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WO2017179775A1 - Techniques de formulation de microstructure pour toxine botulique - Google Patents

Techniques de formulation de microstructure pour toxine botulique Download PDF

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WO2017179775A1
WO2017179775A1 PCT/KR2016/009672 KR2016009672W WO2017179775A1 WO 2017179775 A1 WO2017179775 A1 WO 2017179775A1 KR 2016009672 W KR2016009672 W KR 2016009672W WO 2017179775 A1 WO2017179775 A1 WO 2017179775A1
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botulinum toxin
microstructure
coating composition
thickener
stabilizer
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PCT/KR2016/009672
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English (en)
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Kyeong-Yeop MOON
Chang-Jin Lee
Dae-Gun Kim
Dong-Hoon Oh
Do-Hyun Kang
Woo-Ran LEE
Jeong-Gyu LEE
Jun-Jin Yoon
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Hugel Inc.
Small'lab Co., Ltd.
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Publication of WO2017179775A1 publication Critical patent/WO2017179775A1/fr

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    • 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
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • 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
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    • 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
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    • 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
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    • 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
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • 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
    • A61K47/38—Cellulose; Derivatives 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
    • A61K47/30—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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0023—Drug applicators using microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0053—Methods for producing microneedles

Definitions

  • the present invention relates to microstructure formulation techniques for botulinum toxin.
  • botulinum toxin is classified into seven subtypes (subtypes A to G) according to their serological characteristics, and inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction in animals having neurological function to thereby cause asthenia universalis.
  • subtypes B, D, F and G of botulinum toxin cleave synaptobrevin at a specific site
  • subtypes A and E cleave SNAP25 at a specific site
  • subtype C cleaves syntaxin at a specific site
  • botulinum toxin is the most lethal substance among known biological toxins, with an estimated human median lethal dose (LD50) of 1.3-2.1 ng/kg intravenously or intramuscularly, and 10-13 ng/kg when inhaled.
  • LD50 human median lethal dose
  • botulinum toxin has great therapeutic effects on various diseases, but is lethal even in a very small amount due to its strong toxicity. For this reason, when botulinum toxin is to be used in a living body, it is necessary to minutely control the concentration of botulinum toxin.
  • botulinum toxin loses its effect in the body after 3-4 months after single administration, a technology enabling low-concentration botulinum toxin to be administered over a long period of time without inconvenience is urgently required.
  • microstructures include microneedles, microblades, microknifes, microfibers, microspikes, microprobes, microbarbs, microarrays or microelectrodes, and among them, the term “microneedle” means a technology that forms a hole through the skin by use of a fineneedle to increase drug penetration.
  • a microneedle is made of a biodegradable material, the microneedle itself containing a pharmacological component will be biodegraded slowly in vivo while it releases the drug.
  • the drug release rate can be controlled depending on the choice of the material and a preparation method for the microneedle.
  • the present invention is directed to microstructure formulation techniques for botulinum toxin, and the microstructures of the present invention release a low-concentration botulinum toxin slowly over a long period of time, and thus are expected to greatly contribute to the safe and convenient medical use of botulinum toxin.
  • the present invention has been made in order to solve the above-described problems occurring in the prior art, and it is an object of the present invention to provide microstructure formulation techniques for botulinum toxin.
  • “botulinum toxin” is a neurotoxic protein produced by the bacterium Clostridium botulinum .
  • Clostridium botulinum has more than 127 species, grouped according to their morphology and functions.
  • the anaerobic, gram-positive bacteria Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism.
  • the spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism.
  • botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores.
  • the botulinum toxin can apparently pass unattenuated through the lining of the gut and shows a high affinity for cholinergic motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty in walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
  • Botulinum toxin type A is known as the most lethal natural biological agent to man. About 50 picograms of a commercially available botulinum toxin type A (purified neurotoxin complex) is an LD50 (i.e., 1unit). Interestingly, on a molar basis, botulinum toxin type A is about 1.8 billion times more lethal than diphtheria, about 600 million times more lethal than sodium cyanide, about 30 million times more lethal than cobra toxin and about 12 million times more lethal than cholera. One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each.
