WO2024181609A1 - Composition comprising beneficial intestinal bacteria for preventing or treating neuropathic pain - Google Patents

Abstract

The present invention relates to: a composition comprising beneficial intestinal bacteria for preventing or treating neuropathic pain; and the like. It has been identified that Lactobacillus bacteria, Bifidobacterium, and Streptococcus, which are beneficial intestinal bacteria, according to the present invention, have excellent analgesia and analgesic longevity in pain behavior experiments of mouse models of neuropathic pain. In addition, the mechanism of relieving pain by reducing the expression of TNF-α has been identified. Therefore, the composition is expected to be widely used in the prevention and treatment of neuropathic pain.

Description

Composition for preventing or treating neuropathic pain containing intestinal beneficial bacteria

The present invention relates to a composition for preventing or treating neuropathic pain, including intestinal beneficial bacteria.

This application claims priority to Korean Patent Application No. 10-2023-0025933, filed February 27, 2023, the entire contents of which are incorporated herein by reference.

Neuropathic pain is a type of acute or chronic pathological pain syndrome in which the pain-prolonging mechanism is due to an abnormality in the somatosensory process of the central or peripheral nervous system. The pathophysiological mechanism of neuropathic pain is a complex of peripheral and central mechanisms, and has been extensively studied in animal models recently. The peripheral mechanism is the abnormal change of sodium channels due to changes in the expression of sodium channel genes in primary afferent neurons and dorsal root ganglia, which has been studied extensively recently, causing electrophysiological changes in the dorsal root ganglion, thereby inducing spontaneous pain and inappropriate pain responses. In some cases, cross-excitation between the sympathetic nervous system and primary afferent neurons is involved. Damage to the peripheral nerves causes changes in the central nervous system. For example, in animal experiments, a decrease or loss of segmental or suprasegmental inhibitory mechanisms; These include structural changes in the terminal portions of the dorsal horn of primary afferent fibers; and central sensitization. The cytological mechanism of central sensitization has been revealed to involve NMDA (N-methyl-D-aspartate) receptors.

The intestines not only absorb and excrete nutrients from food that enters the body, but also have various functions such as developing and regulating the body's immune function, and providing a living environment for intestinal bacteria that help maintain health and prevent diseases. Food contains a large amount of bacteria, viruses, fungi, and toxins that are harmful to health. Accordingly, the intestines have well-developed immune tissues to deal with these harmful substances, and various immune cells and immune-related substances are actively working. The human intestine is about 8 to 9 meters long, and the intestinal mucosa is made up of circular folds and villi, which rapidly increase the surface area, reaching about 200 ^m2 when flattened. About 400 types of microorganisms live in the human intestines, and they weigh about 1 to 2 kg. Beneficial bacteria that produce nutrients, strengthen immunity, and suppress the growth of harmful bacteria coexist and maintain balance. However, it is known that if the balance of intestinal microorganisms is disrupted due to unhealthy eating habits or indiscriminate drug use, harmful bacteria will proliferate excessively, which will increase the risk of various autoimmune diseases, allergies, inflammatory diseases, arteriosclerosis, and even cancer.

Meanwhile, the inventors of the present invention confirmed that intestinal beneficial bacteria have a preventive and therapeutic effect on neuropathic pain, and designed and completed the invention by providing a composition for treating neuropathic pain containing intestinal beneficial bacteria.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci.

Another object of the present invention is to provide a health functional food composition for preventing or improving neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci.

Another object of the present invention is to provide a method for preventing, improving, or treating neuropathic pain, comprising a step of administering to a subject in need thereof lactobacillus bacteria, bifidobacteria, and streptococci, which are intestinal beneficial bacteria.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

The terminology used herein is for the purpose of description only and should not be construed in a limiting sense. The singular expression includes the plural expression unless the context clearly indicates otherwise. As used herein, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but should be understood to not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Terms defined in commonly used dictionaries, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense, unless expressly defined in this application.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci.

In the present invention, intestinal microorganisms account for the majority of microorganisms in the human body, and there are more than 4,000 to 10,000 species. Beneficial bacteria account for 30% of intestinal microorganisms, and harmful bacteria account for 5-10%, and among these, beneficial bacteria are called probiotics. In the present invention, the intestinal beneficial bacteria may include or consist of lactobacillus bacteria, bifidobacteria, and streptococci, but are not limited thereto.

