0067] With respect to component (iv), the term “algitol” as used herein is used synonymously with the term “sugar alcohol.” Sugar alcohols have the general formula HOCH2(CHOH) n It contains CH2OH. Common sugar alcohols include ethylene glycol, glycerol, erythritol, treitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boremitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol.

[0068] Storage of the liquid formulation of the present invention

[0069] 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 apparatus having a partially or completely enclosed space that can be sealed or is sealed and can be used to contain, store, and / or transport the liquid formulation. The container system is preferably a sealed (or enclosed) container made of glass or plastic (such as an organic polymer), or partially or mostly made of glass or plastic, and examples include containers in the form of (i) syringes, (ii) vials, (iii) carpules, or (iv) ampoules. 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 is known in the art.

[0070] The liquid formulation of the present invention does not require reconstitution before injection, can be used immediately, and can be used in the form of, for example, a pre-filled syringe, thus offering advantageous improvements in safety and administration accuracy compared to lyophilized botulinum toxin formulations in powder form. Furthermore, the excellent stability of the liquid formulation simplifies transportation, storage, and handling by physicians. In particular, the excellent photostability of the liquid formulation of the present invention simplifies the manufacturing process, allowing for product filling and packaging without strict light shielding, and reducing the possibility of activity degradation due to storage in exposure to light by physicians before use. Moreover, since the liquid formulation of the present invention does not contain substances that intensify injection pain, its acceptance by physicians and patients is improved, particularly in the field of aesthetics.

[0071] Liquid formulation according to the first embodiment

[0072] In a first aspect of the present invention, the present invention comprises water and the following components: (i) Botulinum toxin, (ii) Human serum albumin (HSA), (iii) Hyaluronic acid, (iv) Algitol The present invention provides a liquid formulation containing the following:

[0073] According to the present invention, the liquid formulation contains a chelating agent or is prepared with a chelating agent.

[0074] In Embodiment (I), the liquid formulation contains a chelating agent.

[0075] In Embodiment (II), the liquid formulation comprises a chelating agent and a metal salt selected from salts of calcium, magnesium, zinc, and mixtures thereof.

[0076] In Embodiment (III), human serum albumin is subjected to the following steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. It is a process that includes [the following].

[0077] In Embodiment (IV), the liquid formulation is prepared by the following steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. It is a process that includes [the following].

[0078] It should be noted that the terms “human serum albumin” and “chelating agent” are not intended to imply any limitation on their physical forms, nor are they intended to exclude the presence of other substances or compounds that are mixed with or contained in HSA and the chelating agent. This means that “human serum albumin” that comes into contact with the chelating agent in steps (a) and (b) or (d) and (e) may be in any form, such as solid or liquid (e.g., aqueous composition or aqueous solution). Similarly, the “chelating agent” may be in any form, such as solid or liquid (e.g., aqueous composition or aqueous solution). Furthermore, the expression "contacting human serum albumin with a chelating agent" in Embodiment III or IV does not exclude the fact that the human serum albumin is in the form of one or more additional components included in the final liquid formulation, for example, an isotonic agent or buffering agent (e.g., a solid composition or a liquid composition, particularly an aqueous composition or aqueous solution), and / or the chelating agent is in the form of one or more additional components included in the final liquid formulation, for example, an isotonic agent or buffering agent (e.g., a solid composition or a liquid composition, particularly an aqueous composition or aqueous solution).

[0079] Preferably, the mixture obtained in step (a) or step (d) is an aqueous mixture. This aqueous mixture can be prepared in various ways. For example, human serum albumin may be in the form of an aqueous composition, such as an aqueous solution, which is mixed with a chelating agent that may exist in solid or liquid form and a chelating agent that may exist, for example, in the form of an aqueous solution of the chelating agent. Alternatively, human serum albumin may be in the form of a solid, such as a freeze-dried material, mixed with the chelating agent and the aqueous solution, or mixed with an aqueous solution of the chelating agent. Preferably, human serum albumin is in the form of an aqueous composition, more preferably an aqueous solution, and the chelating agent is a solid or an aqueous composition (e.g., an aqueous solution).

[0080] In particular, the HSA that comes into contact with the chelating agent in step (a) or the HSA contained in the liquid formulation that comes into contact with the chelating agent in step (d) may be in the form of an aqueous solution containing at least 5% w / v HSA, more preferably 10-30% w / v HSA, and most preferably 20% w / v HSA. Furthermore, the pH of the mixture obtained in step (a) or step (d) may be adjusted to a pH in the range of 6.0-9.0, preferably 6.5-8.5, more preferably 7.0-8.5, and most preferably 7.0-8.0.

[0081] Furthermore, the contact step (a) or step (d) of the method for preparing a liquid formulation according to the present invention may include several substeps. For example, in one embodiment, the contact in step (a) includes, or consists only of, the steps of: mixing a chelating agent (e.g., EDTA) and human serum albumin; incubating the mixture for a predetermined time; and optionally, dialyzing the mixture against a buffer containing a chelating agent, preferably the same as that used in the incubation substep. In another embodiment, the mixture is dialyzed (directly) against a buffer containing a chelating agent without incubation, and the chelating agent is preferably the same as that used in the step of mixing a chelating agent (e.g., EDTA) and human serum albumin. In yet another embodiment, the chelating agent and human serum albumin are not mixed before dialyzing. That is, the contact step (a) may include, or consist only of, the step of dialyzing the mixture against a buffer containing a chelating agent.

[0082] Preferably, step (a) includes, or consists of, adding a chelating agent to a composition containing HSA (e.g., a solution of HSA), or mixing a chelating agent with a composition containing HSA (e.g., a solution of HSA). The resulting mixture is then incubated for some time. For example, by standing for a given time without stirring, or by stirring for a given time.

[0083] The incubation time is not limited to a specific range, but is typically at least 0.5 hours, particularly at least 1 hour, and more particularly at least 2 hours. The upper limit of the incubation time is not significant and could be, for example, 1 hour, 2 hours, 5 hours, or 10 hours. Therefore, the incubation time may be, for example, 0.5 to 5 hours, or 1 to 10 hours. Similarly, the incubation temperature is not particularly limited and may be, for example, in the range of 0°C to 60°C. Preferably, the temperature is 0°C to 30°C. That is, room temperature (20°C or 25°C) is a suitable temperature in this invention. As is known to those skilled in the art, temperature affects the reaction time. Generally, the incubation conditions (e.g., time and temperature) are selected so that the remaining amount of chelating agent (e.g., EDTA) in the final product is 100 μM or less, preferably 10 μM or less, and more preferably 1 μM or less.

[0084] Optionally, the mixture may be further processed following the incubation step, for example, by dialyzing the incubated mixture against a buffer containing a chelating agent, which is typically the same chelating agent used in the incubation step. The chelating agent used in this optional dialyzing step is preferably contained in the dialyzing buffer at a concentration of 0.1 mM to 1000 mM, more preferably 1 mM to 200 mM, and most preferably 10 mM to 100 mM. The buffer used in the dialyzing step is preferably (i) to have a pH of 7.5 to 8.5, (ii) to further contain a buffering agent, preferably a buffering agent corresponding to the final composition, or (iii) to further contain an isotonic agent, preferably 0.9% w / v sodium chloride, or (i) and (ii), or (i) and (iii), or (ii) and (iii), or (i) and (ii) and (iii).

[0085] The contact in process (d) may include similar sub-processes.

[0086] In the present invention, the term "chelating agent" as used herein is not particularly limited, as long as it can bind metal ions. The term "chelating agent" as used herein may also be referred to as "chelator" or "metal ion sequestering agent." Chelating agents for use herein are typically metal ion-binding organic compounds. Metal ions generally form multiple coordination bonds with organic chelating agents that act as polydentate ligands.

[0087] Suitable chelating agents for use herein include, but are not limited to, aminopolycarboxylic acids (e.g., aminopolycarboxylic acids having 3 to 6, preferably 4, carboxylic acid functional groups), as well as other compounds such as citrates, porphyrins, TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), TETA (triethylenetetramine), and mixtures thereof. Examples of aminopolycarboxylic acids include NTA (nitrilotriacetic acid or 2,2',2"-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).

[0088] A particularly preferred chelating agent for use in this specification is one of the following general formulas: (HO2CCH2)2N-RN(CH2CO2H)2(I) Examples of compounds include aminopolycarboxylic acid compounds having at least four carboxylic acid functional groups. The R group is not particularly limited and may not contain a carboxylic acid functional group, or it may contain one or two carboxylic acid functional groups. Preferably, the R group does not contain a carboxylic acid functional group, or contains one. Most preferably, it does not contain a carboxylic acid functional group.

