WO2018038585A1 - Formulation liquide contenant la toxine botulique et un agent stabilisant, et procédé de préparation de cette dernière - Google Patents

Formulation liquide contenant la toxine botulique et un agent stabilisant, et procédé de préparation de cette dernière Download PDF

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WO2018038585A1
WO2018038585A1 PCT/KR2017/009383 KR2017009383W WO2018038585A1 WO 2018038585 A1 WO2018038585 A1 WO 2018038585A1 KR 2017009383 W KR2017009383 W KR 2017009383W WO 2018038585 A1 WO2018038585 A1 WO 2018038585A1
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botulinum toxin
arginine
pharmaceutical formulation
stabilizing
buffer
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PCT/KR2017/009383
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English (en)
Korean (ko)
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이치건
엄지현
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주식회사 에이비바이오
휴젤(주)
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Priority claimed from PCT/KR2016/009706 external-priority patent/WO2018038301A1/fr
Priority to EP20195590.3A priority Critical patent/EP3777837B1/fr
Priority to EP17844005.3A priority patent/EP3505155A4/fr
Priority to EP20195594.5A priority patent/EP3782605A1/fr
Priority to CN201780052253.5A priority patent/CN109640954B/zh
Priority to RU2019104998A priority patent/RU2748653C2/ru
Application filed by 주식회사 에이비바이오, 휴젤(주) filed Critical 주식회사 에이비바이오
Priority to US16/327,390 priority patent/US10772943B2/en
Priority to BR112019003770-5A priority patent/BR112019003770A2/pt
Priority to JP2019532909A priority patent/JP6797305B2/ja
Priority to CA3033729A priority patent/CA3033729A1/fr
Publication of WO2018038585A1 publication Critical patent/WO2018038585A1/fr
Priority to US16/934,654 priority patent/US11224640B2/en
Priority to US16/934,649 priority patent/US11147860B2/en

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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)
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • 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

Definitions

  • the present invention relates to liquid formulations comprising botulinum toxin and stabilizers and methods for their preparation.
  • botulinum toxin has a problem in that it is not easy to store, distribute, and manage the pharmaceutical formulation as a protein preparation. This is due to the instability of the protein, which is a serious problem for protein preparations where the drug is formulated at extremely low concentrations, such as botulinum toxin. Since botulinum toxin protein has a property of adhering to a solid surface, when injected into a container, a portion of the protein adheres to the inner wall of the container causing loss of the active ingredient, and the protein can be easily oxidized or broken down into small fragments.
  • the purified botulinum toxin is distributed in the form of lyophilized powder in the manufacturing process and diluted in a solvent immediately before use in the clinical administration to patients in liquid form.
  • this also has a high risk of medical accidents due to human error, such as a user's dilution error or contamination of the dilution solvent. Therefore, there is an urgent need to develop stabilizers that can prevent protein denaturation even during the manufacture and distribution of botulinum toxin liquid formulations.
  • U.S. Patent Publication No. 2007-0134199 discloses a botulinum toxin composition comprising glutamine and glutamic acid or asparagine and aspartic acid as amino acids
  • Korean Patent Publication No. 1087017 discloses a botulinum toxin composition using methionine as a stabilizer.
  • the present invention relates to a liquid formulation comprising a botulinum toxin and a stabilizer and a preparation method thereof, wherein the pharmaceutical formulation comprising the botulinum toxin of the present invention is an arginine, glutamic acid, or aspart as a stabilizer for the botulinum toxin as an active ingredient.
  • Botulinum toxin as a liquid formulation containing acid or as a stabilizing buffer, gluconolactone buffer, or tartaric acid buffer is easy to store and distribute, and has demonstrated a significant effect of botulinum toxin stabilization under conditions suitable for human temperature and pH. It is expected to be widely used for safe and convenient medical use.
  • the present invention has been made to solve the above problems of the prior art, and relates to a liquid formulation comprising the botulinum toxin and stabilizer and a method for preparing the same.
  • Botulinum Toxin is a neurotoxin protein made by Clostridium botulinum bacteria. There are over 127 species of Clostridium genus, classified according to their form and function. The anaerobic, gram-positive bacterium Clostridium botulinum produces botulinum poison, a potent polypeptide neurotoxin that causes a neuropathic disease called botulism in humans and animals. Spores of Clostridium botulinum are found in the soil and can be cultured in sealed, canned home containers that are not properly sterilized, which causes many botulism.
  • Botulinum toxin can appear to pass through the intestinal lining without reducing toxicity and exhibits high affinity for cholinergic motor neurons. Symptoms of botulinum toxin poisoning can progress to gait disorders, swallowing disorders and speech disorders, paralysis and death of respiratory muscles.
  • Botulinum toxin type A is the most lethal natural biological known to man.
  • the commercially available botulinum toxin type A purified neurotoxin complex
  • botulinum toxin type A Based on molar (M), botulinum toxin type A is 1.8 billion times more than diphtheria, 600 million times more than sodium cyanide, 30 million times more than cobra toxin, and 12 million times more lethal than cholera.
  • Botulinum toxin 1 unit (U) can be defined as LD50 via intraperitoneal injection into female Swiss Webster mice weighing 18-20 g each.
  • botulinum neurotoxins are characterized by neurotoxin serotypes A, B, C1, D, E, F and G, respectively, which are differentiated by neutralization with type-specific antibodies.
  • Different serotypes of botulinum toxin differ depending on the animal species on which they act and the degree and duration of paralysis that results. For example, botulinum toxin type A was determined to be 500 times more potent than botulinum toxin type B, as measured by the paralysis rate occurring in rats.
  • botulinum toxin type B was determined to be nontoxic even when 480 U / kg, approximately 12 times that of botulinum toxin type A primate LD50, was administered to primates.
  • Botulinum toxin binds to cholinergic motor neurons with strong affinity and is believed to enter neurons and inhibit the release of acetylcholine. As well as by phagocytosis and phagocytosis, they can be further absorbed through low affinity receptors.
  • the molecular mechanism of toxin poisoning is similar and appears to consist of at least three stages.
  • the toxin binds to the presynaptic membrane of the target neuron by the specific interaction of the toxin's heavy chain (H chain or HC) with the cell surface receptor, which is the botulinum toxin type and tetanus
  • H chain or HC toxin's heavy chain
  • Hc carboxyl terminal fragment of the heavy chain
  • the toxin passes across the plasma membrane of the poisoned cell.
  • botulinum toxin is wrapped by cells through receptor-mediated endocytosis, resulting in the formation of endosomes containing the toxin.
  • the toxin then exits the endosomes and enters the cytoplasm of the cell.
  • This step is believed to be mediated by the amino terminal fragment of the heavy chain, HN, which causes structural changes in the toxin below about pH 5.5.
  • Endosomes are known to have proton pumps that reduce the endosomal internal pH.
  • Structural relocation exposes the hydrophobic residues of the toxin, allowing the toxin to be surrounded by the endosome membrane.
