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WO2025032220A1 - Formulation de toxine botulique liquide et son utilisation - Google Patents

Formulation de toxine botulique liquide et son utilisation Download PDF

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Publication number
WO2025032220A1
WO2025032220A1 PCT/EP2024/072585 EP2024072585W WO2025032220A1 WO 2025032220 A1 WO2025032220 A1 WO 2025032220A1 EP 2024072585 W EP2024072585 W EP 2024072585W WO 2025032220 A1 WO2025032220 A1 WO 2025032220A1
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WIPO (PCT)
Prior art keywords
liquid formulation
chelating agent
present
concentration
serum albumin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/072585
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English (en)
Inventor
Klaus Fink
Harold Victor Taylor
Karl-Heinz Eisele
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merz Pharma GmbH and Co KGaA
Original Assignee
Merz Pharma GmbH and Co KGaA
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Publication of WO2025032220A1 publication Critical patent/WO2025032220A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/91—Injection

Definitions

  • the present invention relates to liquid formulations comprising (i) botulinum toxin, (ii) human serum albumin (HSA), (iii) hyaluronic acid, and (iv) an alditol.
  • the liquid formulations are characterized by an improved stability against light.
  • the invention further relates to a method of making the liquid formulations and to the use of the liquid formulations in the treatment of therapeutic and cosmetic indications.
  • BoNTs Botulinum neurotoxins
  • BoNT/A-G Two serotypes, type A (BoNT/A) and type B (BoNT/B), are currently in clinical use.
  • BoNTs are produced by Clostridium spp., in particular Clostridium botulinum, in the form of high molecular weight (up to -900 kDa) complexes. These toxin complexes are composed of the active 150 kDa neurotoxin and several complexing proteins (non-toxic neurotoxin-associated proteins, NAPs).
  • BoNT BoNT-containing carboxylates
  • BoNT is highly susceptible to various conditions such as heat and alkaline pH.
  • the challenge regarding the preparation of a medical dosage form of BoNT is to formulate a composition that protects the BoNT from inactivation or partial loss of biological activity during production, storage or use of the product.
  • BoNT due to the extremely high potency of BoNT, with the human lethal dose being in the range of only about 0.1 -1 ng/kg, pharmaceutical formulations of BoNT comprise extremely small quantities of the toxin in the range of only about 1 ng per vial. This aggravates the known problem of loss of toxin activity due to surface denaturation. Therefore, one of the main challenges in the formulation of BoNTs is minimizing activity loss during manufacture and storage.
  • BoNT formulations of BoNTs must be suitable for medical use in a wide range of different indications. This means that the BoNT formulation must be compatible with injection into very different types of tissue, such as muscles, different layers of skin (dermis, subcutis), or glands (e.g., salivary gland).
  • the main BoNT products currently available are provided as lyophilized powders, i.e., in a form that is stable over a long period of time when stored at 2-8 °C or even at room temperature. Lyophilized forms of the BoNT/A complex were first introduced on the market in 1989 (Botox®, Allergan) and 1991 (Dysport®, Ipsen). In 2005, the first stable dosage form of the pure 150 kDa BoNT/A neurotoxin without complexing proteins was approved (Xeomin®; Merz Pharmaceuticals). However, these lyophilized products need to be reconstituted prior to use, a process potentially leading to dosing errors and sterility issues. Therefore, major efforts have been made to develop liquid formulations of BoNTs, which are more convenient to use and can be easily administered.
  • BoNT A commercial liquid formulation of BoNT is available under the trademark Neurobloc® (Eisai). It is a sterile solution of the BoNT/B complex formulated in a buffer containing disodium succinate, sodium chloride, human serum albumin (HSA), sodium caprylate, and sodium N-acetyltryptophanate.
  • HSA human serum albumin
  • Other commercially available liquid formulations are sold under the trademark Innotox® (Medytox), and Alluzience® (Ipsen/Galderma).
  • Both these liquid formulations contain the BoNT/A toxin complex and, in addition to water, sodium chloride, a detergent (Innotox®: polysorbate 20; Alluzience®: polysorbate 80), an amino acid (Innotox®: methionine; Alluzience®: histidine) and an additional excipient (Innotox®: sodium phosphate as a buffer; Alluzience®: sucrose).
  • a detergent Innotox®: polysorbate 20; Alluzience®: polysorbate 80
  • an amino acid Innotox®: methionine; Alluzience®: histidine
  • an additional excipient Innotox®: sodium phosphate as a buffer; Alluzience®: sucrose.
  • US 8 372 645 discloses a method for stabilizing a botulinum neurotoxin from Clostridium botulinum 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 is free of the complexing proteins which naturally form complexes with botulinum neurotoxins, comprising intermixing the neurotoxin with a non-proteinaceous stabilizing agent in an aqueous solution in an amount effective to retain the biological activity of the neurotoxin, wherein the aqueous solution does not comprise mammalian derived proteins.
  • the non-proteinaceous stabilizing agent 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 comprises a pH buffer.
  • the composition may be freeze- dried.
  • US 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 wherein the pharmaceutical composition has a reduced toxicity.
  • the high molecule weight polysaccharide is selected from the group consisting of hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch, hydroxybutyl starch, and hydroxypentyl starch.
  • US 2012/0141532 discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a botulinum neurotoxin and a viscous carrier for the botulinum neurotoxin, wherein the viscous carrier is selected from the group of viscous carriers consisting of hyaluronic acid, carbomer, polyacrylic acid, cellulose polycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharide, polyacrylamide, polyvinyl alcohol, polyvinyl acetate, chitosans, algenates and derivatives and mixtures thereof.
  • the viscous carrier is selected from the group of viscous carriers consisting of hyaluronic acid, carbomer, polyacrylic acid, cellulose polycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharide, polyacrylamide, polyvinyl alcohol, polyvinyl acetate, chitosans, algenates and derivatives and mixtures thereof
  • WO 2010/090677 discloses an animal-protein free, solid-form Clostridial toxin pharmaceutical composition
  • a Clostridial toxin active ingredient an effective amount of a sugar excipient and an effective amount of surfactant excipient, wherein the sugar excipient is a monosaccharide, a disaccharide or a trisaccharide, and wherein the surfactant excipient is a poloxamer, a polysorbate, a polyoxyethylene glycol dodecyl ether, or a polyoxyethylene octyl phenyl ether.
  • the composition is buffered to about pH 5.5 to about pH 6.5 using a citrate buffer, a phosphate buffer, a histidine buffer, or a histidine phosphate buffer.
  • the composition may further comprise an effective amount of sodium chloride.
  • the non-protein polymer excipient is a dextran, a polyethylene glycol, a polyethylene imine, a polyvinyl pyrrolidone, a polyvinyl acetate, an inulin, a starch, or a starch derivative.
  • the composition further comprises an effective amount of a non-protein polymer excipient.
  • the Clostridial toxin active ingredient is stable for at least one-year when stored at either ambient or below freezing temperatures.
  • WO2017/148915 finally discloses a botulinum toxin containing composition comprising non-crosslinked hyaluronic acid and human serum albumin.
  • the present invention is based on the unexpected finding that the use of a pretreated protein stabilizer based on human serum albumin (HSA) in the formulation of a liquid botulinum toxin formulation results in a reduced light sensitivity and, thus, in an improved light stability. Furthermore, it was unexpectedly found that very low amounts of iron ions (Fe 3+ ions) drastically increase the light sensitivity of liquid botulinum toxin formulations containing human serum albumin (HSA). This is surprising since iron is relatively common and ubiquitous, and moreover, it was found that other metal ions such as copper, cobalt or nickel do not show this destabilizing effect.
  • HSA human serum albumin
  • the present invention is based on the surprising finding that tryptophan and N-acetyl-tryptophan decrease the light stability of liquid botulinum toxin formulations containing human serum albumin (HSA).
  • HSA human serum albumin
  • This finding is highly surprising since commercial HSA products, in addition to water and sodium chloride, usually contain sodium caprylate and N-acetyl-tryptophan to stabilize HSA at high temperatures, which in this way get into the botulinum toxin formulation. It is also surprising because tryptophan is described as stabilizing additive for botulinum toxin (see, e.g., EP 3 679 946).
