Microstructure preparation technology of botulinum toxin

Abstract

This invention relates to a microstructure preparation technique for botulinum toxin. The microstructure preparation technique of this invention allows for precise control of the botulinum toxin concentration, reducing pain during administration and ensuring accurate injection at the target site. Therefore, this technique is expected to be widely used in the safe and convenient medical application of botulinum toxin.

Description

[Technical Field]

[0001] This invention relates to the microstructure preparation technology of botulinum toxin. [Background Technology]

[0002] Since the 1890s, various Clostridium species have been discovered that secrete neurotoxic toxins, and the characteristics of these toxins have been studied over the past 70 years (Schant, EJ et al., Microbiol. Rev., 56:80, 1992). Among these toxins, botulinum toxin is classified into seven subtypes, A through G, based on its serological characteristics. In animals with nervous system function, it causes general weakness by inhibiting the extracellular secretion of acetylcholine at the cholinergic presynapse of the neuromuscular junction. Botulinum toxin subtypes B, D, F, and G are known to cleave small synaptic vesicle proteins at specific sites, subtypes A and E cleave SNAP25 at specific sites, and subtype C cleaves synaptic fusion proteins at specific sites. Therefore, the neurotoxicity of botulinum toxin has recently been investigated for cosmetic or therapeutic purposes. Treatments using botulinum toxin technology have been proposed or attempted to address various autonomic nervous system disorders, including optic nerve disorders, pain and hyperhidrosis, migraines, postoperative pain and visceral pain, psoriasis and dermatitis, various cancers, and neurogenic inflammation.

[0003] However, botulinum toxin is the most lethal known biological toxin. The median lethal dose (LD50) for intravenous or intramuscular injection in humans is 1.3-2.1 ng / kg, and the LD50 for inhalation is 10-13 ng / kg. As mentioned above, botulinum toxin has strong therapeutic effects on various diseases, but its toxicity is also high; even small amounts can be fatal. Therefore, when using botulinum toxin in vivo, precise concentration control and administration to precise sites are essential.

[0004] Meanwhile, microstructures include microneedles, microblades, microknife, microfibers, microspikes, microprobes, microbarbs, microarrays, or microelectrodes. Microneedles, in particular, refer to a technique that enhances drug penetration by piercing the skin with fine needles. Specifically, when using microneedles to deliver botulinum toxin, the toxin is injected through dozens to hundreds of microneedles, thus reducing injection pain and overcoming side effects caused by the toxin not being precisely applied to the desired site.

[0005] Therefore, this invention relates to microstructure formulation technology for botulinum toxin, wherein the microstructure of this invention is used to reduce pain during botulinum toxin administration and facilitates the precise application of small amounts of toxin at precise sites. Therefore, this invention is expected to have wide applications in safe and convenient medical settings. [Summary of the Invention]

[0006] [Technical Issues]

[0007] To address the aforementioned existing technical problems, this invention is provided, and a microstructure preparation technique for botulinum toxin is also provided.

[0008] However, the technical problems to be solved by the present invention are not limited to the above-mentioned problems, and those skilled in the art can fully understand other problems not mentioned above from the following description.

[0009] [Technical Solution]

[0010] In the following description, the present invention will be described with reference to various embodiments. To provide a comprehensive understanding of the invention, numerous specific details, such as specific forms, compositions, and processes, are set forth in the description. However, certain embodiments may be implemented without one or more of these specific details, or in combination with other known methods and forms. In another embodiment, known processes and preparation techniques have not been specifically described in detail to avoid unnecessarily obscuring the invention. Throughout this specification, references to "an embodiment" or "an embodiment" mean that one or more embodiments of the invention include a specific feature, form, combination, or property described in connection with that embodiment. Therefore, the expressions "an embodiment" or "an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, specific features, forms, combinations, or properties may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0012] In one embodiment of the invention, "botulinum toxin" is a neurotoxic protein produced by *Clostridium botulinum*. The genus *Clostridium* comprises 127 or more species, classified according to morphology and function. The anaerobic Gram-positive bacterium *Clostridium botulinum* produces botulinum toxin, a potent polypeptide neurotoxin that can cause neuroparalysis in humans and animals, a condition known as botulism. Spores of *Clostridium botulinum* exist in soil and can grow in poorly sterilized and sealed household canned food containers, leading to numerous botulism incidents. Typically, symptoms of botulism appear 18 to 36 hours after consuming food infected with cultures or spores of *Clostridium botulinum*. The toxicity of botulinum toxin does not appear to diminish as it passes through the intestinal wall and exhibits a high affinity for cholinergic motor neurons. Symptoms of botulism can include gait disturbances, dysphagia and speech impairment, respiratory muscle paralysis, and death.

[0013] Botulinum toxin type A is the most lethal natural biological agent known to humans. The LD50 of commercially available botulinum toxin type A (purified neurotoxin complex) is approximately 50 picograms (one unit). Interestingly, based on molar amounts, botulinum toxin type A is 1.8 billion times more lethal than diphtheria toxin, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobra venom, and 12 million times more lethal than cholera. One unit (U) of botulinum toxin can be defined as the LD50 of intraperitoneal injection in female Swiss Webster mice weighing 18 to 20 grams.

[0014] Typically, seven immunologically distinct botulinum neurotoxins are characterized by neurotoxin serotypes A, B, C1, D, E, F, and G, and are differentiated by neutralization with specific types of antibodies. Different serotypes of botulinum toxins vary in the types of animals affected and the degree and duration of paralysis induced. For example, based on the speed of paralysis in rats, botulinum toxin type A is 500 times stronger than botulinum toxin type B. Furthermore, botulinum toxin type B has been shown to be non-toxic in primates even at a dose of 480 U / kg (approximately 12 times the primate LD50 of botulinum toxin type A). Botulinum toxin is believed to bind with high affinity to cholinergic motor neurons, entering the neurons and inhibiting the release of acetylcholine. It can be further absorbed not only through phagocytosis and endocytosis but also through low-affinity receptors.

[0015] Regardless of serotype, the molecular mechanisms of botulinum toxin poisoning are similar and appear to involve at least three steps. In the first step of this process, the toxin binds to the presynaptic membrane of the target neuron via a specific interaction between the toxin heavy chain (H chain or HC) and cell surface receptors. The receptors for different types of botulinum toxin and tetanus toxin are believed to be different. The carboxyl-terminal fragment of the heavy chain and Hc are considered important for targeting botulinum toxin to the cell surface.

[0016] In the second step, the toxin crosses the cytoplasmic membrane of the poisoned cell. First, botulinum toxin is engulfed by the cell via receptor-mediated endocytosis, forming an endosome containing the toxin. The toxin then exits the endosome and enters the cytoplasm. This step is thought to be mediated by the amino-terminal (HN) region of the heavy chain, which causes a conformational change in the toxin at pH approximately 5.5 or lower. Endosomals are known to possess proton pumps that lower the pH within the endosome. The conformational change exposes hydrophobic residues in the toxin, allowing it to bind to the endosome membrane. Subsequently, the toxin (or at least the light chain of the toxin) is transported across the endosome membrane into the cytoplasm.