  • the toxin (or at least the light chain) is then translocated to the cytosol through the endosomal membrane.
  • the final step of the botulinum toxin activation mechanism is believed to include the reduction of disulfide bonds connecting the heavy and light chains.
  • the total toxic activity of botulinum and tetanus toxins is included in the light chain of the holotoxin;
  • Light chains are zinc (Zn ++ ) endopeptidase that selectively cleave proteins necessary for recognition, docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the plasma membrane with the vesicles.
  • Tetanus neurotoxin, botulinum toxin types B, D, F and G cause the breakdown of synaptobrevin (or vesicle-associated membrane protein (VAMP)), synaptosome membrane protein.
  • VAMP vesicle-associated membrane protein
  • VAMPs present on the cytoplasmic surface of synaptic vesicles are mostly eliminated as a result of one of these degradations.
  • Longitudinal types A and E cleave SNAP-25.
  • Serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Except for type B (and tetanus toxin) that cleave the same bond, each toxin specifically cleaves different bonds. Each such cleavage disrupts the process of vesicle-membrane docking to prevent exocytosis of the vesicle contents.
  • Botulinum toxin is used in the clinic for the treatment of neuromuscular disorders (ie, movement disorders) characterized by hyperactive skeletal muscle.
  • the botulinum toxin type A complex was approved by the US Food and Drug Administration for the treatment of essential blepharospasm, strabismus, and lateral spasm.
  • botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and the treatment of glabellar fold
  • botulinum toxin type B was also approved for the treatment of cervical dystonia.
  • Botulinum serotypes other than toxin type A appear to have a lower potency and / or shorter duration in activity compared to botulinum toxin type A.
  • botulinum toxin type A injected peripherally intramuscularly usually appear within a week after injection.
  • the typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A is about 3 months on average, but therapeutic activity has been reported for significantly longer periods.
  • botulinum toxin serotypes appear to inhibit the release of the neurotransmitter acetylcholine at neuromuscular junctions, they act on different neurosecretory proteins and cleave these proteins at different sites.
  • botulinum types A and E both cleave 25 kDa synaptosome related protein (SNAP-25), but target different amino acid sequences of this protein.
  • Botulinum toxin types B, D, F and G act on vesicle-related proteins (VAMP, so-called synaptobrevin), each serotype cleaves different sites of this protein.
  • VAMP vesicle-related proteins
  • botulinum toxin type C1 appears to cleave both syntaxin and SNAP-25. The difference in this mechanism of action will affect the relative potency and / or duration by the action of the various botulinum toxin serotypes.
  • the substrate of botulinum toxin can be found in several different cell types.
  • the molecular weight of the botulinum toxin protein molecule is about 150 kDa in all seven known botulinum toxin serotypes.
  • Botulinum toxin is released as a complex comprising 150kDa botulinum toxin protein molecules along with related nontoxic proteins by Clostridium bacteria. Therefore, botulinum toxin type A complexes can be produced in 900kDa, 500kDa and 300kDa types by Clostridium-based bacteria.
  • Botulinum toxin types B and C1 appear to be produced only as a 700kDa or 500kDa complex.
  • Botulinum toxin type D is produced as 300kDa and 500kDa complexes.
  • botulinum toxin types E and F are produced only as about 300 kDa complexes. These complexes (ie, those having a molecular weight greater than about 150 kDa) are believed to include non-toxic hemagglutinin proteins and non-toxic and non-toxic hemagglutinin proteins. These two non-toxin proteins (including the related neurotoxin complex with the botulinum toxin molecule) will act to provide stability against botulinum toxin molecule denaturation and protection against digestive acids when the toxin is ingested.
  • the botulinum toxin may include both a form containing no complexing protein or a complex form containing complexing protein.
  • the botulinum toxin protein which does not contain naturally forming complexing proteins and is derived from A, B, C, D, E, F or G Clostridium botulinum, has a molecular weight of approximately 150 kDa.
  • the toxin protein is produced by the Clostridium botulinum bacteria, it is produced by forming various complexes with various hemagglutinin and non-hemagglutinin proteins that assist and protect the action of the botulinum toxin protein. do.
  • Botulinum toxin serotype A containing a naturally occurring complexed protein is in the form of a complex having a molecular weight of approximately 900 kDa, 500 kDa, or 300 kDa
  • serotypes B and C are in the form of a complex having a molecular weight of approximately 500 kDa
  • serotypes E and F are in the form of a complex having a molecular weight of approximately 300 kDa.
  • botulinum toxin inhibits potassium cation induced release of acetylcholine and norepinephrine from primary cell cultures of brain stem tissue.
  • botulinum toxin inhibits the induced release of glycine and glutamate in primary cultures of spinal cord neurons and inhibits neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP, substance P (substance P) and glutamate, respectively, in brain synaptosome samples. It has been reported to inhibit release.
  • botulinum toxin inhibits the stimulus-induced release of most neurotransmitters.
  • Botulinum toxin type A can be obtained by making and culture a culture of Clostridium botulinum in a fermenter, and then harvesting and purifying the fermentation mixture by known methods. Initially all botulinum toxin serotypes are synthesized as inactive single chain proteins that must be cleaved or nicked by proteases and must be neuroactivated. Bacterial strains producing botulinum toxin serotypes A and G have endogenous proteases, so serotypes A and G can be recovered primarily from bacterial cultures as the active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by non-proteolytic strains and are therefore typically inactivated when recovered from culture.
  • serotypes B and F are produced by both proteolytic and non-proteolytic strains, they can be recovered in active or inactive form.
  • proteolytic strains that produce botulinum toxin type B serotypes cleave only a portion of the toxin produced.
  • the exact ratio of nicked molecules to unnicked molecules depends on the incubation time and the temperature of the culture. Therefore, as already known, botulinum toxin type B is significantly less potent than botulinum toxin type A, for example, a certain percentage of any sample of botulinum toxin type B toxin will be inactive.
  • botulinum toxin type B at the same dosage level is known to have a shorter duration of activity and less potency upon intramuscular injection compared to botulinum toxin type A.
  • High-quality crystalline botulinum toxin with ⁇ 3 ⁇ 10 7 U / mg from Clostridium botulinum, A260 / A278 of 0.60 or less, and a characteristic band pattern in gel electrophoresis Type A can be obtained.
  • the known Shantz process can be used to obtain crystalline botulinum toxin type A.
  • botulinum toxin type A complexes can be isolated and purified from anaerobic fermentation cultured Clostridium botulinum type A in a suitable medium.
  • Such known processes also include, for example: purifying botulinum toxin type A having a molecular weight of about 150 kDa and a specific potency of at least 1-2 ⁇ 10 8 LD50 U / mg; Purifying botulinum toxin type B having a molecular weight of about 156 kDa and a specific potency of at least 1-2 ⁇ 10 8 LD50 U / mg; It can be used to obtain pure botulinum toxins from non-toxin proteins, such as purifying botulinum toxin type F having a molecular weight of about 155 kDa and a specific potency of 1-2 ⁇ 10 7 LD50 U / mg or more.