[0089] Examples of compounds of general formula (I) include, for example, EDTA, EGTA, BAPTA, DTPA, and TTHA. Particularly preferred for use herein are EDTA, EGTA, and DTPA, more preferred are EDTA and DTPA, and most preferred is EDTA. Any mixture of the above chelating agents can be used in the present invention, for example, as a solution, as a solid, or bound to a matrix.

[0090] In another embodiment, the chelating agent is 2,2',2''-nitrilotriacetic acid, or comprises the same.

[0091] In step (a), the HSA comes into contact with a chelating agent in an amount such that the chelating agent is present in the liquid formulation at a concentration of 0.1 mM to 1000 mM or 0.1 mM to 500 mM, more preferably 0.5 mM to 500 mM or 1 mM to 500 mM, and most preferably 10 mM to 100 mM.

[0092] In step (b), the chelating agent can be removed by any suitable technique, such as dialysis (conventional dialysis using a dialysis bag, counterflow dialysis, etc.), reverse osmosis, filtration, cross-flow filtration, ultrafiltration, and chromatography (e.g., ion exchange chromatography or gel filtration chromatography).

[0093] Preferably, the chelating agent is removed by dialysis. Dialysis is typically performed for 0.5 to 48 hours, particularly 1 to 24 hours or 1 to 12 hours, at a temperature of 0°C to 30°C, particularly 2°C to 30°C or 4°C to 25°C, for example, at room temperature. Furthermore, dialysis is typically performed with 10 to 1000 times the amount of dialysis buffer compared to the incubated HSA / chelating agent mixture, and the dialysis buffer is usually changed at least once. The molecular weight cutoff of the dialysis membrane used may be, for example, 10 kDa.

[0094] Generally, dialysis conditions (e.g., time, temperature, amount of buffer, number of buffer changes) are selected so that the remaining amount of chelating agent (e.g., EDTA) in the final product is at a concentration of 100 μM or less, preferably 10 μM or less, and more preferably 1 μM or less.

[0095] According to a preferred embodiment of the present invention, the HSA starting material is first pretreated by contacting it with a chelating agent, and then the chelating agent is removed. The pretreated HSA thus obtained is then used to prepare the liquid botulinum toxin preparation of the present invention. As used herein, the term “human serum albumin starting material” is intended to refer to commercially available or conventionally available donor HSA material (HSA derived from human blood, more precisely from human plasma) or recombinant HSA material that has not undergone the pretreatment described herein. A mixture of donor HSA material and recombinant HSA material is also included in the term “human serum albumin starting material” as used herein.

[0096] More specifically, according to this preferred embodiment (III), a liquid formulation comprising (i) botulinum toxin and (ii) human serum albumin is (a) A step of contacting human serum albumin (starting material) with a chelating agent to obtain a mixture of human serum albumin (starting material) and a chelating agent; or (b) A step of contacting human serum albumin (starting material) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; (c) a step of removing the chelating agent from the mixture to obtain pre-treated human serum albumin material; and (g) A step of mixing botulinum toxin with pre-treated human serum albumin material. It is prepared by a method that includes [the following].

[0097] In the presence of EDTA, the addition of a salt selected from calcium, magnesium, or zinc, or a mixture thereof, in step (b) forms an EDTA complex, i.e., EDTA complexed with the added metal cation as the central atom. The properties of the salt anion are not important; the only requirement is that the salt is soluble in the aqueous formulation of the present invention. For example, the anion may be anion of a pharmaceutically acceptable inorganic or organic acid. Alternatively, the chelating agent itself may form an anion of a metal salt, and components (iii) and (iv) may be added to the liquid formulation as, for example, Na2CaEDTA.

[0098] Suitable counteranions include, for example, acetate, aspartate, benzenesulfonate, benzoate, besilate, bicarbonate, tartrate, bromide, cansilate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, glycolyl arsanylate, hexanoate, hydravamin, hydroxynaphthoate, iodide, lactobionate, nitrate, malate, maleate, mandelate, mesilate, methyl bromide, methyl nitrate, methyl sulfate, mutinate, napsylate, nitrate, octanoate, oleate, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, basic acetate, succinate, sulfate, tartrate, and theoclate.

[0099] Preferably, the salt is a chloride salt. Particularly preferred salts include calcium chloride (i.e., CaCl2 and its hydrate), magnesium chloride (i.e., MgCl2 and its hydrate), and zinc chloride (i.e., ZnCl2 and its hydrate). When these salts are added in the presence of EDTA, an EDTA complex is formed, i.e., EDTA complexed with the added metal cation as the central atom.

[0100] Generally, the concentration of metal ions in the salt is assumed to be 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.

[0101] Surprisingly, the inventors of the present invention discovered that adding salts of alkaline earth metals or transition metals, particularly salts of calcium, magnesium, or zinc, significantly reduced the injection pain of the liquid formulation of the present invention while simultaneously maintaining its storage stability and photostability. This discovery was unexpected because (i) the liquid botulinum toxin formulation described herein was found to cause unforeseen and unpredictable unpleasant injection pain, (ii) surprisingly, the addition of salts of alkaline earth metals or transition metals, such as calcium chloride, dramatically reduced the injection pain, and (iii) despite the belief that a free (non-complexing) chelating agent was necessary to obtain the photostability effect, the storage stability and photostability of the liquid formulation were maintained, i.e., the central metal atom (e.g., Ca) 2+ This is because chelating agents containing (e.g., EDTA) have surprisingly been found to still be able to protect botulinum toxin from light.

[0102] According to another preferred embodiment of the present invention, a liquid preliminary formulation is brought into contact with a chelating agent, and then the chelating agent is removed to obtain a liquid formulation. As used herein, the term “liquid preliminary formulation” refers to a liquid formulation comprising at least components (i) and (ii) (i.e., botulinum toxin and HSA), and preferably all components and substances contained in the final liquid composition, particularly components (iii) and (iv). In the latter case, the treatment and removal of the chelating agent results in the final liquid formulation.

[0103] More specifically, according to this preferred embodiment, a liquid formulation comprising (i) botulinum toxin and (ii) human serum albumin is (d) contacting a liquid preliminary formulation containing botulinum toxin and human serum albumin with a chelating agent to obtain a mixture of the liquid preliminary formulation and the chelating agent, or (e) contacting a liquid preliminary formulation containing botulinum toxin and human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) removing the chelating agent from the mixture to obtain a pretreated human serum albumin material. It is prepared by a method comprising

[0104] Advantageously, the pretreated HSA provides low concentrations of Fe 3+ ions.

[0105] In a preferred embodiment, the liquid formulation contains Fe 3+ ions at a concentration of less than 1 μM.

[0106] Preferably, the Fe 3+ concentration in the liquid formulation is less than 1000 nM, less than 750 nM, less than 500 nM, or less than 250 nM, more preferably, the Fe 3+ concentration in the liquid formulation is less than 200 nM, less than 150 nM, or less than 100 nM, particularly preferably, the Fe 3+ concentration in the liquid formulation is less than 50 nM or less than 10 nM, and most preferably, the Fe 3+ concentration in the liquid formulation is less than 1 nM, less than 100 pM, less than 10 pM, or less than 1 pM.

[0107] Advantageously, the pretreated HSA does not contain or contains only low concentrations of tryptophan and N-acetyltryptophan.

[0108] In a preferred embodiment, the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0109] As used herein, the term “free of tryptophan and N-acetyltryptophan” means that the liquid formulation does not contain tryptophan and N-acetyltryptophan, or does not contain any tryptophan and N-acetyltryptophan at all. Specifically, it means that tryptophan and N-acetyltryptophan have not been added to the formulation. Alternatively, it means that the concentration of tryptophan and N-acetyltryptophan in the liquid formulation is 0 μM (i.e., <0.5 μM according to general rounding rules), in particular ≤0.1 μM or ≤0.01 μM or ≤0.001 μM, and more specifically 0 nM (i.e., <0.5 nM according to general rounding rules). As used herein, the term "containing less than or equal to" tryptophan and N-acetyltryptophan means that the total amount of tryptophan and N-acetyltryptophan may be ≤50 μM, preferably ≤20 μM, more preferably ≤10 μM, even more preferably ≤1 μM, even more preferably ≤0.1 μM, most preferably ≤0.01 μM, or ≤0.001 μM. Accordingly, as used herein, the term "not containing, or containing less than or equal to" means that the total amount of tryptophan and N-acetyltryptophan in the liquid formulation is 0 μM to X, or 0 nM to X, where X is 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM.