[0017] The final step in the activation mechanism of botulinum toxin is thought to involve the reduction of disulfide bonds connecting the heavy and light chains. The full toxic activity of botulinum and tetanus toxins is contained in the light chain of the whole toxin; this light chain is a zinc (Zn++) endopeptidase that selectively cleaves proteins essential for the recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic membrane and for vesicle fusion with the membrane. Tetanus neurotoxin, as well as botulinum toxins types B, D, F, and G, cause the degradation of synaptic vesicle proteins (also known as vesicle-associated membrane proteins (VAMPs)), which are synaptosome membrane proteins. Due to one of these cleavage processes, VAMPs present on the cytoplasmic surface are typically removed. Serotypes A and E cleave SNAP-25. Serotype C1 was initially thought to cleave synaptic fusion proteins, but it was later found to cleave both synaptic fusion proteins and SNAP-25. Each botulinum toxin specifically cleaves different bonds, except for type B (and tetanus toxin), which cleaves the same bonds. These cuts all interfere with the vesicle-membrane docking process, thereby preventing the secretion of vesicle contents into the extracellular space.

[0018] Botulinum toxin has been clinically used to treat neuromuscular disorders (i.e., movement disorders) characterized by skeletal muscle hyperactivity. In 1989, the U.S. FDA approved botulinum toxin type A complex for the necessary treatment of blepharospasm, strabismus, and hemifacial spasm. Subsequently, the FDA also approved botulinum toxin type A for the treatment of cervical dystonia and glabellar lines, and botulinum toxin type B was approved for the treatment of cervical dystonia. Compared to botulinum toxin type A, other botulinum toxin serotypes appear to have lower potency and / or shorter duration of action. Peripheral intramuscular injection of botulinum toxin type A typically shows clinical efficacy within one week after injection. The typical duration of symptom relief from a single intramuscular injection of botulinum toxin type A is approximately 3 months, but significantly longer durations of action have also been reported.

[0019] Although all serotypes of botulinum toxin appear to inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction, they exert their effects by influencing different neurosecretory proteins and / or cleaving these proteins at different sites. For example, both botulinum toxin types A and E cleave the 25kD synaptosome-associated protein (SNAP-25), but they target different amino acid sequences of this protein. Botulinum toxin types B, D, F, and G act on VAMP (also known as synaptic vesicle protein), with each serotype cleaving this protein at different sites. Finally, botulinum toxin type C1 appears to cleave both synaptic fusion protein and SNAP-25. These differences in mechanisms of action may affect the relative potency and / or duration of action of various botulinum toxin serotypes. In particular, substrates for botulinum toxin can be found in a wide variety of cell types.

[0020] For all seven known botulinum toxin serotypes, the molecular weight of the botulinum toxin protein molecule is approximately 150 kDa. Interestingly, botulinum toxin is released by Clostridium bacteria as a complex containing a 150 kDa botulinum toxin protein molecule and associated non-toxin proteins. Therefore, type A botulinum toxin complexes can be produced by Clostridium bacteria in 900 kDa, 500 kDa, and 300 kDa forms. Types B and C1 botulinum toxin appear to be produced only as 700 kDa or 500 kDa complexes. Type D botulinum toxin is produced as a 300 kDa or 500 kDa complex. Finally, types E and F botulinum toxin are produced only as complexes of approximately 300 kDa. Complexes (i.e., those with a molecular weight greater than approximately 150 kDa) are thought to contain non-toxic hemagglutinin proteins and non-toxic non-hemagglutinin proteins. These two non-toxin proteins (including the associated neurotoxin complex and the botulinum toxin molecule) provide stability against the denaturation of the botulinum toxin molecule and protect it from digestive acid damage when ingested. Additionally, due to the relatively large size of the botulinum toxin complex (molecular weight - approximately 150 kDa or greater), the botulinum toxin diffuses slowly from the site of intramuscular injection.

[0021] Furthermore, in vitro studies have shown that botulinum toxin inhibits potassium-induced release of acetylcholine and norepinephrine in primary brainstem cell cultures. Additionally, botulinum toxin has been reported to reduce the induced release of glycine and glutamate in primary spinal cord neuron cultures, as well as the release of various neurotransmitters (acetylcholine, dopamine, norepinephrine, CGRP, substance P, and glutamate) in brain synaptosomes. Therefore, when applied at appropriate concentrations, botulinum toxin can inhibit the stimulus-induced release of most neurotransmitters.

[0022] Type A botulinum toxin can be obtained by culturing Clostridium botulinum cultures in a fermenter, followed by collecting and purifying the fermentation mixture, according to known methods. First, all botulinum toxin serotypes are synthesized as inactive single-chain proteins, which must be cleaved or split by proteases to acquire neuroactive properties. Bacterial strains that produce botulinum toxin serotypes A and G possess endogenous proteases, therefore serotypes A and G can often be recovered from bacterial cultures in their active form. In contrast, botulinum toxin serotypes C1, D, and E are typically inactive when recovered from cultures because they are synthesized by non-proteolytic strains. Serotypes B and F can be produced by both proteolytic and non-proteolytic strains, and therefore can be recovered in either active or inactive form. However, for example, proteolytic strains producing botulinum toxin serotype B may only cleave a portion of the produced toxin. The exact ratio of cleaved to uncleaved molecules depends on the culture time and temperature. Therefore, as mentioned earlier, since botulinum toxin type B is significantly less potent than botulinum toxin type A, for example, a certain proportion of botulinum toxin type B in any sample will be inactive. The presence of inactive botulinum toxin molecules in clinical samples increases the overall protein load of the sample, which is not beneficial to the clinical efficacy of the toxin but is associated with increased antigenicity. Furthermore, it is known that at the same dose level, when injected intramuscularly, botulinum toxin type B has a shorter duration of activity and lower potency compared to botulinum toxin type A.

[0023] The Hall A strain of Clostridium botulinum can produce high-quality crystalline botulinum toxin type A with a specific titer ≥3×10⁻⁶. 7 U / mg, A260 / A278 < 0.60, characterized by separated bands on gel electrophoresis. Crystalline botulinum toxin type A can be obtained using the known Schantz method. Typically, *Clostridium botulinum* type A is cultured in a suitable medium to obtain an anaerobic ferment, from which the botulinum toxin type A complex is isolated and purified. This known process can be used to isolate pure subtypes of botulinum toxin from non-toxic proteins, for example, to purify botulinum toxin type A with a molecular weight of approximately 150 kDa and a specific titer of 1–2 × 10⁻⁶. 8 LD50 U / mg or higher; purified botulinum toxin type B with a molecular weight of approximately 156 kDa and a specific potency of 1-2 × 10⁻⁶ kDa. 8 LD50 U / mg or higher; or purified botulinum toxin type F with a molecular weight of approximately 155 kDa and a specific potency of 1-2 × 10⁻⁶. 7 LD50 U / mg or higher.

[0024] Botulinum toxin and / or botulinum toxin complexes are available from known compound manufacturers in the art, and pure botulinum toxin can also be used to prepare pharmaceutical compositions.

[0025] Typically, like enzymes, the biological activity of botulinum toxin (an intracellular peptidase) depends at least in part on, for example, its three-dimensional conformation. Therefore, the toxicity of botulinum toxin type A can be removed by heating, various chemicals, surface scratching, and surface drying. Furthermore, when known toxin complexes obtained through culturing, fermentation, and purification are diluted to very low toxin concentrations and used to prepare pharmaceutical compositions, the toxicity of the known toxins is rapidly removed without a suitable stabilizer. Since the specific toxicity of a toxin complex is rapidly lost when diluted in large quantities, diluting a few milligrams of toxin into a solution containing a few nanograms of toxin per milliliter is quite difficult. Because pharmaceutical compositions containing toxins may be used months or years after formulation, the toxins should be stabilized with a suitable stabilizer. Therefore, in this invention, it is necessary to develop optimal stabilizer technology to stably control the release of botulinum toxin in vivo.