  • Botulinum toxin and / or botulinum toxin complexes are also commercially available from compound manufacturers known in the art, and pure botulinum toxin can also be used to prepare pharmaceutical compositions.
  • botulinum toxins which are intracellular peptidase, depends, at least in part, on their three-dimensional structure. Therefore, botulinum toxin type A can be eliminated by heat, various chemicals, surface scratches and surface drying. It is also known that when toxin complexes obtained by known culture, fermentation and purification are diluted to very low toxin concentrations and used in the preparation of pharmaceutical compositions, the toxicity of toxins is quickly eliminated without suitable stabilizers. Due to the rapid loss of specific toxicity when diluted in large quantities, it is quite difficult to dilute several milligrams of toxin into a solution containing several nanograms per milliliter. Toxins must be stabilized using suitable stabilizers or stabilization buffers, as they can be used for months or years after the preparation of pharmaceutical compositions containing toxins.
  • botulinum toxin type A As described below, the use of botulinum toxin type A in clinical settings has been reported:
  • the general duration of intramuscular injection is typically about 3-4 months. However, in some cases, subtype A can last for more than 12 months (European J. Neurology 6 (Supp 4): S111-S1150: 1999), and when used in the treatment of gland such as hyperhidrosis, Can last for 27 months.
  • Botulinum toxin not only exhibits pharmacological action in the peripheral area, but may also have an inhibitory effect in the central nervous system.
  • Weigand et al., Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292,161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281,47-56 show that botulinum toxin can be traced back to the spinal cord region by retrograde transport.
  • botulinum toxin administered, eg, intramuscularly to the peripheral site can be retrograde transported to the spinal cord.
  • Botulinum toxin is also used in various autonomic nervous system disorders (US Pat. No. 5,766,605 and Goldman (2000), Aesthetic Plastic Surgery, including skin, bone and tendon wounds (see US Pat. No. 6,447,787), pain (see US Pat. No. 6,113,915), hyperhidrosis).
  • Jul-Aug 24 (4): 280-282 tension headache (see US Pat. No. 6,458,365), migraine pain (see US Pat. No. 5,714,468), post-operative pain and visceral pain (see US Pat. No. 6,464,986), hair Growth and hair maintenance (see US Pat. No. 6,299.893), psoriasis and dermatitis (see US Pat. No.
  • US Pat. No. 5,989,545 may be used to treat pain by administering a modified Clostridial neurotoxin or fragment thereof, preferably botulinum toxin, chemically conjugated or recombinantly fused to a specific target residue, to the spinal cord Is disclosed. It is also disclosed that targeted botulinum toxins (ie, having non-natural binding moieties) can be used in the treatment of various diseases (WO 96/33273; WO 99/17806; WO 98/07864; WO 00). / 57897; WO 01/21213; WO 00/10598).
  • Botulinum toxin which weakens the biting or biting muscles of the mouth, heals the wounds and the resulting ulcers (Payne M., et al, Botulinum toxin as a novel treatment for self mutilation in Lesch-Nyhan syndrome, Ann Neurol 2002 Sep; 52 (3 Supp 1): S157), treating benign gall bladder injury or tumor (Blugerman G., et al., Multiple eccrine hidrocystomas: A new therapeutic option with botulinum toxin, Dermatol Surg 2003 May; 29 (5): 557-9), to treat dentition (Jost W., Ten years' experience with botulinum toxin in anal fissure, Int J Colorectal Dis 2002 Sep; 17 (5): 298-302), and To treat atopic dermatitis (Heckmann M., et al. Botulinum toxin type A injection in the treatment of Lichen simplex: An open pilot study, J Am Acad Dermatol 2002
  • botulinum toxin may have the effect of reducing inflammatory pain induced in rat formalin models (Aoki K., et al, Mechanism of the antinociceptive effect of subcutaneous Botox: Inhibition of peripheral and central nociceptive processing, Cephalalgia 2003 Sep; 23 (7): 649.).
  • botulinum toxin nerve inhibition may result in a decrease in epithelial thickness (see Li Y, et al., Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin, Exp Neurol 1997; 147: 452-462). .
  • Tetanus toxin and derivatives thereof can also be used for treatment.
  • Tetanus toxin is very similar to botulinum toxin.
  • tetanus toxin and botulinum toxin are both polypeptides produced by closely related species of Clostridium (Clostridium tetanini and Clostridium botulinum, respectively).
  • tetanus toxin and botulinum toxin are both double chain proteins composed of light chain (molecular weight about 50 kDa) covalently bound to heavy chain (molecular weight about 100 kDa) by a single disulfide bond.
  • the molecular weight of tetanus toxin and each of the seven botulinum toxins (non-complex) is about 150 kDa.
  • the light chain contains domains with intracellular biological (protease) activity, while the heavy chain contains receptor binding (immunogenic) and cell membrane translocation domains.
  • both tetanus and botulinum toxins exhibit large, specific affinity for ganglioside receptors on the surface of presynaptic cholinergic neurons.
  • Receptor-mediated endocytosis of tetanus toxins by peripheral cholinergic neurons results in axon retrograde transport, blockade of inhibitory neurotransmitter release from central synapses, and convulsive paralysis.
  • receptor mediated endocytosis of botulinum toxin, by peripheral cholinergic neurons rarely causes retrograde transport, inhibition of acetylcholine exocytosis from addicted peripheral motor neurons, and relaxation paralysis.
  • tetanus toxin and botulinum toxin resemble each other in both biosynthesis and molecular composition.
  • tetanus toxin and botulinum toxin type A have 34% identical protein sequences throughout and 62% identical sequences in some functional domains (Binz T. et al., The Complete Sequence of Botulinum Neurotoxin Type A and Comparison). with Other Clostridial Neurotoxins, J Biological Chemistry 265 (16); 9153-9158: 1990).
  • acetylcholine is a neurotransmitter first discovered as an ester of choline and acetic acid, distributed throughout the neuron, the chemical formula is C 7 H 16 NO 2 , molecular weight is 146.21.
  • neuromodulators can be released by the same neuron, but typically in the mammalian nervous system, only a single type of small molecule neurotransmitter is released by each type of neuron.
  • This neurotransmitter acetylcholine is a neuronal in several parts of the brain, especially large pyramidal cells in the motor cortex, several different neurons in the cerebral nucleus, motor neurons distributed in skeletal muscle, and ganglia in the autonomic nervous system (both sympathetic and parasympathetic). It is secreted by previous neurons, bag 1 fibers of the proximal spindle fibers, neurons after the ganglia of the parasympathetic nervous system and several neurons after the ganglion of the sympathetic nervous system.
  • acetylcholine has an excitatory effect.
  • acetylcholine is known to have an inhibitory effect at some peripheral parasympathetic nerve endings (eg, inhibition of the heartbeat by the vagus nerve).
  • the centrifugal signal of the autonomic nervous system is transmitted to the body through the sympathetic nervous system or parasympathetic nervous system.