[0110] Metal ions (for example, Ca 2+ Co 2+ Cu 2+ Ni 2+ , or Fe 3+The concentration of ) can be determined by measurement methods known to those skilled in the art, such as atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and inductively coupled plasma atomic emission spectroscopy (ICP-AES) (also called inductively coupled plasma atomic emission spectroscopy (ICP-OES)). Preferably, the concentration of metal ions can be measured using ICP-MS or ICP-OES, particularly ICP-OES (Second Supplement to USP38-NF 33, Chemical Tests / <233> Elemental Impurities-Procedure 1(ICP-OES)and Procedure 2(ICP-MS),2015Second Supplement to USP38-NF 33,Chemical Tests / <233> See also Elemental Impurities - Procedure 1 (ICP-OES) and Procedure 2 (ICP-MS), 2015.

[0111] The concentrations of the amino acids tryptophan and N-acetyltryptophan can be determined by various techniques (e.g., DC, HPLC, LC-MS, GC-MS, CE, etc.), as is known to those skilled in the art. For example, N-acetyltryptophan can be determined by separation using liquid chromatography on a reversed-phase column and UV detection at 280 nm, as described by Nelis et al. (Nelis et al., J. Chromatogr., 1985, 333(2):381-387), or by a method based on UV spectroscopy of the acid-soluble fraction remaining after protein precipitation, as described by Yu and Finlayson (Yu, MW and Finlayson, JS, J. Pharm. Sci., 1984, 73(1):82-86). Tryptophan can be quantitatively determined using methods involving liquid chromatography-tandem mass spectrometry, for example, as described by Wentao et al. (Analytical and Bioanalytical Chemistry, 2011, 401:3249-3261).

[0112] As used herein, the term “tryptophan” refers to L-tryptophan, D-tryptophan, or a mixture of L-tryptophan and D-tryptophan (D / L-tryptophan). Similarly, as used herein, the term “N-acetyltryptophan” refers to N-acetyl-L-tryptophan, N-acetyl-D-tryptophan, or a mixture of N-acetyl-L-tryptophan and N-acetyl-D-tryptophan (N-acetyl-D / L-tryptophan).

[0113] The human serum albumin used in the preparation of the liquid formulation is a human serum albumin material that preferably contains 50 mM or less, more preferably 20 mM or less or 10 mM or less, even more preferably 1 mM or less or 0.1 mM or less, and most preferably 0.01 nM or less or 0 mM of tryptophan and N-acetyltryptophan.

[0114] It has been noted that all commercially available donor HSA products contain a considerable amount (>10 mM) of N-acetyltryptophan. Therefore, such products need to be purified to reduce the amount of N-acetyltryptophan to a desired level before being used in the formulation of the liquid formulation of the present invention. Preferred methods for preparing "purified HSA" are described below in detail in relation to a third aspect of the present invention. Briefly, human serum albumin (also referred to herein as "purified HSA") that is free of or contains small amounts of tryptophan and N-acetyltryptophan can be obtained by removing tryptophan and / or N-acetyltryptophan from human serum albumin starting material by dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, field flow fractionation, or precipitation (e.g., salting out, ethanol precipitation).

[0115] Although not limited to theory, the HSA material used to prepare the liquid formulation of the present invention, particularly donor HSA derived from human blood containing a large amount of iron ions, is at least partially responsible for the photosensitive induction properties of unpurified (untreated) HSA. 3+ It is thought to contain a considerable amount of ions.

[0116] In addition to the advantages described above, this aspect of the present invention is also advantageous in the development of manufacturing processes and novel formulations. For example, equipment (e.g., containers) and materials (e.g., excipients) can be selected so that the final product contains as little iron as possible.

[0117] Furthermore, the present invention is based on the surprising discovery that tryptophan and N-acetyltryptophan reduce the photostability of liquid botulinum toxin preparations containing human serum albumin (HSA). This discovery is indeed surprising because commercially available HSA products typically contain sodium caprylate and N-acetyltryptophan to stabilize HSA at high temperatures, in addition to water and sodium chloride, which can lead to contamination of the botulinum toxin preparation. What is even more surprising is that tryptophan is listed as a stabilizing additive for botulinum toxin (see, for example, European Patent No. 3679946).

[0118] Preferably, the method for preparing a liquid formulation according to a second aspect of the present invention is the same as the method described in relation to a first aspect of the present invention. Furthermore, a liquid formulation according to a first aspect of the present invention may have the same composition as the liquid formulation according to a second aspect of the present invention. Therefore, all descriptions, comments, disclosures, definitions, etc., given for a liquid formulation according to a first aspect of the present invention apply equally to a liquid formulation according to a second aspect of the present invention, unless otherwise explicitly stated.

[0119] Liquid formulation according to the second embodiment

[0120] In a second aspect, the present invention relates to water and the following components: (ii) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Algitol, which is sorbitol This relates to liquid formulations, including [the specified term].

[0121] In a preferred embodiment, the aqueous formulation according to the second embodiment contains Fe 3+ Contains ions at a concentration of less than 1 μM.

[0122] Preferably, Fe in the liquid formulation 3+ The concentration is less than 1000 nM, less than 750 nM, less than 500 nM, or less than 250 nM, and more preferably Fe in the liquid formulation. 3+ The concentration is less than 200 nM, less than 150 nM, or less than 100 nM, and is particularly preferably Fe in the liquid formulation. 3+ The concentration is less than 50 nM or less than 10 nM, and most preferably Fe in the liquid formulation. 3+ The concentration is less than 1 nM, less than 100 pM, less than 10 pM, or less than 1 pM.

[0123] In a preferred embodiment, the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0124] In a further embodiment, the present invention relates to water and the following components: (iii) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Algitol This relates to liquid formulations, including [the specified term].

[0125] In a preferred embodiment, the aqueous formulation according to the second embodiment contains Fe 3+ Contains ions at a concentration of less than 1 μM.

[0126] Preferably, Fe in the liquid formulation 3+ The concentration is less than 1000 nM, less than 750 nM, less than 500 nM, or less than 250 nM, and more preferably Fe in the liquid formulation. 3+ The concentration is less than 200 nM, less than 150 nM, or less than 100 nM, and is particularly preferably Fe in the liquid formulation. 3+ The concentration is less than 50 nM or less than 10 nM, and most preferably Fe in the liquid formulation. 3+ The concentration is less than 1 nM, less than 100 pM, less than 10 pM, or less than 1 pM.

[0127] In a preferred embodiment, the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0128] According to the present invention, the liquid formulation described in the first or second embodiment or a further embodiment comprises components (v) and (vi): (v) tonicity agent; (vi) buffer It further includes one or both of the above.

[0129] As used herein, the term “isotonic agent” refers to a substance added to an injectable formulation to make its osmotic properties similar to those of a physiological solution. Isotonic agents are sometimes also called “osmotic regulators.” Isotonic agents are not particularly limited and may be selected from the group consisting of, for example, sugars, salts, polymers, and mixtures thereof.

[0130] Examples of isotonic agents include sucrose, glucose, sodium carbonate, amino acids, polyethylene glycol (PEG), dextran, cyclodextrin, and colloids (e.g., colloidal polysaccharides). Typically, the concentration of the isotonic 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, and even more specifically, 0.6–1.2% w / v.

[0131] Preferably, the isotonic agent is sodium chloride (NaCl). Sodium chloride may be present in the liquid formulation of the present invention in an amount of 0.01 to 2.0% w / v, preferably 0.1 to 1.5% w / v, more preferably 0.5 to 1.2% w / v or 0.8 to 1.0% w / v, and most preferably 0.9% w / v.

[0132] As used herein, the term “buffer” means 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 buffer is one that does not chemically react with other components and is present in an amount sufficient to provide the desired pH buffering degree. Such buffers include, for example, amino acids, acetates, malic acid, ascorbates, citrates, tartrates, fumarates, succinates, phosphates, bicarbonates, TRIS, Bis-TRIS, ACES, MES, BES, MOPS, HEPES, TES, PIPES, trichine, and imidazoles.

