KR20170141068A - A pharmaceutical composition comprising botulinum toxin for treating thermal pain - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising botulinum toxin as an effective component for treating heat-induced pain. The pharmaceutical composition for the treatment of heat-induced pain of the present invention is expected to be widely used in the medical field to improve the quality of life of a patient by being effective in alleviating heat-induced pain upon peripheral or central injection.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition comprising botulinum toxin for treating thermal pain comprising botulinum toxin as an active ingredient,

The present invention relates to a pharmaceutical composition for treating heat-induced pain comprising botulinum toxin as an active ingredient.

Pain is an unpleasant sensory or emotional experience that can be described as actual, potential tissue damage or damage, adverse effects, and the like. Pain allows us to avoid potential danger situations, protects the damaged body parts until they are recovered, and allows us to avoid situations that may arise in the future. Pain is caused by stimulation of sensory receptors located in the peripheral nervous system, or by damage or dysfunction of the peripheral nervous system or central nervous system. Sensory receptors can be categorized according to position, shape, or adaptation rate, depending on the stimulus, among which temperature receptors contain crouze bodies and respond to temperature change stimuli such as hot or cold. In general, humans do not accept the temperature of 18 to 43 캜 as the temperature to feel the pain, but sometimes the sensation receptive neurons are found to have a potential higher than the threshold value even in the temperature range of 18 to 43 캜. The allodynia caused by pain caused by a strong temperature stimulation of 17 ° C or lower or 44 ° C or higher or an abnormality occurring within a temperature range of 18 to 43 ° C is referred to as heat-induced pain or heat pain. Among the categories of pain, there is little known about heat-induced pain. In 1999, Eewards and Fillingim studied ethnic differences in the response to heat-induced pain (Ethnic Differences in Thermal Pain Responses, RR EDWARDS AND RB FILLINGIM, Psychosomatic Medicine 61: 346-354, 1999) In the present study, we used the fMRI to assess the changes of the brain during hypersensitivity stimulation (FMRI of Thermal Pain: Effects of Stimulus Laterality and Attention, Jonathan CW Brooks et al., NeuroImage 15, 293-301 2002) There are very few researches on the mechanism of the onset and the treatment.

On the other hand, botulinum toxin is a kind of neurotoxin produced by Clostridium botulinum, which acts on the endogenous nerve endings of neurons in mammals and inhibits the extracellular excretion of synaptic vesicles. This poison does not affect the synthesis or storage of transporters and the conduction of action potentials. It consists of a heavy chain of 100 kDa and a light chain of 50 kDa, folded into one SS bond. The heavy chain is necessary because this venom specifically binds to the neural membrane and the light chain enters the cell. In the cell, the reduced light chain after bisected by proteolytic enzyme acts as a peptide internal hydrolase with Zn ^{2 +} . The extracellular excretion is mediated by the NSF-SNAP (N-ethyl-maleimide-desensitized fusion protein-soluble NSF attachment protein) complex involved in the membrane fusion in the cell, suggesting that the synaptic vesicle is required to bind to the cell membrane do. There is synaptobrevin as a receptor on the synaptic vesicle, and there are synaptic and SNAP25 on the cell membrane side. These toxins are classified into seven groups A to G by serotypes, A and E being SNAP25, B, D and F being synaptobrevin and C being specific to syntactic, It was found that the extracellular excretion of the lost action was stopped.

Accordingly, the present invention relates to a pharmaceutical composition for treating heat-induced pain comprising botulinum toxin as an active ingredient, and the pharmaceutical composition of the present invention is expected to be effectively utilized for medical use because it effectively alleviates heat-induced pain.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it relates to a microstructure formulation technique for botulinum toxin.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

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

In one embodiment of the present invention, " Botulinum Toxin " is a neurotoxin protein produced by Clostridium botulinum bacteria. The Clostridium genus is more than 127 species and is classified according to its form and function. The anaerobic, gram-positive bacterium Clostridium botulinum produces botulinum toxin, a potent polypeptide neurotoxin that causes botulism in humans and animals. Spores of Clostridium botulinum are found in soil and can be cultured in sealed, home-canned food containers that are not properly sterilized, which causes many botulism. Symptoms of botulism typically occur 18 to 36 hours after eating food from Clostridium botulinum culture or spore-infected food. The botulinum toxin appears to pass through the intestinal lumen without decreasing its toxicity and exhibits high affinity for cholinergic motor neurons. Symptoms of botulinum toxin poisoning may include symptoms of walking, dysphagia and speech disorders, respiratory muscle paralysis and death.

Botulinum toxin type A is the most deadly natural biologic agent known to humans. The LD50 of commercially available botulinum toxin type A (purified neurotoxin complex) is about 50 picograms (i.e., one unit). Interestingly, on a molar basis, botulinum toxin type A is 1.8 billion times more damaging than diphtheria, 600 million times more damaging to sodium cyanide, 30 million times more damaging than cobra toxin, and 12 million times more damaging than cholera. Botulinum toxin 1 unit (U) can be defined as an LD50 via intraperitoneal injection in female Swiss Webster mice weighing 18-20 g each.

Seven botulinum neurotoxins, which are generally immunologically distinctive, are characterized by their respective neuropeptide serotypes A, B, C1, D, E, F and G, which are differentiated by type-specific antibodies. The different vertical types of botulinum toxin differ depending on the species of animal and the degree and duration of paralysis they cause. For example, botulinum toxin type A was measured to be 500 times more potent than botulinum toxin type B when measured by ratios occurring in rats. In addition, botulinum toxin type B was determined to be non-toxic when administered to primates at 480 U / kg, which is about 12 times that of botulinum toxin type A primate LD50. Botulinum toxin binds with strong affinity to cholinergic motor neurons, and enters neurons and is thought to inhibit the release of acetylcholine. Can be further absorbed through low affinity receptors as well as by phagocytic and acellular effects.

Regardless of the vertical type, the molecular mechanism of toxic poisoning is similar and appears to consist of at least three stages. In the first step of the process, the specific interaction of the cell surface receptor with the heavy chain (H chain or HC) of the toxin binds the toxin to the synaptic membrane of the target neuron, which is the botulinum toxin type and the tetanus It is considered different depending on each type of toxin. It is believed that the carboxyl end fragment of the heavy chain, Hc, is important for targeting the toxin to the cell surface.