  • the ganglion neurons of the sympathetic nervous system extend from ganglion sympathetic neuronal cell bodies located in the medial lateral angle of the spinal cord.
  • Pre-ganglion sympathetic fibers extending from the cell body form synapses with post-ganglion neurons located in the paraspinal sympathetic ganglia or pre-vertebral ganglion. Since pre-ganglion neurons of the sympathetic and parasympathetic nervous system are both cholinergic, applying acetylcholine to the ganglion will excite the neurons after sympathetic and parasympathetic ganglia.
  • Acetylcholine activates two types of receptors, muscarinic receptors and nicotinic receptors.
  • Muscarinic receptors are found in all effector cells stimulated by post-ganglion neurons in the parasympathetic nervous system and cholinergic neurons in the sympathetic nervous system. Nicotinic receptors are found not only in the adrenal medulla but also in the autonomic ganglia on the cell surface of post-ganglion neurons at synapses between pre- and post-ganglion neurons in both the sympathetic and parasympathetic nervous systems. Nicotinic receptors are also found in the membranes of many non-autonomic nerve endings, such as skeletal muscle fibers of neuromuscular junctions.
  • Acetylcholine is released from cholinergic neurons when small, transparent intracellular vesicles fuse with presynaptic neuronal cell membranes.
  • a wide range of non-neuronal secretory cells such as the adrenal medulla (also PC12 cell line) and islet cells of the pancreas, release catecholamines and parathyroid hormones from large, large densecore vesicles, respectively.
  • the PC12 cell line is a clone of rat chromophiloma cells widely used as a tissue culture model for the study of sympathoadrenal develpoment.
  • botulinum toxin When toxins are permeable to denervated cells (eg by electroshock) or by direct injection, botulinum toxin inhibits the release of both compound types in both cell types in vitro. Botulinum toxin is also known to inhibit the release of the neurotransmitter glutamate from cortical synaptosome cell cultures.
  • Neuromuscular junctions are formed in skeletal muscle by peripheral axons of muscle cells. Signals transmitted through the nervous system become action potentials at the terminal axons and activate ion channels, resulting in the neurotransmitter acetylcholine from synaptic vesicles in neurons, for example, in the motor endplates of neuromuscular junctions. Is released. Acetylcholine binds to the acetylcholine receptor protein on the surface of muscle endplates across the extracellular space. Once sufficiently bound, the action potential of muscle cells causes specific membrane ion channel changes, resulting in the contraction of muscle cells. Acetylcholine is then released from muscle cells and metabolized by cholinesterase in the extracellular space. The metabolite is recovered back to the terminal axon for regeneration with acetylcholine.
  • stabilizer is any additive which is added to increase the stability of the active ingredient and to prevent the active ingredient from being randomly altered to oxidize, crystallize or denature into a flexible substance. It means, and if it is pharmaceutically acceptable, it is not greatly limited. Evaluation of the stabilizing efficacy of the stabilizer can be carried out without limiting the temperature, but it is typically understood that the stabilizing effect is maintained in the long term at lower temperatures than at higher temperatures. Thus, evaluating long-term stabilization efficacy at low temperatures can be replaced by "acceleration experiments" performed at high temperatures for short periods of time. For example, the result of evaluating the stabilizing effect of a specific stabilizer at 37 ° C.
  • the stabilizer is added to preserve or maintain the biological activity of the Clostridial neurotoxin protein including the botulinum toxin, preferably arginine, methionine, aspartic acid, glutamic acid, gluconolactone, tartaric acid, and And / or sodium hyposulfite, more preferably arginine, aspartic acid, and / or glutamic acid, but are not limited thereto.
  • the botulinum toxin preferably arginine, methionine, aspartic acid, glutamic acid, gluconolactone, tartaric acid, and And / or sodium hyposulfite, more preferably arginine, aspartic acid, and / or glutamic acid, but are not limited thereto.
  • stabilization buffer or “stabilization buffering agent” as having both the effect as a stabilizer and the effect as a buffer, can be prepared by adding a material having a stabilizing effect to a general buffer, and stabilized buffer Stabilizing synergic effects can be expected with additional stabilizers.
  • the stabilizing buffer is preferably, but is not limited to, gluconolactone buffer, and / or tartaric acid buffer.
  • arginine is a kind of basic amino acid, the molecular formula is represented by C 6 H 14 N 4 O 2 , molecular weight 174.21, is water-soluble, the abbreviation of the residue is 'Arg', For more simplicity, we express it as 'R'.
  • fish are contained in a large amount of protein cruffin and white porcelain. It was isolated from the sprouting of lupine (one of the beans) whitened by MJS Schulze and E. Steiger. The nitrate was white as argent and named arginine.
  • L-arginine exists as one of the amino acids that make up protein, and belongs to the protein protamine in fish sperm.
  • arginine In herring and salmon, about 70% of the constituent amino acids are arginine. It is also in the free state in plant seeds. Arginine residues are strongly basic due to their guanidino groups. Alkaline action of ⁇ -naphthol and hypochlorous acid gives a distinctive red color and can be quantified. As a metabolic pathway in vivo, it is a constituent of the ornithine cycle discovered by HA Krebs and the like, and is decomposed into urea and ornithine by the action of arginase. It is produced from citrulline and ispartamic acid. It is a non-essential amino acid for adults but an essential amino acid for infants. It protects against the poisoning effect of ammonia and large amounts of amino acids.
  • Arginase exists in the brain and regulates the amount of arginine, a precursor of ⁇ -guanidinobutyric acid.
  • arginine phosphate In invertebrates, in the form of arginine phosphate, it plays an important role in the contraction of muscle as a phosphogen, and is widely present as a precursor of a specific guanidine base (magmatin octopine).
  • Glutamic acid is a kind of amino acid, also called “glutamic acid”, represented by the residue abbreviation "Glu", or "E”, and the molecular formula is C 5 H 9 NO 4 . It was first discovered in the hydrolyzate of gluten in wheat, which is one of the most widely used amino acids in protein. Especially, it is contained in wheat glyazine at 43.7%, and saturated hydrogen chloride in the hydrolyzate of protein such as wheat and soybean It can be separated by hydrochloride.
  • “Aspartic acid” is a kind of amino acid, also called “aspartic acid”, represented by the residue abbreviation "Asp", or “D”, the molecular formula is C 4 H 7 NO 4
  • One of the amino acids constituting the protein it is an acidic amino acid having two carboxy groups (-COOH) in the molecule, and is classified nutritionally as non-essential amino acid.
  • glutamine it is known to play a central role in in vivo amino group transfer reactions.
  • Gluconolactone is a white crystalline or crystalline powder which has no odor or has a slight odor, and initially has a sweet taste but a slight sour taste.
  • the chemical formula is C 6 H 10 O 6 .