  • the present invention provides for a liquid formulation comprising water and the following components:
  • the liquid formulation comprises a chelating agent
  • the liquid formulation comprises a chelating agent and a metal salt selected from a salt of calcium, magnesium, zinc, and mixtures thereof; or
  • said human serum albumin has been subjected to the following process comprising steps (a) and (c) or steps (b) and (c):
  • step (c) removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis; or (IV) the liquid formulation has been subjected to the following process comprising steps (d) and (f) or steps (e) and (f):
  • step (f) removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis.
  • alditol wherein the alditol is sorbitol.
  • the liquid formulation further comprises one or both of components (v) and (vi):
  • the botulinum toxin is a botulinum neurotoxin complex; or the botulinum toxin is botulinum neurotoxin free of complexing proteins; or wherein the botulinum toxin is of serotype A; or the botulinum toxin is botulinum neurotoxin of serotype A free of complexing proteins; or the botulinum toxin is of serotype A and is present at a concentration of 1 -1000 U/ml; or the botulinum toxin is botulinum neurotoxin of serotype A free of complexing proteins and is present at a concentration of 1 -1000 U/ml.
  • the human serum albumin is present in the liquid formulation at a concentration of 0.001 -1 .00% w/v.
  • the chelating agent is selected from the group consisting of aminopolycarboxylic acids having three to six carboxylic acid functional groups, citrate, porphyrins, N,N,N’,N’- tetrakis(2-pyridinylmethyl)-1 ,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof, preferably wherein the chelating agent is an aminopolycarboxylic acids of general formula (I):
  • R comprises no carboxylic acid group or one or two carboxylic acid groups
  • the aminopolycarboxylic acid preferably being selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(a-aminoethylether)- N,N,N’,N’-tetraacetic acid (EGTA), 1 ,2-bis(o-aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetramine-hexaacetate (TTHA), and mixture thereof; or wherein the chelating agent is 2,2’,2”-nitrilotriacetic acid.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis(a-aminoethylether)- N,N,N’,N’-tetraacetic acid
  • BAPTA di
  • the metal salt is present in the liquid formulation in an amount of 0.01 -100 mM; or wherein the metal salt is calcium chloride and is present in the liquid formulation in an amount of 0.01 -100 mM.
  • the liquid formulation contains Fe 3+ ions in a concentration of less than 1 pM, preferably less than 500 nM, and more preferably less than 250 nM.
  • the concentration of tryptophan and N-acetyl-tryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N- AcTrp).
  • the hyaluronic acid is not crosslinked; or hyaluronic acid is present at a concentration of 2 to 10 mg/ml and wherein the hyaluronic acid is not crosslinked.
  • hyaluronic acid is present at a concentration of 2 to 10 mg/ml and wherein the hyaluronic acid is crosslinked.
  • the alditol is selected from glycerol, mannitol, isomalt, lactitol, sorbitol, xylitol, threitol, erythritol and arabitol, preferably from mannitol and sorbitol.
  • the alditol is present in a concentration of from 20 to 100 mg/ml.
  • the tonicity agent is present in the liquid formulation in an amount of 0.01 -2.0 % w/v, or the tonicity agent is sodium chloride, or the tonicity agent is sodium chloride and is present in the liquid formulation in an amount of 0.01 -2.0 % w/v;
  • the buffer agent is present in the liquid formulation in a concentration of 1 -100 mM, or the buffer agent is citric acid, an amino acid, phosphate, or a mixture thereof, or the buffer agent is histidine, or the buffer agent is histidine, phosphate, or a mixture thereof, and the histidine and phosphate are present in the liquid formulation in a concentration of 1 -100 mM.
  • the pH of the liquid formulation is in the range of from of 5.0 to 8, preferably from 5.5 to 6.5.
  • the invention in a third aspect, relates to a method of making the liquid formulation as defined in the first aspect or any of the embodiments referring thereto, comprising: mixing components (i) to (iv) and optionally (v) and/or (vi) in a liquid comprising water; and
  • step (c) removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis; or
  • step (f) removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis; preferably wherein the contacting in step (a) or step (b) is carried out by mixing the composition comprising human serum albumin with the chelating agent or the chelating agent and the metal salt or wherein the contacting in step (d) and (e) is carried out by mixing a liquid formulation comprising components (i) to (iv) with the chelating agent or the chelating agent and the metal salt such that the concentration of the chelating agent in the liquid formulation is 1 mM - 500 mM; or the invention relates to a method of making the liquid formulation as defined in the second aspect or any of the embodiments referring thereto, comprising: mixing components (i) to (iv) and optionally (v) and/or (vi) in a liquid comprising water.
  • the invention relates to the liquid formulation as defined in the first and second aspect or any of the embodiments referring thereto for use in therapy, particularly for use in the treatment of neuromuscular diseases, pain, sialorrhea, hyperhidrosis, urological disorders, and neurological disorders,
  • the invention relates to the use of the liquid formulation as defined in the first or second aspect or any of the embodiments referring thereto in a cosmetic I non-therapeutic application.
  • the invention in a sixth aspect, relates to a method of treating a disease or condition, comprising administering an effective amount of the liquid formulation as defined in the first or second aspect or any of the embodiments referring thereto to a person in need thereof.
  • the aqueous solution may be a buffered solution, with or without a saline solution, and may be a physiological saline solution such as a buffered (e.g., phosphate and/or histidine buffered) physiological saline solution.
  • a buffered e.g., phosphate and/or histidine buffered physiological saline solution.
  • the botulinum toxin is not particularly limited and includes botulinum toxin of any serotype (e.g., BoNT/A-G).
  • the botulinum toxin may be of serotype A or B (BoNT/A, BoNT/B).
  • the botulinum toxin is of serotype A, more preferably of serotype A1 (BoNT/A1 ), and most preferably BoNT/A1 produced by Clostridium botulinum Hall strain.
  • botulinum toxin may be a natural neurotoxin obtainable from the bacteria Clostridium botulinum or any other botulinum toxin such as a botulinum toxin obtainable from alternative sources, including recombinant technologies and genetic or chemical modification.
  • botulinum toxin (“BT) and the synonymously used term “botulinum neurotoxin” (“BoNT”) are intended to refer to the pure botulinum neurotoxin and/or any complex thereof, i.e., any complex of the pure botulinum neurotoxin and complexing proteins (referred to as the "toxin complex").
  • the term "pure botulinum neurotoxin”, as used herein, means the botulinum neurotoxin free of complexing proteins (sometimes also referred to as the "neurotoxic component"), or more precisely, the botulinum neurotoxin without neurotoxin- associated complexing proteins (NAPs).
  • the pure botulinum neurotoxin is the (active) neurotoxic polypeptide that ultimately inhibits acetylcholine release. It is a di-chain protein comprised of a light chain (LC; about 50 kDa) and a heavy chain (HC; about 100 kDa), held together by a disulfide bond.
  • the active neurotoxic polypeptide may therefore also be referred to herein as the "150 kDa neurotoxin", “Clostridium botulinum neurotoxin (150 kD)” or "neurotoxic component”.
  • toxin complex refers to a high-molecular complex of the neurotoxic component and a set of complexing proteins (NAPs).
  • NAPs complexing proteins
  • the term “toxin complex” includes the 900 kDa, 500 kDa, and 300 kDa Clostridium botulinum type A toxin complexes.
  • the complexing proteins are nontoxic nonhaemagglutinin (NTNHA) and, in strains of serotypes A-D, different haemagglutinins (HAs).
  • NTNHA nontoxic nonhaemagglutinin
  • HAs haemagglutinins
  • the 900 kDa complex is included in onabotulinumtoxin A (BotoxO/Vistabel®, Allergan, Inc., Irvine, CA, USA).
  • a toxin complex as active agent is contained in Dysport® (Azzalure®, Ipsen, Paris, France), Alluzience® (Ips
  • the concentration of the botulinum toxin in the liquid formulations of the present invention may be in the range of 1 -1000 U/ml, preferably in the range of I Q- 200 U/ml, more preferably in the range of 20-150 U/ml, such as 50 U/ml or 100 U/ml.
  • the term "units" or "U”, as used herein refers to the biological activity (biological potency) of the toxin and relates to the dose that is lethal to 50% of mice tested (LD50). More specifically, within the context of the present invention, the LDso is measured using the mouse bioassay (MBA), unless otherwise stated.
