KR101448155B1 - Composition containing 3-methyladenine for inhibiting or treating neuropathic pain - Google Patents

Abstract

The present invention relates to a composition containing 3-methyladenine (3-MA) as an effective component for inhibiting or treating neuropathic pain. The 3-methyladenine according to the present invention inhibits an autophagic reaction of a gamma-aminobutyricacid (GABA) neuron by attentively inhibiting revelation of LC3 and becline 1 in a neuron of dorsal horn, and effectively inhibits pain by hindering signal transmission of pain which induces overexcitabilities of the dorsal horn, thereby being useful for inhibiting or treating neuropathic pain.

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting or treating neuropathic pain comprising 3-methyladenine as an active ingredient. BACKGROUND ART Composition containing 3-methyladenine for inhibiting or treating neuropathic pain

The present invention relates to a composition for inhibiting or treating neuropathic pain comprising 3-methyladenine (3-MA) as an active ingredient.

Pain is the most common cause for patients to visit the hospital. Pain is a medical imperative that must be resolved because it can ruin an individual's life and lead to social and economic losses. Most medical illnesses are accompanied by pain as a major symptom. Depending on the etiology of each disease, different treatments should be chosen for the treatment of pain. For example, abdominal pain due to appendicitis can only be suppressed by surgically removing the inflamed cecum, and joint pain caused by arthritis can be suppressed only by the use of anti-inflammatory analgesics and physical therapy. On the other hand, severe pain caused by nerve damage (for example, neuropathic pain) is not controlled by conventional pain suppression method, and a new treatment method is required.

Pain is a phenomenon that is felt by physical stimulation or neural stimulation, and there is a difference in pain level depending on subjective experience. Pain is similar in many respects to neurobiological responses such as learning and memory. Pain is recognized in certain areas of the central nervous system. Pain generally has a mechanism of pain caused by pain, that is, a peripheral stimulus (harmful) that is transmitted to the central nervous system, but is not always triggered by pain associated with pain. There are various types of broad clinical pain, which are known to be caused by different underlying pathological mechanisms.

Pain can be classified into acute pain, chronic pain, and neuropathic pain according to the origin of cognition.

Neuropathic pain (NPP) is a complex intractable chronic pain caused by nerve damage and abnormal neurological function, and it is known to cause persistent pain due to a disease already existing by neurological disorders, unlike acute pain. In addition, neuropathic pain is mostly associated with tissue damage, damaging the nerve fibers themselves, and causing nerve damage or injury. Neuropathic diseases are known to be caused by trauma, inflammation, ischemic injury, and nerve damage caused by metabolites.

Neuropathic pain is allodynia, which complains of pain even in the presence of hairy stimuli; Hyperalgesia, which shows increased pain to the painful stimulus; It is known that these symptoms are more complex symptoms rather than a single symptom, such as stinging pain, stinging symptoms, and electrical anomalies that cause an abnormal sensation.

Neuropathic pain is divided into peripheral neuropathic pain and central neuropathic pain. Peripheral neuropathic pain is caused by damage or infection of peripheral sensory nerves such as nerve damage, trigeminal neuralgia, sensory neuropathy, chronic back pain, post-operative pain, and central neuropathic pain is caused by brain / spinal trauma, stroke, Is known to be caused by damage to the central nervous system (CNS) and / or spinal cord such as sclerosis. On the other hand, peripheral neuropathic pain and central neuropathic pain can be induced without definite initial nerve damage, and the underlying pathological mechanism is not constant.

In the International Society of Pain Medicine (IASP, Seattle, Washington, USA), peripheral neuropathic pain was defined as a pain initiated or induced by a primary injury or disorder of the peripheral nervous system, and central neuropathic pain was classified as primary injury Or pain initiated or induced by a disorder.

Peripheral neuropathic pain or central neuropathic pain is not only distinguished by the anatomical location of the injury site, but also by the mechanism of pain. In other words, neuropathic pain is difficult to treat because there is no clear relationship between the specific pain symptoms or the mechanism of action between drugs and effects.