  • Synthetic expanding agent well soluble in water and slightly soluble in ethanol, but insoluble in ether. The aqueous solution is slowly hydrolyzed to equilibrate Gluconic acid with ⁇ -lactone and ⁇ -lactone, and the higher the temperature or pH, the faster hydrolysis occurs. After about 2 hours at room temperature of 25 °C completely hydrolyzed to a solution of 55-60% gluconic acid, 40-50% lactone.
  • glucono- ⁇ -lactone is not an acid, when dissolved in water, it is hydrolyzed to show acidity. Therefore, it is recommended to be used as an acidity regulator for swelling agent, and it does not react with each other when combined with sodium bicarbonate (heavy bath). In addition, since hydrolysis occurs gradually, when used as an acidity regulator for swelling agent, it can react slowly with sodium bicarbonate to make a product with a very fine texture. It also has antioxidant properties because it forms complex salts with metals. Although it is not acid, it is used to lower the pH of soft products because aqueous solution shows acidity by heating.
  • “Tartaric Acid” is an organic compound obtained by treating sulfuric acid on a precipitate formed by adding calcium carbonate to tin, and is also referred to as tin acid because it is contained in tin precipitated when making wine. Also called dioxysuccinic acid. It is represented by the formula C 4 H 6 O 6 .
  • m-tartaric acid that do not have optical activity, in addition to right-rotating L-tartaric acid, left-rotating D-tartaric acid, and their equivalent mixtures of racemic tartaric acid (also known as folate).
  • L-tartaric acid is a dominant substance, and it is widely distributed in a plant system as an acid, calcium salt, and potassium salt of free state.
  • pharmaceutical composition means a composition to be administered for a specific purpose.
  • the pharmaceutical composition of the present invention is a botulinum toxin composition comprising arginine, glutamic acid, or aspartic acid as a stabilizing agent, or gluconolactone buffer, or tartaric acid buffer as a stabilizing buffer, and a protein and It may include a pharmaceutically acceptable carrier, excipient or diluent.
  • Said "pharmaceutically acceptable" carrier or excipient means that which has been approved by the governmental regulatory authority, or listed in government or other generally approved pharmacopoeia for use in vertebrates, and more particularly in humans. do.
  • the pharmaceutical compositions of the present invention may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, may be prepared in solid or semisolid form, but most preferably in liquid form. .
  • the pharmaceutical composition may be stable under the conditions of manufacture and storage, and may be preserved against the contaminating action of microorganisms such as bacteria or fungi.
  • the pharmaceutical compositions of the present invention may be in sterile powder form for reconstitution with a suitable carrier prior to use.
  • the pharmaceutical compositions may be in unit-dose form, in microneedle patches, in ampoules, or in other unit-dose containers, or in multi-dose containers.
  • the pharmaceutical composition may be stored in a freeze-dried (freeze-dried) state, which requires the addition of a sterile liquid carrier, eg, water for injection just before use.
  • a sterile liquid carrier eg, water for injection just before use.
  • Immediately injectable solutions and suspensions may be prepared as sterile powders, granules or tablets.
  • the pharmaceutical compositions of the present invention may be formulated in a liquid or included in the form of microspheres in the liquid.
  • the pharmaceutical compositions of the present invention, or pharmaceutically acceptable compounds and / or mixtures thereof may be included at concentrations between 0.001 and 100,000 U / kg.
  • the pharmaceutical compositions of the present invention may be formulated as a suspending agent, preservative, solubilizing agent and / or dispersing agent, dye, buffer, antimicrobial agent, antifungal agent, isotonic agent (e.g. sugar or sodium chloride).
  • additional stabilizer refers to the stabilizer of the present invention (arginine, glutamic acid, or aspartic acid) or to the stabilizing buffer of the present invention (gluconolactone buffer, or tartaric acid buffer). Further included stabilizers may be used without limitation as long as they are generally known in the art.
  • the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers, and the carrier may be a solvent or a dispersion medium.
  • pharmaceutically acceptable carriers include water, saline, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols), oils, and suitable mixtures thereof.
  • the pharmaceutical compositions of the present invention are sterile, and non-limiting examples of sterilization techniques applied to the pharmaceutical compositions of the present invention include filtration through bacterial-suppressing filters, terminal sterilization, incorporation of sterile preparations, irradiation, sterile gas irradiation Heating, vacuum drying and freeze drying.
  • “administration” means introducing the composition of the invention to the patient in any suitable way, the route of administration of the composition of the invention via any general route as long as it can reach the target tissue. May be administered.
  • intramuscular injection administration in the form of liquid formulations is most preferred, but is not limited thereto.
  • the method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount.
  • the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently.
  • a pharmaceutical formulation comprising a neurotoxin and a stabilizer, wherein the neurotoxin is any one or more selected from the group consisting of botulinum toxin, tetanus toxin, cholera toxin, and pertussis toxin.
  • the botulinum toxin provides a pharmaceutical formulation, characterized in that the botulinum toxin type A, wherein the botulinum toxin is in a form containing no complexing protein or a complex form containing a complexing protein
  • the stabilizer is any one or more selected from the group consisting of arginine, glutamic acid, and aspartic acid, wherein the stabilizer is provided in the form of a stabilizing buffer to provide a pharmaceutical formulation
  • the stabilization buffer is gluconolactone buffer, Or it provides a pharmaceutical formulation which is a tartaric acid buffer, the stabilizer provides a pharmaceutical formulation, characterized in that it comprises 0.01 to 1,000 mM per 100 units of botulinum toxin, the pH of the pharmaceutical formulation is characterized in that 5.5 to 7.0
  • the pharmaceutical formulation provides a pharmaceutical formulation further comprises a local anesthetic, the local anesthetic provides a pharmaceutical formulation that is lidocaine, the local ane
  • the buffer provides a method for preparing a pharmaceutical formulation, which is a gluconolactone buffer, or tartaric acid buffer, and the pharmaceutical formulation provides a method for preparing a pharmaceutical formulation in a liquid form.
  • Botulinum toxins inhibit systemic acetylcholine exocytosis at the cholinergic synapses of neuromuscular tumors in animals with nerve function, causing systemic asthenia.
  • Botulinum toxin has a therapeutic effect in various diseases, but the neurotoxic function is very toxic and extremely fatal, so it is essential to finely control the concentration when used in vivo.
  • the current botulinum toxin pharmaceutical composition is manufactured and distributed as a lyophilized formulation due to protein denaturation, so that the user dilutes in a liquid solvent immediately before use in clinical practice, and thus the risk of medical accidents due to human error such as dilution error or contamination of the dilution solvent. There was this high issue.
  • the present invention relates to a botulinum toxin composition
  • a botulinum toxin composition comprising arginine, glutamic acid, or aspartic acid as a stabilizer, or a gluconolactone buffer, or tartaric acid buffer as a stabilizing buffer, wherein the composition of the present invention is botulinum even when distributed in liquid form.
  • toxin stabilization As the significant effects of toxin stabilization have been demonstrated, it is expected to be widely utilized for the safe and convenient medical use of botulinum toxin.