  • the MBA determines the mean lethal dose (LD50) of toxin/neurotoxin after intraperitoneal injection in mice, i.e., the dose of toxin/neurotoxin capable of killing 50 % of a group of mice.
  • 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, e.g., Pearce, L. B.; Borodic, G. E.; First, E. FL; MacCallum, FL D.
  • botulinum toxin activity Evaluation of the lethality assay. Toxicol. AppL Pharmacol. 1994, 128:69-77). A person skilled in the art will be able to determine suitable botulinum toxin concentrations depending on the serotype and the intended use.
  • a cell-based assay can be used to determine botulinum toxin activity, as described in WO 2009/1 14748, WO 2013/049508 or WO201 4/207109.
  • a person skilled in the art will be able to correlate botulinum toxin activity results obtained with a cell-based assay with results obtained in the mouse LD50 assay by calibration using a LD50 reference standard.
  • the conversion rate of ONA and INCO is 1 :1.
  • the conversion rate of ONA/INCO:ABO is 1 :2.5.
  • the conversion rate of ONA/INCO:RIM is 1 :50, and the conversion rate of ONA/INCO:TBD is 1 :1.5.
  • HSA Human serum albumin
  • the human serum albumin acts as a stabilizing protein.
  • stabilizing protein generally refers to a polypeptide that results in an increased stability of the botulinum toxin.
  • the HSA may be present in the liquid formulation in an amount of 0.001 -2.0 % w/v, preferably in an amount of 0.001 -1 .00 % w/v, more preferably in amount of 0.01 - 0.5 % w/v, still more preferably in an amount of 0.02-0.3 % w/v, and most preferably in an amount of 0.03-0.15 % w/v.
  • human serum albumin or "HSA” is intended to refer to donor HSA (HSA derived from human blood or, more precisely, from human plasma) and recombinant HSA.
  • the human serum albumin is donor HSA.
  • the human serum albumin is recombinant HSA.
  • HSA or a pre-formulation containing HSA is subjected to a chelating agent to bind or remove metal cations, in particular Fe 3+ ions, which may negatively affect stability, in particular light stability.
  • a chelating agent to bind or remove metal cations, in particular Fe 3+ ions, which may negatively affect stability, in particular light stability.
  • hyaluronic acid as used within the context of the present invention, also called HA, hyaluronate, or hyaluronan, is an anionic, nonsulfated glycosaminoglycan which can be represented by the follow structural formula: [0057]
  • hyaluronic acid can be regarded as a polymer of disaccharides, wherein said disaccharides are composed of two glucose derivatives, namely D- glucuronic acid and D-N-acetylglucosamine.
  • glucuronic acid is glycosidically [3(1 — >3)-linked to N-acetylglucosamine which in turn is glycosidically [3(1 — >4)-linked to the next glucuronic acid.
  • hyaluronic acid comprises all the pharmaceutically acceptable salts, hydrates and/or solvates thereof.
  • hyaluronic acid is present in form of the sodium salt.
  • hyaluronic acid may be understood in the broadest sense as any moiety of hyaluronic acid known in the art.
  • Hyaluronic acid can be present in the form of linear chains, which are not crosslinked and in the form of crosslinked hyaluronic acids.
  • the hyaluronic acid used in the liquid formulations according to the invention is not crosslinked; or hyaluronic acid is present at a concentration of 2 to 10 mg/ml and wherein the hyaluronic acid is not crosslinked.
  • hyaluronic acid is present at a concentration of 2 to 10 mg/ml and wherein the hyaluronic acid is crosslinked.
  • hyaluronic acid used in the liquid formulations according to the invention may also be subjected to a chelating agent analogously to the purification of HSA in order to remove metal ions that may negatively affect light stability.
  • the hyaluronic acid used in the liquid formulations according to the invention has an average molecular weight of 100 KDa to 8 MDa, preferably from 500 KDa to 7 MDa, more preferably from 1 MDa to 6 MDa, more preferably from 2 MDa to 5 MDa, more preferably from 2 MDa to 4,5 MDa, more preferably from 2,5 to 4,5 MDa, more preferably from 2,5 MDa to 4.0 MDa, in particular from 3.0 to 3.8 MDa.
  • the hyaluronic acid used in the liquid formulations according to the invention is not crosslinked, and has an average molecular weight of 100 KDa to 8 MDa, preferably from 500 KDa to 7 MDa, more preferably from 1 MDa to 6 MDa, more preferably from 2 MDa to 5 MDa, more preferably from 2 MDa to 4,5 MDa, more preferably from 2,5 to 4,5 MDa, more preferably from 2,5 MDa to 4.0 MDa, in particular from 3.0 to 3.8 MDa.
  • alditol as used herein is synonymously used with the term “sugar alcohol”.
  • Sugar alcohols have the general formula HOCH2(CHOH) n CH2OH. Common sugar alcohols are ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol.
  • the liquid formulation of the present invention can be stored in any suitable container system.
  • a suitable container system for storing the liquid formulation of the present invention is any device having a partially or fully enclosed space that can be sealed or is sealed and can be used to contain, store, and/or transport liquid formulations.
  • a container system is preferably a closed (or sealed) container made of, or partially or predominantly made of, glass or plastic (e.g., organic polymers) and includes, for example, containers in the form of (i) a syringe, (ii) a vial, (iii) a carpule, or (iv) an ampoule.
  • the liquid formulation is stored in a syringe in the form of a prefilled syringe, as known in the art.
  • the liquid formulation of the present invention advantageously provides an improved safety and dosing accuracy compared to lyophilized botulinum toxin preparations in powder form since the formulation does not need to be reconstituted prior to injection but is ready-to-use and can be used, e.g., in the form of a prefilled syringe.
  • the excellent stability of the liquid formulation facilitates and simplifies the transport and storage as well as the handling by the physician.
  • the superior light stability of the liquid formulation of the present invention simplifies the manufacturing process, allows the product to be filled and packaged without extensive light protection, and reduces potential loss of activity due to light-exposed storage at the physician's place prior to use.
  • the liquid formulation of the present invention does not contain substances which results in enhanced injection pain, thereby increasing acceptance of the formulation by physicians and patients, particularly in the aesthetic field.
  • the present invention provides for a liquid formulation comprising water and the following components:
  • HSA human serum albumin
  • the liquid formulation contains or is prepared by means of a chelating agent.
  • the liquid formulation comprises a chelating agent.
  • the liquid formulation comprises a chelating agent and a metal salt selected from a salt of calcium, magnesium, zinc, and mixtures thereof.
  • said human serum albumin has been subjected to the following process comprising steps (a) and (c) or steps (b) and (c):
  • step (b) 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) removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis.
  • liquid formulation has been subjected to the following process comprising steps (d) and (f) or steps (e) and (f):
  • step (f) removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis.
  • human serum albumin and chelating agent are not intended to imply any restrictions as to their physical form or to exclude the presence of other substances or compounds that are mixed with, or included in, the HSA and the chelating agent.
  • the "human serum albumin” that is contacted in steps (a) and (b) or (d) and (e) with the chelating agent may be present in any form, such as in the form of a solid or a liquid (e.g., an aqueous composition or aqueous solution).
  • the "chelating agent” may be present in any form, such as in the form of a solid or a liquid (e.g., an aqueous composition or aqueous solution).
  • the expression "contacting human serum albumin with a chelating agent" in embodiments III or IV does not exclude that the human serum albumin is in the form of a composition (e.g., a solid composition or liquid composition, in particular an aqueous composition or aqueous solution) that contains one or more additional components included in the final liquid formulation, such as a tonicity agent or a buffer agent or, and/or the chelating agent is in the form of a composition (e.g., a solid composition or liquid composition, in particular an aqueous composition or aqueous solution) that contains one or more additional components included in the final liquid formulation, such as a tonicity agent or a buffer agent.
  • a composition e.g., a solid composition or liquid composition, in particular an aqueous composition or aqueous solution
  • the chelating agent is in the form of a composition (e.g., a solid composition or liquid composition, in particular an aqueous composition or aqueous solution) that contains one or more additional components
  • the mixture obtained in step (a) or step (d) is an aqueous mixture.
  • This aqueous mixture can be prepared by different ways.