  • botulinum neurotoxin serotypes A, B, C1, D, E, F and G are distinguished by neutralization with type-specific antibodies.
  • the different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than botulinum toxin type B.
  • botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for botulinum toxin type A.
  • Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine. Additional uptake can take place through low affinity receptors, as well as by phagocytosis and pinocytosis.
  • the molecular mechanism of toxin intoxication appears to be similar and to involve at least 3 steps.
  • the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain (the H chain or HC), and a cell surface receptor.
  • the receptor is thought to be different for each type of botulinum toxin and for tetanus toxin.
  • the carboxyl end segment of the HC appears to be important for targeting of the botulinum toxin to the cell surface.
  • the botulinum toxin crosses the plasma membrane of the target cell.
  • the botulinum toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the botulinum toxin is formed.
  • the toxin escapes the endosome into the cytoplasm of the cell.
  • This step is thought to be mediated by the amino end segment of the heavy chain, the HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH.
  • the conformational shift exposes hydrophobic residues in the toxin, which permits the botulinum toxin to embed itself in the endosomal membrane.
  • the botulinum toxin (or at least the light chain of the botulinum toxin) then translocates through the endosomal membrane into the cytoplasm.
  • the last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain and the light chain.
  • the entire toxic activity of botulinum and tetanus toxins is contained in the light chain of the holotoxin; the light chain is a zinc (Zn ++ ) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane.
  • Serotype A and E cleave SNAP-25.
  • Serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25.
  • Each of the botulinum toxins specifically cleaves a different bond, except type B (and tetanus toxin) which cleave the same bond. Each of these cleavages blocks the process of vesicle-membrane docking, thereby preventing exocytosis of vesicle content.
  • Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles (i.e. motor disorders).
  • a botulinum toxin type A complex was approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm.
  • a botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and for the treatment of glabellar lines
  • a botulinum toxin type B was approved for the treatment of cervical dystonia.
  • Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A.
  • Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection.
  • the typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about 3 months, although significantly longer periods of therapeutic activity have been reported.
  • botulinum toxins serotypes Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and cleaving these proteins at different sites.
  • botulinum types A and E both cleave the 25 kDa synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein.
  • Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site.
  • VAMP vesicle-associated protein
  • botulinum toxin type C1 appears to cleave both syntaxin and SNAP-25.
  • the molecular weight of the botulinum toxin is about 150 kDa.
  • the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kDa botulinum toxin protein molecule along with associated non-toxin proteins.
  • the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kDa, 500 kDa or 300 kDa forms.
  • Botulinum toxin types B and C1 are apparently produced as only a 700 kDa or 500 kDa complex.
  • Botulinum toxin type D is produced as 300 kDa or 500 kDa complexes.
  • botulinum toxin types E and F are produced as only approximately 300 kDa complexes.
  • the complexes i.e. molecular weight greater than about 150 kDa
  • the complexes are believed to contain a non-toxin hemagglutinin proteins, a non-toxin, and non-toxic non-hemagglutinin protein.
  • These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when a botulinum toxin is ingested.
  • botulinum toxin complexes result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
  • botulinum toxin inhibits potassium cation-induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue.
  • botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP, substance P, and glutamate.
  • the stimulus-evoked release of most neurotransmitters can be blocked by botulinum toxin.
  • Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can, therefore, be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D, and E are synthesized by nonproteolytic strains and are therefore typically inactivated when recovered from culture.
  • Serotypes B and F are produced by both proteolytic and nonproteolytic strains, and thus can be recovered in either the active or inactive form.
  • the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced.
  • the exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin is likely to be inactive, possibly accounting for the known significantly lower potency of botulinum toxin type B as compared to botulinum toxin type A.
  • botulinum toxin type B has, upon intramuscular injection, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level.
  • High-quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of ⁇ 3 ⁇ 10 7 U/mg, an A260/A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis.
  • the known Schantz process can be used to obtain crystalline botulinum toxin type A.
  • the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium.