The treatment of neuropathic pain is currently combined with medication, sympathetic nerve therapy, nerve stimulation, and psychotherapy, among which medication is said to be the most effective. However, drug therapy has problems such as toxicity and side effects due to drug resistance, burden due to high cost, and development of new drugs is continuously required. In addition, neuropathic pain can not be suppressed in spite of these various complex treatment methods, and it can be suffered from unbearable pain for a lifetime. In this case, the patient becomes an extreme choice of depression, social isolation, suicide, etc., resulting in social problems.

Currently, neuropathic pain inhibitors include anticonvulsants such as gabapentin, pregabalin, which act on the calcium channel; Anticonvulsants such as carbamazepine acting on the sodium channel; Serotonin norepinephrine reuptake inhibitor (SNRI) antidepressants; And opioids, which are recommended only in emergencies, are all clinically used, and they are all known to be involved only in response to pain. Most of these inhibitors are used in combination, and all of them cause similar side effects, so that the use of the inhibitor for a long period of time may deteriorate the condition of the patient. In addition, the use of such inhibitors has a problem that the therapeutic effect of pain is relatively low as compared with the risk of side effects.

Because neuropathic pain suppressants must be administered to the patient over a long period of time, the toxicity and side effects of the drug itself should be low and oral administration possible. Therefore, it is urgent to develop new medicines and functional foods which are excellent in inhibiting neuropathic pain and have less toxicity and side effects.

On the other hand, apoptosis is a process of predetermined cell death, and it is known that it plays an important role in maintaining various physiological functions such as homeostasis, development, and cell differentiation normally. In addition, apoptosis is known to cause an in vivo defense function to remove virus-infected cells and malignant cells such as cancer. The disorder or failure of apoptosis is known to act as a pathological cause for various diseases such as cancer, degenerative neurological diseases, immune disorder diseases and the like. The autophagy activity, which is closely related to the apoptosis process, refers to the action of the cell itself to decompose unnecessary wastes, proteins, and cell organelles in the cell using lysozyme, which is a digestive enzyme.

The inventors of the present invention found that 3-methyladenine was found to inhibit neuropathic pain in LC3 and Becklin 1 in neurons of spinal cord olfactory, while solving the above problems and having less toxicity and side effects and inhibiting neuropathic pain Inhibited the autophagic action of gamma-aminobutyricacid (GABA) -sensitive neurons and inhibited the signal transduction of the pain inducing hyperactivity of the spinal cord, confirming the excellent effect of inhibiting pain , Thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for inhibiting or treating neuropathic pain containing 3-methyladenine as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating neuropathic pain containing 3-methyladenine as an active ingredient.

The present invention provides a pharmaceutical composition for inhibiting or treating neuropathic pain comprising 3-methyladenine as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating neuropathic pain containing 3-methyladenine as an active ingredient.

The 3-methyladenine of the present invention significantly inhibits the expression of LC3 and beclin 1 in neurons of the spinal cord, thereby suppressing the self-stimulation of GABA (gamma-aminobutyricacid) neurons, And inhibits the signal transmission of the induced pain, so that the effect of suppressing the pain is excellent, so that it can be usefully used for suppressing or treating neuropathic pain.