[0133] Preferably, the buffer is a phosphate (i.e., a phosphate buffer), an amino acid, or a mixture thereof. As used herein, the term “phosphate” generally means both the unprotonated and protonated forms, and any salts thereof. The amino acid may be selected from aspartic acid, glycine, glutamic acid, histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof, preferably selected from histidine, proline, taurine, methionine, serine, tyrosine, and mixtures thereof.

[0134] In one embodiment of the present invention, component (iv) of the liquid formulation is sorbitol, and buffering agent (vi) is a phosphate.

[0135] Most preferably, the amino acid is histidine.

[0136] The buffers most preferred for use in this specification are histidine, phosphates, or mixtures thereof.

[0137] The concentration of the buffering agent in the liquid formulation of the present invention is preferably 1 to 100 mM, preferably 2 to 50 mM, and more preferably 5 to 20 mM. If the buffering agent is an amino acid (e.g., histidine), it 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. If the buffering agent is a phosphate, it 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.

[0138] 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, 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.

[0139] The viscosity of the liquid formulation of the present invention is typically in the range of 0.1 to 100 Pa·s, preferably 0.3 to 50 Pa·s, more preferably 0.5 to 100 Pa·s, and most preferably 0.8 to 5 Pa·s. In a particularly preferred embodiment of the present invention, the viscosity of the liquid formulation is in the range of 1 to 3 Pa·s. As used herein, the term “viscosity” means “complex viscosity” at 1 Hz and is typically determined by a shear rheometer, for example, an oscillating mode cone-plate shear rheometer.

[0140] Furthermore, unless otherwise specified or intended, the liquid formulations of the present invention may further contain one or more additional pharmaceutically acceptable excipients. For example, the liquid formulations may contain one or more of sucrose, lactose, dextran, polyvinylpyrrolidone, 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 pharmaceutically acceptable excipients include those well known in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0141] On the other hand, this specification also intends for the liquid formulations of the present invention to specifically lack certain components (i.e., compounds, materials, or substances), such as chelating agents, surfactants, polysaccharides, amino acids, stabilizing peptides, etc. (including any combination thereof).

[0142] As used herein, the term “detergent” is used synonymously with “surfactant” and is intended to include nonionic and ionic surfactants. As used herein, the term “stabilized peptide” generally means a peptide consisting of 5 to 50 amino acids, e.g., a peptide of 10 to 40 amino acids, or a peptide of 15 to 30 amino acids. Therefore, the term “stabilized peptide” excludes HSAs.

[0143] In one embodiment, the liquid formulation of the present invention is free of surfactants, particularly polysorbates, and more particularly, polysorbate 20 and / or polysorbate 80. In another embodiment, the liquid formulation of the present invention is free of alginates. In another embodiment, the liquid formulation of the present invention is free of succinates. In another embodiment, the liquid formulation of the present invention is free of 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 is free of sugars, such as monosaccharides, oligosaccharides, or polysaccharides, or mixtures thereof. In particular, the liquid formulation of the present invention may be free of one or more (e.g., 2, 3, or 4) of sucrose, lactose, maltose, and trehalose. In another embodiment, the liquid formulation of the present invention is free of chelating agents, particularly chelating agents described herein in connection with the present invention. In the present invention, it is also intended that the liquid formulation may be free of some or all of the above-mentioned compounds.

[0144] In one embodiment, the liquid formulation of the present invention comprises: (i) a surfactant, and monosaccharides, oligosaccharides and polysaccharides; (ii) a surfactant, and any amino acid, or a surfactant, and all amino acids except histidine; (iii) a surfactant and a stabilizing peptide; (iv) a monosaccharide, oligosaccharide and polysaccharide, and any amino acid, or a monosaccharide, oligosaccharide and polysaccharide, and all amino acids except histidine; (v) a monosaccharide, oligosaccharide and polysaccharide, and a stabilizing peptide; (vi) any amino acid and a stabilizing peptide, or all amino acids except histidine and a stabilizing peptide; (vii) a surfactant, monosaccharide, oligosaccharide and polysaccharide, and any amino acid, or a surfactant, monosaccharide, (viii) Lacking ligosaccharides and polysaccharides, and all amino acids except histidine; (ix) Surfactants, any amino acids and stabilized peptides, or surfactants, all amino acids except histidine and stabilized peptides; (x) Monosaccharides, oligosaccharides and polysaccharides, any amino acids and stabilized peptides, or monosaccharides, oligosaccharides and polysaccharides, all amino acids except histidine and stabilized peptides; (xi) Lacking surfactants, monosaccharides, oligosaccharides and polysaccharides, any amino acids and stabilized peptides, or surfactants, monosaccharides, oligosaccharides and polysaccharides, all amino acids except histidine and stabilized peptides.

[0145] In another embodiment, the liquid formulation of the present invention contains no amino acids other than histidine. In another embodiment, the liquid formulation of the present invention contains no monosaccharides, disaccharides, or trisaccharides. In another embodiment, the liquid formulation of the present invention contains no stabilizing peptides or proteins other than HSA. In another embodiment, the liquid formulation of the present invention does not contain phosphates, for example, phosphate buffers.

[0146] In further embodiments, the liquid formulation of the present invention comprises (i) succinate and surfactant (e.g., polysorbate), (ii) succinate and methionine, (iii) succinate and sucrose, (iv) surfactant (e.g., polysorbate) and methionine, (v) surfactant (e.g., polysorbate) and sucrose, (vi) methionine and sucrose, (vii) succinate, surfactant (e.g., polysorbate), and methionine, (xiii) succinate Lacking salts, surfactants (e.g., polysorbate), and sucrose; (ix) succinates, methionine, and sucrose; (x) surfactants (e.g., polysorbate), methionine, and sucrose; and (xi) succinates, surfactants (e.g., polysorbate), methionine, and sucrose; (xii) surfactants (e.g., polysorbate and histidine); (xiii) surfactants (e.g., polysorbate), histidine, and sucrose).

[0147] Furthermore, any of the liquid formulations of the present invention lacking one or more components (i.e., compounds, materials, or substances) may further lack a chelating agent, in particular the chelating agents described herein.

[0148] Furthermore, a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 1.0% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 10 to 100 mg / ml, (v) an isotonic agent in an amount of 0.01 to 2.0% w / v, preferably sodium chloride, and (vi) a buffer in an amount of 1 to 100 mM.

[0149] A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 0.5% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 10 to 100 mg / ml, (v) an isotonic agent in an amount of 0.1 to 1.5 w / v, preferably sodium chloride, and (vi) a buffer in an amount of 1 to 100 mM.

[0150] A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 10 to 100 mg / ml, (v) an isotonic agent in an amount of 0.6 to 1.2% w / v, preferably sodium chloride, and (vi) a buffer in an amount of 5 to 90 mM.

[0151] A further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 0.5% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 20 to 90 mg / ml, (v) sodium chloride as an isotonic agent in an amount of 0.9% w / v, and (vi) a buffer in an amount of 5 to 90 mM.

[0152] A further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 20 to 90 mg / ml, (v) sodium chloride as an isotonic agent in an amount of 0.9% w / v, and (vi) a buffer in an amount of 5 to 90 mM.

[0153] Furthermore, in this specification, preferred liquid formulations described in the above and following paragraphs are defined as Fe 3+ It is also intended that the ion concentration may be characterized as less than 1 μM, preferably less than 1000 nM or less than 500 nM, more preferably less than 250 nM or less than 100 nM, and most preferably less than 10 nM or less than 1 nM.

[0154] Furthermore, it is intended that the liquid formulations described in the above and below paragraphs may be characterized in that the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0155] A further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 1.0% w / v, (v) an isotonic agent in an amount of 0.01 to 2.0% w / v, preferably sodium chloride, and (vi) a buffer at a concentration of 1 to 100 mM. A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 0.5% w / v, (v) an isotonic agent in an amount of 0.01 to 2.0% w / v, preferably sodium chloride, and (vi) a buffer at a concentration of 1 to 100 mM. A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (v) an isotonic agent in an amount of 0.1 to 2.0% w / v, preferably sodium chloride, and (vi) a buffer at a concentration of 1 to 100 mM. A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (v) an isotonic agent in an amount of 0.6 to 1.3% w / v, preferably sodium chloride, and (vi) a buffer at a concentration of 2 to 50 mM. A preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (v) sodium chloride in an amount of 0.9% w / v, and (vi) a buffer at a concentration of 5 to 20 mM. In each case, (iii) the concentration of hyaluronic acid may be in the range of 2 to 10 mg / ml, and (iv) the concentration of alditol may be in the range of 10 to 100 mg / ml.