[0026] According to reports, botulinum toxin type A has already been used clinically in the following ways:

[0027] Typically, the duration of action for a single intramuscular injection of botulinum toxin is about 3 to 4 months. However, in some cases, subtype A can be effective for 12 months or more, and in some cases, up to 27 months, when used to treat glandular disorders such as hyperhidrosis.

[0028] In addition to its pharmacological effects at peripheral sites, botulinum toxin can also exhibit inhibitory effects in the central nervous system. There are reports of botulinum toxin being able to retrograde back to the spinal cord region. Therefore, botulinum toxin injected at peripheral sites (e.g., intramuscularly) can be retrogradely transported to the spinal cord.

[0029] Botulinum toxin has also been suggested or used to treat skin, bone and tendon injuries, pain, various autonomic nervous system disorders (including hyperhidrosis, tension headaches, migraines, postoperative pain and visceral pain), hair growth and hair maintenance, psoriasis and dermatitis, muscle injuries, various cancers, smooth muscle disorders, nerve entrapment syndromes, acne, neurogenic inflammation, eye diseases, pancreatic diseases, prostate diseases (including benign prostatic hyperplasia, prostate cancer and urinary incontinence), fibromyalgia and piriformis syndrome.

[0030] It is known that modified chemically coupled or recombinant fused to a specific target portion of clostridium neurotoxin or fragments thereof (preferably botulinum toxin) can be used to treat pain by administration to the spinal cord, and it is known that targeted botulinum toxin (i.e. having a non-naturally bound portion) can be used to treat a variety of conditions.

[0031] Furthermore, the technique of injecting botulinum toxin into the pectoral muscles to control chest spasms is known, and controlled-release toxin implants in the case of percutaneous botulinum toxin administration are also known. Botulinum toxin is known to be used for: atrophying the chewing or biting muscles of the mouth, allowing self-inflicted wounds and resulting ulcers to heal; treating benign cystic lesions or tumors; treating anal fissures; and treating certain types of atopic dermatitis.

[0032] In addition, botulinum toxin can reduce induced inflammatory pain in a rat formalin model. Furthermore, it has been reported that botulinum toxin nerve blockade can lead to a reduction in epidermal thickness. Finally, botulinum toxin application to the feet is known to treat excessive sweating, toe cramps, idiopathic toe gait, and foot dystonia.

[0033] Tetanus toxin and its derivatives (i.e., those with non-natural targeting components), fragments, hybrids, and chimeras can also be used for treatment. Tetanus toxin is very similar to botulinum toxin. Therefore, both tetanus toxin and botulinum toxin are polypeptides produced by closely related clostridium species (Clostridium tetani and Clostridium botulinum). Furthermore, both tetanus toxin and botulinum toxin are double-stranded proteins composed of a light chain (molecular weight: approximately 50 kDa) and a heavy chain (molecular weight: approximately 100 kDa), covalently linked by a single disulfide bond. Therefore, the molecular weight of both tetanus toxin and seven botulinum toxins (non-complexes) is approximately 150 kDa. Additionally, in both tetanus toxin and botulinum toxin, the light chain contains domains exhibiting intracellular biological (protease) activity, while the heavy chain contains receptor-binding (immunogenic) and cell membrane transport domains.

[0034] Furthermore, both tetanus toxin and botulinum toxin exhibit high specific affinity for ganglioside receptors on the surface of presynaptic cholinergic neurons. Receptor-mediated endocytosis of tetanus toxin by peripheral cholinergic neurons leads to retrograde axonal transport, blockade of central synaptic release of inhibitory neurotransmitters, and spastic paralysis. Conversely, receptor-mediated endocytosis of botulinum toxin by peripheral cholinergic neurons hardly leads to retrograde transport, inhibition of extracellular acetylcholine secretion from poisoned peripheral motor neurons, or flaccid paralysis.

[0035] Finally, tetanus toxin and botulinum toxin share similar biosynthesis and molecular structure. Therefore, there is an overall 34% protein sequence identity between tetanus toxin and botulinum toxin type A, with sequence identity reaching as high as 62% for certain functional domains.

[0036] In one embodiment of the invention, the "acetylcholine" is a neurotransmitter, first discovered as an ester of choline and acetic acid, which is distributed throughout neurons. The chemical formula of acetylcholine is C7H. 16 NO2 has a molecular weight of 146.21.

[0037] Although there is evidence that several neuromodulators can be released by the same neuron, typically in the mammalian nervous system, each type of small-molecule neurotransmitter is released by only one type of neuron. The neurotransmitter acetylcholine is secreted by neurons in multiple brain regions, particularly the large pyramidal cells of the motor cortex, several different neurons in the basal ganglia, certain types of motor neurons distributed in skeletal muscle, preganglionic neurons of the autonomic nervous system (sympathetic and parasympathetic systems), bag-1 fibers of muscle spindle fibers, postganglionic neurons of the parasympathetic nervous system, and postganglionic neurons of the sympathetic nervous system. Normally, most postganglionic neurons of the sympathetic nervous system secrete norepinephrine; only the postganglionic sympathetic fibers leading to sweat glands, arrector pili muscles, and certain blood vessels are cholinergic. In most cases, acetylcholine has an excitatory effect. However, acetylcholine is known to have an inhibitory effect on some peripheral parasympathetic nerve endings (e.g., inhibiting heart rate via the vagus nerve).

[0038] Signals from the autonomic nervous system are transmitted throughout the body via the sympathetic or parasympathetic nervous system. Preganglionic neurons of the sympathetic nervous system originate from the cell bodies of preganglionic sympathetic neurons located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers extending from these cell bodies form synapses with postganglionic neurons in the paravertebral sympathetic ganglia or the anterior vertebral ganglia. Since both sympathetic and parasympathetic preganglionic neurons are cholinergic, the application of acetylcholine to the ganglia can stimulate both sympathetic and parasympathetic postganglionic neurons.

[0039] Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. Muscarinic receptors are found in all effector cells stimulated by postganglionic neurons of the parasympathetic nervous system and postganglionic cholinergic neurons of the sympathetic nervous system. Nicotinic receptors are found in the adrenal medulla and in the autonomic ganglia on the surface of postganglionic neurons located at synapses between preganglionic and postganglionic neurons of the sympathetic and parasympathetic nervous systems. Nicotinic receptors are also found in many involuntary nerve endings, such as in the skeletal muscle fiber membrane at the neuromuscular junction.

[0040] When small, transparent intracellular vesicles fuse with the presynaptic neuron cell membrane, acetylcholine is released from the cholinergic neuron. Various non-neuronal secretory cells, such as adrenal medulla (and the PC12 cell line) and pancreatic islet cells, release catecholamines and parathyroid hormone from large, dense core vesicles, respectively. The PC12 cell line, a clone of rat pheochromocytoma cells, is widely used as a tissue culture model for studying sympathetic adrenal gland development. When botulinum toxin is infiltrated (via electroporation) or injected directly into denervated cells, it inhibits the release of both compounds from both cell types in vitro. Furthermore, botulinum toxin is known to inhibit the release of the neurotransmitter glutamate from cortical synaptic vesicle cell cultures.

[0041] The neuromuscular junction is formed in skeletal muscle via axons adjacent to muscle cells. Signals transmitted through the nervous system induce action potentials at the terminal axons, activating ion channels and causing the neurotransmitter acetylcholine to be released from synaptic vesicles of neurons at the motor endplate of the neuromuscular junction. Acetylcholine crosses the extracellular space and binds to acetylcholine receptor proteins on the surface of the muscle endplate. Once sufficient binding occurs, the action potential of the muscle cell induces changes in specific membrane ion channels, leading to muscle cell contraction. Acetylcholine is then released from the muscle cell and metabolized in the extracellular space by cholinesterases. The metabolites are recycled back to the terminal axons for reprocessing into acetylcholine.