In the second step, the toxin passes across the plasma membrane of the poisoned cell. Initially, botulinum toxin is enveloped by cells through receptor-mediated endocytosis, resulting in endosomes containing toxins. The toxin then exits the endosome and enters the cytoplasm of the cell. This step is believed to be mediated by the amino terminal fragment, HN, of the heavy chain that causes structural changes in the toxin at about pH 5.5 or lower. Endosomes are known to have proton pumps that reduce endogenous internal pH. The structural displacement can expose the hydrophobic residues of the toxin, allowing the toxin to be surrounded by the endosomal membrane. The toxin (or at least light chain) is then displaced to the cytosol through the endosomal membrane.

The last step in the botulinum toxin activation mechanism is believed to involve the reduction of disulfide bonds linking the heavy and light chains. The total toxic activity of botulinum and tetanus toxins is included in the light chain of holotoxin, light chains are recognized, and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, It is a zinc (Zn ⁺⁺ ) endopeptidase that selectively cleaves the protein necessary for fusion. Tetanus neurotoxins, botulinum toxin types B, D, F and G cause the degradation of synaptobrevin (or vesicle-associated membrane protein (VAMP)) and synaptosome membrane proteins. VAMP present on the cytoplasmic surface of synaptic vesicles is largely eliminated as a result of one of these degradation actions. Vertical types A and E cut SNAP-25. Vertical type C1 was originally thought to cleave syntactic ginsenosides but was found to cleave syntactic and SNAP-25. Except for type B (and tetanus toxin) that cleaves the same linkage, each toxin specifically cleaves a different linkage. Each such cleavage interferes with the process of vesicle-membrane docking and blocks exocytosis of vesicle contents.

Botulinum toxin has been used clinically for the treatment of neuromuscular disorders characterized by activity-mediated skeletal muscles (i. E., Movement disorders). In 1989, the botulinum toxin type A complex was approved by the US Food and Drug Administration to be used in the treatment of essential blepharospasm, strabismus, and hemifacial spasm. Subsequently, botulinum toxin type A was also approved for the treatment of cervical dilatation and the treatment of wrinkles from the FDA, and botulinum toxin type B was also approved for the treatment of cervical dilatation. Botulinum serotypes other than toxin type A appear to have lower potency and / or shorter duration of activity as compared to botulinum toxin type A. The clinical effect of injected botulinum toxin type A in the peripheral intima usually appears within one week of injection. The typical duration of symptom relief by intramuscular injection of once-a-bottle botulinum toxin type A is on average about 3 months, although therapeutic activity for a significantly longer period has been reported.

Although all botulinum toxin serotypes seem to inhibit the release of the neurotransmitter acetylcholine at neuromuscular junctions, they act on different neurotransmitter proteins and / or they cleave the protein at different sites. For example, botulinum types A and E all cleave 25 kD synaptosome related protein (SNAP-25), but target different amino acid sequences of this protein. Botulinum toxin types B, D, F and G act on vesicle-associated proteins (VAMP, so-called synaptobrevin), each of which truncates different regions of the protein. Finally, botulinum toxin type C1 appears to cleave both syntactic and SNAP-25. This differential mechanism of action will affect the relative potency and / or duration of action of various botulinum toxin serotypes. In particular, substrates for the botulinum toxin can be found in a variety of different cell types.

The molecular weight of the botulinum toxin protein molecule is about 150 kD in all seven known botulinum toxin longitudinal types. Interestingly, by clostridial bacteria, the botulinum toxin is released as a complex comprising a 150 kD botulinum toxin protein molecule with the associated non-toxic protein. Therefore, the botulinum toxin type A complex can be produced by Clostridium bacteria, in the form of 900k, 500Dk and 300kD. Botulinum toxin types B and C1 appear to be produced only as a 700 kD or 500 kD complex. Botulinum toxin type D is produced as a 300 kD and 500 kD complex. Finally, botulinum toxin types E and F are generated only as a 300 kD complex. These complexes (i.e., those with a molecular weight greater than about 150 kD) are believed to include non-toxic erythrocyte aggregate proteins and non-toxin and non-toxic non-red cell aggregate proteins. These two non-toxin proteins (including related neurotoxin complexes along with botulinum toxin molecules) will serve to provide stability to botulinum toxin molecule denaturation and protection against peptic acid when the toxin is ingested. In addition, the greater the botulinum toxin complex (molecular weight greater than about 150 kD), the more slowly the botulinum toxin will diffuse from the site of injection of the botulinum toxin complex.

Also, in vitro studies have shown that botulinum toxin inhibits potassium cation-induced release of acetylcholine and norepinephrine from primary cell cultures of brain stem tissue. In addition, the botulinum toxin inhibits the induced release of glycine and glutamate in primary cultures of spinal cord neurons and inhibits the release of glycine and glutamate from the brain synaptosomal specimens in the presence of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP, substance P and glutamate Lt; / RTI > Thus, when used at moderate concentrations, the stimulatory-induced release of most neurotransmitters is inhibited by the botulinum toxin.

Botulinum toxin type A can be obtained by culturing a culture of Clostridium botulinum in a fermentation tank and then harvesting and purifying the fermented mixture by a known method. Initially, all botulinum toxin longitudinal types are synthesized as inactive single chain proteins, which must be nicked and nerve activated by proteases. The bacterial strains that produce botulinum toxin serotypes A and G carry an endogenous protease, and therefore, types A and G can be recovered primarily from the bacterial culture as an active form. In contrast, botulinum toxin serotypes C1, D, and E are synthesized by non-degradable strains and are therefore typically inactivated when recovered from the culture. Since serotypes B and F are produced by both proteolytic and non-degradable strains, they may be recovered in an active or inactive form. However, for example, a proteolytic strain that produces the botulinum toxin type B serotype only cleaves a portion of the resulting toxin. The exact ratio of nicked molecules to non-nicked molecules depends on the incubation time and the temperature of the culture. Therefore, as is already known, botulinum toxin type B toxins will be inactive, for example, in some specimens of a given percentage, as botulinum toxin type B is much less effective than botulinum toxin type A. The presence of an inert botulinum toxin molecule in a clinical sample will contribute to the overall protein load of the sample, which is associated with an increase in antigenicity without contributing to clinical effects. Also, at the same dose level, botulinum toxin type B is known to have a shorter duration of activity and less efficacy in muscle injection than botulinum toxin type A.