  • the known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kDa molecular weight with a specific potency of 1-2 ⁇ 10 8 LD50U/mg or greater; purified botulinum toxin type B with an approximately 156kDa molecular weight with a specific potency of 1-2 ⁇ 10 8 LD50U/mg or greater, and; purified botulinum toxin type F with an approximately 155kDa molecular weight with a specific potency of 1-2 ⁇ 10 7 LD50U/mg or greater.
  • purified botulinum toxin type A with an approximately 150 kDa molecular weight with a specific potency of 1-2 ⁇ 10 8 LD50U/mg or greater
  • purified botulinum toxin type B with an approximately 156kDa molecular weight with a specific potency of 1-2 ⁇ 10
  • Botulinum toxins and/or botulinum toxin complexes are commercially available from compound manufacturers known in the art, and pure botulinum toxin can also be used to prepare a pharmaceutical composition.
  • botulinum toxin type A is detoxified by heat, various chemicals surface stretching and surface drying. Additionally, it is known that dilution of a botulinum toxin complex obtained by the known culturing, fermentation and purification to the very low toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution.
  • the toxin should be stabilized with a suitable stabilizing agent.
  • a suitable stabilizing agent As disclosed in the present invention, the development of optimal stabilizer technology is necessary to control the in vivo release of botulinum toxin to a slow release form.
  • botulinum toxin type A has been used in clinical settings as follows:
  • botulinum toxin subtype A can have an efficacy for up to 12 months ( European J. Neurology 6 (Supp 4): S111-S1150:1999), and in some circumstances for as long as 27 months, when used to treat glands, such as in the treatment of hyperhydrosis.
  • botulinum toxins may also have inhibitory effects in the central nervous system.
  • Work by Weigand et al, Nauny - Schmiedeberg's Arch . Pharmacol . 1976; 292, 161-165, and Habermann, Nauny - Schmiedeberg's Arch . Pharmacol. 1974; 281, 47-56 showed that botulinum toxin is able to ascend to the spinal area by retrograde transport.
  • a botulinum toxin injected at a peripheral location for example intramuscularly, may be retrograde transported to the spinal cord.
  • a botulinum toxin has also been proposed for or has been used to treat skin bone and tendon wounds (US Patent No. 6,447,787); intrathecal pain (see US Patent No. 6,113,915); various autonomic nerve disorders, including sweat gland disorders (see e.g. US Patent No. 5,766,605 and Goldman (2000), Aesthetic Plastic Surgery July-August 24(4):280-282); tension headache (US Patent No. 6,458,365); migraine headache (US Patent No. 5,714,468); post-operative pain and visceral pain (US Patent No. 6,464,986); hair growth and hair retention (US Patent No. 6,299,893); psoriasis and dermatitis (US Patent No.
  • Botulinum toxin type A causes diffuse and highly selective atrophy of rat prostate , Neurourol Urodyn 1998; 17(4):363); fibromyalgia (US Patent No. 6,623,742), and piriformis muscle syndrome (see Childers et al. (2002), American Journal of Physical Medicine & Rehabilitation, 81:751-759).
  • US Patent No. 5,989,545 discloses that a modified clostridial neurotoxin or fragment thereof, preferably a botulinum toxin, chemically conjugated or recombinantly fused to a particular targeting moiety can be used to treat pain by administration of the agent to the spinal cord. Additionally, it has been disclosed that targeted botulinum toxins (i.e. with a non-native binding moiety) can be used to treat various conditions (see WO 96/33273; WO 99/17806; WO 98/07864; WO 00/57897; WO 01/21213; WO 00/10598).
  • botulinum toxin has been injected into the pectoral muscle to control pectoral spasm (Senior M., Botox and the management of pectoral spasm after subpectoral implant insertion, Plastic and Recon Surg, July 2000, 224-225).
  • Controlled release toxin implants are known (see US Patent Nos. 6,306,423 and 6,312,708) as is transdermal botulinum toxin administration (U.S. Patent Application Serial No. 10/194,805).