  • the human serum albumin may be in the form of an aqueous composition, e.g., an aqueous solution, which is mixed with the chelating agent that may be present in solid form or in liquid form, e.g., in the form of an aqueous solution of the chelating agent.
  • the human serum albumin may be in the form of a solid, e.g., a lyophilized material, which is mixed with a chelating agent and an aqueous solution or with an aqueous solution of the chelating agent.
  • the human serum albumin is in the form of an aqueous composition, more preferably an aqueous solution
  • the chelating agent is a solid or an aqueous composition (e.g., aqueous solution).
  • the HSA that is contacted in step (a) or the HSA that is contained in the liquid formulation is contacted in step (d) with a chelating agent 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.
  • 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.
  • contacting in step (a) comprises, or consists (only) of, the steps of mixing a chelating agent (e.g., EDTA) and human serum albumin, incubating said mixture for a determined time, and optionally subjecting said mixture to dialysis against a buffer containing a chelating agent, the chelating agent being preferably the same as that used in the incubating sub-step.
  • a chelating agent e.g., EDTA
  • human serum albumin e.g., human serum albumin
  • said mixture is not incubated but (directly) subjected to dialysis against a buffer containing a chelating agent, the chelating agent being preferably the same as that used in the step of mixing a chelating agent (e.g., EDTA) and human serum albumin.
  • a chelating agent e.g., EDTA
  • human serum albumin e.g., human serum albumin
  • contacting step (a) may comprise, or may consist (only) of, the step of subjecting said mixture to dialysis against a buffer containing a chelating agent.
  • step (a) comprises, or consists (only) of, a step of adding a chelating agent to a composition comprising HSA (e.g., a solution of HSA) or of mixing a chelating agent with a composition comprising HSA (e.g. a solution of HSA).
  • a chelating agent e.g., a solution of HSA
  • the resulting mixture is then incubated for some time, e.g., allowed to stand for a given time without stirring or stirred for a given time.
  • the time of incubation is not confined 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 time of incubation is not critical and may be, e.g., 1 hour, 2 hours, 5 hours or 10 hours.
  • the incubation time may be, for examples, 0.5 to 5 hours or 1 hour to 10 hours.
  • the incubation temperature is not particularly limited and may, for example, be within the range of 0 °C to 60 °C.
  • the temperature is from 0 °C to 30 °C. This means that room temperature (20 °C or 25 °C) is a suitable temperature within the present invention.
  • the temperature influences the reaction time.
  • the incubation conditions e.g., time and temperature
  • the residual amount of chelating agent e.g., EDTA
  • the residual amount of chelating agent e.g., EDTA
  • the incubation step is followed by further processing such as dialysis of the incubated mixture against a buffer containing a chelating agent, typically the same chelating agent as that used in the incubation step.
  • the chelating agent used in this optional dialyzing step is preferably contained in the dialysis buffer at a concentration of 0.1 mM - 1000 mM, more preferably 1 mM - 200 mM, and most preferably 10 mM - 100 mM.
  • the buffer used in the dialyzing step (i) has a pH of 7.5 - 8.5
  • (ii) further comprises a buffer agent, preferably according to the final composition, or
  • (iii) further comprises a tonicity agent, preferably 0.9% sodium chloride w/v, or (i) and (ii), or (i) and (iii), or (ii) and (iii), or (i) and (ii) and (iii).
  • step (d) may comprise analogous sub-steps.
  • chelating agent is not particularly limited as far as it is capable of binding metal ions.
  • chelating agent may also be referred to "chelator” or "sequestering agent”.
  • the chelating agents for use herein are typically metal ion-binding organic compounds.
  • the metal ions generally form multiple coordinate bonds with the organic chelating agents which act as polydentate ligands.
  • Suitable chelating agents for use herein include, but are not limited to, aminopolycarboxylic acids (e.g., aminopolycarboxylic acids having three to six, preferably four, carboxylic acid functional groups) and other compounds such as citrate, porphyrins, TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1 ,2-ethanediamine), TETA (triethylenetetramine), and mixtures thereof.
  • aminopolycarboxylic acids e.g., aminopolycarboxylic acids having three to six, preferably four, carboxylic acid functional groups
  • other compounds such as citrate, porphyrins, TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1 ,2-ethanediamine), TETA (triethylenetetramine), and mixtures thereof.
  • Exemplary aminopolycarboxylic acids include NTA (nitrilotriacetate, or 2,2’,2”-nitrilotriacetic acid), DOTA (1 ,4,7,10- tetraazacyclododecan-1 ,4,7,10-tetraacetate), TED (ethylenediaminotriacetate), EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis(p-aminoethylether)- N,N,N',N -tetraacetic acid), BAPTA (1 ,2-bis(o-aminophenoxy)ethane-/V,/ ⁇ /,/ ⁇ /',/ ⁇ /'- tetraacetic acid), DTPA (diethylenetriaminepenta-acetic acid), and TTHA (triethylenetetraminehexaacetate).
  • NTA nitrilotriacetate, or 2,2’,2”-nitrilotriacetic acid
  • Particularly preferred chelating agents for use herein include compounds of general formula (I):
  • R group is not particularly limited and may comprise no carboxylic acid functional group or one or two carboxylic acid functional groups.
  • the R group comprises no or one carboxylic acid functional group, most preferably no carboxylic acid functional group.
  • 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.
  • Mixtures of any of the above-mentioned chelating agents may also be used within the present invention, for example as solutions, solids or coupled to matrices.
  • the chelating agent is or comprises 2,2’,2”- nitrilotriacetic acid.
  • step (a) the HSA is preferably contacted with an amount of the chelating agent such that the chelating agent is present in the liquid formulation at a concentration of 0.1 mM - 1000 mM or 0.1 mM - 500 mM, more preferably 0.5 mM - 500 mM or 1 mM - 500 mM, and most preferably 10 mM - 100 mM.
  • the chelating agent may be removed by any appropriate technique such as dialysis (including conventional dialysis using a dialysis bag, counterflow dialysis, etc.), reverse osmosis, filtration, crossflow filtration, ultrafiltration, and chromatographic methods (e.g., ion exchange chromatography or gel filtration chromatography).
  • dialysis including conventional dialysis using a dialysis bag, counterflow dialysis, etc.
  • reverse osmosis filtration, crossflow filtration, ultrafiltration
  • chromatographic methods e.g., ion exchange chromatography or gel filtration chromatography.
  • the chelating agent is removed by dialysis.
  • the dialysis is typically carried out for 0.5-48 hours, in particular 1 -24 hours or 1 -12 hours, at a temperature of 0 °C to 30 °C, particularly at a temperature of 2 °C to 30 °C or 4 °C to 25 °C, including room temperature.
  • the dialysis is typically carried out against a volume of dialyzing buffer of 10 to 1000 times the volume of the incubated HSA/chelating agent mixture, and the dialysis buffer is usually changed at least once.
  • the dialysis membrane used may have a molecular weight cut-off of, e.g., 10 kDa.
  • the dialysis conditions e.g., time, temperature, volume of buffer, number of buffer changes
  • the residual amount of chelating agent e.g., EDTA
  • the concentration of 100 pM or less preferably at a concentration of 10 pM or less, more preferably at a concentration of 1 pM or less.
  • a HSA starting material is first pre-treated by contacting same with a chelating agent, followed by removing the chelating agent.
  • the thus obtained pre-treated HSA is then used to prepare the liquid botulinum toxin formulation of the present invention.
  • the term "human serum albumin starting material” is intended to refer to donor HSA material (HSA derived from human blood or, more precisely, from human plasma) or recombinant HSA material, as commercially or conventionally obtainable, i.e., without having been subjected to a pre-treatment as described herein.
  • a mixture of the donor HSA material and the recombinant HSA material is also encompassed by the term "human serum albumin starting material", as used herein.
  • the liquid formulation comprising (i) botulinum toxin and (ii) human serum albumin is prepared by a method comprising the following steps: (a) contacting a human serum albumin (starting material) with a chelating agent to obtain a mixture of the human serum albumin (starting material) and the chelating agent, or
  • the addition of the salts selected from calcium, magnesium or zinc, and mixtures thereof in step (b) will result in the formation of EDTA complexes, i.e., EDTA with the added metal cation complexed as central atom.
  • EDTA complexes i.e., EDTA with the added metal cation complexed as central atom.