[0156] Furthermore, in this specification, the preferred liquid formulations described above are Fe 3+ It is also intended that the ion concentration may be characterized as less than 1 μM, preferably less than 1000 nM or less than 500 nM, more preferably less than 250 nM or less than 100 nM, and most preferably less than 10 nM or less than 1 nM.

[0157] Furthermore, this specification may also describe the liquid formulation as being characterized in that the concentrations of tryptophan and N-acetyltryptophan in the liquid formulation are 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

[0158] A preferred liquid formulation of the present invention includes a buffer selected from (i) botulinum toxin, (ii) HSA, (iii) hyaluronic acid, (iv) alditol, (v) sodium chloride as an isotonic agent, and (vi) histidine, phosphates and mixtures thereof as a buffering agent, preferably histidine.

[0159] A particularly preferred liquid formulation of the present invention comprises a buffer comprising: (i) botulinum toxin in a concentration of 10 to 200 U / ml; (ii) HSA in an amount of 0.01 to 1.0% w / v; (iii) hyaluronic acid in an amount of 2 to 10 mg / ml; (iv) algitol in an amount of 10 to 100 mg / ml; (v) sodium chloride in an amount of 0.01 to 2.0% w / v, preferably 0.9% w / v; and (vi) a buffer selected from histidine, phosphates, and mixtures thereof in a concentration of 1 to 100 mM, preferably histidine in a concentration of 1 to 100 mM.

[0160] Another particularly preferred liquid formulation of the present invention comprises a buffer comprising (i) botulinum toxin in a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.01 to 0.5% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 10 to 100 mg / ml, (v) sodium chloride in an amount of 0.9% w / v, and (vi) a buffer selected from histidine, phosphates and mixtures thereof in a concentration of 1 to 100 mM, preferably histidine in a concentration of 1 to 100 mM.

[0161] A further particularly preferred liquid formulation of the present invention comprises a buffer selected from (i) botulinum toxin at a concentration of 10 to 200 U / ml, (ii) HSA in an amount of 0.05 to 0.25% w / v, (iii) hyaluronic acid in an amount of 2 to 10 mg / ml, (iv) algitol in an amount of 10 to 100 mg / ml, (v) sodium chloride in an amount of 0.9% w / v, and (vi) histidine, phosphates, and mixtures thereof at a concentration of 1 to 100 mM, preferably 2 mM to 50 mM or more preferably 5 mM to 20 mM, and preferably histidine at a concentration of 1 to 100 mM, preferably 2 mM to 50 mM, more preferably 5 mM to 20 mM.

[0162] Furthermore, the above-mentioned preferred liquid formulations, and particularly preferred liquid formulations, preferably have a pH in the range of 6.0 to 7.5. In addition, the botulinum toxin is preferably of serotype A, and more preferably a neurotoxic component of serotype A.

[0163] Preferred exemplary liquid formulations of the present invention are as follows:

[0164] Formulation 1: BoNT / A (complex-forming protein-free) 50 U / ml HSA (pre-treated with EDTA and dialysis-treated) 0.85 mg / ml Non-crosslinked hyaluronic acid (3.3 MDa) 5 mg / ml Sorbitol 35 mg / ml NaCl 0.9% (9 mg / mL) Sodium phosphate 10 mM pH 6

[0165] Example formulation 1 HSA (EDTA-dialysis-treated) is, The process involves adding 37.2 mg of Na2-EDTA to 1 ml of 20% HSA solution while stirring, and adjusting the pH to 8.0 with NaOH. The process involves incubating at room temperature for 6 hours while stirring. The process involves performing dialysis (with a 100kDa MWCO of the dialysis membrane) on a 100-fold volume of EDTA buffer (100mM Na2-EDTA; 10mM histidine; 0.9% NaCl pH 7.0) at room temperature for 16 hours while gently stirring on a magnetic stirrer. The process involves performing dialysis at room temperature for 24 hours using 100 times the amount of 10 mM histidine and 0.9% NaCl (pH 6.0), then replacing the dialysis buffer with fresh buffer and performing another 24 hours of dialysis, and repeating this process two more times. It is prepared by an exemplary process that includes the following:

[0166] In exemplary formulation 1, EDTA may optionally be replaced with other chelators such as EGTA, BAPTA, or DTPA. Instead of adding solid EDTA to the HSA solution, a concentrated EDTA solution may be used (e.g., 200 mM EDTA, pH 8.0). The initial dialysis step (to the EDTA buffer) may be omitted. Other methods such as cross-flow dialysis or ultrafiltration may be used instead of dialysis.

[0167] Another preferred liquid formulation is Formulation 2.

[0168] Formulation 2: BoNT / A (complex-forming protein-free) 50 U / ml HSA (pre-treated with EDTA and dialysis-treated) 0.85 mg / ml Non-crosslinked hyaluronic acid (3.3 MDa, pre-treated with EDTA and dialyzed) 5 mg / ml Sorbitol 35 mg / ml NaCl 0.2% (2 mg / ml) Sodium phosphate 10 mM pH 6

[0169] Another preferred liquid formulation is formulation 3.

[0170] Formulation 3: BoNT / A (complex-forming protein-free) 50 U / ml HSA (pre-treated with EDTA and dialysis-treated) 0.85 mg / ml Non-crosslinked hyaluronic acid (3.3 MDa) 5 mg / ml Mannitol 70 mg / ml NaCl 0.2% (2 mg / ml) Histidine 10mM pH 6

[0171] The present invention also relates to a liquid formulation containing botulinum toxin, wherein, after storage at a high temperature of 40°C for 4 weeks, the toxin activity does not decrease by more than 20% compared to the initial toxin activity. Furthermore, the present invention relates to a liquid formulation containing botulinum toxin, wherein the liquid formulation is 250 W / m² 2 This relates to a liquid formulation in which, after exposure to a light source of 7 hours, the toxin activity does not decrease by more than 20% compared to the initial toxin activity. Preferably, a photostability test, i.e., 250 W / m² 2 For a 7-hour exposure, a SUNTEST CPS+ instrument (ATLAS Material Testing Technology LLC) can be used, equipped with a window glass filter compliant with ICH Q1B and a filter set that provides a spectral distribution in the wavelength range of 320-800 nm corresponding to ID65 (indoor indirect daylight standard) according to ISO 10977.

[0172] Method for preparing the liquid formulations described in the first and second embodiments according to the third embodiment.

[0173] In a third embodiment, the present invention relates to a method for preparing a liquid formulation as described in the first embodiment or any embodiment referenced therefrom, wherein the method is Water and, (I) Chelating agent or, (II) Chelating agents, and metal salts selected from salts of calcium, magnesium, zinc, and mixtures thereof. This includes mixing components (i) to (iv) and optionally (v) and / or (vi) in a liquid containing, (III) The method is steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; (b) A step of contacting the human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. including, or (IV) The method is steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. This includes further information about the methods.

[0174] Preferably, the contact in step (a) or step (b) is carried out by mixing a composition containing human serum albumin with a chelating agent or a chelating agent and a metal salt, or the contact in steps (d) and (e) is carried out by mixing a liquid formulation containing components (i) to (iv) with a chelating agent or a chelating agent and a metal salt to adjust the concentration of the chelating agent in the liquid formulation to 1 mM to 500 mM.

[0175] Furthermore, the present invention relates to a method for preparing a liquid formulation according to the second embodiment or any embodiment referenced thereto, The present invention relates to a method comprising mixing components (i) to (iv), and optionally (v) and / or (vi), in a liquid containing water.

[0176] The preparation of the liquid formulations of the present invention is not particularly limited, and each formulation technique is known to those skilled in the art. As described above, the liquid formulations of botulinum toxin are generally aqueous solutions, preferably saline solution, more preferably physiological saline solution, and most preferably buffered (e.g., phosphate or histidine buffered) physiological saline solution.

[0177] Preferably, the salt is dissolved first, then HSA is added, the pH is adjusted if necessary, and finally the botulinum toxin is added. This order is not a requirement, but it is thought to maintain the maximum specific activity of BoNT without any problems. Preferably, the method for preparing the liquid botulinum toxin preparation does not involve the reconstitution of the lyophilized botulinum toxin preparation in powder form.

[0178] Use of the liquid formulations described in the first and second embodiments according to the fourth aspect.