A high quality crystalline botulinum toxin having a characteristic of having a band pattern of ≥3 × 10 ⁷ U / mg from Hall A strain of Clostridium botulinum, having a A260 / A278 of not more than 0.60, and exhibiting a unique band pattern in gel electrophoresis Type A can be obtained. The known Shantz process can be used to obtain crystalline botulinum toxin type A. In general, botulinum toxin type A complexes can be isolated and purified from anaerobic fermentations cultured with Clostridium botulinum type A in a suitable medium. Such a known process may also include, for example: purifying botulinum toxin type A having a molecular weight of about 150 kD and a specific potency of 1-2 x 10 ⁸ LD50 U / mg or more; Purifying botulinum toxin type B having a molecular weight of about 156 kD and a specific potency of 1-2 x 10 ⁸ LD50 U / mg or more; Can be used to isolate pure botulinum toxins from non-toxin proteins, such as purifying botulinum toxin type F having a molecular weight of about 155 kD and a specific potency of 1-2 X 10 ⁷ LD50 U / mg or greater.

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

As with enzymes in general, the biological activity of botulinum toxins (which are intracellular peptidases) depends, at least in part, on their three-dimensional structure. Therefore, botulinum toxin type A can be eliminated by toxicity by heat, various chemicals, surface scratching and surface drying. It is also known that when the toxin complexes obtained by known cultivation, fermentation and purification are diluted to very low toxin concentrations and used in pharmaceutical formulation preparations, the toxin toxicity is rapidly eliminated without suitable stabilizing agents. Diluting large quantities with a solution containing a few nanograms per milliliter is quite difficult because of the rapid loss of specific toxicity. The toxin must be stabilized using a suitable stabilizing agent since it can be used for several months or years after producing the pharmaceutical composition containing the toxin. Therefore, as in the present invention, development of an optimal stabilizer technology is essential for controlling the release of botulinum toxin in vivo in a sustained release form.

As described below, the use of botulinum toxin type A in clinical settings has been reported.

The typical duration of intramuscular injection in a botulinum toxin administered in a single dose in vivo is typically about 3 to 4 months. However, in some cases, the A subtype may persist for more than 12 months (European J. Neurology 6 (Supp 4): S111-S1150: 1999), when used in the treatment of glands such as hyperhidrosis, Can last for 27 months.

Botulinum toxin not only exhibits pharmacological action at the peripheral site, but can also inhibit the central nervous system. Weigand et al., Nauny-Schmiedeberg ' s Arch. Pharmacol. 1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg ' s Arch. Pharmacol. 1974; 281, 47-56 show that botulinum toxin can be traced back to the spinal cord area by retrograde transport. Thus, for example, a botulinum toxin administered intramuscularly at a peripheral site can be retrograde to the spinal cord.

The botulinum toxin can also be used to treat a variety of autonomic nervous system dysfunctions (US Pat. No. 5,766,605 and Goldman (2000), Aesthetic Plastic Surgery (US Pat. No. 6,447,787), intrathoracic pain (see US Patent 6,113,915) (U.S. Patent No. 6,458,365), migraine pain (U.S. Patent No. 5,714,468), post-surgical pain and visceral pain (U.S. Patent No. 6,464,986), hair growth and hair (U.S. Patent No. 6,299,893), psoriasis and dermatitis (U.S. Patent No. 5,670,484), injured muscles (U.S. Patent No. 6,423,319), various cancers (U.S. Patent Nos. 6,139,845 and 6,063,768), smooth muscle disorders (U.S. Patent No. 5,437,291), nerve entrapment syndrome (U.S. Patent No. 6,063,768), eye diseases (see U.S. Patent No. 6,265,379), pancreatic diseases (see U.S. Patent Nos. 6,143,306 and 6,261,572); Prostate diseases including prostate hyperplasia, prostate cancer and urinary incontinence (see U.S. Patent Nos. 6,365,164 and 6,667,041 and Doggweiler R. et al. Botulinum toxin type A causes diffuse and highly selective atrophy of rat prostate, Neurourol Urodyn 1998; 17 (4): 363 ); (US Pat. No. 6,623,742), Isoge's syndrome (see Childers et al. (2002), American Journal of Physical Medicine & Rehabilitation, 81: 751-759).

U.S. Patent No. 5,989,545 may be used to treat pain by administering to a spinal cord a modified Clostridium neurotoxin, or a fragment thereof, preferably botulinum toxin, chemically conjugated, recombinantly fused to a specific target moiety . It is also disclosed that a targeted botulinum toxin (i.e., having a non-native binding moiety) can be used in treating various diseases (WO 96/33273; WO 99/17806; WO 98/07864; WO 00 / RTI > WO 01/21213; WO 00/10598).

In addition, techniques for controlling the thoracic spasm by injecting botulinum toxin into the pectoral muscles have been known (Senior M., Botox and the management of pectoral spasm after subpectoral implant insertion, Plastic and Recon Surg, July 2000, 224-225), transcutaneous botulinum toxin Controlled release toxin implants (see U.S. Patent Nos. 6,306,423 and 6,312,708) are known, as is the case for administration (U.S. Patent Application No. 10 / 194,805). Tullinum toxin is used to treat the wound and resulting ulcer by weakening the biting muscles of the mouth (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), treatment of benign gallbladder injuries or tumors (Blugerman G., et al., Multiple eccrine hydrocystomas: a new therapeutic option with botulinum toxin, Dermatol Surg 2003 May; 29 (5): 557-9), treating the teeth (Jost W., Ten years' experience with botulinum toxin in anal fissure, Int J Colorectal Dis 2002 Sep; 17 (5): 298-302) In order to treat atopic dermatitis (Heckmann M., et al. Botulinum toxin type A injection in the treatment of Lichen simplex: An open pilot study, J Am Acad Dermatol 2002 Apr; 46 (4): 617-9) ≪ / RTI >

It has also been reported that botulinum toxin nerve inhibition can lead to a reduction in epithelial thickness (Li Y, et al., Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin, Exp Neurol 1997; 147: 452-462). Finally, sweating of excessive foot (Katsambas A., et al., Cutaneous diseases of the foot: Unapproved treatments, Clin Dermatol 2002 Nov-Dec; 20 (6): 689-699; Sevim, S., et al, Botulinum toxin (A), A., A., Local Botulinum toxin type A injections in the treatment of spastic toes, Am. J Phys Med Rehabil 2002 Oct; 81 (10): 770-5), idiopathic toe walking (Tacks, L., et al., Idiopathic toe walking: Treatment with botulinum toxin A injection, Dev Med Child Neurol 2002; 91): 6), and foot dystonia (Rogers J., et al., Injections of botulinum toxin A in foot dystonia, Neurology 1993 Apr; 43 (4 Suppl 2) Is known.