FIG. 1 is a graph showing the results of measurement of pain thresholds (PWTs) of SNL (spinal nerve ligation) treatment group and sham treatment group according to the POD after spinal nerve ligation according to the present invention. (Expressed as the mean ± SD of 50% of the pain threshold, normalized to n = 6, *** P <0.001 for each animal's baseline).
FIG. 2 is a graph showing immunohistochemical analysis of the expression of LC3 and Becklin 1 in the spinal cord olfactory of the SNL treatment group and the sham treatment group according to the present invention at 14 days after the spinal nerve ligation surgery (Scale bar is Aa -d, 50 탆 for Ba-d, and 20 탆 for Ae, f and Be, f).
FIG. 3 is a graph showing quantitative analysis of LC3 and Becklin 1-IR cells in the spinal cord olfactory of the SNL-treated group according to the present invention by image analysis using a dark-field microscope ((*, P <0.05) , Mean ± standard deviation, n = 6, paired t-test).
Figure 4 shows the expression of LC3 and Becklin 1 in the SNL-treated group according to the present invention by double immunofluorescence staining analysis using LC3 and Becklin 1 as an antibody against NeuN as a neuron marker and GFAP as an astrocytic marker Fig.
Figure 5 shows the expression of LC3 in the SNL-treated group according to the present invention by double immunofluorescence staining analysis using an antibody against LC3 as a microglia cell marker Iba1 and colonic polyposis coli (APC) And Fig.
FIG. 6 is a graph showing the results of double immunofluorescence staining analysis (scale bar: 20 μm) of LC3 and Bechlin 1-IR neurons in the SNL-treated group according to the present invention.
FIG. 7 is a graph showing quantitative analysis of LC3, Bechlin 1 and calretinin IR cells of the SNL-treated group according to the present invention at 14 days after surgery of spinal nerve ligation (mean ± SD, mean Differences were analyzed by paired t-test, * P <0.05, ** P <0.01, *** P <0.001).
FIG. 8 is a graph showing the results of measuring the pain thresholds (PWTs) according to the POD after spinal cord ligation after 3-methyladenine was injected into the SNL-treated group according to the present invention (pain threshold Values are expressed as mean ± standard deviation of 50% of values, normalized to n = 6, * P <0.05, *** P <0.001 for each animal's baseline).

The present invention provides a composition for inhibiting or treating neuropathic pain comprising 3-methyladenine as an active ingredient.

The composition comprises a pharmaceutical composition and a food composition.

Hereinafter, the present invention will be described in detail.

The 3-methyladenine of the present invention significantly suppresses the expression of LC3 and beclin 1 in neurons of the spinal cord, thereby suppressing the self-stimulation of GABA (gamma-aminobutyricacid) neurons, And inhibits the signal transmission of the induced pain, so that the effect of suppressing the pain is excellent, so that it can be usefully used for suppressing or treating neuropathic pain.

The neuropathic pain preferably includes, but is not limited to, central neuropathic pain and peripheral neuropathic pain.

The composition of the present invention may contain at least one known active ingredient having an effect of preventing or treating neuropathic pain together with 3-methyladenine.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the 3-methyladenine of the present invention may be administered in an amount of 0.001 mg / kg to 10000 mg / kg per day, and may be administered once a day or divided into several times.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of neuropathic pain.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The composition of the present invention may be added to a health functional food for the purpose of preventing or improving neuropathic pain. When 3-methyladenine of the present invention is used as a food additive, the 3-methyladenine may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the 3-methyladenine of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments and examples of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples and preparative examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and preparation examples.

[Example 1] Preparation of co-localized animal model causing neuropathic pain by L5 spinal nerve ligation (SNL)

Sprague-Dawley male rats (rats, 180-200 g, Koatech, Korea) were used as experimental animal models for neuropathic pain (NPP) And were individually housed in cages in circulating environmental conditions. All experiments were conducted with approval from the Animal Care and Use Committee of the National Chungnam National University and were conducted in accordance with the ethical guidelines of the International Association of Pain Research and the National Institute of Health. One hour before the experiment, the animals were separated from the breeding environment and transported to the laboratory. Water and food were fed unlimitedly until transported to the laboratory. The rats were anesthetized by administering sodium pentobabital (50 mg / kg ip) and the left lumbar vertebra (L) 6 transversus was carefully removed to identify the L4 and L5 spinal nerves Respectively. The left L5 spinal nerve was lined with 3-0 silk, and after complete seizure was confirmed, the wound was sutured and spinal nerve ligation (SNL) induced neuropathic pain Animal models were prepared (SNL treated group). In addition, all the procedures except spinal nerve ligation were performed in the same manner as the SNL-treated group to produce an animal model without spinal nerve ligation (sham-treated group).