[0042] In one embodiment of the present invention, "microstructure" includes, but is not limited to, microneedles, microblades, microknives, microfibers, microthorns, microprobes, microbarbs, microarrays, or microelectrodes. In particular, in the present invention, the microstructure is preferably a microneedle. Furthermore, when manufacturing the microneedles of the present invention for medical purposes, their components are preferably "biocompatible or biodegradable materials." Here, the term "biocompatible material" refers to a material that is non-toxic and chemically inert to the human body. Furthermore, the term "biodegradable material" refers to a material that can be degraded in vivo by bodily fluids, enzymes, or microorganisms.

[0043] In this invention, the microstructure is manufactured to comprise the botulinum toxin composition of this invention, which contains botulinum toxin, a thickener, and a stabilizer as active ingredients, or is coated with the botulinum toxin composition of this invention. Here, the microstructure includes any microstructure manufactured by microstructure manufacturing methods used in the art, but this invention is not limited thereto.

[0044] In one embodiment of the invention, the term "microneedle" refers to a technique that enhances drug penetration by using fine needles to puncture the skin. Microneedles are primarily used for in vivo drug delivery, blood collection, and in vivo analyte detection.

[0045] In 1998, the first known microstructure array was fabricated at the Georgia Institute of Technology, using semiconductor processing technology to make silicon elements, and its potential as an alternative to subcutaneous injection was proposed.

[0046] Unlike other existing needles, microneedles are characterized by painless and non-invasive skin penetration. To achieve this painless penetration, a smaller tip diameter that minimizes invasiveness is crucial. Furthermore, since microneedles must penetrate 10-20 μm of the stratum corneum (the skin's strongest barrier), they require sufficient physical rigidity. Additionally, an appropriate length must be considered to reach capillaries and improve drug delivery efficiency.

[0047] Following the initial development of in-plane type microneedles, various other types of microneedles have been developed. Solid silicon microneedles with diameters of 50-100 μm and 500 μm were fabricated using an etching method, but these methods failed to achieve painless skin penetration and made it difficult to deliver drugs and cosmetic ingredients to the desired areas.

[0048] Meanwhile, Prausnitz of the University of Georgia proposed a method for preparing biodegradable polymer microneedles by etching glass or by photolithography to form molds. Furthermore, in 2006, a method was proposed to prepare biodegradable solid microneedles by loading capsule-formed substances onto the ends of a mold manufactured using photolithography. While this method has the advantage of freely loading drugs prepared in capsule form, the rigidity of the microneedles decreases with increasing drug loading, thus limiting its application for drugs requiring large-volume administration.

[0049] In 2005, Nano Device & Systems proposed an absorbent microneedle. This absorbent microneedle is intended for drug delivery or cosmetic treatments without the need to remove the inserted microneedle from the skin. The method involves preparing the microneedle by injecting a composition made of maltose and a drug into a mold and allowing it to solidify. This Japanese patent proposed using absorbent microneedles for transdermal drug absorption; however, these microneedles cause pain when penetrating the skin. Furthermore, due to limitations in mold manufacturing technology, it is impossible to manufacture microneedles with a suitable tip diameter that does not cause pain and a length (i.e., 1 mm or more) required for effective drug delivery.

[0050] In 2008, Prausnitz of the University of Georgia produced biodegradable microneedles made from a mixture of polyvinylpyrrolidone (PVP) and methacrylic acid (MAA) in a polydimethylsiloxane (PDMS) mold. Alternatively, microneedles can be prepared by placing carboxymethyl cellulose into a conical mold. However, the limitations of mold-based manufacturing methods include the need for complex processes to create new molds to control the diameter and length of the microneedles, and the complexity and time-consuming process of placing the material into the mold to manufacture the microneedles.

[0051] In 2008, an apparatus and method for manufacturing skin needles using pin structures were reported. This method involves pulling a substrate with pins on a base by heating or using the tensile force of an adhesive material. Because this method involves using pin structures to pull molten or adhesive material, it cannot overcome the increased cost due to the need to manufacture new pin structures according to the desired pattern. Furthermore, due to the heating process, it is difficult to load various heat-sensitive biological drugs (such as hormones, vaccines, and other protein drugs).

[0052] Meanwhile, the skin, starting from its surface, consists of the stratum corneum (<20 μm), epidermis (<100 μm), and dermis (300-2500 μm). Therefore, to deliver drugs and physiologically active substances to specific skin layers without causing pain, microneedles prepared with a tip diameter of approximately 30 μm, an effective length of 200-2000 μm, and sufficient rigidity to penetrate the skin can effectively deliver drugs and cosmetic ingredients. Furthermore, to deliver drugs or physiologically active substances via biodegradable solid microneedles, any processes that might destroy the activity of the drugs or physiologically active substances, such as high-temperature treatment or organic solvent treatment, should be avoided during the manufacturing process.

[0053] Due to limitations in manufacturing methods, traditional solid microneedles are made from specific materials (such as silicon, polymers, metals, and glass). The manufacturing process using mold technology is complex and time-consuming, resulting in drawbacks such as drug denaturation, insufficient hardness, and drug loss. Therefore, there has been a need for a microneedle manufacturing technology that allows the microneedles to have a sufficiently small diameter to penetrate the skin without causing pain, a sufficiently long length to penetrate deeply, sufficient hardness without specific material limitations, and minimizes drug loss.

[0054] In this invention, the microstructure is characterized by being coated with a composition containing botulinum toxin, and can be pretreated with a polymeric compound to improve the stability and efficiency of the coating. When the microstructure is a microneedle, the polymeric compound is preferably polylactic acid (PLLA), and the polymeric compound used for pretreatment is preferably polyvinyl alcohol (PVA), but the invention is not limited thereto. The concentration of PVA is preferably 1-10%, more preferably 1-5%, further preferably 1-3%, and even more preferably 2%.

[0055] In one embodiment of the invention, the term "pharmaceutical composition" refers to a composition administered for a specific purpose. For the purposes of this invention, the pharmaceutical composition precisely administers botulinum toxin at a desired location at a precisely controlled concentration, and the pharmaceutical composition may include proteins and pharmaceutically acceptable carriers, excipients, or diluents. The term "pharmaceuticalally acceptable" carriers or excipients refers to those approved by a government regulatory authority or listed in a government-approved pharmacopoeia or other recognized pharmacopoeia for use in mammals, particularly humans.

[0056] The microstructure containing botulinum toxin as the active ingredient for parenteral administration can be prepared as a suspension, solution, or emulsion in an oily or aqueous carrier, or as a solid or semi-solid form, and may contain formulations such as suspending agents, stabilizers, solubilizers, and / or dispersants. This form can be sterile and can be fluid. It can be stable under manufacturing and storage conditions and can be protected against contamination by microorganisms such as bacteria or fungi. Alternatively, the microstructure containing botulinum toxin as the active ingredient can be a sterile powder for reconstitution with a suitable carrier prior to use. The pharmaceutical composition can be present in unit dose form, in microneedle patches, in ampoules, or in other unit dose or multi-dose containers. Alternatively, the pharmaceutical composition can be stored under lyophilized (freeze-dried) conditions and requires immediate addition to a sterile liquid carrier, such as water for injection, prior to use. Immediate-use solutions and suspensions can be prepared from sterile powders, granules, or tablets.