  • a botulinum toxin can be used to: weaken the chewing or biting muscle of the mouth so that self inflicted wounds and resulting ulcers can be healed (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); permit healing of benign cystic lesions or tumors (Blugerman G., et al., Multiple eccrine hidrocystomas: A new therapeutic option with botulinum toxin, Dermatol Surg 2003 May; 29(5):557-9); treat anal fissure (Jost W., Ten years' experience with botulinum toxin in anal fissure, Int J Colorectal Dis 2002 September; 17(5):298-302); and treat certain types of atopic dermatitis (Heckmann M., et al., Botulinum toxin type A injection in the treatment
  • a botulinum toxin may have the effect of reducing induced inflammatory pain in a rat formalin model (Aoki K., et al, Mechanisms of the antinociceptive effect of subcutaneous Botox: Inhibition of peripheral and central nociceptive processing, Cephalalgia 2003 September; 23(7):649). Furthermore, it has been reported that botulinum toxin nerve blockage can cause a reduction of epidermal thickness (Li Y, et al., Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin, Exp Neurol 1997; 147:452-462).
  • Tetanus toxin as wells as derivatives (i.e. with a non-native targeting moiety), fragments, hybrids and chimeras thereof can also have therapeutic utility.
  • the tetanus toxin bears many similarities to the botulinum toxins.
  • both the tetanus toxin and the botulinum toxins are polypeptides made by closely related species of Clostridium ( Clostridium tetani and Clostridium botulinum , respectively).
  • both the tetanus toxin and the botulinum toxins are dichain proteins composed of a light chain (molecular weight: about 50 kDa) covalently bound by a single disulfide bond to a heavy chain (molecular weight: about 100 kDa).
  • the molecular weight of tetanus toxin and of each of the 7 botulinum toxins (non-complexed) is about 150 kDa.
  • the light chain bears the domain which exhibits intracellular biological (protease) activity, while the heavy chain comprises the receptor binding (immunogenic) and cell membrane translocational domains.
  • both the tetanus toxin and the botulinum toxins exhibit a high, specific affinity for gangliocide receptors on the surface of presynaptic cholinergic neurons.
  • Receptor-mediated endocytosis of tetanus toxin in peripheral cholinergic neurons results in retrograde axonal transport, blocking the release of inhibitory neurotransmitters from central synapses, and causing a spastic paralysis.
  • receptor-mediated endocytosis of botulinum toxin in peripheral cholinergic neurons hardly results in retrograde transport, inhibition of acetylcholine exocytosis from the central synapses, and a flaccid paralysis.
  • botulinum toxin also can undergo retrograde transport along axons and possibly inhibit the release of acetylcholine in central synapse (Bomba-Warczak et al., Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons, Cell Reports, 2016 August; 16, 1974-1987).
  • tetanus toxin and the botulinum toxins resemble each other in both biosynthesis and molecular architecture.
  • there is an overall 34% identity between the protein sequences of tetanus toxin and botulinum toxin type A, and a sequence identity as high as 62% for 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 an ester of choline and acetic acid, which is the first known neurotransmitter. It is distributed throughout neurons, and has a chemical formula of C 7 H 16 NO 2 and a molecular weight of 146.21 kDa.
  • neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, specifically by the large pyramidal cells of the motor cortex, several different neurons in the basal ganglia, the motor neurons that innervate the skeletal muscles, the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), the bag 1 fibers of the muscle spindle fiber, the postganglionic neurons of the parasympathetic nervous system, and some of the postganglionic neurons of the sympathetic nervous system.
  • acetylcholine has an excitatory effect.
  • acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings (for example, inhibition of heart rate by the vagal nerve).
  • the efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system.
  • the preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord.
  • the preganglionic sympathetic nerve fibers, extending from the cell body synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
  • Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors.
  • the muscarinic receptors are found in all effector cells stimulated by the postganglionic, neurons of the parasympathetic nervous system as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system.
  • the nicotinic receptors are found in the adrenal medulla, as well as within the autonomic ganglia, that is on the cell surface of the postganglionic neuron at the synapse between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic systems. Nicotinic receptors are also found in many nonautonomic nerve endings, for example in the membranes of skeletal muscle fibers at the neuromuscular junction.
  • Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane.
  • a wide variety of non-neuronal secretory cells, such as adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and parathyroid hormone, respectively, from large dense-core vesicles.
  • the PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development.
  • Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro , when the denervated cells are permeabilized (as by electroporation) or directly injected with the toxin. Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
  • a neuromuscular junction is formed in skeletal muscle by the proximity of axons to muscle cells.
  • a signal transmitted through the nervous system results in an action potential at the terminal axon, with activation of ion channels and resulting release of the neurotransmitter acetylcholine from intraneuronal synaptic vesicles, for example at the motor endplate of the neuromuscular junction.
  • the acetylcholine crosses the extracellular space to bind with acetylcholine receptor proteins on the surface of the muscle end plate. Once sufficient binding has occurred, an action potential of the muscle cell causes specific membrane ion channel changes, resulting in muscle cell contraction.
  • the acetylcholine is then released from the muscle cells and metabolized by cholinesterases in the extracellular space. The metabolites are recycled back into the terminal axon for reprocessing into further acetylcholine.
  • microstructures include microneedles, microblades, microknifes, microfibers, microspikes, microprobes, microbarbs, microarrays or microelectrodes, but are not limited thereto.
  • the microstructures that are used in the present invention are preferably microneedles.
  • these microneedles are preferably made of a "biocompatible or biodegradable material".
  • biocompatible material refers to a material that is non-toxic to the human body and is chemically inactive.
  • biodegradable material refers to a material that can be degraded in vivo by body fluids, enzymes or microorganisms.
  • microneedle refers to a technology that forms a hole in the skin by use of a microneedle to increase drug penetration.
  • the microneedles are mainly used for in vivo drug delivery, blood collection, in vivo analyte detection, etc.
  • microneedles should not cause pain when they penetrate the skin, and for such painless skin penetration, the diameter of the top for the minimum sharpness is important.
  • the microneedle should have a physical hardness sufficient to penetrate a 10-20 ⁇ m thick stratum corneum that is the strongest barrier in the skin.
  • the microneedle should have a suitable length so as to reach capillaries to thereby increase the efficiency of drug delivery.
  • in-plane microneedles were suggested (Silicon-processed Microneedles, Journal of microelectrochemical systems 8, 1999), various types of microneedles have been developed.
  • a solid silicon microneedle array made using an etching method was suggested as an out-of-plane microneedle array (US Patent Publication No. 2002138049).
  • the solid silicon microneedle according to this method has a diameter of 50-100 ⁇ m and a length of 500 ⁇ m, but it is impossible to realize painless skin penetration, and in vivo delivery of a drug or a cosmetic component to the target site is not reliably achieved.
  • biodegradable polymer microneedles which comprises preparing a mold by etching of glass or by photolithography
  • a method of making biodegradable microneedles by loading a capsule-type material onto the end of a mold made by a photolithography method was proposed (Polymer Microneedles for Controlled-Release Drug Delivery, Pharmaceutical Research 23, 2006, 1008).
  • the use of this method has an advantage in that a capsule-type drug is easily loaded.
  • the hardness of the microneedles decreases, indicating that the application of this method to a drug that needs to be administered in large amounts is limited.
  • an absorption type microneedle was suggested by Nano Device and Systems Inc. (Japanese Unexamined Patent Application Publication No. 2005154321). This absorption type microneedle is to be used in drug delivery or cosmetic care without removing the microneedle inserted into the skin.
  • a microneedle was made by applying a composition containing maltose to a mold and solidifying the composition.
  • the above-described Japanese patent publication suggests transdermal absorption of a drug through the absorption-type microneedle, but the microneedle caused pain when penetrating the skin.
  • the making method using the mold has a limitation in that a new template and mold need to be made through a complicated process to control the diameter and length of a microneedle.
  • Mukai et al. disclosed an apparatus and method of manufacturing a skin needle using a pin structure (US Patent Publication No. 20080157421A1).
  • This method includes heating a viscous material on a base of a substrate and pulling the viscous material using tensile force by means of a pin. Because the method involves pulling a material, which is melted by heat or is viscous, using a pin structure, it further requires a process of making a new pin structure according to a desired pattern, resulting in an increased production cost, and the heating process makes it difficult to load various heat-sensitive biodrugs (hormones, vaccines, other protein drugs, etc.).