Tetanus toxin and its derivatives (i.e., with non-natural target residues), fragments, hybrids and chimeras can also be used for treatment. Tetanus toxins are very similar to botulinum toxins. Thus, tetanus toxin and botulinum toxin are all polypeptides produced by Clostridium (Clostridium tetany and Clostridium botulinum, respectively) of closely related species. In addition, both tetanus toxin and botulinum toxin are dual chain proteins composed of light chains (molecular weight about 50 kD) covalently linked to a heavy chain (molecular weight about 100 kD) by a single disulfide bond. And, the molecular weight of the tetanus toxin and each of the seven botulinum toxins (non-complex) is about 150 kD. Further, in both tetanus toxin and botulinum toxin, light chains include domains with intracellular biological (protease) activity, while heavy chains include receptor binding (immunogenicity) and cell membrane potential domains.

In addition, both tetanus toxin and botulinum toxin exhibit a large, specific affinity for the ganglioside receptor on the surface of pre-synaptic cholinergic neurons. Due to receptor-mediated endocytosis of the tetanus toxin by peripheral cholinergic neurons, axillary dendritic transport, blockade of inhibitory neurotransmitter release from the central synapse, and spastic paralysis occur. In contrast, receptor-mediated endocytosis of the botulinum toxin by peripheral cholinergic neurons causes little inhibition of acetylcholine exocytosis and relaxation paralysis from retrograde transport, poisoned peripheral motor neurons.

Finally, the tetanus toxin and the botulinum toxin resemble each other in both biosynthesis and molecular organization. Thus, tetanus toxin and botulinum toxin type A are 34% identical throughout the protein sequence and 62% identical in some functional domains (Binz T. et al., The Complete Sequence of Botulinum Neurotoxin Type A and Comparison with Other Clostridial Neurotoxins, J Biological Chemistry 265 (16); 9153-9158: 1990).

In one embodiment of the present invention, " acetylcholine " is the first neurotransmitter found as an ester of choline and acetic acid. It is distributed throughout the neurons and has the formula C ₇ H ₁₆ NO ₂ , with a molecular weight of 146.21.

There is evidence to suggest that some neurotransmitter can be released by the same neuron, but typically in the mammalian system, only a single form of small molecule neurotransmitter is released by each type of neuron. This neurotransmitter, acetylcholine, is involved in the neurons of various parts of the brain, especially the large pyramidal cells of the motor cortex, several different neurons of the cerebral nucleus, motor neurons distributed in the skeletal muscle, ganglia of the autonomic nervous system (both sympathetic and parasympathetic) Previous neurons, bag 1 fibers of the spinal fibers, postganglionic neurons of the parasympathetic nervous system, and some postnegative neurons of the sympathetic nervous system. Originally, most post-ganglion neurons in the sympathetic nervous system secrete norepinephrine, norepinephrine, whereas only the sympathetic nerve fibers after the ganglion to the sweat glands, the paraplegia and some blood vessels are cholinergic. In most cases, acetylcholine exhibits an exciting effect. However, it is known that acetylcholine has an inhibitory effect on some peripheral parasympathetic nerve terminals (e. G., Inhibition of vasopressing by the vagus nerve).

The efferent signal of the autonomic nervous system is transmitted to the body through the sympathetic or parasympathetic nervous system. The preganglionic neurons of the sympathetic nervous system extend from the ganglion neuron cell body located at the medial lateral angle of the spinal cord. The preganglionic sympathetic nerve fibers extending from the cell body synapse with the posterior ganglion neurons located in the perierves sympathetic ganglia or the spinal ganglia. Because the preganglionic neurons of the sympathetic and parasympathetic nerves are all cholinergic, applying acetylcholine to the ganglia will excite neurons after sympathetic and parasympathetic ganglia.

Acetylcholine activates two types of receptors, muscarinic receptors and nicotinic receptors. Muscarinic receptors are found in all effector cells stimulated by post-ganglion neurons of the parasympathetic nervous system and post-ganglionic cholinergic neurons of the sympathetic nervous system. Nicotinic receptors are found in the autonomic ganglia on the cell surface of postganglionic neurons in synapses between preganglionic and postganglionic neurons as well as adrenal medulla, both sympathetic and parasympathetic. Nicotinic receptors are also found in the membranes of many non-autonomic nerves, for example skeletal muscle fibers of neuronal junctions.

Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with pre-synaptic neuronal cell membranes. Extensive non-neuronal secretory cells, such as the adrenal medulla (also the PC12 cell line) and pancreatic islet cells, release catecholamines and parathyroid hormones from large, large densecore vesicles, respectively. The PC12 cell line is a clone of rat chromaffin cell line extensively used as a tissue culture model for the study of sympathoadrenal develpoment. Botulinum toxin inhibits the release of both types of compounds in both cell types in vitro by allowing the toxin to permeate into the denervated cells (by electroporation) or by direct injection. Botulinum toxin is also known to inhibit the release of glutamate, a neurotransmitter, from synaptosomal cell cultures of the cortex.

The neuromuscular junction is formed in the skeletal muscle by the peripheral axons of muscle cells. Signals transmitted through the nervous system become action potentials in the distal axons and activate the ion channels. As a result, for example, in the motor end plate of the neuromuscular junction, the neurotransmitter acetylcholine from neuronal synaptic vesicles . Acetylcholine binds to the acetylcholine receptor protein on the surface of the muscle endplate across the extracellular space. Once fully conjugated, the action potential of the muscle cell causes a specific membrane ion channel change, which results in muscle cell contraction. The acetylcholine is then released from the muscle cells and metabolized by the cholinesterase in the extracellular space. Metabolite products are recovered again as apical axons for regeneration with acetylcholine.

In one embodiment of the present invention, the "central" means a passage through which the central nervous system passes in an individual. In the present specification, the central cerebral cortex represents the cerebellum, the chin, and the cranial membrane.