[0057] In some non-limiting embodiments, the microstructure containing botulinum toxin as an active ingredient can be formulated as a liquid or contained in the form of microspheres. In one non-limiting embodiment, the liquid composition of the microstructure containing botulinum toxin as an active ingredient contains 0.001-100,000 U / kg of botulinum toxin or a pharmaceutically acceptable compound and / or mixture thereof. Furthermore, in one non-limiting embodiment, excipients suitable for the microstructure composition containing botulinum toxin as an active ingredient include preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, such as sugars or sodium chloride. Here, the term "stabilizer" refers to a compound optionally used in the pharmaceutical compositions of the present invention to increase shelf life. In one non-limiting embodiment, the stabilizer can be a sugar, amino acid, compound, or polymer, wherein the sugar can be a monosaccharide, disaccharide, or polysaccharide, and preferably a disaccharide. Disaccharides are classified into reducing disaccharides and non-reducing disaccharides. Reducing disaccharides have a free "hemiacetal" unit, in which one of the two "reducing sugars" can act as a reducing "aldehyde." Non-reducing disaccharides are formed by acetal bonding between the shell-less carbons of the two monosaccharides, and therefore do not contain a hemiacetal that can act as a reducing agent. Examples of reducing disaccharides include cellobiose (Cel) and maltose (Mal), and examples of non-reducing disaccharides include trehalose (Tre) and sucrose (Suc). Pharmaceutical compositions may contain one or more pharmaceutically acceptable carriers. The carrier can be a solvent or a dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, physiological saline, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, and suitable mixtures thereof.

[0058] Parenteral preparations can be sterilized. Non-limiting examples of sterilization techniques include the use of bacterial inhibition filters, terminal sterilization, addition of sterile reagents, irradiation, application of sterile gases, heating, vacuum drying, and freeze drying.

[0059] In one embodiment of the invention, the term "application" refers to introducing the composition of the invention into a patient by any suitable method, and the route of application of the composition of the invention can be any conventional route that enables the composition of the invention to reach the target tissue. The route of application can be oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, intranasal, intrapulmonary, rectal, intracavitary, or intrathecal administration; however, for the microstructure of the invention containing the botulinum toxin composition as the active ingredient, intradermal administration via microneedles is preferred.

[0060] The treatment methods of the present invention may include administering a pharmaceutically effective amount of a pharmaceutical composition. The effective amount of the present invention can be adjusted according to various factors, including the type or severity of the disease, the type and content of the active ingredient and other components contained in the composition, the type of formulation, the patient's age, weight, general health condition, sex, and diet, the time of administration, the route of administration, the release rate of the composition, the duration of treatment, and concurrent medications.

[0061] In one embodiment of the present invention, a botulinum toxin composition is provided, comprising botulinum toxin as an active ingredient, a thickener, and a stabilizer, wherein the botulinum toxin is any one or more selected from the group consisting of: type A, type B, type C, type D, type E, type F, and type G botulinum toxin; the thickener is any one or more selected from the group consisting of: carboxymethyl cellulose, sodium salt; sodium alginate; hyaluronic acid; methyl cellulose; hydroxyethyl cellulose, and polyvinylpyrrolidone; and its content is 0.05-10 wt%; the stabilizer is any one or more selected from the group consisting of: trehalose, sucrose, a mixture of trehalose and sucrose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride; and its content is 0.03-50 wt%. As a stabilizer, the mixture of trehalose and sucrose is a mixture of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% trehalose and about 95%, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5% sucrose.

[0062] In another embodiment of the invention, a microstructure comprising a botulinum toxin composition is provided, which is any one or more selected from the group consisting of: microneedles, microblades, microknives, microfibers, microspurs, microprobes, microbarbs, microarrays, and microelectrodes.

[0063] In another embodiment of the present invention, a method for preparing a botulinum toxin composition is provided, the botulinum toxin composition comprising a mixture of botulinum toxin, a thickener, and a stabilizer, wherein the botulinum toxin is any one or more selected from the group consisting of: type A, type B, type C, type D, type E, type F, and type G botulinum toxin; the thickener is any one or more selected from the group consisting of: carboxymethyl cellulose, sodium salt; sodium alginate; hyaluronic acid; methyl cellulose; hydroxyethyl cellulose, and polyvinylpyrrolidone; and is mixed in an amount of 0.05-10 wt%; the stabilizer is any one or more selected from the group consisting of: trehalose, sucrose, a mixture of trehalose and sucrose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride; and is mixed in an amount of 0.03-50 wt%.

[0064] In another embodiment of the present invention, a method for coating a target object with a botulinum toxin composition is provided, comprising (a) pretreating the target object with a polymeric compound; (b) mixing botulinum toxin, a thickener, and a stabilizer; and (c) adding the mixture of (b) dropwise to the pretreated target object added in (a), followed by drying. In this method, the polymeric compound may be polyvinyl alcohol used in an amount of 0.5-10 wt%; the botulinum toxin is botulinum toxin type A; the thickener is one or more selected from the group consisting of: carboxymethyl cellulose, sodium salt; sodium alginate; hyaluronic acid; methyl cellulose; hydroxyethyl cellulose; and polyvinylpyrrolidone; the stabilizer is any one or more selected from the group consisting of: trehalose and / or sucrose, a mixture of trehalose and sucrose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride; and the target object is a microstructure, wherein the microstructure is a microneedle.

[0065] In another embodiment of the invention, a composition for coating microstructures is provided, comprising botulinum toxin, hydroxyethyl cellulose, and sucrose, wherein the botulinum toxin is one or more selected from the group consisting of botulinum toxin type A, type B, type C, type D, type E, type F, and type G, and the microstructure is one or more selected from the group consisting of microneedles, microblades, microknife, microfibers, microspiky, microprobes, microbarbs, microarrays, and microelectrodes.

[0066] In the composition used for coating microstructures, the mixing ratio (w:w) of hydroxyethyl cellulose and sucrose can be greater than 30 to less than 90, 50 to 80, 60 to 80, or 60 to 70 relative to 1 part hydroxyethyl cellulose.

[0067] In the composition used for coating microstructures, the mixing ratio (w:w) of hydroxyethyl cellulose and sucrose can be greater than 30 to less than 90 parts of sucrose relative to 1 part of hydroxyethyl cellulose, preferably 50 to 80 parts, more preferably 60 to 80 parts, and even more preferably 60 to 70 parts.

[0068] In another embodiment of the invention, a microstructure coated with a composition for coating microstructures is provided. In this method, the microstructure is any one or more selected from the group consisting of: microneedles, microblades, microknife, microfibers, microspiky, microprobes, microbarbs, microarrays, and microelectrodes.

[0069] In another embodiment of the invention, a method for preparing a composition for coating microstructures is provided, comprising mixing botulinum toxin, hydroxyethyl cellulose, and sucrose. In this method, the botulinum toxin is one or more selected from the group consisting of botulinum toxin types A, B, C, D, E, F, and G, and the microstructure is one or more selected from the group consisting of microneedles, microblades, microknife, microfibers, microspiky, microprobes, microbarbs, microarrays, and microelectrodes.

[0070] In another embodiment of the invention, a method is provided for coating a microstructure using a composition containing botulinum toxin, comprising (a) mixing botulinum toxin, hydroxyethyl cellulose, and sucrose; and (b) coating the surface of the microstructure with the mixture of (a). In this method, the botulinum toxin is one or more selected from the group consisting of botulinum toxin types A, B, C, D, E, F, and G, and the microstructure is one or more selected from the group consisting of microneedles, microblades, microknife, microfibers, microspiky, microprobes, microbarbs, microarrays, and microelectrodes.

[0071] The invention will be described step by step in detail below.