  • the nature of the anion of the salt is immaterial, the only requirement being that the salt is soluble in the aqueous formulation of the present invention.
  • the anion may be an anion of pharmaceutically acceptable inorganic and organic acids.
  • the chelating agent itself can form the anion of the metal salt, and components (iii) and (iv) are added to the liquid formulation as, e.g., Na2CaEDTA.
  • Suitable counter anions include, for example, acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, glycollylarsanilate, hexanoate, hydrabamine, hydroxynaphthoate, iodide, lactobionate, nitrate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, octanoate, oleate, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, s
  • the salts are chloride salts.
  • Particularly preferred salts include calcium chloride (i.e., CaCh and hydrates thereof), magnesium chloride (i.e., MgCk and hydrates thereof) and zinc chloride (i.e., ZnCh and hydrates thereof).
  • CaCh and hydrates thereof calcium chloride
  • magnesium chloride i.e., MgCk and hydrates thereof
  • zinc chloride i.e., ZnCh and hydrates thereof.
  • the concentration of the metal ion of the salt is about equimolar to the concentration of the chelating agent.
  • the salt may 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.
  • the salt is present in the liquid formulation at a concentration of 0.01 -100 mM, preferably 0.1 -50 mM, more preferably 0.05-20 mM, and most preferably 1 -10 mM.
  • liquid pre-formulation is contacted with a chelating agent, followed by removing the chelating agent to obtain the liquid formulation.
  • liquid pre-formulation refers to a liquid formulation which contains at least components (i) and
  • the liquid formulation comprising (i) botulinum toxin and (ii) human serum albumin is prepared by a method comprising the following steps:
  • the pretreated HSA provides for a low concentration of Fe 3+ ions.
  • the liquid formulation contains Fe 3+ ions in a concentration of less than 1 pM.
  • 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 preferable 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.
  • the pre-treated HSA contains no or only low concentrations of tryptophan and N-acetyl-tryptophan.
  • the concentration of tryptophan and N-acetyl- tryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).
  • the term contains no" tryptophan and N-acetyl-tryptophan means that the liquid formulation is free of, or completely free of, tryptophan and N- acetyltryptophan. Specifically, it means that the formulation contains no added tryptophan and N-acetyl-tryptophan. Alternatively, it means that the concentration of tryptophan and N-acetyl- tryptophan in the liquid formulation is 0 pM (i.e.
  • ⁇ 0.5 pM in particular ⁇ 0.1 pM or ⁇ 0.01 pM or ⁇ 0.001 pM, more particularly 0 nM (i.e., according to general rules of rounding ⁇ 0.5 nM.
  • the term "contains no more than" of tryptophan and N-acetyl-tryptophan, as used herein, means that the total amount of tryptophan and N-acetyl-tryptophan may be ⁇ 50 pM, and is preferably ⁇ 20 pM, more preferably ⁇ 10 pM, still more preferably ⁇ 1 pM, yet more preferably ⁇ 0.1 pM, and most preferably ⁇ 0.01 pM or ⁇ 0.001 pM.
  • the term "contains no or no more than”, as used herein, means that the total amount of tryptophan and N-acetyl-tryptophan in the liquid formulation is between 0 pM and X or between 0 nM and X, with X being 50 pM, 20 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM and 0.001 pM.
  • the concentration of metal ions can be determined by measuring 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 referred to as inductively coupled plasma optical emission spectrometry (ICP-OES)).
  • AAS atomic absorption spectroscopy
  • ICP- MS inductively coupled plasma mass spectrometry
  • ICP-AES inductively coupled plasma atomic emission spectroscopy
  • ICP-OES inductively coupled plasma optical emission spectrometry
  • ICP-MS or ICP-OES may be used for measuring the concentration of metal ions (see also Second Supplement to USP38-NF 33, Chemical Tests / ⁇ 233> Elemental Impurities - Procedure 1 (ICP-OES) and Procedure 2 (ICP-MS), 2015).
  • the concentration of the amino acids tryptophan and N-acetyl-tryptophan can be determined by various techniques (e.g., DC, HPLC, LC-MS, GC-MS, CE etc.), as known to those skilled in the art.
  • N-acetyl-tryptophane can be determined by separation using liquid chromatography on a reversed-phase column and UV detection at 280 nm as described in Nelis et al. (Nelis et aL, J.
  • Tryptophane can, e.g., be quantitatively determined using a method involving liquid chromatographytandem mass spectrometry as described by Wentao et al. (Wentao et aL, Analytical and Bioanalytical Chemistry, 201 1 , 401 :3249- 3261 ).
  • tryptophan refers to L-tryptophan, D-tryptophan or a mixture of L-tryptophan and D-tryptophan (D/L-tryptophan).
  • 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).
  • the human serum albumin used for preparing the liquid formulation is preferably a human serum albumin material containing no more than 50 mM, more preferably no more than 20 mM or 10 mM, still more preferably no more than 1 mM or 0.1 mM, and most preferably no more than 0.01 nM or 0 mM tryptophan and N-acetyl- tryptophan.
  • human serum albumin with no, or a low content of, tryptophan and N-acetyl-tryptophan can be obtained by subjecting a human serum albumin starting material to dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, field flow fractionation or precipitation (e.g., salt precipitation, ethanol precipitation), resulting in the removal of tryptophan and/or N-acetyl-tryptophan.
  • the HSA material used for preparing the liquid formulation of the present invention in particular the donor HSA derived from human blood that contains large amounts of iron ions, contains a significant amount of Fe 3+ ions which are responsible - at least in part - for the photosensitivity-inducing properties of unpurified (untreated) HSA.
  • this aspect of the present invention is also advantageous in the development of manufacturing processes and new formulations.
  • equipment e.g., vessels
  • materials e.g., excipients
  • the present invention is based on the surprising finding that tryptophan and N-acetyl-tryptophan decrease the light stability of liquid botulinum toxin formulations containing human serum albumin (HSA).
  • HSA human serum albumin
  • This finding is highly surprising since commercial HSA products, in addition to water and sodium chloride, usually contain sodium caprylate and N-acetyl-tryptophan to stabilize HSA at high temperatures, which in this way get into the botulinum toxin formulation. It is also surprising because tryptophan is described as stabilizing additive for botulinum toxin (see, e.g., EP 3 679 946).
  • the method for preparing the liquid formulation according to the second aspect of the present invention is the same as the method described in connection with the first aspect of the present invention.
  • the liquid formulation according to the first aspect of the present invention may have the same composition as the composition of the liquid formulation according to the second aspect of the present invention. Therefore, all explanations, comments, disclosures, definitions and the like given for the liquid formulation according to the first aspect of the present invention equally apply to the liquid formulation according to the second aspect of the present invention, unless explicitly stated otherwise.
  • the present invention relates to a liquid formulation, comprising water and the following components:
  • alditol wherein the alditol is sorbitol.
  • the aqueous formulation according to the second aspect contains Fe 3+ ions in a concentration of less than 1 pM.
  • 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 preferable 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.
  • the concentration of tryptophan and N-acetyl- tryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).
  • the present invention relates to a liquid formulation, comprising water and the following components:
  • the aqueous formulation according to the second aspect contains Fe 3+ ions in a concentration of less than 1 pM.
  • 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 preferable 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.
  • the concentration of tryptophan and N-acetyltryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).
  • liquid formulation as defined in the first or the second aspect or in the further aspect further comprises one or both components (v) and (vi):
  • tonicity agent refers to an agent that is added to injectable formulations to render the formulations similar in osmotic characteristics to physiologic fluids.
  • the tonicity agent may also be referred to as "osmotic regulator”.
  • the tonicity agent is not particularly limited and may, for example, be selected from the group consisting of sugars, salts, polymers, and mixtures thereof.
  • Exemplary tonicity agents include sucrose, glucose, sodium carbonate, amino acids, polyethylene glycol (PEG), dextran, cyclodextrin, and colloids (e.g., colloidal polysaccharides).
  • concentration of the tonicity agent is in the range of 0 - 2.0 % w/v, in particular 0.01 - 2.0% w/v or 0.1 -1 .5 % w/v, more particularly 0.6-1 .2 % w/v.
  • the tonicity agent is sodium chloride (NaCI).