In one embodiment of the present invention, " peripheral " is a passage through which a peripheral nerve passes in an individual. In the present specification, " peripheral " means subcutaneous.

In one embodiment of the present invention, the term " cerebellomedullary cistern " refers to a place where the subterranean space is enlarged between the lower part of the cerebellum and the back of the breathing brain. It is the widest of the descending diaphragm. From the ceiling of this lumen, the outer wall is made from the crust of the cerebellum extending from the top of the cerebellum to the tongue tail, and the floor is the smoke film of the fourth ventricle. The vessel is punctured and the cerebrospinal fluid is collected (the larynx).

In one embodiment of the present invention, the term " atlanto-occipital membrane " refers to a membrane surrounding two joints and is rich in elastic fibers. Two joints are connected to the first cervical vertebra and the posterior bones. The joints are protrusions on the lateral sides of the occipital laryngeal larynx. The joints are the upper joints of the cholesteatoma. Since the shape is the hypoglossal joint, the motion of this joint can be anterior, posterior and lateral bending. The membranes are divided into two groups: pre- and post-transfection.

In the present invention, " dure membrane " is a kind of meninges. The brain surrounding the brain consists of a membrane on the third layer, which surrounds the brain outside the brain in the skull. It is also called the dura mater or dura. The hard membrane wrinkles into the intracranial space, dividing the brain into several zones. Generally, the hard film corrugation is composed of a two-ply hard film layer. The cerebral sickle, cerebellar tent, cerebellar sickle, and saddle barriers are the wrinkles of the dura mater. The dementia of the brain has many sensory and autonomic nerves. Sensory nerves are the tertiary nerves and the first to third hard nerves, and most of the autonomic nerves are distributed in the meningeal blood vessels. Because the brain itself does not have sensory nerves and does not feel pain, most headaches originate from nerves distributed in the brain or blood vessels.

In one embodiment of the present invention, the term " sensory receptor " refers to the primary receptors that transmit sensations in response to stimulation of the environment within and outside the organism, at the sensory nerve endings. Sensory receptors transmit stimuli to the central nervous system by creating differential potentials or action potentials in the same cell or adjacent cells when stimulated by the senses. Sensory receptors are classified according to ① a stimulus, ② position, ③ type, or ④ adaptation speed.

Specifically, according to a fit stimulus, a baroreceptor responds to pressure on the blood vessel, a chemical receptor responds to chemical stimulation, an electromagnetic radiation receiver reacts to electromagnetic waves, an infrared receiver reacts to infrared rays, The receptacle reacts to visible light, the ultraviolet receptor reacts to ultraviolet light, the electric receptor reacts to the electric field, the magnetoreceptors respond to the magnetic field, the mechanical receptors respond to physical flux and deformation, nociceptor responds to threats to injure or injure body tissues, the osmotic receptor responds to the osmotic concentration of the solution, the proprioceptor responds to the posture sensation, and the temperature receptor responds to hot or cold.

Depending on the location, the skin receptors present in the skin include skin mechanoreceptors, temperature receptors, nociceptors, and chemoreceptors, and skin mechanoreceptors include rupinis, meister, pachinis, , The lupini body accepts the kidney stimulation of the skin, the meister body senses the slow vibration, the stroking, the texture change, the pachini body senses the strong pressure and the rapid vibration, and the Merkel disc has constant pressure and texture Lt; / RTI > The temperature receptor contains the corpuscle bodies and senses temperature changes. Apart from the skin, muscles have a unique receptor that is involved in skeletal muscle reflexes, which are mechanical receptors in representative muscles. When the muscle contracts or relaxes, it sends information about the muscle length to the spinal cord and brain. In addition, the olfactory tendon organ responds to the muscle tension that causes contraction of the length, and the joint receptors are activated by distortions that vary with the location of the bones.

③ Depending on the shape, the somatosensory receptors near the surface of the skin are divided into two main classes: the end of the neuron is a dehydrated, free nerve ending capsule and a receptive receptor.

Depending on the rate of adaptation, tonic receptors and phasic receptors can be distinguished. Tension receptors are sensory receptors that adapt slowly to stimuli. Continuously generate action potentials during the duration of the stimulus so that information about the duration of the stimulus can be communicated. Some tonic receptors are always active. Pain receptors, capsules, and receptors located in the muscular spine are typical stress receptors. Phased receptors are sensory receptors that adapt quickly to stimuli. For stimulation, cells respond and adapt quickly, so they can not provide information about the duration of the stimulus. Instead, you can communicate information about the intensity and speed changes of rapidly changing stimuli. An example of a phase sensitive receiver is a pachinizer.

In one embodiment of the invention " pain " means an unpleasant sensory or emotional experience that can be described as actual, potential tissue damage or damage, adverse effects, and the like. Pain allows us to avoid potential danger situations, protects the damaged body parts until they are recovered, and allows us to avoid situations that may arise in the future. Pain is caused by stimulation of sensory receptors located in the peripheral nervous system, or by damage or dysfunction of the peripheral nervous system or central nervous system. Most pain disappears as the stimulus disappears or the body 's damage recovers, but sometimes it lasts until then. In addition, there is no stimulation, and there is pain in some cases even if there is no pathological cause. On the other hand, it is reported that the pain is not born naturally. Pain is a major symptom in many medical conditions and can have a profound effect on human life and basic functioning.