  • the skin is composed of stratum corneum ( ⁇ 20 ⁇ m), epidermis ( ⁇ 100 ⁇ m), and dermis (300-2,500 ⁇ m), which are sequentially stacked from the outer layer of the skin. Therefore, to deliver a drug and a physiologically active substance to a specific skin layer without causing pain, making a microneedle to have a top diameter of 30 ⁇ m or less, an effective length of 200-2,000 ⁇ m and a hardness sufficient for skin penetration is effective in delivering the drug and the skin care component.
  • any process that may destroy the activity of the drug and the physiologically active substance such as high-temperature treatment, organic solvent treatment, etc., should be eliminated from the microneedle making process.
  • the term "pharmaceutical composition” refers to a composition that is administered for a specific purpose.
  • the pharmaceutical composition according to the present invention is to administer a low-concentration botulinum toxin in vivo over a long period of time, and may include a protein and a pharmaceutically acceptable carrier, excipient or diluent, which are involved in this administration.
  • the "pharmaceutically acceptable” carrier or excipient means the one approved by a regulatory agency of a government or listed in the Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
  • Microstructures containing a botulinum toxin as an active ingredient may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, or be prepared in the form of solid or semi-solid, and may contain formulating agents such as suspending agents, stabilizers, solubilizing agents and/or dispersing agents.
  • This form may be sterilized and may be fluid. It can be stable under the conditions of manufacture and storage, and can be preserved against the contamination with microorganisms such as bacteria or fungi.
  • microstructures containing a botulinum toxin as an active ingredient may be in the form of sterile powder for reconstitution with suitable carriers before use.
  • compositions may be present in unit-dose form, microneedle patches, in ampoules, or other unit-dose containers or in multi-dose containers.
  • the pharmaceutical compositions can be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example, saline for injections, immediately prior to use.
  • sterile liquid carrier for example, saline for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules or tablets.
  • microspheres containing a botulinum toxin as an active ingredient may be formulated as liquid, or may be contained in the form of microspheres in liquid.
  • a microstructure liquid composition containing a botulinum toxin as an active ingredient contains a botulinum toxin or a pharmaceutically acceptable compound and/or mixture at a concentration of 0.001-100,000 U/kg.
  • Excipients suitable for microsphere compositions containing a botulinum toxin as an active ingredient include preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugars or sodium chloride.
  • stabilizer refers to a compound optionally used in the pharmaceutical compositions of the present invention in order to increase storage life.
  • stabilizers may be sugars, amino acids or polymers.
  • the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers.
  • the carrier can be a solvent or dispersion medium.
  • pharmaceutically acceptable carriers include water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), oils, and suitable mixtures thereof.
  • the parenteral formulation can be sterilized.
  • sterilization techniques include filtration through a bacterial-retaining filter, terminal sterilization, incorporation of sterilizing agents, irradiation, heating, vacuum drying, and freeze drying.
  • the term "administration" means introducing the composition of the present invention into a patient by any suitable method.
  • the composition of the present invention may be administered via any general route, as long as it can reach a target tissue.
  • the composition can be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intracutaneously, intranasally, intrapulmonarily, intrarectally, or intrathecally.
  • microstructures containing a botulinum toxin as an active ingredient according to the present invention are preferably administered intracutaneously as microneedles.
  • the treatment method according to the present invention may comprise administering a pharmaceutically effective amount of the pharmaceutical composition.
  • the effective amount can vary depending on various factors, including the kind of disease, the severity of the disease, the kinds and contents of active ingredient and other ingredients contained in the composition, the kind of formulation, the patient’s age, weight, general health state, sex and diet, administration time, the route of administration, the secretion rate of the composition, the period of treatment, and drugs used concurrently.
  • a coating composition containing: a botulinum toxin; a thickener; and a stabilizer.
  • the botulinum toxin may be selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G.
  • the botulinum toxin is preferably botulinum toxin type A.
  • the thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and the thickener may be contained in an amount of 0.05-10 wt%.
  • the stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride, and the stabilizer may be contained in an amount of 0.03-30 wt%.
  • the coating composition may be coated on the surface of a microstructure, and the microstructure may be a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray or a microelectrode.