  • the sodium chloride may be present in the liquid formulation of the present invention in an amount of 0.01 - 2.0 % w/v, preferably 0.1 - 1.5 % w/v, more preferably 0.5 - 1 .2 % w/v or 0.8 - 1 .0 % w/v, and most preferably 0.9 % w/v.
  • buffer agent means an agent which maintains the pH of the liquid formulation in an acceptable range, i.e., an agent capable of controlling the pH of the formulation.
  • Suitable buffer agents are those that are not chemically reactive with other ingredients and are present in amounts sufficient to provide the desired degree of pH buffering.
  • buffer agents include, for example, amino acids, acetate, malic acid, ascorbate, citrate, tartrate, fumarate, succinate, phosphate, bicarbonate, TRIS, Bis-TRIS, ACES, MES, BES, MOPS, HEPES, TES, PIPES, tricine, and imidazole.
  • the buffer agent is phosphate (i.e., a phosphate buffer), an amino acid, or a mixture thereof.
  • phosphate generally means unprotonated and protonated forms and any salts thereof.
  • the amino acid may be selected from aspartate, glycine, glutamate, histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof, and is preferably selected from histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof.
  • component (iv) of the liquid formulation is sorbitol and the buffer agent (vi) is phosphate.
  • amino acid is histidine.
  • the most preferred buffer agent for use herein is histidine, phosphate or a mixture thereof.
  • the concentration of the buffer agent in the liquid formulation of the present invention is preferably 1 -100 mM, preferably 2-50 mM, more preferably 5-20 mM.
  • the buffer agent is an amino acid (e.g., histidine), it may be present in the liquid formulation at a concentration of 1 -100 mM, preferably 2-50 mM, more preferably 5-20 mM, and most preferably 10 mM.
  • the buffer agent is phosphate, it may be present in the liquid formulation at a concentration of 1 -100 mM, preferably 2-50 mM, more preferably 5-20 mM, and most preferably 10 mM.
  • the pH of the liquid formulation of the present invention is typically in the range of 5.0-8.0, particularly in the range of 5.5-7.5, and preferably in the range of 5.5- 7.0 or 6.0-7.5, more preferably in the range of 6.0-7.0, and most preferably in the range of 6.0-6.5.
  • the viscosity of the liquid formulation of the present invention is typically in the range of from 0.1 to 100 Pa»s, preferably 0.3 to 50 Pa»s, more preferably 0.5 to 010 Pa»s, and most preferably in the range of from 0.8 to 05 Pa»s. In a particularly preferred embodiment of the present invention the viscosity of the liquid formulation is in the range of from 1 to 3 Pa*s.
  • the term “viscosity” as used herein means “complex viscosity” at 1 Hz and is typically determined in a shear rheometer, e.g., by a cone and plate shear rheometer in oscillating mode.
  • the liquid formulations of the present invention may further comprise one or more additional pharmaceutically acceptable excipients, unless otherwise stated or intended.
  • the liquid formulations may contain one or more of sucrose, lactose, dextran, polyvinylpyrrolidone, lactic acid, citric acid, amino acid(s), benzyl alcohol, lidocaine, gelatine, hydroxyethyl starch (HES), polyethylene oxide, and polysorbate (e.g., polysorbate 20, polysorbate 80).
  • suitable pharmaceutically acceptable excipients comprise those well known in the art, see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.
  • liquid formulations of the present invention specifically lack certain components (i.e., compounds, materials or substances), for example chelating agents, detergents, polysaccharides, amino acids, stabilizing peptides, and the like, including any combination thereof.
  • the term “detergent”, is synonymously used with “surfactant” and is intended to include non-ionic and ionic detergents.
  • the term “stabilizing peptide”, as used herein, generally means a peptide consisting of 5 to 50 amino acids, such as a peptide of 10 to 40 amino acids or 15 to 30 amino acids. Hence, the term “stabilizing peptide” excludes HSA.
  • the liquid formulation of the present invention does not contain detergents, particularly does not contain polysorbate, more particularly does not contain polysorbate 20 and/or polysorbate 80.
  • the liquid formulation of the present invention does not contain alginate.
  • the liquid formulation of the present invention does not contain succinate.
  • the liquid formulation of the present invention does not contain one or more (e.g., 2, 3, 4 or 5) amino acids selected from the group consisting of: arginine, glutamic acid, methionine, tryptophane, and serine.
  • the liquid formulation of the present invention does not contain a saccharide, such as a mono-, oligo- or polysaccharide or a mixture thereof.
  • the liquid formulation of the present invention may not contain one or more (e.g., 2, 3 or 4) of sucrose, lactose, maltose, and trehalose.
  • the liquid formulation of the present invention does not contain a chelating agent, in particular those described herein in connection with the present invention. It is also contemplated within the present invention that the liquid formulation lacks more than one, or all of the compounds mentioned above.
  • the liquid formulation of the present invention lacks (i) detergents and mono-, oligo- and polysaccharides, (ii) detergents and any amino acid, or detergents and all amino acids except histidine, (iii) detergents and stabilizing peptides, (iv) mono-, oligo- and polysaccharides and any amino acid, or mono-, oligo- and polysaccharides and all amino acid except histidine, (v) mono-, oligo- and polysaccharides and stabilizing peptides, (vi) any amino acids and stabilizing peptides, or all amino acids except histidine and stabilizing peptides, (vii) detergents, mono-, oligo- and polysaccharides and any amino acid, or detergents, mono-, oligo- and polysaccharides and all amino acids except histidine, (viii) detergents, mono-, oligo- and polysaccharides, and stabilizing peptides, (ix)
  • the liquid formulations of the present invention do not contain any other amino acid than histidine. In another embodiment, the liquid formulation of the present invention does not contain any mono-, di- and trisaccharides. In another embodiment, the liquid formulation of the present invention does not contain any other stabilizing peptide or protein than HSA. In another embodiment, the liquid formulations of the present invention do not contain phosphate such as in the form of a phosphate buffer.
  • the liquid formulations of the present invention lack (i) succinate and a detergent (e.g., polysorbate), (ii) succinate and methionine, (iii) succinate and sucrose, (iv) a detergent (e.g., polysorbate) and methionine, (v) a detergent (e.g., polysorbate) and sucrose, (vi) methionine and sucrose, (vii) succinate, a detergent (e.g., polysorbate) and methionine, (xiii) succinate, a detergent (e.g., polysorbate) and sucrose, (ix) succinate, methionine and sucrose, (x) a detergent (e.g., polysorbate), methionine and sucrose, and (xi) succinate, a detergent (e.g., polysorbate), methionine and sucrose, and (xi) succinate, a detergent (e.g., polysorbate), methionine and sucrose, (x
  • any of the liquid formulations of the present invention that lacks one or more components (i.e., compounds, materials or substances) may further lack a chelating agent, in particular the chelating agents described herein.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -1 .0 % w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 10 - 100 mg/ml, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.01 - 2.0% w/v, and (vi) a buffer agent in an amount of 1 -100 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -0.5% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 10 - 100 mg/ml, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.1 -1 .5% w/v, and a buffer agent in an amount of 1 -100 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 10 - 100 mg/ml, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.6-1 .2% w/v, and (vi) a buffer agent in an amount of 5-90 mM.
  • a further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -0.5% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 20 - 90 mg/ml, (v) sodium chloride as a tonicity agent in an amount of 0.9% w/v, and (vi) a buffer agent in an amount of 5-90 mM.
  • a further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 20 - 90 mg/ml, (v) sodium chloride as a tonicity agent in an amount of 0.9% w/v, and (vi) a buffer in an amount of 5-90 mM.
  • the preferred liquid formulations described in the preceding paragraph as well as in the following paragraphs may be characterized by a concentration of Fe 3+ ions of less than 1 pM, 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.
  • the liquid formulations described in the preceding paragraph as well as in the following paragraphs may be characterized in that the concentration of tryptophan and N-acetyl-tryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).
  • a further preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -1 .0% w/v, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.01 -2.0% w/v, and (vi) a buffer agent in a concentration of 1 -100 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -0.5% w/v, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.01 -2.0% w/v, and (vi) a buffer agent in a concentration of 1 -100 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.1 -2.0% w/v, and (vi) a buffer agent in a concentration of 1 -100 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (v) a tonicity agent, preferably sodium chloride, in an amount of 0.6-1 .3% w/v, and (vi) a buffer agent in a concentration of 2-50 mM.