[Example 2: Identification of mechanical allodynia by spinal nerve ligation in an animal model causing neuropathic pain]

1. Assessment of pain sensitivity by spinal nerve ligation

In order to confirm the induction of seasonal allodynia by spinal nerve ligation in an animal model in which neuropathic pain was induced, the paw withdrawal thresholds for the SNL treatment group and the sham treatment group prepared in Example 1 were measured. PWTs (or degree of painfulness) were measured according to post-surgery day (POD).

Specifically, the PWTs were measured using an up-down testing paradigm (Chaplan, SR, Bach, FW, Pogrel, JW, Chung, JM, Yaksh, TL, 1994. Quantitative assessment of tactile allodynia in the rat paw . In the SNL-treated group, spinal cord nerve ligation was performed on the day before surgery, on the day of surgery, at 3 days, 7 days, 10 days, and 14 days after surgery using the Neurosci Methods. In the sham-treated group without ligature surgery, it was measured on the day of surgery. The Von Frey hairs (39337500, Touch Test ™ Sensory Evaluator Kit of 20) were tested with the force of log units (0.007, 0.016, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, ) Were applied to the posterior lateral part of the hind paw corresponding to the fifth segment of the lumbar spine of the white rat for 4 seconds to 6 seconds, respectively, and mechanical stimulation was applied to measure the PWTs. First, mechanical stimulation of 2 g log units was applied. When a positive avoidance response was identified, a small bona fide hair was used, and when the negative avoidance response was confirmed, the experiment was performed with a higher force applied to measure the degree of hypersensitivity to the pain. The results are shown in Fig.

As shown in FIG. 1, the SNL-treated group and the sham-treated PWTs of the present invention were measured according to the post-surgery day (POD) of spinal nerve ligation, The PWTs of the ipsilateral side were significantly lower than those of the reference (n = 6). On the other hand, sham - treated group showed no significant change in PWTs compared to standard. In addition, in the SNL treatment group of the present invention, the PWTs on the ipsilateral side of the POD 7 day were significantly lower than those of the sham treatment group (ipsilateral and contralateral side), and it was confirmed that the PWTs were maintained until 14 days.

Therefore, it was confirmed that mechanical allodynia was caused by spinal nerve injury in the ipsilateral side because the PWTs on the ipsilateral side were significantly lowered by spinal nerve ligation surgery.

2. Statistical analysis

Behavioral studies and immunohistochemical analyzes were statistically analyzed using Paired t-test. Values were expressed as mean ± standard deviation, with significance set at P <0.05. Statistical software package SigmaStat (Systat, San Jose, Calif.) Was used to perform all statistical analyzes.

[Example 3] In the neuropathic pain-induced animal model, the spinal nerve ligation LC3 and Becklin 1]

 In order to examine the expression of LC3 and Becklin 1 in the spinal cord olfactory by spinal nerve ligation in an animal model inducing neuropathic pain, immunohistochemical analysis was performed on SNL treatment group and sham treatment group prepared in Example 1 above Were performed on the 14th day after neural ligament surgery.