In the present invention, the intestinal beneficial bacteria can be an analgesic for neuropathic pain and can have a continuous analgesic effect, but is not limited thereto. In addition, the intestinal beneficial bacteria can suppress the expression of TNF-α, and can restore the intestinal microbial environment or improve intestinal microbial imbalance. The TNF-α (Tumor Necrosis Factor-α) activates spinal neurons to increase the release of glutamate before the synapse and activates the NMDA (N-methyl-D-aspartate) receptor of the postsynaptic membrane that contributes to neuropathic pain. Therefore, the intestinal beneficial bacteria of the present invention improve and treat neuropathic pain by suppressing the expression of TNF-α.

In the present invention, Lactobacillus bacteria ( Lactobacillus *?*) are called lactic acid bacteria or lactic acid bacteria and are representative probiotics. The above Lactobacillus bacteria are Lactobacillus brevis , Lactobacillus acetotolerans, Lactobacillus acidifarinae , Lactobacillus acidipiscis, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarus, Lactobacillus amylolyticus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus animalis . The bacterial strain may be selected from the group consisting of Lactobacillus animalis, Lactobacillus antri, Lactobacillus apodemi, Lactobacillus plantarum , Lactobacillus acidophilus, Lactobacillus paracasei, or Lactobacillus helveticus , preferably selected from the group consisting of Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus paracasei , and Lactobacillus helveticus , but is not limited thereto. Specifically, the Lactobacillus bacteria may be, but are not limited to, L. plantarum BP06 (LMG S-29189), L. acidophilus BA05 (LMG P-21904), or L. paracasei BP07 (LMG S-29188), L. helveticus BD08 (LMG S-29187).

In the present invention, Bifidobacterium is a gram-positive bacterium and a branched anaerobic bacterium, and is one of the major genera of bacteria that constitute the gastrointestinal microflora of mammals. The above bifidobacterium may be Bifidobacterium longum , the bifidobacterium may be Bifidobacterium catenulatum , Bifidobacterium adolescentis , Bifidobacterium breve , or Bifidobacterium animalis subsp. lactis, and may be preferably selected from the group consisting of Bifidobacterium breve and Bifidobacterium animalis subsp. lactis, but is not limited thereto. Specifically, the above bifidobacterium may be B. animalis subsp . lactis BL03 (LMG P-17502), B. animalis subsp . lactis BI04 (LMG P-17502), or B. breve BB02 (LMG P-29190), but is not limited thereto.

In the present invention, streptococci refer to bacteria of the genus Streptococcus . They are also called streptococci because the shape of the colony formed is chain-shaped. The streptococci may be Streptococcus salivarius , Streptococcus oralis , Streptococcus mitis , Streptococcus sanguinis, Streptococcus parasanguinis , or Streptococcus thermophilus , and are preferably Streptococcus thermophilus, but are not limited thereto. Specifically, the streptococcus may be, but is not limited to, S. thermophilus BT01 (LMG S-29186).

In the present invention, the intestinal beneficial bacteria are B. animalis subsp . lactis BL03 (LMG P-17502), S. thermophilus BT01 (LMG S-29186), B. animalis subsp . lactis BI04 (LMG P-17502), L. plantarum BP06 (LMG S-29189), L. acidophilus BA05 (LMG P-21904), L. paracasei BP07 (LMG S-29188), L. helveticus BD08 (LMG S-29187), and B. breve BB02 (LMG P-29190).

The intestinal beneficial bacteria of the present invention can be used as bacteria included in VSL#3 ^ⓡ (Nutrilinea Srl inc). The VSL#3 contains four types of lactic acid bacteria, three types of bifidobacteria, and one type of streptococcus.

In the present invention, neuropathic pain is a pain that occurs when the central or peripheral nerves themselves are damaged for some reason, causing them to send an incorrect signal to the center that controls pain, and the nerves themselves cause pain. The neuropathic pain may be selected from the group consisting of peripheral neuropathic pain, diabetic neuropathic pain, post-herpetic neuralgia, neuropathic pain due to spinal cord injury, pain syndrome after spinal surgery, trigeminal neuralgia, complex regional pain syndrome (CRPS), phantom pain, terminal pain, cancer neuropathic pain, and central pain after stroke, and may be pain due to central nerve damage, but is not limited thereto.