  • 1 is a result of measuring the remaining titers after adding arginine or methionine to botulinum toxin and culturing for 28 to 56 days according to an embodiment of the present invention.
  • FIG. 2 is a result of measuring the residual titer after culturing the botulinum toxin composition to which arginine or methionine is added for 56 days at pH6.0 to pH7.0 conditions according to an embodiment of the present invention.
  • FIG. 2A shows the result of pH 6.0
  • FIG. 2B shows the pH 6.5
  • FIG. 2C shows the pH 7.0.
  • Figure 3 is a result of measuring the remaining titers after culturing the botulinum toxin composition containing various antioxidants for 28 days according to an embodiment of the present invention.
  • Figure 4 is a result of measuring the residual titer after culturing the botulinum toxin composition containing various buffer solutions for 28 days according to an embodiment of the present invention.
  • FIG. 5 is a step-by-step schematic diagram of the DESCR assay for measuring the botulinum toxin titer according to an embodiment of the present invention.
  • FIG. 6 is a result comparing the efficacy of arginine and methionine to stabilize the liquid botulinum formulation according to an embodiment of the present invention.
  • FIG. 6A is a case where no local anesthetic is included
  • FIG. 6B is a result when a local anesthetic (0.3% lidocaine) is included.
  • FIG. 7 is a result of evaluating the effect of the liquid formulation container material on the stabilization of BoNT / A efficacy according to an embodiment of the present invention.
  • FIG. 7A shows a case where no local anesthetic is included
  • FIG. 7B shows a result when a local anesthetic (0.3% lidocaine) is included.
  • FIG. 8 is a result of comparing the stabilizing efficacy of arginine in a formulation containing tartaric acid or gluconolactone as a buffering agent, according to an embodiment of the present invention using a glass container.
  • FIG. 8A shows a case where no local anesthetic is included
  • FIG. 8B shows a result when a local anesthetic (0.3% lidocaine) is included.
  • FIG. 9 is a result of evaluating the effect on the efficacy of arginine as a stabilizer when glutamic acid is additionally included in tartaric acid or gluconolactone buffering agent according to an embodiment of the present invention.
  • FIG. 9A shows a case where no local anesthetic is included
  • FIG. 9B shows a result when a local anesthetic (0.3% lidocaine) is included.
  • FIG. 10 is a result of evaluating an optimal concentration of glutamic acid that contributes to stabilization efficacy of arginine in a formulation prepared with a gluconolactone buffering agent according to one embodiment of the present invention.
  • FIG. 10A shows a case where no local anesthetic is included
  • FIG. 10B shows a case where a local anesthetic (0.3% lidocaine) is included.
  • FIG. 11 is a result of evaluating the effect of aspartic acid on the BoNT / A stabilizing efficacy of arginine according to an embodiment of the present invention.
  • FIG. 11A is a case where no local anesthetic is included
  • FIG. 11B is a result when a local anesthetic (0.3% lidocaine) is included.
  • the stabilizing efficacy of arginine and methionine on botulinum toxin showed a pH-dependent result.
  • the acidity of the liquid BoNT / A formulation was fixed at 6.0 and BoNT / A was added at an initial titer of 80 units / ml in a composition with varying arginine concentration, followed by incubation at 37 degrees for 8 weeks.
  • the titer was measured by DESCR Assay.
  • the control group without stabilizer showed 10% of remaining titer of BoNT / A after 2 weeks and 67% of test group with 50 mM methionine added after 2 weeks, 4 weeks, and 8 weeks, respectively.
  • Gluconolactone (Sigma G2164); Tartaric acid (L (+)-tartaric acid, Merck 100804); Lidocaine hydrochloride monohydrate (Sigma L5647); Octanoic acid (Sigma C2875); Methionine (L-methionine, Merck K45023607 414); Arginine (L-arginine, Merck K45895542 534); Glycine (Glycine, Bioshop GLN001-1); Glutamic acid (L-Glutamic acid, Merck 100291); Aspartic acid (Merk K45895542 534); Maleic acid (Merk S6858580 534); Butylated hydroxyanisole (Sigma SLBM1210V); Propyl gallate (Sigma P3130); Sodium bisulfite (Sigma MKBR6468V); Thioglycolic acid (Sigma T3758); Cysteine (L-cystein hydrochloride, K46446495 513); Succi
  • Botulinum toxin in the present invention was produced by Hugel Co., Ltd. (Korea), and used at a concentration of 0.1 mg / ml and titer of 1,529 units / ⁇ l (BoTest standard). All experiments for the detection of additives with stabilizing potency were carried out in the “protein dilution buffer” of the composition (50 mM NaPO 4 , pH7.0, 1 mM DTT, 0.05 wt% polysorbate, and 20 wt% glycerol) recommended by Hugel Co., Ltd. Dilution at 50 units / ⁇ l.
  • each experimental group (100ul) for the detection of stabilizing efficacy additive candidates was added to 200units botulinum toxin and stabilizer candidate additives in the "stabilization composition (10mM NaPO 4 (pH5.5-7.0), 0.01 wt% polysorbate, 130mM NaCl)". Prepared by dilution. Then, each experimental group was incubated for 1 to 11 weeks at 37 °C, a portion (25%) was used for measuring the remaining titer. The titer of botulinum toxin was measured using BoTest (BoTest® Botulinum Neurotoxin Detection Kits, BioSentinel, USA).
  • Example 1-2 Comparison of Stabilization Effects of Arginine and Methionine with Different pH
  • the stabilization efficacy of botulinum toxin was compared and verified when arginine and methionine were used as stabilizers in the pH range of 5.5 to 7.0.
  • botulinum toxin was added to the stabilizing composition described in Example 1, and 50 mM arginine or methionine was further added. The remaining titers were measured after incubation at 37 ° C. for 28 to 56 days. The results are shown in Table 1 and FIG.
  • the negative control group to which the stabilization candidate was not added tended to be unstable in the botulinum toxin at various pH conditions, and almost no residual titer was detected after 28 days.
  • botulinum toxin showed a residual titer of up to about 10% in the range of pH 5.5 to 7.0, whereas the residual titer of up to about 30% was measured in the experimental group to which arginine was added.
  • arginine exhibited higher efficacy in stabilizing botulinum toxin in the pH range of 5.5 to 6.0 than in pH 6.5 to 7.0.
  • Example 1-3 Comparison of stabilization efficacy of arginine and methionine
  • botulinum toxin compositions containing arginine (50mM) or methionine (50mM) as stabilizing additives were cultured for 56 days, and the results of three independent experiments were measured. The results were statistically compared to verify the efficacy of each. As a control, a sample containing no additives was used. One-way ANOVA method was used as a method for confirming the significance between the three experimental groups. When the probability of significance was less than 0.05, it was judged that there was a significant difference between the three groups and the post-test was performed by the LSD method. The results are shown in Table 2 and FIG.
  • arginine and methionine have the highest stabilizing efficacy at pH 6.0, which is consistent with the results of the previous experiment.
  • arginine showed higher stabilizing efficacy than methionine in all experimental groups.