Specifically, SD male rats were anesthetized by administering sodium pentobarbital (50 mg / kg intraperitoneal injection) and then perfused with heparin phosphate buffer solution (PBS, pH 7.4) by perfusion perfusion. At this time, the phosphate buffer solution was obtained by perfusion with 4% paraformaldehyde for 15 minutes. The lumbar spine (L4-L6) area of the spinal cord was immediately removed, placed in the same fixative overnight, and then placed in paraffin. The 4-μm section of the paraffin-embedded tissue array was rehydrated and deparaffinized in a series of alcohol grades. Antigen was diluted in 0.01 M citrate buffer (pH (pH)) by heating the sample to a microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) 6.0). For immunohistochemical analysis, the activity of endogenous peroxidase was blocked using 0.3% hydrogen peroxide. The sections were treated with protein-blocking solution (Dako, Glostrup, Denmark) for 20 minutes and then incubated for a period of 20 minutes against most LC3 (1: 200, # AP1801a, ABGENT) and Becklin 1 (1: 500, # AP1818a, ABGENT) Specific polyclonal antiserum was incubated overnight at 4 ° C in a humid chamber. After washing with PBS, the tissues were exposed to biotin anti-rabbit IgG and streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, Calif.). Immunostaining was visualized with diaminobenzidine (DAB) and the specimens were fixed using Polymount (Polysciences, Inc., Washington, PA). The results are shown in Fig.

As shown in Fig. 2, immunohistochemical analysis of LC3 and Bechlin 1 expression in the spinal cord olfactory of the rat at 14 days after the neural ligation showed that the expression of both LC3 and Bechlin 1 . In particular, LC3 and Bechlin 1 expression were significantly induced in the superficial lumbar dorsal horn. In addition, SNL treatment group significantly induced LC3 and Becklin 1 expression compared to naive and sham treated groups. On the other hand, naive and sham-treated groups showed low levels of LC3 and beclin 1, and no significant difference was observed between naive and sham-treated groups for LC3 and Becklin 1 expression.

Therefore, the level of LC3 and beclinal 1 protein in the olfactory olfactory margin of the L5 spinal cord was significantly increased by spinal nerve ligation, and it was confirmed that spontaneous predation was induced by ipsilateral spinal nerve injury.

[Example 4] In an animal model inducing neuropathic pain, in the spinal cord nerve ligation, Quantitative analysis of LC3 and Becklin 1-IR cells]

In order to quantitate LC3 and Bechlin 1-immunoreactive cells (IR cells) in the spinal cord olfactory by spinal nerve ligation in an animal model inducing neuropathic pain, a dark field microscope ) (Zeiss Axioscope, Hallbergmoos, Germany) for image analysis of the spinal cord segments.

LC3 and Becklin 1-IR cells were quantified both on the ipsilateral side and on the contralateral side of the spinal cord segment. Quantitative analysis of the LC3 and Becklin 1-IR cells revealed that the gray matter landmark defined each spinal lobe and determined segment levels (Abbadie, C., Besson, JM, 1994. Chronic treatments with aspirin or acetaminophen reduce both the development of polyarthritis and Fos-like immunoreactivity in rat lumbar spinal cord. Pain 57, 45-54.).

Specifically, five spinal segments were randomly selected from the lumbar spinal cord segment, and each slice was analyzed using Axioscope A1 (Carl Zeiss, Germany) connected to a computer-assisted image analysis system (Image J, NIH, USA) Digitized to 4096 gray levels. To maintain a threshold for each image to be measured and to account for subtle differences in immuno staining, background subtraction and shading correction were performed, and then the average gray level of each image was adjusted at least 85% Were measured. The settings for microscope illumination and data collection were fixed through the entire analysis procedure. The mean number of LC3 and Becklin 1-IR cells per slice was obtained from the rats. At this time, the average number obtained was the average for at least 5 rats in each group and presented as group data. LC3 and Becklin 1-IR-positive cells were quantified in various superficial dorsal horn regions (SDH, laminae I and II). This site was identified based on cytoarchitectonic criteria as previously defined. All numerical results related to the IR-positive cell profile in each olfactory region were expressed as number of LC3 and Becklin 1-IR cells per unit area, and the results are shown in FIG.

As shown in Fig. 3, quantitative analysis of LC3 and Becklin 1-IR cells in the spinal cord olfactory of white rat induced neuropathic pain by spinal nerve ligation was carried out using image analysis. As a result, , And the number of LC3 and Becklin 1-IR cells in the spinal cord olfactory bulb was significantly increased compared with the contralateral side. On the other hand, several small numbers of LC3 and Bechlin 1-positive-IR cells were found to be found in the contralateral spinal cord smell.