In the present invention, the "complex regional pain syndrome (CRPS)" is a disease that exhibits severe burning pain, allodynia, hyperalgesia, etc. centered around the injured area after various types of trauma such as damage to soft tissue, fracture, nerve damage, etc., and the connection with the sympathetic nerve is related to the induction of pain.

In the present invention, the above "post-herpetic neuralgia" refers to a case where pain persists even after shingles has healed. Shingles refers to a nerve disease in which the virus that caused chickenpox in childhood is hidden in specific nerve cells and regenerates when the body's resistance is temporarily weakened and moves along nerve fibers, causing blisters and severe pain on the skin distributed by the nerve.

In the present invention, the "trigeminal neuralgia" refers to a disease in which a momentary pain like an electric shock or a stabbing occurs in the facial area, especially the teeth, forehead, cheeks, upper jaw, and lower jaw.

In the present invention, the "phantom pain" means that an amputee feels a phantom sensation and feels pain in the amputated part as if it were real. The "palpitation" means that there is pain in the amputated part of the limb.

In the present invention, "diabetic neuropathy" is caused by damage to peripheral nerves due to diabetes, and pain accompanied by decreased sensation appears in the hands, upper limbs, feet and lower limbs, and in severe cases, causes motor disorders.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipses, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigants, ointments, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

Additives for tablets, powders, granules, capsules, pills and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethyl cellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinyl pyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

Additives that can be used in the liquid formulation according to the present invention include water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

The syrup according to the present invention may use a solution of white sugar, other sugars or sweeteners, and may also use a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, etc., as needed.

Purified water may be used in the emulsion according to the present invention, and an emulsifier, a preservative, a stabilizer, a fragrance, etc. may be used as needed.

The suspension according to the present invention may use suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like. Surfactants, preservatives, stabilizers, colorants, and fragrances may also be used as needed.

The injection according to the present invention includes: solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, nonvolatile oils such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nitrile acid amide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cisplatinum, sodium alginate, Tween 80, and aluminum monostearate.

The suppository according to the present invention comprises cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranette wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method. In the present invention, “prevention” means any act of inhibiting or delaying the onset of a target disease, “treatment” means any act of improving or beneficially changing a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any act of reducing a parameter related to a target disease, for example, the degree of symptoms, by administering a composition according to the present invention.

The present invention provides a health functional food composition for preventing or improving neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci.

In the present invention, "food" means a natural product or processed product containing one or more nutrients, preferably a product that has undergone a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

In the present invention, the "functional food" is the same term as food for special health use (FoSHU), and means a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to nutritional supply, and can be manufactured in the form of tablets, capsules, pills, granules, powder, liquid, flakes, paste, syrup, gel, jelly, bars, or films. Here, "function" means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological actions.

When the lactobacillus bacteria, bifidobacteria, and streptococci of the present invention are used as food additives, they can be added as they are or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredients can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the lactobacillus bacteria, bifidobacteria, and streptococci of the present invention can be added in an amount of 15 wt% or less, or 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the effective ingredients can also be used in an amount greater than the above range.

There are no special restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for preventing, improving, or treating neuropathic pain, comprising a step of administering to a subject in need thereof lactobacillus bacteria, bifidobacteria, and streptococci, which are intestinal beneficial bacteria.

In addition, the present invention provides a use of intestinal beneficial bacteria, such as Lactobacillus bacteria, Bifidobacterium bacteria, and Streptococcus bacteria, for preventing, improving, or treating neuropathic pain.

The present invention also provides the use of intestinal beneficial bacteria, such as Lactobacillus bacteria, Bifidobacterium bacteria, and Streptococcus bacteria, for producing a drug for preventing, ameliorating, or treating neuropathic pain.

The present invention confirmed that the intestinal beneficial bacteria of the present invention have excellent therapeutic effects on neuropathic pain and have excellent analgesic durability, and confirmed a mechanism for alleviating pain by reducing the expression of TNF-α. Therefore, the present invention can be utilized as a pharmaceutical composition or a health functional food composition for preventing, improving, and treating neuropathic pain.