[0072] [Beneficial Effects]

[0073] In animals with neurological function, botulinum toxin inhibits the extracellular secretion of acetylcholine at the cholinergic presynapse of the neuromuscular junction, thereby causing systemic weakness. Botulinum toxin is highly neurotoxic; even small amounts can be fatal. Therefore, when used in vivo, precise concentration control and targeted administration techniques are essential. This invention relates to a microstructure preparation technique for botulinum toxin, in which the microstructure of this invention is used to reduce pain during botulinum toxin administration and facilitates the precise application of small amounts of toxin at precise sites. Therefore, this invention holds promise for widespread application in safe and convenient medical settings. [Image Description]

[0074] Figure 1 This demonstrates the recovery rate of the absolute potency of botulinum toxin from the pretreated coating material in one embodiment of the invention.

[0075] Figure 2 This invention illustrates the recovery rate of the absolute potency of botulinum toxin based on the type and concentration of thickener mixed with botulinum toxin in one embodiment of the invention.

[0076] Figure 3 This illustrates the recovery rate of the absolute potency of botulinum toxin based on the type and concentration of stabilizers mixed with botulinum toxin in one embodiment of the invention.

[0077] Figure 4 The results show the determination of the long-term stability of the combination of SA or HA thickener and stabilizer in one embodiment of the invention.

[0078] Figure 5 The results show the determination of the long-term stability of the combination of PVP or HEC thickener and stabilizer in one embodiment of the invention.

[0079] Figure 6 The results show the results of confirming the coating uniformity of the coating composition containing botulinum toxin in one embodiment of the invention.

[0080] Figure 7 The results of regression analysis on the measured viscosity values ​​of the coating composition based on the mixing ratio of the HEC:Suc composition are shown in one embodiment of the present invention.

[0081] Figure 8 The results of regression analysis of the measured surface tension of the coating composition based on the mixing ratio of the HEC:Suc composition in one embodiment of the present invention are shown.

[0082] Figure 9 A set of images shows the coating composition uniformity results of HEC:Suc combinations in various mixing ratios according to one embodiment of the invention.

[0083] Figure 10 The results of the coating uniformity of the coating composition in one embodiment of the present invention are shown, expressed as the results of regression analysis of the relative standard deviation of the total number of pixels with respect to the mixing ratio of the HEC:Suc composition.

[0084] Figure 11 The results show the long-term stability of the coating compositions of HEC:Suc combinations in various mixing ratios in one embodiment of the invention.

[0085] [Invention Embodiments]

[0086] To test the long-term stability of the combination of PVP or HEC thickeners with disaccharides or amino acid stabilizers, an accelerated stability test was conducted at 37°C for 5 weeks after the preparation and dropwise addition of the botulinum toxin-coated composition. Trehalose (Tre) and / or sucrose (Suc) were used as disaccharide stabilizers, and methionine (Met) and glycine (Gly) were used as amino acid stabilizers. The results confirmed that using PVP or HEC as a thickener and disaccharides or methionine as stabilizers was the optimal stable combination for preparing the botulinum toxin-coated composition. [Example]

[0087] The present invention will now be described in further detail with reference to embodiments. These embodiments are provided only to explain the invention more specifically, and it will be apparent to those skilled in the art that the scope of the invention is not limited to the embodiments based on the essential points of the invention.

[0088] Example 1. Determination of dilution factor in botulinum toxin potency test

[0089] Since the loss of botulinum toxin potency when the 2× coating composition and the 2× botulinum toxin composition are mixed together is unknown, preliminary experiments were conducted to determine the change in botulinum toxin potency immediately after the preparation of the botulinum toxin composition.

[0090] Therefore, 2× coating composition and 2× botulinum toxin composition were prepared separately and mixed in a 1:1 ratio to prepare the mixed composition shown in Table 1 below.

[0091] [Table 1]

[0092]

[0093] For this mixed composition, an in vitro potency assay kit was used ( The Matrix Botulinum Neurotoxin Assay Kit, Biosentinel, Inc., was used to detect botulinum toxin potency. The specific method was performed according to the same procedure described in existing literature by Dunning FM (J Vis Exp. 2014.3.3; (85). doi:10.3791 / 51170.). Furthermore, considering the immediate decrease in potency after preparation, two dilution factors (75 and 112.5) were applied. The results are shown in Table 2 below.

[0094] [Table 2]

[0095]

[0096] Botulinum toxin potency measurements of the mixed compositions showed a recovery of 50% or higher absolute potency. However, for CMC, the potency exceeded the range of the standard curve when a dilution factor of 112.5 was applied, and the potency of CMC and sodium phosphate was lower than that of HAS or Tween 20 when measured immediately after preparation.

[0097] Example 2. Selection of pre-coating materials

[0098] Experiments were conducted to examine changes in botulinum toxin potency, depending on whether the PLA (poly(lactic acid)) matrix was pre-coated and the type of pre-coating material.

[0099] A mixture of 6.5 μl each of the coating composition HSA+NaCl, Tween 20, CMC, and sodium phosphate (pH 6.0) and botulinum toxin was added dropwise to a PLA matrix (uncoated (N), 2% PVA pre-coated (PVA), or a corresponding thickener (e.g., when using 1% HSA+1.8% NaCl as a thickener, the PLA matrix was also coated with 1% HSA+1.8% NaCl)) and then dried overnight at room temperature (O / N). After drying, each matrix was placed in a 1.5 mL tube containing 0.3 mL of 0.9% NaCl and eluted for toxin at room temperature for 30 minutes. After elution, assuming the botulinum toxin potency remained at 100% during recovery after addition, each composition was diluted with a dilution factor of 83.3 for in vitro potency determination. The measurement results are shown in Table 3 below. Figure 1 As shown.

[0100] [Table 3]

[0101]

[0102] As the results above show, during the recovery process after addition, when the potency exceeds the range of the standard curve, it is expressed as N / D. Regarding the sodium phosphate results, it was found that the potency was stably maintained when the coated matrix was pretreated with 2% PVA.

[0103] Tween 20 was found to have low potency in all matrices, indicating that it is not suitable as a coating composition. For CMC, the situation was similar to that of sodium phosphate, with no potency detected in any matrices except those pre-coated with PVA.

[0104] Example 3. Selection of the optimal thickener for the coating composition containing botulinum toxin

[0105] To select the thickener to be included in the coating composition from the alternative materials, eight coating candidates (2% CMC, 1% SA, 1% HA, 1% MC, 1% HEC, 2% PVP, 1% HSA + 1.8% NaCl and 0.05M phosphate) were prepared at 2× concentrations and mixed with 2× botulinum toxin solution (5,000 U / 3.25 μl) to prepare a 1:1 solution. 6.5 μl of the resulting solution was added dropwise to a matrix pretreated with 2% PVA, and the resulting matrix was then dried overnight at room temperature (O / N). After drying, each matrix was placed in a 1.5 mL tube containing 0.3 mL of 0.9% NaCl, and the toxin was eluted at room temperature for 30 minutes. After elution, assuming the potency remained at 100% during recovery after addition, each composition was diluted with a dilution factor of 83.3 for in vitro potency determination. The results are shown in Table 4 below. Figure 2 As shown.

[0106] [Table 4]

[0107]

[0108]

[0109]

[0110] As shown in Table 4, two samples (sample 1 and 2) were prepared for each test group, and the toxins eluted from each sample were measured twice. The results showed that the recoveries of the CMC, MC, HEC, PVP, and HSA+NaCl coated compositions relative to the target potency were as high as 80% or more. In particular, the recoveries of MC and HSA+NaCl were 90% or more. In addition, the average potency recoveries of SA and HA were determined to be 60% or higher, which was higher than the average potency recoveries of sodium phosphate.