Pain can be classified into physical pain, mental pain, and fantasy pain. Physical pain means that it is caused by physical change. Mental pain means that it is caused by a change of mind. Body pain can be divided into sensory pain caused by activation of the sensory organs and neural pain caused by the nervous system. Sensory pain is the process by which peripheral nerve fibers respond to stimuli received in sensory receptors. Sensory pain is caused by thermal stimuli (hot or cold), mechanical irritants (bumps or tears), or chemical irritations (when disinfecting the wound area or red pepper powder in the eyes). Neurogenic pain is caused by damage or dysfunction of the nervous system. It is largely divided into peripheral nervous system abnormality and central nervous system abnormality. Neurogenic pain refers to allodynia, which is not explained by a single etiology or a specific site of injury, but also by pain that continues to develop into a sensory disorder or a nontoxic stimulus below the threshold. Neuralgia may last for a long time or may occur intermittently, such as an electric shock. Sensory pain is tingling or numbness, while neuralgia patients express the pain as itching, numbness, numbness, burning sensation, pain, stinging with a needle. About 7% to 8% of the population are suffering from this pain, and 5% of them are reported to have severe pain. In particular, in cancer patients, the tumor often suffers from a peripheral nervous system stress or a side effect of chemotherapy, radiotherapy, or surgery. Mental pain is referred to as psychalgia or somatoform pain and is a pain that occurs as the pain is caused by mental, emotional, and behavioral factors and gradually increases and continues. Occasionally, somatic pain such as headache, back pain, or abdominal pain is diagnosed as a result of mental pain. Psychiatric pain is rather rare in patients with mental illnesses and is more common in modern people, and is more frequently induced in emotional events such as feeling socially alienated, injured, or sad in life . Mental pain is caused by the inability to express stress or emotional conflict, and is caused by psychosocial problems or various mental problems. Some scholars have interpreted this pain as a protective process to prevent anger from being unconsciously emitted. People who have had long-term pain often have mental disorders such as hysteria, depression, and hypochondriasis. Scholars have argued that neuroses resulting from extreme injuries lead to chronic illnesses, and it is reported that chronic diseases can lead to neurosis. High neurotic triad, anxiety, and low self-esteem are gradually reduced and returned to normal levels in the course of chronic disease recovery by medical treatment. A phantom tube is a kind of neurogenic pain due to the absence of a part of the body or the pain that the brain feels no longer receiving a signal for stimulation. The phantom tube is a common symptom in amputation patients, and one study found that the amputation started in 8 days after amputation and that 72% of the amputees experienced this experience. Six months later, 65% of patients continued to experience fantasy. The fantasy barrel is described as a stinging sensation, a crushing burden, a burning sensation, and a feeling of cramps. If this pain persists for a long period of time, the whole body may be sensitive to stimuli, and touching may cause hallucinations and may cause urination or defecation. Local anesthetics may be given to sensitive areas near the nerve or amputation site to relieve pain from days to weeks or permanently, but only during the duration of the effect of the anesthetic.

Pain can also be classified as peripheral pain and central pain according to neurotransmission mechanisms. Peripheral pain is most often caused by partial impairment of tissue adjacent to a particular nerve fiber, a pain stimulus. These changes in tissues release chemicals that activate the intracellular fibers in the skin to act on the nerve endings, such as neuropeptides, serotonin, and histamine. In analgesics studies these materials are considered very important. The action potential generated from neurons by the combination of chemicals is linked to the dorsal horn of the spinal cord, which synapses in the spinal cord to stimulate pain fibers to secrete glutamate, substance P (Substance P). Thus, the pain is transmitted to the brain's thalamus. Pain fibers are divided into two types, one enclosed by myelin fibers (Aα, Aβ, Aδ) and one not enclosed (C) depending on the axon type. These are classified into skeletal muscle receptors, , Pain and temperature receptors, pain, temperature, and itching receptors. Each fiber has different characteristics depending on the type of stimulus as well as its diameter, conduction velocity, and threshold level. Studies on capsaicin have supported this theory of pain fibers characterized by stimulation. Scientists studying capsaicin-responsive receptors, an ingredient that makes pungent taste when consumed in pepper, have found that this receptor also responds to a sudden rise in temperature. This receptor is called vanilloid receptor 1 (VR1). VR1 receptor-deficient mice responded to mechanical stimulation pain but did not respond to stimulation by heat or capsaicin. According to this, the reason why it feels hot when you eat spicy red pepper can be explained by capsaicin contained in red pepper activates VR1 receptor. Absorption of capsaicin into the skin also alleviates pain in the joints, suggesting that the neurotransmitters were temporarily depleted as the pain fibers became hyperactive.

Central pain in the central nervous system is transmitted along a specific pathway. Afferent fibers of the peripheral nerves that transmit neuroinformation activate cells using a transmitter such as glutamate, which is a neuromodulator that secretes neuropeptide P, which is a neuropeptide in the spinal cord do. For example, when capsaicin is injected into the skin, certain pain stimuli result in the release of substance P in the spinal cord, which is transmitted as it binds to receptors in post-synaptic neurons. The steps are as follows. Pain information is transmitted rapidly by Aδ fibers wrapped in myelin, slowly transmitted through C fibers without myelin, and synapses form in the spinal cord. ② The intersection of the axons of neurons in the posterior part of the stomach occurs, and the stimulation rises along the spinal cord through the training and pneumothorax. ③ Other pain information on the pons is transmitted to the brainstem area, which controls the behavior of the pain, such as screaming. ④ The pain information from the midbrain is transmitted to many regions of the thalamus and cerebral cortex. ⑤ Pain information activates the cingulate cortex area by forming a synapse again in the whole brain.

In one embodiment of the present invention, " thermal pain " means pain caused by temperature stimulation. In general, humans do not accept the temperature of 18 to 43 캜 as the temperature to feel the pain, but sometimes the sensation receptive neurons are found to have a potential higher than the threshold value even in the temperature range of 18 to 43 캜. Thus, in the present specification, heat-induced pain or heat pain includes allodynia caused by pain caused by strong temperature stimulation of 17 DEG C or lower or 44 DEG C or higher, or abnormality occurring within a temperature range of 18 to 43 DEG C .

In one embodiment of the present invention, " Iba1 (Ionized calcium binding adapter molecule 1) " is a marker marker protein of microglia.

In one embodiment of the present invention, " GFAP (glia fibrillary acidic protein) " is a marker marker protein of astrocyte.

&Quot; Pharmaceutical composition " in one embodiment of the present invention means a composition to be administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention is to administer a composition comprising botulinum toxin as an active ingredient into a living body, and may include a protein involved therein, a pharmaceutically acceptable carrier, an excipient or a diluent. The above "pharmaceutically acceptable" carrier or excipient as referred to above means approved by the regulatory agency of the government or listed in government or other generally approved pharmacopoeias for use in vertebrates, and more particularly in humans do.

Compositions comprising botulinum toxin as an active ingredient suitable for parenteral administration may be in the form of a suspension, solution or emulsion in an oily or aqueous carrier, and may be prepared in the form of a solid or semi-solid, , Solubilizing agents and / or dispersing agents. This form can be sterilized and liquid. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria or fungi. Alternatively, a composition comprising the botulinum toxin as an active ingredient may be in the form of a sterile powder for reconstitution with a suitable carrier before use. The pharmaceutical composition may be in unit-dose form, in a micro needle patch, in an ampule, or in other unit-dose containers, or in multi-dose containers. Alternatively, the pharmaceutical composition may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, e. G., A vehicle immediately prior to use. Immediately, the injection solutions and suspensions may be prepared from sterile powders, granules or tablets.