According to the present invention, the intestinal beneficial bacteria, Lactobacillus bacteria, Bifidobacterium bacteria, and Streptococcus bacteria, were confirmed to have excellent analgesic effects and sustained analgesia in pain behavior experiments on a neuropathic pain mouse model. In addition, the mechanism of alleviating pain by reducing the expression of TNF-α was confirmed. Therefore, it is expected that it can be widely used in the prevention and treatment of neuropathic pain.

Figure 1 is a schematic diagram showing the administration of intestinal beneficial bacteria and pain behavior experiment.

Figure 2 is a graph showing the analgesic effect of intestinal beneficial bacteria on neuropathic pain.

Figure 3 is a graph showing the inhibitory effect of intestinal beneficial bacteria on TNF-α expression in the analgesic effect of neuropathic pain.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Experimental method]

1. experimental animals

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. Male C57B/L6 mice (8–10 weeks old) were purchased from Daehan Biolink (DBL, Eumseong, Korea). All animals were housed in a specific pathogen-free environment at 22–24°C, 55% humidity, and a 12-h light/dark cycle. All protocols were performed in accordance with the guidelines of the International Association for the Study of Pain (IASP).

2. Neuropathic pain model

Adult male mice (C57B/L6) older than 8 weeks of age were injected intraperitoneally with an anesthetic (sodium pentobarbital) at 50 mg/kg, and neuropathic pain was induced through L5 spinal nerve transection (L5 SNT). Specifically, the skin from L4 to S2 of the spinal cord was incised, and the L6 transverse process was removed to expose the L5 nerve. After the exposed L5 spinal nerve was transected, the surgical site was disinfected, and sutured with a surgical stapler. Then, the body temperature of the operated mice was maintained on a warming pad until they recovered from anesthesia.

3. Pain behavior experiment

First, each mouse was placed individually in a transparent acrylic cage on a wire mesh and allowed to adapt to the environment for two hours. After that, a behavioral experiment was performed, and only mice within the baseline threshold (1.0-1.5 g) were selected and divided into groups of 5-6 mice. Neuropathic pain was induced the next day through L5 SNT surgery. On days 3, 7, 14, 21, and 28 after surgery, the mice were allowed to adapt to the environment for 30 minutes using the same method as above, and a pain behavior experiment was performed. The pain behavior experiment was performed using an up and down method using a von-Frey filament (North Coast Medical, Inc., USA). Specifically, when the mouse's sole was pricked with the filament, if it responded with a rapid avoidance response or licking, the sole was pricked with a filament of a weaker stimulus. If there was no response, the sole was pricked with a filament of a stronger stimulus. In addition, to maintain consistency of the experiment, all behavioral experiments were conducted between 10:00 AM and 2:00 PM. The 50% probability of avoidance response was quantified and statistically processed.

4. Intestine Beneficial bacteria Dosage

The intestinal beneficial bacteria used in the present invention are VSL#3 products, which are composed of four types of lactic acid bacteria; three types of bifidobacteria; and one type of streptococcus ( B. animalis) . subsp . lactis BL03, S. thermophilus BT01, B. animalis subsp . lactis BI04, L. plantarum BP06, L. acidophilus BA05, L. paracasei BP07 , L. helveticus BD08, B. breve BB02). 450 Х 10 ⁹ (CFU) of probiotic bacteria, VSL#3 (Nutrilinea Srl inc), was dissolved in 1 L of water to prepare a concentration of 4.5 g/L. After placing it in a 200 ml water bottle, the mice were placed in the cages so that they could drink freely, and the water was changed every two days. In addition, intestinal beneficial bacteria were administered from 3 weeks before neuropathic pain surgery to 4 weeks after the surgery. Each mouse received approximately 22.5 Х 10 ⁸ (CFU)/day of intestinal beneficial bacteria when drinking 5 ml of water per day.