  • the control group without stabilizer did not measure the titer of botulinum toxin under all conditions after 56 days of incubation, whereas the experimental group with methionine added had a residual titer of 22.6% at pH 6.0 and the addition of arginine.
  • the experimental group had a residual titer of 69.1% under the same conditions.
  • stabilization efficacy value was significant in all experimental groups except the experimental group cultured for 14 days under pH6.0.
  • Least Significant Difference (LSD) method it was confirmed that the stabilization efficacy of arginine was significantly superior to methionine at a level of p ⁇ 0.05 to p ⁇ 0.001.
  • the measured values after 28 days incubation at pH7.0 were 17.1% in the methionine-added test group and 55.8% in the arginine-added test group, and there was a difference in stabilization efficacy at the significance level of p ⁇ 0.001.
  • the measured values after incubation for 56 days were 22.6% in the methionine-added test group and 69.1% in the arginine-added test group.
  • Example 1-4 Development of a New Stabilizer for Botulinum Toxin Liquid Formulations
  • Examples 1-2 and 1-3 show that arginine has superior stabilizing efficacy than methionine in the liquid formulation of botulinum toxin.
  • detection of new additives showing similar efficacy with arginine enables various product development, and for this, the present inventors compared and investigated the botulinum toxin stabilizing efficacy of the stabilizing candidates described in Table 3 below. The results are shown in FIGS. 3 and 4.
  • FIG. 3 shows the remaining titers of botulinum toxin after 28 days of botulinum toxin composition containing sodium bisulfite, propyl gallate, thioglycolate, and antioxidants in Table 3
  • Experimental results showed that all the antioxidant additives used in the verification showed lower stabilizing efficacy than the control group methionine.
  • Figure 4 in the case of the buffer solution, gluconolactone showed a significant level of stabilizing effect at pH 6.5, tartaric acid showed a stabilizing effect in the range of pH5.5 to 6.5. The results suggest the possibility of developing new additives with significant effects.
  • Example 1 is a sample of a liquid formulation prepared by mixing a relatively high titer of botulinum toxin (200 units / 0.1 ml) was prepared in a polypropylene tube, and the residual titer was measured using a BoTest assay.
  • the present invention is a formulation similar to the composition of the solid or liquid botulinum products currently on the market; That is, the study of the stabilization formulation of the botulinum toxin was performed using a formulation having an initial titer of botulinum toxin of 40 to 80 units / ml.
  • BoTest Assay requires at least 50 units of titer, the remaining titers of liquid formulations prepared under these conditions cannot be measured. Therefore, the present inventors have developed a small amount of the likely quantitative measurement of the activity of BoNT / A DESCR (D irect E LISA coupled with in vitro S NAP25 c leavage r eaction) method.
  • the DESCR method is described in detail in Example 2-2.
  • BoNT / A For simplicity, 200 units of BoNT / A were added to 100 ⁇ l of liquid formulation in the sample of all formulations for measuring residual titer by BoTest Assay of Example 1, but the residual titer was measured by the newly developed DESCR method. 40-80 units / mL of BoNT / A was added to 0.1-1 mL of the liquid formulation.
  • DESCR Direct ELISA coupled with in vitro SNAP25 cleavage reaction
  • the extent of the reaction is detected by a chromogenic reaction, which is a primary antibody (HRP) and a HRP (Horseradish peroxidase) -bound secondary antibody (HRP) that specifically react to the form truncated by BoNT / A (SNAP25197). It can be detected based on the color reaction using conjugated secondary antibody.
  • HRP primary antibody
  • HRP Hexeradish peroxidase
  • the botulinum toxin used in the experiment was diluted to various concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6 units), 20 ⁇ l of buffer solution (20 mM HEPES-NaOH, pH 7.1), 0.1% Tween 20, 10
  • the enzyme reaction was carried out for 21 hours at 37 ° C. in ⁇ M ZnCl 2, and 1 ⁇ g GST-SNAP25).
  • Example 2-3 Liquid botulinum formulation Stabilizing Comparison of the efficacy of arginine and methionine
  • Example 1 the stabilizing effect of arginine and methionine on botulinum toxin showed a pH-dependent result. That is, in the experimental group to which methionine was added as a stabilizer, botulinum toxin showed a residual titer of up to about 10% in the range of pH 5.5 to 7.0, whereas the residual titer of up to about 30% was measured in the experimental group to which arginine was added. In addition, arginine showed higher efficacy in stabilizing BoNT / A at pH 5.5-6.0 than pH 6.5-7.0. Negative controls without stabilizing candidate additives showed unstable tendency of BoNT / A under various pH conditions, resulting in little residual titer detected after 28 days.
  • the control group without stabilizer showed 10% of remaining titer of BoNT / A after 2 weeks and 67% of test group with 50 mM methionine added after 2 weeks, 4 weeks, and 8 weeks, respectively. Remaining titers of 47%, 47% and 27% showed that methionine had a significant stabilizing effect.
  • arginine a stabilizing effect of methionine or higher was shown at a concentration of 50 to 100 mM, and residual titers of 31 to 65% were measured even after 8 weeks.
  • Example 2-4 Determine the effect of liquid formulation container material on stabilizing BoNT / A efficacy
  • Example 2-3 is the result obtained by preparing a formulation comprising botulinum toxin in a polypropylene tube.
  • the botulinum toxin liquid formulation which is actually distributed for clinical use, is prepared in a glass container, the remaining potency was re-validated using a glass container for stabilizing efficacy of methionine and arginine.
  • BoNT / A was prepared in a liquid formulation containing 20 mM methionine or 100 mM arginine at an initial titer of 40 units / ml, and the remaining titer was measured by DESCR Assay while incubating for 8 weeks at 37 degrees. The results are shown in FIG.
  • BoNT / A residual titers of methionine-containing formulations were determined to be 41%, 15%, and 8% after 2, 4, and 8 weeks, respectively.
  • the BoNT / A residual titers of the arginine-containing formulations were measured to be 84%, 64%, and 43% after the same period, showing a significant difference from the BoNT / A residual titers of the formulations containing menionine.
  • Example 2-5 With buffering agents Glutamic acid Evaluation of the Effect on Arginine as a Stabilizer
  • the stabilization efficacy of arginine showed a similar pattern in all liquid formulations, and after 8 weeks, the remaining titer of BoNT / A was measured to be 57-70%.
  • the stabilizing effect of arginine was significantly higher in the formulation containing gluconolactone, and after 8 weeks, the remaining titer of BoNT / A was measured to be about 10% higher than that of other samples. It was. The same trend was observed for formulations containing lidocaine.
  • Example 2-3 when 50 mM glutamic acid was added to a BoNT / A liquid formulation composed of sodium phosphate buffer and 50 mM arginine, BoNT / A residual titer was found to be 65% after 8 weeks. 32% of glutamic acid was not included. Similar results were obtained with the formulation containing lidocaine, with BoNT / A residual titers of 71% measured after 8 weeks. In this case, 45% of residual titer was measured when glutamic acid was not included (see FIG. 6).