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (v) sodium chloride in an amount of 0.9% w/v, and (vi) a buffer agent in a concentration of 5-20 mM.
  • the concentration of (iii) hyaluronic acid may be in the range of from 2 to 10 mg/ml, and the concentration of (iv) alditol may be in the range of from 10 - 100 mg/ml, respectively.
  • the preferred liquid formulations described above may be characterized by a concentration of Fe 3+ ions of less than 1 pM, 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.
  • liquid formulations described above may be characterized that th concentration of tryptophan and N-acetyl-tryptophan in the liquid formulation is no more than 50 pM, preferably no more than 20 pM, more preferably no more than 10 pM, still more preferably no more than 1 pM, and most preferably 0 pM, of tryptophan and N-acetyltryptophan (total concentration of both Trp and N-AcTrp).
  • a preferred liquid formulation of the present invention comprises (i) botulinum toxin, (ii) HSA, (iii) hyaluronic acid, (iv) alditol, (v) sodium chloride as a tonicity agent, and (vi) a buffer agent selected from histidine, phosphate and a mixture thereof as a buffer agent, wherein the buffer agent is preferably histidine.
  • a particularly preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 - 1 .0% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) alditol in an amount of 10 - 100 mg/ml, (v) sodium chloride in an amount of 0.01 -2.0% w/v, preferably 0.9% w/v, and (vi) a buffer agent selected from histidine, phosphate and a mixture thereof at a concentration of 1 -100 mM, wherein the buffer agent is preferably histidine at a concentration of 1 -100 mM.
  • Another particularly preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.01 -0.5% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) an alditol in an amount of 10-100 mg/ml, (v) sodium chloride in an amount of 0.9% w/v, and (vi) a buffer agent selected from histidine, phosphate and a mixture thereof at a concentration of 1 -100 mM, wherein the buffer agent is preferably histidine at a concentration of 1 -100 mM.
  • Yet another particularly preferred liquid formulation of the present invention comprises (i) botulinum toxin at a concentration of 10-200 U/ml, (ii) HSA in an amount of 0.05-0.25% w/v, (iii) hyaluronic acid in an amount of 2 to 10 mg/ml, (iv) alditol in an amount of 10 - 100 mg/ml, (v) sodium chloride in an amount of 0.9% w/v, and (vi) a buffer agent selected from histidine, phosphate and a mixture thereof at a concentration of 1 -100 mM, preferably at a concentration of 2 mM to 50 mM or, more preferably 5 mM to 20 mM, wherein the buffer agent is preferably histidine at a concentration of 1 -100 mM, preferably at a concentration of 2 mM to 50 mM, more preferably 5 mM to 20 mM,
  • the preferred and particularly preferred liquid formulations describe above have a pH that is preferably in the range of 6.0-7.5.
  • the botulinum toxin is preferably of serotype A and, more preferably, is the neurotoxic component of serotype A.
  • a preferred exemplary liquid formulation of the present invention is the following:
  • Non-crosslinked Hyaluronic acid (3.3 MDa) 5 mg /ml
  • the HSA (EDTA-dialyzed) of exemplary Formulation 1 is prepared by an exemplary process including the following steps: addition of 37.2 mg Na2-EDTA per ml of 20% HSA solution with stirring and adjustment to pH 8.0 with NaOH, incubation for 6 hours at room temperature with stirring, dialysis against 100-fold volume of EDTA buffer (100 mM Na2-EDTA; 10 mM histidine; 0.9% NaCI pH 7.0) for 16 h at room temperature with gentle stirring on magnetic stirrer (MWCO of dialysis membrane 10 kDa) dialysis against 100-fold volume of 10 mM histidine, 0.9% NaCI pH 6.0 for 24 h at room temperature, followed by replacing dialysis buffer with fresh buffer and dialyzing for another 24 h, and repeating two times more.
  • EDTA buffer 100 mM Na2-EDTA; 10 mM histidine; 0.9% NaCI pH 7.0
  • EDTA can optionally be replaced by other chelators such as EGTA, BAPTA or DTPA.
  • a concentrated EDTA solution can be used (e.g., 200 mM EDTA, pH 8.0).
  • the first dialysis step (against EDTA buffer) can also be omitted.
  • Other methods such as crossflow dialysis or ultrafiltration, can also be used instead of dialysis.
  • Formulation 2 Another preferred liquid formulation is Formulation 2:
  • HSA pretreated with EDTA and dialyzed 0.85 mg/ml
  • Non-crosslinked Hyaluronic acid 3.3 MDa
  • Mannitol 70 mg/ml NaCI 0.2% (2 mg/ml)
  • the present invention also relates to a liquid formulation comprising a botulinum toxin, wherein the toxin activity is not reduced by more than 20%, relative to the initial toxin activity, upon storage of the liquid formulation for 4 weeks at an elevated temperature of 40°C. Furthermore, the present invention relates to a liquid formulation comprising a botulinum toxin, wherein the toxin activity is not reduced by more than 20%, relative to the initial toxin activity, upon exposure of the liquid formulation for 7 hours to a light source at 250 W/m 2 .
  • a SUNTEST CPS+ instrument (ATLAS Material Testing Technology LLC) can be used, equipped with a filter set to provide a spectral distribution in the wavelength range of 320-800 nm corresponding to the ID65 (indoor indirect daylight standard) per ISO 10977 with a window glass filter according to ICH Q1 B.
  • the present invention further relates to a method for preparing the liquid formulation as defined in the first aspect or any of the embodiments referring thereto, comprising: mixing components (i) to (iv) and optionally (v) and/or (vi) in a liquid comprising water; and
  • step (c) removing the chelating agent from the mixture obtained in step (a) or step (b) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis; or
  • step (f) removing the chelating agent from the mixture obtained in step (d) or step (e) by dialysis, filtration, crossflow filtration or ultrafiltration, preferably by dialysis.
  • step (a) or step (b) is carried out by mixing the composition comprising human serum albumin with the chelating agent or the chelating agent and the metal salt or wherein the contacting in step (d) and (e) is carried out by mixing a liquid formulation comprising components (i) to (iv) with the chelating agent or the chelating agent and the metal salt such that the concentration of the chelating agent in the liquid formulation is 1 mM - 500 mM.
  • the invention relates to a method of making the liquid formulation as defined in the second aspect or any of the embodiments referring thereto, comprising: mixing components (i) to (iv) and optionally (v) and/or (vi) in a liquid comprising water.
  • the preparation of the liquid formulations of the present invention is not particularly limited and the respective formulation techniques are known to those skilled in the art.
  • the liquid formulation of botulinum toxin is generally an aqueous solution, preferably a saline solution, more preferably a physiological saline solution, and most preferably a buffered (e.g., phosphate or histidine buffered) physiological saline solution.
  • the salts are dissolved first, then HSA is added, the pH is adjusted if necessary, and finally the botulinum toxin is added.
  • This order is not a mandatory requirement but is believed to safely preserve maximum specific activity of the BoNT.
  • the method for preparing the liquid botulinum toxin formulation does not comprise the reconstitution of a lyophilized botulinum toxin preparation in powder form.
  • the present invention relates to a liquid formulation of the present invention for use in therapy.
  • the liquid formulation of the present invention may be used in the treatment of neuromuscular diseases, pain, sialorrhea, hyperhidrosis, urological disorders, and neurological disorders.
  • neuromuscular diseases include dystonia, spasm, tremor, hyperkinetic movement disorders, and cerebral palsy.
  • the urological disorders include, among others, conditions characterized by detrusor overactivity, overactive bladder, neurogenic bladder and interstitial cystitis, treatment of vulvodynia and chronic pelvic pain, benign prostate hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD).
  • BPH benign prostate hyperplasia
  • DSD detrusor sphincter dyssynergia
  • Exemplary neurological disorders include chronic or episodic migraines, cervical dystonia, peripheral neuropathic pain, (post-stroke) spasticity, and blepharospasm.
  • the liquid formulation of the present invention is administered intramuscularly, subcutaneously, subdermally or intradermally.
  • the present invention relates to a cosmetic use of the liquid formulation of the present invention for the treatment of a cosmetic condition.
  • the use of the present invention may be a cosmetic use, thus may also be a non-therapeutic use.