[0179] In a fourth embodiment, the present invention relates to a liquid formulation of the present invention for use in therapeutic purposes.

[0180] In particular, the liquid formulations of the present invention can be used to treat neuromuscular diseases, pain, salivation, hyperhidrosis, urinary tract disorders, and neurological disorders. Exemplary neuromuscular diseases include dystonia, convulsions, tremors, hyperactivity disorder, and cerebral palsy. Urinary tract disorders include, in particular, conditions characterized by detrusor overactivity, overactive bladder, neurogenic bladder and interstitial cystitis, treatment of vulvar pain and chronic pelvic pain, benign prostatic hyperplasia (BPH), and detrusor-sphincter dyssynergia (DSD). Exemplary neurological disorders include chronic or recurrent migraines, cervical dystonia, peripheral neuropathic pain, spasticity (post-stroke), and blepharospasm.

[0181] Generally, the liquid formulations of the present invention are administered intramuscularly, subcutaneously, subdermally, or intradermally.

[0182] Use of the liquid formulations described in the first and second embodiments according to the fifth aspect

[0183] In a fifth embodiment, the present invention relates to the cosmetic use of the liquid formulation of the present invention for the treatment of cosmetic conditions.

[0184] In one embodiment, the use of the present invention may be for cosmetic purposes and therefore may also be for non-therapeutic purposes. The use of the present invention may be carried out by cosmetics manufacturers, beauty professionals, or medical professionals.

[0185] This aspect of the present invention relates to the purely aesthetic use of the liquid formulation of the present invention. Preferred cosmetic conditions to be treated include the treatment of skin conditions, particularly wrinkles, and especially facial wrinkles.

[0186] As used herein, the term “wrinkle” is to be interpreted broadly to include not only wrinkles but also lines, rhythids, creases, furrows, and folds. The terms “line,” “wrinkle,” “rhythid,” “crease,” and “fold” share similar definitions and are often used interchangeably. In this invention, “line” is generally interchangeable with “wrinkle,” but may preferably refer to a shallower depression in the skin than a “wrinkle.” “Fold” is interchangeable with wrinkles and lines, and is preferably a linear depression. “Crease” is interchangeable with wrinkles, lines, and folds. This preferably refers to a mild wrinkle and may describe a specific wrinkle in a particular location. As used herein, “rhythid” has essentially the same meaning as wrinkle. However, “rhythid” preferably refers to a skin structure formed by a collection of irregular lines. "Deep wrinkles" are deep sagging or deep lines in the skin.

[0187] Preferably, the wrinkles treated according to the present invention are facial wrinkles, including, for example, horizontal lines on the forehead, lines between the eyebrows (e.g., vertical lines between the eyebrows), periorbital lines, crow's feet, lines at the base of the nose (i.e., lines that extend downward on both sides of the nose), nasolabial folds, perilabial lines, radial lines of the upper lip, radial lines of the lower lip, corners of the mouth, marionette lines, perilabial lines, oral commissures, labiomental crease, and cobblestone chin.

[0188] To treat the facial wrinkles mentioned above, botulinum toxin is usually administered by intramuscular injection into the following muscles: the frontalis muscle (horizontal lines on the forehead), the procerus and corrugator supercilii muscles (vertical wrinkles between the eyebrows), the orbicularis oculi muscle (crow's feet / circumorbital lines), the bridge of the nose, the procerus muscle on the side of the bridge of the nose (lines at the base of the nose), the levator labii superioris alaeque nasi muscle (nasolabial folds), the orbicularis oris muscle (radial lines of the upper and lower lip), the depressor anguli oris muscle (angular lines of the mouth, marionette lines, labial commissure, labial groove), and the mentalis muscle (perioral lines, cobblestone jaw).

[0189] A more preferred cosmetic use of the liquid formulation of the present invention relates to use for cosmetic purposes, including rejuvenation and / or improvement of the skin quality of the face and / or body.

[0190] In a further preferred embodiment, rejuvenation and / or improvement of the skin quality of the face and / or body includes improvement and / or reduction and / or filling and / or prevention of wrinkles, particularly wrinkles resulting from muscular activity such as facial expression imitation; skin smoothing; improvement of skin laxity; lifting effect; moisturizing and / or softening of the skin; improvement and / or reduction and / or filling of facial lines; soft tissue augmentation; improvement of subcutaneous support of the eyebrows, cheekbones and cheek fat pads; improvement of the tear trough; improvement of the appearance of the nose; elimination of facial asymmetry; improvement of the jawline; or a combination of two or more of these.

[0191] Generally, the liquid formulations of the present invention are administered intramuscularly, subcutaneously, subdermally, or intradermally.

[0192] Use of the liquid formulations described in the first and second aspects according to the sixth aspect.

[0193] In a sixth aspect, the present invention relates to a method for treating a disease or condition, comprising administering an effective amount of the liquid formulation of the present invention to a body in need thereof.

[0194] A disease or condition may be any one of the diseases and conditions described herein, regardless of whether it is a therapeutic or cosmetic indication. Accordingly, in one embodiment, the present invention relates to a method for treating a condition to be treated, comprising administering an effective amount of the liquid formulation of the present invention to a body in need. In another embodiment, the present invention relates to a (non-therapeutic) method for treating a cosmetic (aesthetic) condition, preferably a skin condition, comprising injecting an effective amount of the liquid formulation of the present invention to a body in need.

[0195] The subjects of treatment are not particularly limited, other than having a disease or condition that can be treated according to the present invention. Those skilled in the art will be able to determine an appropriate administration regimen for treating a given therapeutic or cosmetic indication.

[0196] In particular, depending on the disease or condition to be treated, the injection may be administered intradermally, subcutaneously (subdermally), or intramuscularly.

[0197] Further characteristics

[0198] Furthermore, the present invention relates to the use of human serum albumin (HSA) to improve the light and / or temperature stability of a liquid composition containing botulinum toxin, wherein the HSA is (a) A step of contacting a composition containing human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; or, (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; and (c) A step of removing the chelating agent from the mixture, Regarding the use of HSA, which is prepared by a method including the following.

[0199] Furthermore, the present invention provides (a) A step of contacting a composition containing human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; or, (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; and (c) A step of removing the chelating agent from the mixture, This relates to human serum albumin (HSA) that can be obtained by a method including the following.

[0200] Furthermore, the present invention relates to a method for stabilizing a liquid botulinum toxin preparation, comprising combining botulinum toxin with human serum albumin (HSA), wherein the HSA is (a) A step of contacting a composition containing human serum albumin with a chelating agent to obtain a mixture of human serum albumin and a chelating agent; or, (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, a chelating agent, and a metal salt; and (c) A step of removing the chelating agent from the mixture, The present invention relates to a method that includes, and is prepared by, a method. [Examples]

[0201] The following examples illustrate the liquid botulinum toxin preparation and method of preparation according to the present invention. Percentages are expressed as weight / volume (w / v) unless otherwise specified.

[0202] Furthermore, it will be understood that the range encompasses the provided numerical value as a generally rounded value, including the entire limit of rounding. For example, the range "1 mg" encompasses the range from 0.50 to 1.49 mg.

[0203] However, the numerical values ​​of the present invention also disclose more detailed values, including those with one or more digits. Therefore, for example, "1 mg" may also include a specific disclosure of "1.0 mg".

[0204] The biological activity of botulinum toxin was determined using a cell-based titer assay (CBA), as described in International Publication No. 2014 / 207109. Briefly, nerve cells were incubated with a neurotoxin-containing sample and a reference standard of known titer. After incubation, 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 to that of cells treated with the reference standard. Example 1 Light and storage stability of liquid botulinum toxin preparations containing HSA pretreated by conventional dialysis (not according to the present invention)

[0205] In initial experiments, it was found that the biological activity of a liquid formulation of 150 kDa botulinum toxin type A (also referred to herein as "150 kDa BoNT / A") without complex-forming proteins decreased significantly after exposure to light (daylight or indoor light), and that photosensitivity increased with increasing human serum albumin (HSA) concentration. In further experiments, the inventors unexpectedly found that the photostability and storage stability at 40°C of the liquid botulinum toxin formulation could be significantly improved by adding a complexing agent such as EDTA, even when the EDTA was present in the form of its magnesium, calcium, or zinc complex (results not shown).