In some non-limiting embodiments, a composition comprising botulinum toxin as an active ingredient may be formulated as a liquid, or may be included in the form of a microsphere in a liquid. In a non-limiting embodiment, a composition comprising botulinum toxin as an active ingredient comprises a botulinum toxin, or a pharmaceutically acceptable compound and / or mixture thereof, at a concentration of between 0.001 and 100,000 U / kg . In addition, in certain non-limiting embodiments, suitable excipients for compositions comprising botulinum toxin as an active ingredient include preservatives, suspending agents, stabilizers, dyes, buffers, antimicrobial agents, antifungal agents, and isotonic agents, for example, . As used herein, the term "stabilizer" refers to a compound that is optionally used in a pharmaceutical composition of the invention to increase shelf life. In a non-limiting embodiment, the stabilizer is a sugar, amino acid, . The pharmaceutical compositions may comprise one or more pharmaceutically acceptable carriers. The carrier may be a solvent or a dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols), oils, and suitable mixtures thereof.

Parenteral formulations can be sterilized. Non-limiting examples of sterilization techniques include filtration through a bacteria-inhibiting filter, sterilization of the terminals, incorporation of sterile preparations, irradiation, sterilization gas irradiation, heating, vacuum drying and freeze drying.

In one embodiment of the present invention, " administration " means introducing the composition of the present invention to a subject by any suitable method, and the administration route of the composition of the present invention may be administered via any conventional route ≪ / RTI > Intravenous, intramuscular, subcutaneous, intracutaneous, intranasal, intrapulmonary, intrathecal, intracavitary, intraperitoneal, and intrathecal administration may be made, but the botulinum toxin of the present invention In the case of a pharmaceutical composition containing the toxin as an active ingredient, injection administration is preferable.

The method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount. In the present invention, the effective amount may be determined depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, body weight, general health condition, sex and diet, And the fraction of the composition, the duration of the treatment, the drug being co-administered, and the like.

In one embodiment of the invention, there is provided a pharmaceutical composition for treating pain comprising botulinum toxin as an active ingredient, wherein the pain is a heat-sensitive pain, wherein the heat-sensitive pain is less than or equal to 17 & Wherein the heat-sensitive pain provides a pharmaceutical composition that is allodynic due to a temperature stimulation of 18 to < RTI ID = 0.0 > 43 C < / RTI > and wherein the heat-sensitive pain is a pain caused by laser stimulation Wherein the botulinum toxin provides a pharmaceutical composition that is an A toxin, and wherein the botulinum toxin is administered at a concentration of 0.1 to 10 U / kg.

In another embodiment of the present invention, there is provided a pharmaceutical preparation comprising the above pharmaceutical composition, wherein the pharmaceutical preparation is in the form of a tablet, pill, powder, capsule, syrup or emulsion Wherein the pharmaceutical preparation further comprises at least one member selected from the group consisting of a pharmaceutically acceptable carrier, a reinforcing agent and an excipient, wherein the pharmaceutical preparation is administered by injection And a pharmaceutically acceptable carrier.

Hereinafter, the present invention will be described in detail.

Pain is caused by stimulation of sensory receptors located in the peripheral nervous system, or by damage or dysfunction of the peripheral nervous system or central nervous system. Most pain disappears as the stimulus disappears or the body 's damage recovers, but sometimes it lasts until then. In addition, there is no stimulation, and there is pain in some cases even if there is no pathological cause. Pain is a major symptom of many medical conditions, but medical efforts are being made to alleviate pain as much as possible, since it can have a profound effect on human life and functioning. However, there are very few researches on the mechanism and mechanism of the heat - induced pain caused by the temperature change stimulation.

The present invention relates to a pharmaceutical composition for treating heat-induced pain comprising botulinum toxin as an active ingredient, and the pharmaceutical composition of the present invention is expected to effectively utilize for medical use because it effectively alleviates heat-induced pain.

Brief Description of the Drawings Fig. 1 is a graphical representation of pain relief effects of botulinum toxin as a central or peripheral site in a heat-affected pain patient according to an embodiment of the present invention. Fig.
FIG. 2 is an immunohistochemical staining diagram showing changes in microglial cells and astrocytes in normal or heat-sensory pain-expressing individuals according to an embodiment of the present invention.
FIG. 3 is a view showing immunohistochemical staining patterns of microglial cells and astrocytes in the case of botulinum toxin peripherally administered to a heat-induced pain patient according to an embodiment of the present invention.
FIG. 4 is a diagram showing immunohistochemical staining patterns of microglia and astrocytes in the case of administration of a botulinum toxin as a backbone to a heat-induced pain patient according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One. Heat pain  Manufacture of animal models

Experimental animals were male Sprague-Dawley (230-280 g), and animals were allowed to freely feed and water for laboratory animals in an animal room of Kyungpook National University with a constant temperature and a 12-hour light / And fed. This study was approved by the Experimental Animal Committee of the Graduate School of Dentistry of Kyungpook National University and complied with the ethical regulations of the World Institute of Pain Research on conscious animal experiments.

Experimental animals were anesthetized with 3% isoflurane and mixed with CFA oil (Sigma-Aldrich, St. Louis, MO) and physiological saline at a ratio of 1: 1 to induce inflammatory chronic pain in the facial area And injected subcutaneously in the area of 40 [mu] l. When CFA is injected subcutaneously into the facial area, heat-sensitive pain occurs from the first day and continues until the 10th day after the injection. On the 14th day, CFA is restored to the same level as before CFA injection.

Example  2. Heat pain  Botulinum toxin administration to animal models

Example  2-1. Botulinum toxin administration to the backbone

The drug was injected into the cerebellum, the choroid, or the central region of the cranial nerves to inject facial sensory information into the projected trigeminal nucleus group. A catheter was inserted into the experimental animal to inject the drug into the backbone. Anesthetized rats were anesthetized with ketamine (0.5 ml / kg) and the head was fixed to a brain fixator (David Kopf Instruments, Tujunga, CA, USA), and a polyethylene tube (PE10) Intestinal, or intracranial menstrual region. The polyethylene tube was pulled to the skull and secured to the head using metal screws and dental resin (Dentsply Caulk, Milford, USA). The animals were restored for 72 hours postoperatively.