  • the use of the present invention may be conducted by cosmetics, cosmetic professionals or health care professionals.
  • This aspect of the present invention relates to a purely aesthetic use of the liquid formulation of the present invention.
  • Preferred cosmetic conditions to be treated include skin conditions, in particular the treatment of wrinkles of the skin, in particular of the face.
  • wrinkles is to be broadly construed to not only include wrinkles, but also lines, rhytids, creases, furrows, and folds.
  • lines are generally interchangeable with “wrinkles” but may preferably refer to a cutaneous depression that is less deep than a "wrinkle”.
  • a "fold” is interchangeable with wrinkles and lines and is preferably a linear depression.
  • a "crease” is interchangeable with wrinkles, lines and folds.
  • rhytid preferably refers to a mild form of wrinkles and may describe the specific wrinkle in certain locations.
  • a “rhytid”, as used herein, has essentially the same meaning of wrinkle.
  • a “rhytid” preferably refers to a skin structure that is formed by irregular aggregation of lines.
  • a “furrow” is a deep fold or deep line in the skin.
  • the wrinkles treated in accordance with the present invention are facial wrinkles including horizontal forehead lines, glabellar lines (e.g. glabellar frown lines), periorbital lines, Crow’s feet, bunny lines (i.e. , downward radiating lines on the sides of nose), nasolabial folds, perioral lip lines, upper radial lip lines, lower radial lip lines, corner of the mouth lines, marionette lines, perioral lip lines, oral commissures, labiomental crease and cobblestone chin.
  • glabellar lines e.g. glabellar frown lines
  • periorbital lines e.g. glabellar frown lines
  • Crow’s feet e.g. glabellar lines
  • bunny lines i.e. , downward radiating lines on the sides of nose
  • nasolabial folds i.e. , downward radiating lines on the sides of nose
  • perioral lip lines i.e. , downward radiating
  • botulinum toxin is usually administered by intramuscular injection to the following muscles: frontalis muscle (horizontal forehead lines), procerus and corrugator muscles (glabellar frown lines), lateral orbicularis oculi muscle (Crow’s feet/periorbital lines), nasalis, procerus for transverse nasal muscles (bunny lines), levator labii superioris alaeque nasi (nasolabial folds), orbicularis oris (upper and lower radial lip lines), depressor anguli oris (corner of the mouth lines, marionette lines, oral commissures, labiomental crease), and mentalis muscles (perioral lip lines, cobblestone chin).
  • a further preferred cosmetic use of the liquid formulation of the present invention relates to the use for cosmetic applications comprising rejuvenation and/or improvement of the skin quality of the face and/or body.
  • the rejuvenation and/or improvement of the skin quality of the face and/or body comprises improving and/or reducing and/or filling and/or preventing wrinkles, in particular of wrinkles resulting from muscular activity such as mimic activity, skin smoothing, improving skin laxity, lifting effect, moisturizing and/or softening the skin, improving and/or reducing and/or filling facial lines, soft-tissue augmentation, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs, improving nose appearance, resolving facial asymmetries, improving jawlines, or a combination of two or more thereof.
  • the liquid formulation of the present invention is administered intramuscularly, subcutaneously, subdermally or intradermally.
  • the present invention relates to a method of treating a disease or condition, comprising administering an effective amount of the liquid formulation of the present invention to a person in need thereof.
  • the disease or condition may be any one of the diseases and conditions mentioned hereinabove, irrespective of whether it is a therapeutic or cosmetic indication.
  • the present invention relates to a method of treating a therapeutic condition, comprising administering an effective amount of the liquid formulation of the present invention to a person in need thereof.
  • the present invention relates to a (non-therapeutic) method of treating a cosmetic (aesthetic) condition, preferably a skin condition, comprising injecting an effective amount of the liquid formulation of the present invention into a person in need thereof.
  • the person to be treated is not particularly limited other than by having a disease or condition that can be treated in accordance with the present invention. Those skilled in the art will be able to determine appropriate administration regimens for the treatment of a given therapeutic or cosmetic indication.
  • the injection may be intradermal, subdermal (subcutaneous), or intramuscular, depending on the disease or condition to be treated.
  • the present invention relates to a use of a human serum albumin (HSA) for increasing the light and/or the temperature stability of a liquid composition comprising a botulinum toxin, wherein the HSA is prepared by a method comprising the following steps:
  • the present invention relates to a human serum albumin (HSA) which is obtainable by
  • the present invention relates to a method for stabilizing a liquid botulinum toxin formulation, the method comprising combining botulinum toxin with a human serum albumin (HSA), wherein the HSA is prepared by a method comprising the following steps:
  • scope embraces the number values provided as commonly rounded values that embrace the whole rounding limits.
  • scope of “1 mg” embraces the range of from 0.50 to 1 .49 mg.
  • the biological activity of botulinum toxin was determined using a cell-based potency assay (CBA) as described in WO 2014/207109.
  • CBA cell-based potency assay
  • neuronal cells were incubated with the neurotoxin containing sample and a reference standard of known potency. After the incubation period, the cells were lyzed and the amount of cleaved SNAP25 protein was determined by an immunoassay. The biological activity of the sample was then calculated by comparing the cleavage rate of the cells treated with the sample with those treated with the reference standard.
  • CBA cell-based potency assay
  • a concentrated stock solution of HSA e.g., 20%
  • HSA e.g., 20%
  • the dialyzed HSA and the nondialyzed HSA were then used to prepare the following liquid formulations of botulinum toxin (150 kDa BoNT/A without complexing proteins):
  • Formulation 1 65 U/mL botulinum toxin, 0.1% HSA (non-dialyzed), 10 mM histidine, 0.9% NaCI, pH 6.0
  • Formulation 2 65 U/mL botulinum toxin, 0.1% HSA (dialyzed), 10 mM histidine,
  • Example 1 Despite the findings from Example 1 (i.e., EDTA or EDTA complexes required for light stability; dialyzed HSA does not result in a significant improvement of light and storage stability), the present inventors have continued to research for a liquid botulinum toxin formulation that is light and storage stable despite being free of a chelating agent such as EDTA.
  • the dialysis membrane used was a membrane with a MWCO of 10 kDa and the fill volume was about 0.2 ml per cm 2 dialysis membrane.
  • the volume of the dialysis buffer was about 100 times the volume of the sample volume.
  • EDTA-dialyzed HSA was obtained by dialysis for 24 hours at room temperature against a volume of EDTA-free dialysis buffer (10 mM histidine, 0.9% NaCI, pH 6.0) of about 100 times that of the sample volume. The dialysis buffer was then replaced by fresh dialysis buffer and again dialyzed for 24 hours. This was repeated two times to obtain the pretreated HSA ("EDTA-dialyzed HSA").
  • Formulation s 65 U/mL botulinum toxin, 0.085% HSA (EDTA-dialyzed), 10 mM histidine, 0.9% NaCI, pH 6.0
  • Table 4 Storage stability at 40°C of liquid botulinum toxin formulations containing non-dialyzed HSA or EDTA-dialyzed HSA [00219] The results show that the use of EDTA-treated and dialyzed HSA results in a significantly higher biological toxin activity compared to Formulation 1 containing untreated (non-dialyzed) HSA.
  • the chelating agent e.g., EDTA
  • the HSA is pre-treated with a chelating agent followed by removal of the chelating agent by, e.g., dialysis.
  • the resulting formulation is not only light and temperature (storage) stable but also exhibits reduced injection pain when injected compared to liquid formulations containing a chelating agent such as EDTA.
  • HSA stock solution was pre-treated with EDTA and dialyzed as described in Example 2.

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Abstract

La présente invention concerne des formulations liquides comprenant (i) une toxine botulique, (ii) de l'albumine sérique humaine (HSA), (iii) de l'acide hyaluronique, (iv) de l'alditol, et éventuellement (v) un agent de tonicité et/ou (vi) un agent tampon. Dans un mode de réalisation préféré, la HSA a été soumise à un agent chélatant. De plus, la présente invention concerne l'utilisation des formulations liquides dans le traitement d'indications thérapeutiques et cosmétiques.
PCT/EP2024/072585 2023-08-10 2024-08-09 Formulation de toxine botulique liquide et son utilisation Pending WO2025032220A1 (fr)

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