  • the microstructure is preferably a microneedle.
  • a microstructure containing: a botulinum toxin; a thickener; and a stabilizer.
  • the botulinum toxin may be selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G.
  • the botulinum toxin is preferably botulinum toxin type A.
  • the thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and the thickener may be contained in an amount of 0.05-10 wt%.
  • the stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride, and the stabilizer may be contained in an amount of 0.03-30 wt%.
  • the microstructure may be a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray or a microelectrode.
  • the microstructure is preferably a microneedle.
  • a method provided for preparing a coating composition comprising a mixture of a botulinum toxin, a thickener and a stabilizer.
  • the botulinum toxin may be botulinum toxin type A
  • the thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone.
  • the stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride.
  • a method provided for making a microstructure comprising the addition of a botulinum toxin, a thickener and a stabilizer to a metal or a polymer.
  • the botulinum toxin may be botulinum toxin type A
  • the thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone.
  • the stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride.
  • Botulinum toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction in animals having neurological function to thereby cause asthenia.
  • Botulinum toxin has great therapeutic effects on various diseases due to its neurotoxic function, but is lethal even in a very small amount due to its strong toxicity. For this reason, when botulinum toxin is to be used in a living body, it is necessary to minutely control the concentration of botulinum toxin. Thus, a technology enabling a low-concentration botulinum toxin to be administered over a long period of time without inconvenience is urgently required.
  • the present invention is directed to microstructure formulation techniques for botulinum toxin, and the microstructures of the present invention release a low-concentration botulinum toxin slowly over a long period of time, and thus are expected to greatly contribute to the safe and convenient medical use of botulinum toxin.
  • FIG. 1 shows the results of performing an accelerated stability test for a combination of a thickener and a stabilizer in a coating composition on microneedles precoated with 2% PVA.
  • the present invention is provided a coating composition, containing: a botulinum toxin; a thickener; and a stabilizer.
  • the botulinum toxin may be selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G.
  • the botulinum toxin is preferably botulinum toxin type A.
  • the thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and the thickener may be contained in an amount of 0.05-10 wt%.
  • the stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride, and the stabilizer may be contained in an amount of 0.03-30 wt%.
  • the coating composition may be coated on the surface of a microstructure, and the microstructure may be a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray or a microelectrode.
  • the microstructure is preferably a microneedle.
  • botulinum toxin potency which occurs when a 2X coating composition and a 2X botulinum toxin composition are mixed with each other, cannot be seen, a preliminary experiment was performed in order to examine the change in botulinum toxin potency immediately after preparation of a botulinum toxin composition.
  • a 2X coating composition and a 2X botulinum toxin composition were prepared individually and mixed at a ratio of 1:1, thereby preparing a mixture composition as shown in Table 1 below.
  • the potency was measured by an in vitro potency measurement method. Considering the case in which the potency decreases immediately after preparation, two dilution factors (75 and 112.5) were applied. The results of the measurement are shown in Table 2 below.
  • the results of measurement of the botulinum toxin potency for the mixture composition indicated that the values for CMC was out of the range of the standard curve when a dilution factor of 112.5 was applied, and that the values for CMC and sodium phosphate measured immediately after preparation were lower than that for HSA or Tween 20.
  • 6.5 ⁇ l of coating composition comprising each of HSA+NaCl, Tween 20, CMC and sodium phosphate (pH 6.0) was dropped onto each of a PLA substrate (non-coated), a 2% PVA precoated substrate and a corresponding thickener coated substrate (e.g., a PLA substrate coated with 1% HSA + 1.8% NaCl when 1% HSA + 1.8% NaCl was used as the thickener), and then dried at room temperature overnight. After completion of drying, each of the substrate was placed in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes.
  • each composition was diluted at a factor of 83.3 and subjected to in vitro potency measurement. The results of the measurement are shown in Table 3 below.