[0111] Example 4. Selection of the optimal stabilizer for the coating composition containing botulinum toxin

[0112] To select the stabilizer to include in the coating composition from the alternative materials, as shown in Table 5 below, a 2× botulinum toxin solution (5,000 U / 3.25 μl) was mixed with each coating composition prepared as shown in Table 5 to prepare a 1:1 solution. 6.5 μl of this solution was added dropwise to a 2% PVA pre-coated matrix, and then dried overnight at room temperature (O / N). After drying, each matrix was placed in a 1.5 mL tube containing 0.3 mL of 0.9% NaCl, and the toxin was eluted at room temperature for 30 minutes. After elution, assuming the potency remained at 100% during recovery after addition, each composition was diluted with a dilution factor of 83.3 for in vitro potency determination.

[0113] [Table 5]

[0114]

[0115]

[0116] Experimental results showed that, under all stabilizer mixing conditions, the PVP and HEC groups, except for CMC, exhibited recoveries of 90% or higher compared to values ​​detected immediately after HSA preparation. The results are as follows: Figure 3 As shown.

[0117] Example 5. Validation of the long-term stability of the botulinum toxin-containing coating composition

[0118] The long-term stability of the mixture of thickeners (SA, HA, PVP, and HEC) and stabilizers (Tre and Met) selected based on the results of Examples 1 to 4 above was confirmed.

[0119] First, to assess the long-term stability of the SA and HA thickener and stabilizer combinations, PLA matrices were pre-coated with 2% PVA, and 6.5 μl of a 1:1 mixture of 2× botulinum toxin solution (5,000 U / 3.25 μl) and each coating composition prepared as shown in Table 6 was added. The resulting matrices were then dried overnight at room temperature (O / N). After drying, each matrix was placed in a 1.5 mL tube containing 0.3 mL of 0.9% NaCl to assess its potency recovery up to day 8. Since the thickeners and stabilizers in the various combinations of coating compositions affect the measurement of botulinum toxin potency, the potency measured on day 1 for each test group was used as a reference value (100), and the potency measured on day 8 was converted based on the potency measured on day 1, as shown below. Figure 4 As shown. Experimental results indicate that in most test groups using SA or HA thickeners, the potency remained at 50% or higher relative to the potency on day 1 until day 8.

[0120] [Table 6]

[0121] Preparation Examples Remark 1. SA+Tre 10% 0.5% sodium alginate, 10% trehalose 2. SA+Tre 15% 0.5% sodium alginate, 15% trehalose 3. HA + Tre 10% 0.5% hyaluronic acid, 10% trehalose 4. HA+Tre 15% 0.5% hyaluronic acid, 15% trehalose 5. HA+Tre 20% 0.5% hyaluronic acid, 20% trehalose 6. HA+Met 10mM 0.5% hyaluronic acid, 10mM methionine 7. HA+Met 20mM 0.5% hyaluronic acid, 20mM methionine 8. HA+Met 30mM 0.5% hyaluronic acid, 30mM methionine 9. PVP + Tre 10% 1% polyvinylpyrrolidone, 10% trehalose 10. PVP + Met 20mM 1% polyvinylpyrrolidone, 20mM methionine 11. HAS + NaCl 0.5% human serum albumin, 0.9% sodium chloride 12. NaP 25mM sodium phosphate, pH 6.0

[0122] In addition, to test the long-term stability of the combination of PVP and HEC thickeners and disaccharide or amino acid stabilizers, botulinum toxin-coated compositions were prepared in the same manner as the SA and HA tests, and each composition was subjected to accelerated stability testing at 37°C for 5 weeks after dropwise addition. Trehalose (Tre) and / or sucrose (Suc) were used as disaccharide stabilizers, and methionine (Met) and glycine (Gly) were used as amino acid stabilizers. To accurately compare the assay values, the potency measured immediately after each sample preparation was converted to (100%), and the potency recovery rate was compared over 5 weeks. Furthermore, a standard product was used as a positive control. The value was chosen to minimize the error between tests. The results are shown in Table 7 below. Figure 5 As shown.

[0123] [Table 7]

[0124] Preparation Examples Test immediately after preparation 1 day 8 days 22 days 36 days 1% PVP 100.0 92.3 71.2 61.1 N / D 1% PVP + 10% Tre 100.0 107.9 85.1 70.2 57.2 1% PVP + 30% Suc 100 239.5 154.7 154.7 68.1 1% PVP+Met 20mM 100.0 108.4 105.9 87.7 55.0 1% PVP+Gly 20mM 100 79.1 N / D N / D N / D 0.5% HEC 100.0 80.7 65.2 N / D N / D 0.5% HEC + 10% Tre 100.0 92.5 88.9 66.0 53.4 0.5% HEC + 30% Suc 100.0 81.6 55.7 58.9 53.1 0.5% HEC+Met 20mM 100.0 104.0 59.9 N / D N / D 0.5% HEC+Gly 20mM 100 83.2 N / D N / D N / D HSA + NaCl 100.0 112.7 86.0 61.5 45.6 Sodium phosphate (pH 6.0) 100.0 105.2 89.4 67.9 -

[0125] Experimental results confirmed that, compared to the positive control HSA, the optimal stabilizing combination for preparing botulinum toxin-coated compositions was the use of PVP or HEC as thickeners and disaccharides or methionine as stabilizers.

[0126] Example 6. Confirmation of coating uniformity of the coating composition containing botulinum toxin

[0127] Uniform coating of the composition onto the substrate is crucial for botulinum toxin-containing compositions intended for use on substrates such as microneedles, as well as for the potency stability of the botulinum toxin within the composition. Therefore, a 2% PVA pre-coated substrate was coated using a composition containing a thickener and stabilizer, which was evaluated in Example 5 as having excellent botulinum toxin potency stability. Coating uniformity was assessed using average pixel count and magenta percentage. More specifically, images were set to CMYK instead of RGB, the needle size was adjusted uniformly in each image, the background (excluding the needle tip) was removed, the entire area of ​​the needle tip was measured, and the pixel count and magenta percentage of the stained area in the needle tip were measured. The results are shown in Tables 8 and 9. Figure 6 As shown.

[0128] [Table 8]

[0129] Preparation Examples Average number of pixels HEC+Suc, dip the packing once, immersion for 10 seconds. 392 PVP+Tre, soak the packing once, immersion for 10 seconds. 366 HEC+Suc, dip packing once, immersion for 15 seconds. 605 PVP+Tre, soak the packing once, immersion for 15 seconds. 402

[0130] [Table 9]

[0131]

[0132]

[0133] Based on the experimental results, when the microneedles were immersed in each composition for 10 or 15 seconds, the HEC+Suc combination showed a higher average pixel count compared to the PVP+Tre combination in all cases. Furthermore, when the coating time was the same, the percentage of magenta area relative to the total microneedle area in the HEC+Suc combination was significantly higher (HEC+Suc combination: average 89%, PVP+Tre combination: average 71%). These results indicate that the HEC+Suc combination achieved uniform coating compared to the PVP+Tre combination.

[0134] Example 7. Confirmation of the optimal mixing ratio of HEC+Suc combination

[0135] As can be seen from Examples 5 and 6, when coated on the microstructure, the HEC+Suc combination exhibits superior viscosity and stability compared to other combinations of thickeners and stabilizers. Therefore, experiments were conducted to determine the optimal mixing ratio of the HEC+Suc combination.