  • BoNT / A potency in formulations containing sodium phosphate, tartaric acid, or gluconolactone is selected to select optimal buffering agents for stabilization of BoNT / A liquid formulations when both arginine and glutamic acid are included. Comparison was made using the containers. As a result, the remaining titers of BoNT / A measured by DESCR after 2, 4, and 8 weeks were significantly higher in the formulation containing gluconolactone as a buffering agent. After 4 weeks and 8 weeks, the remaining titers reached 96%, 87%, 71%, and 96%, 86%, and 68% for lidocaine.
  • Glutamic acid has the effect of stabilizing BoNT / A in liquid formulations, but the effect of glutamic acid alone is less than that of arginine alone.
  • Formulations containing both glutamic acid and arginine do not show a synergistic effect after a relatively long shelf life of 8 weeks and remain at a constant level (60-80%).
  • Example 2-6 Evaluation of the Effect of Aspartic Acid on Stabilization Effect of Arginine as Stabilizer
  • Residual titers of BoNT / A after 8 weeks in formulations with 10 to 50 mM aspartic acid and 50 mM arginine were measured: 61% in formulations with arginine alone and concentrations in formulations with additional aspartic acid. It was measured at a level of 58-73% without showing a significant relationship to. Unlike the results of residual titers measured after 8 weeks, the residual titers of BoNT / A measured after 2 to 4 weeks show statistically significant differences. Formulations containing only arginine measured 82% and 76% residual titers over 2 weeks and 4 weeks, but 92 to 100% and 85 to 94% residual titers when aspartic acid was added.
  • the formulations containing glutamic acid showed similar stabilizing efficacy over the same period but were relatively low and the residual titers were measured at 83-98%, and 76-88%.
  • Table 4 shows the results when the aspartic acid is added and Table 5 shows the results when glutamic acid is added.
  • BoNT / A The novel liquid composition of BoNT / A established through the above results of systematic comparison of various liquid injectable additives confirmed by the present inventors is 10 mM gluconolactone (pH 6.0), 100 mM sodium chloride, 50 mM Arginine, 50 mM aspartic acid, 0.05% polysorbate.
  • BoNT / A formulations of this composition were prepared using a glass vessel with an initial titer of 40 units / ml.
  • the stability of the product was compared / investigated using a liquid formulation product containing methionine instead of arginine as a control, and the results are shown in FIG. 12.
  • Experimental results showed that the stability of BoNT / A products prepared in the new liquid composition is very similar to the results obtained in in vitro studies.
  • the remaining titers of the control group were 41%, 15% and 8%.
  • botulinum toxin inhibits acetylcholine's exocytosis in cholinergic synapses of neuromuscular endings in animals with nerve function, it causes systemic asthenia, and there has been an effort to use botulinum toxin for cosmetic or therapeutic purposes.
  • botulinum toxin has a problem in that it is not easy to store, distribute, and manage the pharmaceutical formulation as a protein preparation. This is due to the instability of the protein, which is a serious problem for protein preparations where the drug is formulated at extremely low concentrations, such as botulinum toxin.
  • the formulation technology comprising the botulinum toxin and stabilizer of the present invention is a liquid formulation, which is easy to store and distribute, and demonstrates the remarkable effect of botulinum toxin stabilization under conditions suitable for human temperature and pH. It is expected to be widely used for medical use.

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Abstract

La présente invention concerne une formulation liquide contenant la toxine botulique et un agent stabilisant, et le procédé de préparation de cette dernière. La technologie de formulation de la présente invention, contenant la toxine botulique et un agent stabilisant, facilite le stockage et la distribution d'une formulation liquide, et a montré un effet remarquable sur la stabilisation de la toxine botulique dans des conditions correspondant à la température et au pH du corps humain, ce qui lui permettra d'être largement utilisée pour une utilisation médicale sûre et aisée de la toxine botulique.
PCT/KR2017/009383 2016-08-26 2017-08-28 Formulation liquide contenant la toxine botulique et un agent stabilisant, et procédé de préparation de cette dernière WO2018038585A1 (fr)

Priority Applications (11)

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CA3033729A CA3033729A1 (fr) 2016-08-26 2017-08-28 Formulation liquide contenant la toxine botulique et un agent stabilisant, et procede de preparation de cette derniere
EP17844005.3A EP3505155A4 (fr) 2016-08-26 2017-08-28 Formulation liquide contenant la toxine botulique et un agent stabilisant, et procédé de préparation de cette dernière
EP20195594.5A EP3782605A1 (fr) 2016-08-26 2017-08-28 Formulation liquide contenant la toxine botulique et un agent stabilisant et son procédé de préparation
CN201780052253.5A CN109640954B (zh) 2016-08-26 2017-08-28 含有肉毒杆菌毒素和稳定剂的液体制剂及其制备方法
RU2019104998A RU2748653C2 (ru) 2016-08-26 2017-08-28 Жидкая композиция, содержащая ботулотоксин и стабилизирующий агент, и способ её получения
EP20195590.3A EP3777837B1 (fr) 2016-08-26 2017-08-28 Formulation liquide contenant la toxine botulique et un agent stabilisant et procédé de préparation de cette dernière
US16/327,390 US10772943B2 (en) 2016-08-26 2017-08-28 Liquid formulation containing botulinum toxin and stabilizing agent, and preparation method therefor
BR112019003770-5A BR112019003770A2 (pt) 2016-08-26 2017-08-28 formulação líquida que contém toxina botulínica e agente de estabilização, e método de preparação para a mesma
JP2019532909A JP6797305B2 (ja) 2016-08-26 2017-08-28 ボツリヌス毒素および安定化剤を含む液状剤形およびその製造方法
US16/934,654 US11224640B2 (en) 2016-08-26 2020-07-21 Liquid formulation containing botulinum toxin and stabilizing agent, and preparation method therefor
US16/934,649 US11147860B2 (en) 2016-08-26 2020-07-21 Liquid formulation containing botulinum toxin and stabilizing agent, and preparation method therefor

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KR10-2016-0109558 2016-08-26
KRPCT/KR2016/009706 2016-08-31
PCT/KR2016/009706 WO2018038301A1 (fr) 2016-08-26 2016-08-31 Formulation liquide stabilisée de toxine botulique et procédé pour sa préparation
KR10-2017-0108180 2017-08-25
KR10-2017-0108181 2017-08-25
KR1020170108181A KR101919299B1 (ko) 2016-08-26 2017-08-25 보툴리눔 독소 및 안정화 완충액을 포함하는 액상 제형 및 이의 제조방법
KR1020170108180A KR101919298B1 (ko) 2016-08-26 2017-08-25 보툴리눔 독소 및 안정화제를 포함하는 액상 제형 및 이의 제조방법

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CN113164387A (zh) * 2018-11-30 2021-07-23 汇恩斯生物制药株式会社 稳定肉毒杆菌毒素的液体组合物

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