[0206] Based on these findings, it was hypothesized that an unknown component in HSA caused the photosensitivity to botulinum toxin, and that this component could be inactivated or masked in some way by chelating agents such as EDTA. However, further experiments conducted by the inventors surprisingly revealed that EDTA intensified injection pain. Accordingly, the inventors attempted to remove the unknown component causing photoinstability in HSA by dialysis, in order to avoid the use of chelating agents such as EDTA.

[0207] For this purpose, a concentrated stock solution of HSA (e.g., 20%) was carefully dialyzed at room temperature for 4 × 12 hours (3 buffer changes, with 100 times the amount of buffer as the sample) in a buffer of 10 mM histidine and 0.9% NaCl (pH 6.0) to obtain "dialysis-treated HSA". Subsequently, the following liquid formulations of botulinum toxin (150 kDa BoNT / A without complex-forming proteins) were prepared using the dialyzed and undialysis-treated HSA: Preparation 1: 65 U / mL botulinum toxin, 0.1% HSA (non-dialysis), 10 mM histidine, 0.9% NaCl (pH 6.0) Preparation 2: 65 U / mL botulinum toxin, 0.1% HSA (dialysis-treated), 10 mM histidine, 0.9% NaCl (pH 6.0)

[0208] Regarding the photostability of these two formulations, compared to a control sample stored in the dark, they exhibited a photostability of 250 W / m². 2 The relative botulinum toxin activity was determined by measuring the activity after 7 hours of exposure to light at 250 W / m². 2 For the 7-hour exposure to light, a SUNTEST CPS+ instrument (ATLAS Material Testing Technology LLC) was used, equipped with a window glass filter compliant with ICH Q1B and a filter set that provided a spectral distribution in the wavelength range of 320-800 nm corresponding to ID65 (indoor indirect daylight standard) according to ISO 10977. The results are shown in Table 1. [Table 1]

[0209] As is clear from Table 1, formulation 2, which contains dialyzed HSA, showed only slight limitations in its effect on photostability compared to formulation 1, which contains undialysis HSA.

[0210] Furthermore, the storage stability of formulations 1 and 2 was determined by measuring the relative botulinum toxin activity after storage at 40°C for 2 weeks and 4 weeks, compared to a control sample at T0 (measured immediately after preparation of formulations 1 and 2). The results are shown in Table 2. [Table 2]

[0211] As described above, dialysis does not affect the storage stability of botulinum toxin. Example 2 Light and storage stability of liquid botulinum toxin preparations containing chelating agents and HSA pretreated by dialysis.

[0212] Despite the findings from Example 1 (i.e., EDTA or an EDTA complex is necessary for photostability; dialyzed HSA does not result in a significant improvement in photostability and storage stability), the inventors continued their research into liquid botulinum toxin formulations that are photostability and storage stability despite not containing chelating agents such as EDTA.

[0213] First, EDTA was added to a concentrated stock solution of HSA (e.g., 20%) (100 mM = 37.2 mg of Na2-EDTA per 1 ml of HSA solution (pH 8.0)), and incubated at room temperature for 6 hours with stirring. Then, the incubated mixture was dialyzed at room temperature for 16 hours in a buffer containing EDTA (100 mM EDTA, 10 mM histidine, 0.9% NaCl (pH 7.0)). The dialysis membrane used was a membrane with a MWCO of 10 kDa, and the packing volume was 1 cm² of dialysis membrane. 2 The amount was approximately 0.2 ml per sample. The amount of dialysis buffer was approximately 100 times the amount of sample.

[0214] Subsequently, EDTA was removed by dialysis at room temperature for 24 hours using approximately 100 times the amount of sample volume in EDTA-free dialysis buffer (10 mM histidine, 0.9% NaCl (pH 6.0)). Then, the dialysis buffer was replaced with fresh dialysis buffer, and dialysis was performed again for 24 hours. This was repeated twice to obtain pre-treated HSA ("EDTA-dialyzed HSA").

[0215] Subsequently, this pre-treated HSA was used to prepare liquid formulation 3 of botulinum toxin (150 kDa BoNT / A without complex-forming proteins): Formulation 1: Same as Formulation 1 of Example 1 (65 U / mL botulinum toxin, 0.1% HSA (non-dialysis), 10 mM histidine, 0.9% NaCl (pH 6.0)) Preparation 3: 65 U / mL botulinum toxin, 0.085% HSA (EDTA-dialysis), 10 mM histidine, 0.9% NaCl (pH 6.0)

[0216] For the photo-stability of Formulations 1 and 3, it was determined by measuring the relative botulinum toxin activity after 7 hours of exposure to light of 250 W / m 2 compared with the control samples stored in the dark. The results are shown in Table 3.

Table 3

[0217] As is clear from Table 3, in Formulation 3 containing EDTA-dialyzed HSA, a significant improvement in photo-stability was observed compared with Formulation 1 containing non-pretreated HSA.

[0218] Also, for storage stability, it was determined by measuring the relative botulinum toxin activity after storage at 40 °C for 2 weeks and 4 weeks compared with the control samples at T0 (measured immediately after the preparation of Formulations 1 and 3). The results are shown in Table 4.

Table 4

[0219] From the results, it was shown that when using EDTA-treated and dialyzed HSA, the biological toxin activity was significantly higher compared with Formulation 1 containing untreated (non-dialyzed) HSA.

[0220] Overall, this example shows that when HSA is pretreated with a chelating agent and then the chelating agent is removed, for example by dialysis, unexpectedly, the chelating agent (e.g., EDTA) can be omitted from the liquid formulation. The resulting formulation has not only photo and temperature (storage) stability but also reduced pain upon injection compared with the liquid formulation containing a chelating agent such as EDTA. Example 3 Effect of Iron Ions on the Photo-Stability of Liquid Botulinum Toxin Formulations

[0221] Furthermore, an experiment was conducted to examine the effect on the photosensitivity of metal ions. For this purpose, calcium (1 mM Ca 2+ ), cobalt (1 mM Co 2+ ), copper (1 mM Cu2+ ), nickel (1mM Ni 2+ ), or iron (1mM Fe 3+ The ) was added to a liquid formulation (pH 6.0) containing 50 U / ml of 150 kDa BoNT / A, 0.085% pre-treated HSA (i.e., EDTA-treated and dialyzed HSA) prepared according to Example 2, 10 mM histidine, and 0.9% NaCl. The liquid formulation was prepared using high-purity water, histidine, and NaCl, without the use of iron or steel utensils. The resulting formulation was exposed to light (250 W / m²). 2 (After 7 hours of storage in the dark), the product was compared with a control formulation.

[0222] Compared to a formulation without added calcium and cobalt (79% residual toxin activity compared to a control formulation stored in the dark), calcium (1 mM Ca) 2+ ) Addition and cobalt (1 mM Co 2+ The addition of copper (1 mM Cu) was found not to affect photostability (residual toxin activity of 74% and 77% compared to the control formulation stored in the dark). 2+ ) and nickel (1mM Ni 2+ The addition of iron (1 mM Fe) slightly reduced photostability. In contrast, the addition of iron (1 mM Fe) reduced photostability to a relatively small extent. 3+ The addition of ) dramatically reduces photostability and exposure to light (250 W / m²). 2 The residual toxin activity after 7 hours was only 1% compared to the control formulation stored in the dark (results not shown).

[0223] Based on these results, Fe 3+ The effects of ions were investigated in more detail in the concentration range of 10 pM to 100 μM. Similar to the experiment described above, the same preparation containing pre-treated HSA (50 U / ml botulinum toxin, 0.085% pre-treated HSA, 10 mM histidine, 0.9% NaCl (pH 6.0)) was used, and a stock solution (50 mM Fe(NO3)3) was also used. 3+ The concentration was adjusted to the desired final concentration: 250 W / m². 2Table 5 shows the biological activity measured after 7 hours of exposure to light, expressed as a percentage of the biological activity measured for the corresponding sample stored in the dark. [Table 5]

[0224] As can be seen from Table 5, at a concentration of 316 nM, Fe 3+ Ions significantly affected the stability of BonT / A, with a decrease in stability of over 15%. At a concentration of 1 μM, photostability decreased significantly, but it was still higher than at concentrations above 1 μM. At concentrations above 1 μM, the added Fe 3+ Ions have a very adverse effect on the stability of BoNT / A in liquid formulations under the influence of light.