Therefore, the spinal nerve ligation significantly increased the number of LC3 and Bechlin1 protein IR cells in the ipsilateral L5 spinal cord olfactory.

[Example 5: Measurement of expression of LC3 and Bechlin 1-expressing IR cells by spinal nerve ligation in an animal model causing neuropathic pain]

In order to determine whether colocalized LC3 and Becklin 1 in nerve cells and astrocytes by spinal nerve ligation in an animal model inducing neuropathic pain, antibodies against LC3 and Becklin 1 were used as neuronal marker NeuN, an astrocytic marker, GFAP, and double immunofluorescence staining analysis using an antibody against LC3 as a microglia cell marker Iba1 and a mature rare prominent glomerular cell marker, colonic polyposis coli (APC).

First, double immunofluorescence staining analysis was performed using NeuN, an astrocytic marker, and GFAP, an astrocytic marker, as an antibody against LC3 and beclinc1.

Specifically, to demonstrate the presence of a pair of antigens simultaneously in the same section, rabbit LC3 and beclin 1 used one of the monoclonal antibodies. The monoclonal antibodies were incubated with antiglial fibrillary acidic protein (GFAP, 1: 1000, # AM020, Biogenex), anti-NeuN (anti-NeuN) (1: 100, MAB377, Millipore) Calretinin (1: 500, # 6B3, Swant). As described above, the sections were immunoreactive for LC3 and Becklin 1, but a CY3-conjugated anti-rabbit secondary antibody was used (1: 500, Amersham, Buckinghamshire, UK). Segments were then treated once more for GFAP, NeuN and calretinin immunocyte chemistry and CY2-conjugated anti-mouse secondary antibody (1: 200, Amersham) and 4 ', 6-diamidino- - phenylindole (4 ', 6-diamidino-2-phenylindole; DAPI). Double staining segments were observed using an Axiophot microscope (Carl Zeiss, Oberkochen, Germany). The results are shown in Fig.

As shown in FIG. 4, the expression of LC3 and Bechlin 1 was markedly increased in most neurons, confirming that LC3 and Becklin 1-IR cells were co-localized with NeuN, a neuronal cell marker in the olfactory surface. In addition, the expression of LC3 and beclin 1 was increased in some astrocytes, confirming that some LC3 and Becklin 1-IR cells were co-localized with the astrocytic marker GFAP.

Thereafter, double immunofluorescence staining analysis was performed using the above-described method using Iba1 as a microglia cell marker and LCM (Coliform Polyposis coli) (APC) as an antibody against LC3, and the results are shown in the following Respectively.

As shown in FIG. 5, LC3 expression was significantly increased in most neurons, confirming that LC3 IR-cells were co-localized with Iba1, a microglial cell marker in the olfactory surface. In addition, LC3 expression was increased in some astrocytes, confirming that some LC3 IR-cells were co-localized with APC.

Thus, LC3 and Becklin 1-IR cells were co-localized in most neurons in the neuropathic pain-inducing animal model and co-localized in some minor astrocytes to induce autophagy.

[Example 6: Increased expression of LC3 and Becklin 1 in GABA-mediated neurons of spinal cord olfactory by spinal nerve ligation in an animal model of neuropathic pain]

To determine the type of GABA (gamma-aminobutyricacid) neurons in the spinal cord by spinal nerve ligation in an animal model of neuropathic pain, double immunofluorescent staining for LC3, beclin 1 and calretinin IR cells Quantitative analysis was performed. GABA-mediated interneurons located in the spinal lobes (I) to (III) are neurons involved in pain signaling and hyperactivity activation, and Calretinin is a neuronal cell in the lumbar spine, It was used as a marker for the population.