[0136] Example 7-1. Confirmation of viscosity and surface tension of HEC+Suc combinations at various mixing ratios.

[0137] As shown in Table 10, the mixing ratio of HEC and Suc was set at w:w. The remaining portion, excluding HEC and sucrose, was filled with distilled water and stirred at 200-400 rpm for 1 hour or more. The resulting product was sent to the Korean Polymer Testing Laboratory for analysis, where its viscosity and surface tension were measured.

[0138] [Table 10]

[0139]

[0140] During the preparation of compositions using HEC+Suc combinations in various mixing ratios, a stirrer was used to prepare HS0160, HS0130, HS0110, HS0101, and HS1001 into homogeneous solutions. However, for the HS3001 ratio, the viscosity increased dramatically, potentially causing the magnetic rod to fail to stir, thus hindering the successful preparation of the composition. Therefore, the HS3001 and HS6001 formulations were excluded from viscosity and surface tension testing. During preparation, the HS1001 solution was excluded from surface tension measurement experiments because its viscosity was too high compared to other formulations, making surface tension measurement impossible.

[0141] The regression analysis results of viscosity and surface tension measurements are as follows: Figure 7 and 8 As shown. A one-way ANOVA analysis of the viscosity measurement results showed that a higher sucrose content correlated with higher viscosity (R² = 96.54%, P = 0.000, viscosity = 39.53 + 0.8806 sucrose). Surface tension measurements, also analyzed using one-way ANOVA, showed that a higher sucrose content correlated with lower surface tension (R² = 87.27%, P = 0.000, surface tension = 64.838 - 0.05101 sucrose).

[0142] Example 7-2. Confirmation of coating uniformity of HEC+Suc combinations at various mixing ratios.

[0143] Based on the viscosity and surface tension measurement results of Example 7-1, the mixing ratio of the HEC+Suc combination was set as shown in Table 11 below to detect the coating uniformity.

[0144] [Table 11]

[0145]

[0146] The coating uniformity test was performed in the same manner as in Example 6. However, while formulations HS0101, HS0110, HS0130, HS0160, HS0170, and HS0180 were easily prepared, the preparation quality of formulation HS0190 was uncontrolled, resulting in a small amount of sucrose precipitating from the saturated solution the day after preparation, rendering the formulation unusable. Images of coatings at other ratios are shown below. Figure 9 As shown, image analysis confirms that the dye coating patterns are irregular at HS0101 to HS0130 ratios. With increasing sucrose content, the dye coating becomes more uniform, and the dye color becomes more concentrated.

[0147] The results of regression analysis on the relative standard deviation of the number of pixels in the captured image are as follows: Figure 10 As shown in the figure. Experimental results confirm that the number of pixels in the coated portion is relatively large (5529) in HS0101, which has the lowest sucrose ratio, while the number of pixels is smallest (3682) in HS0110, which has the second lowest sucrose ratio. Subsequently, the number of pixels gradually increases with the sucrose mixing ratio, reaching a maximum of 5967 pixels in HS0170. Then, with the increase of sucrose content, the number of pixels tends to decrease again (HS0180 = 5207, HS0190 = 4955). In addition, regression analysis results confirm that the sucrose mixing ratio makes the coating pattern between needles in the patch more uniform.

[0148] Example 7-3. Testing the long-term stability of HEC+Suc combinations at various mixing ratios.

[0149] To confirm whether the HEC:Suc combination compositions at each mixing ratio could maintain botulinum toxin potency on PLLA patches, botulinum toxin was measured and applied to PLLA patches, followed by a 5-week accelerated stability assessment at 37°C. For this purpose, 2× formulations of the HEC:Suc combination as shown in Table 12 were prepared, mixed 1:1 with botulinum toxin, and uniformly sprayed onto PLLA patches pretreated with 2% PVA solution, followed by complete drying at 37°C in an incubator. PLLA patches were collected on days 0, 1, 8, 22, and 36 to measure botulinum toxin potency. Potency measured immediately after each sample preparation was based on 100% conversion, and potency recovery rates were compared over 5 weeks. Results are shown in Table 13 and... Figure 11 As shown.

[0150] [Table 12]

[0151]

[0152] [Table 13]

[0153] Preparation Example (Name) Immediately after preparation Day 1 Day 8 Day 22 Day 36 HS0170 100.0 88.4 53.6 57.0 61.6 HS0160 100.0 73.0 72.1 79.7 75.8 HS0130 100.0 52.1 42.0 33.3 41.9 Negative 100.0 58.0 42.9 47.6 38.4

[0154] The experimental results show that at an HS0150 ratio or lower, the botulinum toxin potency pattern on day 36 is similar to that of the negative control. When the HEC:Suc ratio is 1:30, the botulinum toxin potency pattern appears similar to the negative control. In the 1:60 and 1:70 experimental groups, the botulinum toxin potency remained at 60% or higher (based on day 36) compared to the negative control. These results confirm that the mixing ratio of hydroxyethyl cellulose (HEC) to sucrose plays a crucial role in the stability of botulinum toxin. It can be seen that the HEC+Suc ratio for stabilizing botulinum toxin should exceed 1:30.

[0155] As described above, specific portions of the specification have been described in detail. Although those skilled in the art will appreciate that this particular technique is merely a preferred embodiment, the scope of the specification is not limited thereto. Therefore, the substantial scope of the specification will be defined by the appended claims and their equivalents.

[0156] [Industrial Applicability]

[0157] This invention relates to a microstructure preparation technique for botulinum toxin, wherein the microstructure of this invention is used to reduce pain during botulinum toxin administration and facilitates the precise application of small amounts of toxin at precise sites. Therefore, this invention holds promise for widespread application in safe and convenient medical settings.

Claims

1. A composition for coating microstructures comprising botulinum toxin, hydroxyethyl cellulose and sucrose, wherein the mixing ratio of hydroxyethyl cellulose and sucrose is w:w, and the amount of sucrose is 60 to 80 relative to 1 part hydroxyethyl cellulose.

2. The composition according to claim 1, wherein the botulinum toxin is one or more selected from the group consisting of botulinum toxins of type A, type B, type C, type D, type E, type F and type G.

3. The composition according to claim 1, wherein the microstructure is one or more selected from the group consisting of microneedles and microblades.

4. A microstructure coated with the composition of claim 1.

5. The microstructure according to claim 4, wherein the microstructure is one or more selected from the group consisting of microneedles and microblades.

6. A method for preparing a composition for coating microstructures, comprising: Botulinum toxin, hydroxyethyl cellulose, and sucrose are mixed. The mixing ratio of hydroxyethyl cellulose and sucrose is w:w, where the amount of sucrose is 60 to 80 per part of hydroxyethyl cellulose.

7. The method of claim 6, wherein the botulinum toxin is one or more selected from the group consisting of botulinum toxins of type A, type B, type C, type D, type E, type F and type G.

8. The method of claim 6, wherein the microstructure is one or more selected from the group consisting of microneedles and microblades.

9. A method of coating a microstructure with a composition containing botulinum toxin, comprising: (a) A mixture of botulinum toxin, hydroxyethyl cellulose, and sucrose; and (b) Coating the microstructure surface with the mixture from (a), The mixing ratio of hydroxyethyl cellulose and sucrose is w:w, where the amount of sucrose is 60 to 80 per part of hydroxyethyl cellulose.

10. The method of claim 9, wherein the botulinum toxin is one or more selected from the group consisting of botulinum toxins of type A, type B, type C, type D, type E, type F and type G.

11. The method of claim 9, wherein the microstructure is one or more selected from the group consisting of microneedles and microblades.

Images