[0225] In another experiment, a liquid botulinum toxin preparation containing pre-treated HSA (i.e., 50 U / ml botulinum toxin, 0.085% pre-treated HSA, 10 mM histidine, 0.9% NaCl (pH 6.0)) was stirred in a stainless steel beaker for 48 hours, resulting in a light exposure (250 W / m²). 2 The relative BoNT / A activity after 7 hours was found to be only about 2%, compared to 78% obtained with the same sample stirred in a polypropylene container for 48 hours. This was an extremely surprising finding, as it involved adding trace amounts of Fe to the liquid botulinum toxin preparation from a stainless steel beaker. 3+ This was likely caused by ion elution, indicating that iron ions are an important factor to consider when preparing liquid botulinum toxin preparations. Example 4 Preparation of a stable liquid botulinum toxin preparation

[0226] The bulk solution of the botulinum toxin preparation was prepared using reagents and materials (e.g., purified water, polypropylene containers, PTFE stirrers, etc.) that contained or released minimal amounts of metallic contaminants.

[0227] 5g of sodium hyaluronate (3.3MDa non-crosslinked) was mixed with 500ml of sterile purified water for 48 hours to allow swelling and uniform dissolution.

[0228] The HSA stock solution was pretreated with EDTA and dialyzed as described in Example 2.

[0229] 2 g of sodium chloride, 1.56 g of monosodium phosphate (NaH2PO4·2H2O), and 35 g of sorbitol were dissolved in 450 ml of purified water, and the pH of this solution was adjusted to pH 6.0 with NaOH. Then, HSA (pre-treated with EDTA and dialyzed) and BoNT / A stock solution were added (e.g., 4.25 ml of 20% HSA and 50 μl of BoNT / A 1 MU / ml). Purified water was added to make a final volume of 500 ml, and the solution was filtered to sterile. Finally, the solution was combined with the hyaluronic acid solution and mixed for 2 hours by gentle stirring.

[0230] The bulk solution was filled into 1 ml syringes and stored at 2-8°C.

Claims

1. Water and the following ingredients: (i) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Algitol A liquid formulation containing, (I) The liquid formulation contains a chelating agent, (II) The liquid formulation contains a chelating agent and a metal salt selected from salts of calcium, magnesium, zinc, and mixtures thereof, (III) The human serum albumin is used in the following steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; (b) A step of contacting human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc and mixtures thereof to obtain a mixture of human serum albumin, the chelating agent, and the metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. Including, or subjected to a process, (IV) The liquid formulation is used in the following steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. A liquid formulation that has been subjected to a process, including [the specified element].

2. Water and the following ingredients: (i) Botulinum toxin, (ii) Human serum albumin, (iii) Hyaluronic acid, (iv) Sorbitol, algitol A liquid formulation containing [the specified ingredients].

3. Components (v) and (vi): (v) an isotonic agent; (vi) buffer A liquid formulation according to claim 1 or claim 2, further comprising one or both of the above.

4. The botulinum toxin is a botulinum neurotoxin complex, or The botulinum toxin is a botulinum neurotoxin that does not contain a complex-forming protein, or the botulinum toxin is of serotype A, or The botulinum toxin is a serotype A botulinum neurotoxin that does not contain a complex-forming protein, or The botulinum toxin is of serotype A and is present at a concentration of 1 to 1000 U / ml, or The liquid formulation according to any one of claims 1 to 3, wherein the botulinum toxin is a serotype A botulinum neurotoxin that does not contain complex-forming proteins and is present at a concentration of 1 to 1000 U / ml.

5. The liquid formulation according to any one of claims 1 to 4, wherein the human serum albumin is present in the liquid formulation at a concentration of 0.001 to 1.00% w / v.

6. The chelating agent is selected from the group consisting of aminopolycarboxylic acids having 3 to 6 carboxylic acid functional groups, citrates, porphyrins, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof. Preferably, the chelating agent is of general formula (I): (HO ２ CCH ２ ) ２ N-R-N(CH ２ CO ２ H) ２ (I) An aminopolycarboxylic acid [wherein R is either free of carboxylic acid groups or contains one or two carboxylic acid groups], preferably an aminopolycarboxylic acid selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-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, or The chelating agent is 2,2',2''-nitrilotriacetic acid. A liquid formulation according to claim 1, or any one of claims 3 to 5 insofar as it is dependent on claim 1.

7. The metal salt is present in the liquid formulation in an amount of 0.01 to 100 mM, or the metal salt is calcium chloride and is present in the liquid formulation in an amount of 0.01 to 100 mM. A liquid formulation according to claim 1, or any one of claims 3 to 6 insofar as it is dependent on claim 1.

8. Fe ３＋ A liquid formulation according to any one of claims 1 to 7, wherein the ions are contained at a concentration of less than 1 μM, preferably less than 500 nM, more preferably less than 250 nM, and / or the concentration of tryptophan and N-acetyltryptophan in the liquid formulation is 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).

9. Hyaluronic acid is not cross-linked, or Hyaluronic acid is present at a concentration of 2 to 10 mg / ml, and the hyaluronic acid is not cross-linked, or Hyaluronic acid is present at a concentration of 2-10 mg / ml, and the hyaluronic acid is cross-linked. A liquid formulation according to any one of claims 1 to 8.

10. The liquid formulation according to claim 1, or any one of claims 3 to 9 as dependent on claim 1, wherein the alditol is selected from glycerol, mannitol, isomalt, lactitol, sorbitol, xylitol, trethitol, erythritol, and arabitol, preferably mannitol and sorbitol.

11. The liquid formulation according to any one of claims 1 to 10, wherein the alditol is present at a concentration of 20 to 100 mg / ml.

12. (v) The isotonic agent is present in the liquid formulation in an amount of 0.01 to 2.0% w / v, or the isotonic agent is sodium chloride, or the isotonic agent is sodium chloride and is present in the liquid formulation in an amount of 0.01 to 2.0% w / v; (vi) The buffer is present in the liquid formulation at a concentration of 1 to 100 mM, or the buffer is citric acid, an amino acid, a phosphate, or a mixture thereof, or the buffer is histidine, or the buffer is histidine, a phosphate, or a mixture thereof, and the histidine and phosphate are present in the liquid formulation at a concentration of 1 to 100 mM. A liquid formulation according to any one of claims 3 to 11.

13. The liquid formulation according to any one of claims 1 to 12, wherein the pH of the liquid formulation is in the range of 5.0 to 8, preferably 5.5 to 6.

5.

14. A method for producing a liquid formulation according to claim 1, or any one of claims 3 to 13 insofar as it is dependent on claim 1, wherein the method is Water and, (I) Chelating agent or, (II) Chelating agents, and metal salts selected from salts of calcium, magnesium, zinc, and mixtures thereof. The method involves mixing components (i) to (iv) and optionally (v) and / or (vi) in a liquid containing, (III) The method is steps (a) and (c) or steps (b) and (c): (a) A step of contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; (b) A step of contacting the human serum albumin with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of human serum albumin, the chelating agent, and the metal salt; (c) A step of removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. including, or (IV) The method is steps (d) and (f) or steps (e) and (f): (d) A step of bringing a liquid formulation containing components (i) to (iv) into contact with a chelating agent to obtain a mixture of the liquid formulation and the chelating agent; (e) A step of contacting a liquid formulation containing components (i) to (iv) with a chelating agent and a metal salt selected from calcium, magnesium, or zinc, or a mixture thereof, to obtain a mixture of the liquid formulation, the chelating agent, and the metal salt; (f) A step of removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, cross-flow filtration, or ultrafiltration, preferably by dialysis. Includes, Preferably, the contact in step (a) or step (b) is carried out by mixing a composition containing human serum albumin with the chelating agent or the chelating agent and the metal salt, or the contact in steps (d) and (e) is carried out by mixing a liquid formulation containing components (i) to (iv) with the chelating agent or the chelating agent and the metal salt to adjust the concentration of the chelating agent in the liquid formulation to 1 mM to 500 mM. method; Or, A method for preparing a liquid preparation according to claim 2, or any one of claims 3 to 5 and 8 to 13, to the extent that it is dependent on claim 2, A method comprising mixing components (i) to (iv) and optionally (v) and / or (vi) in a liquid containing water.

15. A liquid formulation according to any one of claims 1 to 13, for use in the treatment of neuromuscular diseases, pain, salivation, hyperhidrosis, urinary tract disorders, and nerve disorders, or Use of the liquid formulation described in any one of claims 1 to 13 for cosmetic purposes, A method for treating a disease or condition, comprising administering an effective amount of a liquid formulation according to any one of claims 1 to 13 to a body in need thereof.

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