Botulinum toxin (0.3 or 1 U / kg) was injected centrally into the intubated polyethylene tube. Botulinum toxin type A (Botulex, Hugel, Korea) was diluted in physiological saline.

Example  2-2. Botulinum toxin administration to the periphery

To block neurotransmitters secreted from the nerve end, 30 μl of botulinum toxin (1 or 3 U / kg) was injected into the facial skin subcutaneous tissue.

Example  3. Heat pain  Identification of botulinum toxin administration effects on animal models

Example  3-1. Result of facial avoidance test

For the evaluation of heat pain, one animal was placed in a transparent plastic observation bin designed to allow the animal to move freely by pulling its neck to receive thermal stimulation. Animal experiments were carried out after stabilization for at least 30 minutes in a quiet place without being bright. Thermal stimulation was applied at a 90 ° angle from the skin 10 cm away from the skin using a laser stimulator (Infrared Diode Laser, LVI-808-10, LVI tech, Seoul, Republic of Korea). The power and current of the laser stimulation were fixed to 11 W and 18.1 A, and the head withdrawal latency was measured by applying heat stimulus to the facial area and then avoiding facial stimulation. Stimulation was performed twice at 5-minute intervals and the mean value was calculated. The cut-off time was set to 20 seconds to prevent tissue damage.

The result of performing the facial avoidance reaction test is shown in Fig. As a result of the test, the delayed time to facial avoidance was increased in all the groups treated with botulinum toxin, regardless of the place of botulinum toxin administration, either peripheral or central, than CFA alone or injected with CFA and saline appear. In addition, facial avoidance delay was found to be longer as the concentration of administered botulinum toxin was higher. This suggests that botulinum toxin treatment has the effect of alleviating pain caused by thermal stimulation.

Example  3-2. Immunohistochemical staining results

On the 5th day when CFA was injected and pain was highest, heat pain was evaluated in experimental animals (n = 5 per group) to evaluate changes in microglial cells and astrocytes, followed by ketamine 0.5 ml / kg . Anesthetized animals were perfused with 0.9% physiological saline and fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH 7.4). The horn horn was removed and fixed with the same fixative at 4 ° C for 2 hours. The postfixed tissue was placed in a 30% sucrose solution and infiltrated at 4 ° C for 24 hours. After the tissue was frozen, a 30 μm-thick slice was prepared using a round-trip machine and reacted in 2.5% horse serum (horse serum blocking solution, Vector Laboratories, Burlingame, CA, USA) for 1 hour. The tissues were incubated at 4 ° C with rabbit anti-Iba1 (1: 2000, Wako, Osaka, Japan) and mouse anti-GFAP (glial fibrillary acidic protein, 1: 5000, Cell Signaling, Danvers, MA, USA) for 24 hours. The cells were reacted with anti-mouse or anti-rabbit secondary antibody (ImmPRESS reagent kit, Vector Laboratories) and developed with DAB solution (3,3'-diaminobenzidine, Vector Laboratories). Colored sections were observed with a microscope (BX 41 and U-RFL-T, Olympus, Tokyo, Japan) and photographed. Image analysis was performed using I-solution (Innerview Co, Seongnam, Republic of Korea) program. Statistical analysis of the results of immunohistochemical staining was carried out by student t-test to verify the significance of the responses. For statistical comparison, the standard value of statistical significance was set at P <0.05. All results were expressed as mean ± standard error (SEM).

The results of immunohistochemical staining are shown in Figs. 2 to 4. Fig. 2, in comparison with the naive group in which no treatment was performed, the group administered with CFA (vehicle) showed a significant increase in Iba1 as a microcytic marker and GFAP as a astrocytic marker (P <0.05) . These changes were mainly distributed in the caudal nucleus of the same side injected with CFA and mainly distributed in the lamina I and II regions. In addition, the expression of Iba1, a microglial cell marker, and GFAP, an astrocytic cell marker, in the experimental animals injected with a botulinum toxin at a dose of 3 U / kg as subcutaneous tissue compared to the group administered with CFA (vehicle) (P < 0.05, FIG. 3) when compared to a control group. The botulinum toxin group also significantly inhibited the expression of Iba1 and GFAP by CFA injection (P <0.05).

The results of this study showed that the injection of CFA into the facial skin increased the expression of Iba1, the microglia cell marker, and GFAP, the astrocytic cell marker, in the trigeminal nerve spinal cord sensory nucleus. Increased expression of Iba1 and GFAP increased the expression of botulinum Lt; RTI ID = 0.0 &gt; toxin. &Lt; / RTI &gt; These results indicate that the analgesic action induced by botulinum toxin injected into the peripheral or central nervous system suppresses the activity of the glial cells in the central nervous system and that this regulation of neuroglial cells is important for the relief of heat-induced pain .

Claims (11)

A pharmaceutical composition for treating pain comprising botulinum toxin as an active ingredient.
The method according to claim 1,
Wherein said pain is a heat-induced pain.
3. The method of claim 2,
Wherein said heat-sensitive pain is pain caused by temperature stimulation below 17 &lt; 0 &gt; C or above 44 &lt; 0 &gt; C.
3. The method of claim 2,
Wherein said heat-sensitive pain is allodynia due to a temperature stimulation of 18 to &lt; RTI ID = 0.0 &gt; 43 C. &lt; / RTI &gt;
3. The method of claim 2,
Wherein said heat-sensitive pain is pain caused by laser stimulation.
The method according to claim 1,
Wherein the botulinum toxin is type A toxin.
The method according to claim 1,
Wherein the botulinum toxin is administered at a concentration of 0.1 to 10 U / kg.
A pharmaceutical preparation comprising the pharmaceutical composition of claim 1.
9. The method of claim 8,
Wherein the pharmaceutical preparation is in the form of tablets, pills, powders, capsules, syrups or emulsions.
9. The method of claim 8,
Wherein the pharmaceutical preparation further comprises at least one member selected from the group consisting of a pharmaceutically acceptable carrier, a reinforcing agent and an excipient.
9. The method of claim 8,
Wherein the pharmaceutical preparation is administered by injection.

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