First, in order to analyze GABAgic neuron types of LC3 and Becklin 1-IR neurons, the same method as in the double immunofluorescence staining assay described in Example 5 above was used to analyze the fluorescence intensity of double immunofluorescence Dyeing analysis was performed. The results are shown in Fig.

As shown in FIG. 6, LC3 and Becklin 1 IR-cells were found to be co-localized with calretinin, a population marker of GABA nerve cells in the surface olfactory.

Thereafter, quantitative analysis on LC3, beclin 1 and calretinin IR cells was carried out in the same manner as the quantitative analysis described in Example 4 above. On the 14th postoperative day of spinal nerve ligation, the number of IR cells of LC3, Bechlin 1 and calretinin, and their mean cell number were measured on the ipsilateral side and the contralateral side of the spinal cord smell. The results are shown in Fig.

As shown in FIG. 7, the number of LC3, beclin 1 and calretinin IR cells in the ipsilateral and contralateral spinal nerves and their average ratios were measured 14 days after the spinal nerve ligation surgery. As a result, calretinin IR GABA- It was confirmed that more than 90% of LC3 and Becklin 1 were expressed in the olfactory flora compared to the other side.

Thus, we confirmed that calretinin IR-GABA-mediated neurons in most neuropathic pain-induced animal models significantly increased the expression of LC3 and Becklin I, leading to self-predation.

[Example 7: Neuropathic pain-inhibiting activity induced by L5 spinal nerve ligation of 3-methyladenine]

To investigate neuropathic pain inhibitory activity of 3-methyladenine (3-MA), an autoregulation inhibitor, 3-MA was administered to experimental animal models of neuropathic pain and PWTs were measured.

Specifically, 30 μg of 3-MA (Sigma, M9281) was dissolved in 10 μl of saline and administered by intrathecal injection to the SNL-treated group prepared in Example 1 above on the 3rd day of POD. Except for 3-MA administration, the whole process was performed in the same manner as in Example 2. [ The results are shown in Fig.

As shown in FIG. 8, when 3-methyladenine was administered to the SNL-treated group, mechanical allodynia decreased from POD 7 days, and mechanical allodynia decreased on POD 14 days. In addition, it was confirmed that mechanical allodynia was reduced to the highest level on POD 10 days.

Therefore, it was confirmed that the 3-methyladenine of the present invention significantly reduces the mechanical allodynia in the spinal cord olfactory, and thus the neuropathic pain induced by the spinal cord nerve ligation is excellent.

Hereinafter, a pharmaceutical composition for preventing or treating neuropathic pain containing 3-methyladenine of the present invention and a preparation example of a food composition for preventing or ameliorating neuropathic pain will be described, but the present invention is not limited thereto It is just a specific description.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

20 mg of 3-methyladenine

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

10 mg of 3-methyladenine

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

10 mg of 3-methyladenine

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

10 mg of 3-methyladenine

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

20 mg of 3-methyladenine

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

[Formulation example 2. Preparation of food preparation]

1. Manufacture of health food

100 mg of 3-methyladenine

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

100 mg of 3-methyladenine

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (6)

A pharmaceutical composition for inhibiting or treating neuropathic pain, comprising 3-methyladenine as an active ingredient.
The use according to claim 1, wherein the 3-methyladenine inhibits the expression of LC3 (microtubule-associated protein 1 light chain 3) or becline 1 (becline 1) Gt;
The pharmaceutical composition for inhibiting or treating neuropathic pain according to claim 1, wherein the 3-methyladenine inhibits autophagic action of GABA (gamma-aminobutyricacid) neurons.
The pharmaceutical composition according to claim 1, wherein the neuropathic pain is central neuropathic pain or peripheral neuropathic pain.
A food composition for inhibiting or improving neuropathic pain comprising 3-methyladenine as an active ingredient.
6. The food composition of claim 5, wherein the neuropathic pain is central neuropathic pain or peripheral neuropathic pain.

Images