  • Preparation Example Substrate precoating Predicted potency (U) Dilution factor Potency (U) Recovery rate (% ) HSA + NaCl N 5000 83.3 6195.2 123.9 PVA 5000 83.3 6811.5 136.2 Corresponding thickener 5000 83.3 6020.7 120.4 Tween 20 N 5000 83.3 N/D Low PVA 5000 83.3 N/D Low Corresponding thickener 5000 83.3 N/D Low CMC N 5000 83.3 N/D Low PVA 5000 83.3 5950.5 119.0 Corresponding thickener 5000 83.3 N/D Low Sodium phosphate (pH 6.0) N 5000 83.3 N/D Low PVA 5000 83.3 3883.3 77.7
  • Tween 20 showed low potency in all the substrate, indicating that it is not suitable as a coating composition.
  • CMC also showed no potency in all the substrates other than the PVA-precoated substrate.
  • Example 3 Selection of Optimum Thickener for Coating Composition Containing Botulinum Toxin
  • each of seven coating candidate materials was prepared at 2X concentration (2% CMC, 1% SA, 1% MC, 1% HEC, 2% PVP, 1 % HSA+1.8% NaCl, and 0.05M phosphate), and mixed with a botulinum toxin 2X concentration solution (5,000 U/3.25 ⁇ l) at a ratio of 1:1. Then, 6.5 ⁇ l of each of the prepared solutions was dropped onto a 2% PVA-precoated substrate, and then dried at room temperature overnight.
  • 2X concentration 2% CMC, 1% SA, 1% MC, 1% HEC, 2% PVP, 1 % HSA+1.8% NaCl, and 0.05M phosphate
  • each of the substrates was placed in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes. After completion of elution, considering the case in which the potency of the botulinum toxin remains 100% during recovery after dropping, each coating solution was diluted at a factor of 83.3 and subjected to in vitro potency measurement. The results of the measurement are shown in Table 4 below.
  • Example 4 Selection of Optimum Stabilizer for Coating Composition Containing Botulinum Toxin
  • each of coating compositions prepared as shown in Table 5 below was mixed with a botulinum toxin 2X concentration solution (5,000 U/3.25 ⁇ l) at a ratio of 1:1, and 6.5 ⁇ l of each of the prepared solutions was dropped onto a 2% PVA-precoated substrate, and then dried at room temperature overnight. After completion of drying, each of the substrates was placed in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes. After completion of elution, considering the case in which the potency of the botulinum toxin remains 100% during recovery after dropping, each coating solution was diluted at a factor of 83.3 and subjected to in vitro potency measurement.
  • Stabilizer n Predictied Potency Potency Average (U) SD RSD Recovery Rate [%] Recovery rate to HSA 1) [%] (U) (U) HSA(5000U) immediately after preparation 1 5000 3996.5 4138.4 200.7 4.8 82.8 100 2 5000 4280.3 1.
  • PVP + TRE10% 1 5000 5128.2 5036.9 129.2 2.6 100.7 122 2 5000 4945.5 2.
  • PVP + TRE15% 1 5000 3728 3877.5 211.4 5.5 77.5 94 2 5000 4026.9 3.
  • PVP + TRE20% 1 5000 3958.8 3957.1 2.3 0.1 79.1 96 2 5000 3955.5 4.
  • a botulinum toxin-containing coating composition with containing a combination of a thickener and a stabilizer was prepared and dropped onto a 2% PVA-coated PLA substrate, and then an accelerated stability test for the coating composition was performed at 37°C for 5 weeks.
  • the results of the test are shown in Table 7 below and FIG. 1.
  • the present invention is directed to microstructure formulation techniques for botulinum toxin, and the microstructures of the present invention release a low-concentration botulinum toxin slowly over a long period of time, and thus are expected to greatly contribute to the safe and convenient medical use of botulinum toxin.

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Abstract

La présente invention concerne des techniques de formulation de microstructure pour toxine botulique. Les microstructures fabriquées selon les techniques de formulation de microstructure de la présente invention libèrent lentement une toxine botulique à faible concentration sur une longue durée, et peuvent ainsi contribuer fortement à l'utilisation médicale sûre et pratique de la toxine botulique.
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CN114630672A (zh) * 2019-10-31 2022-06-14 秀杰股份公司 肉毒杆菌毒素的微结构制备技术
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US12186515B2 (en) 2020-04-28 2025-01-07 Ticona Llc Microneedle assembly

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