5. Immunohistochemical staining ( Immunohistochemistry )

Mice were transcardially perfused with 0.1 M phosphate buffer (pH 7.4) and 4% paraformaldehyde, and the L5 spinal cords were removed and postfixed overnight at 4°C in the same solution. Spinal cord samples were transferred to 30% sucrose for at least 48 h and sectioned coronally into 16-μm-thick sections using a cryostat (CM1860; Leica, Wetzlar, Germany). Spinal cord sections were blocked and incubated with rabbit anti-TNF-α (1:1000; Invitrogen, Waltham, MS, USA), rabbit anti-Iba-1 (1:1,000; Wako, Osaka, Japan), and mouse anti-GFAP (1:1,000; Millipore, Burlington, MA, USA) antibodies. After rinsing five times with 0.1 M PBS, the samples were incubated with FITC- or CY3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1.5 h at room temperature. The samples were then examined using an LSM800 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

6. Real-time RT- PCR

Real-time RT-PCR experiments were performed using the StepOnePlus real-time PCR system (Applied Biosystem, Foster City, CA, USA) according to the 2 ^-△△ Ct method. Total RNA was extracted from L5 spinal cord tissue and primary microglia with TRIzol reagent (Invitrogen) and reverse transcribed using TOPscript RT DryMIX (Enzynomics, Daejeon, Korea). All △Ct values were normalized to the corresponding GAPDH values and expressed as fold induction. The PCR primers shown in Table 1 below were used in the experiments.

7. Statistical Analysis

Data were analyzed using Student’s t-test for comparison between two groups. One-way analysis of variance (ANOVA) with Bonferroni’s post hoc test was used for statistical analysis of multiple comparisons. All data are expressed as mean ± SEM (standard error of the mean), and differences were considered statistically significant when p<0.05 or p<0.001.

[ Example ]

Example 1. Intestine Beneficial bacteria Neuropathic Analgesic effect on pain check

The analgesic effect of intestinal beneficial bacteria on neuropathic pain was confirmed by administering VSL#3 (experimental group) or water (control group) to mice that had neuropathic pain induced through SNT surgery. Normal mice showed a threshold value of 1 to 1.5 g, while mice with induced neuropathic pain showed a threshold value of 0.2 to 0.4 g. The results are shown in Fig. 2.

As shown in Figure 2, the control group experienced neuropathic pain from the third day after surgery and lasted until the 28th day. On the other hand, the experimental group administered VSL#3 showed a significant analgesic effect from the third day after surgery, and the pain almost disappeared by the 14th day, recovering to the baseline threshold (BL). In addition, it was confirmed that this analgesic effect lasted until the 28th day.

Example 2. Intestinal analgesic effect on neuropathic pain Beneficial bacteria Mechanism of action

To investigate the mechanism of action of intestinal beneficial bacteria, mice were divided into four groups (control group-sham, control group-SNT, experimental group-sham, experimental group-SNT) and experiments were performed, and the mRNA expression of the inflammatory cytokine TNF-α in spinal cord tissue (L4-L6) was measured. The results are shown in Fig. 3.

As shown in Fig. 3, in the control group-SNT, the expression of TNF-α increased about 3-fold compared to the control group-sham, whereas in the experimental group-SNT, the expression of TNF-α did not increase compared to the experimental group-sham, and significantly decreased compared to the control group-SNT. This means that when VSL#3 was administered to mice induced with neuropathic pain, it alleviated the pain by reducing the expression of TNF-α.

Therefore, it was confirmed that the intestinal beneficial bacteria of the present invention have excellent therapeutic effects and analgesic durability for neuropathic pain, and the mechanism of alleviating pain by reducing the expression of TNF-α was confirmed.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (13)

A pharmaceutical composition for preventing or treating neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci. In the first paragraph, A composition wherein the above Lactobacillus bacteria are selected from the group consisting of Lactobacillus plantarum , Lactobacillus acidophilus, Lactobacillus paracasei , and Lactobacillus helveticus . In the first paragraph, The above bifidobacteria are Bifidobacterium breve　A composition selected from the group consisting of Bifidobacterium breve and Bifidobacterium animalis subsp. lactis . In the first paragraph, A composition wherein the above-mentioned streptococcus is Streptococcus thermophilus . In the first paragraph, The above intestinal beneficial bacteria are Lactobacillus plantarum , Lactobacillus acidophilus, Lactobacillus paracasei , Lactobacillus helveticus, and Bifidobacterium breve.　 breve ), Bifidobacterium animalis subspecies lactis　 animalis 　A composition comprising Streptococcus subsp . lactis and Streptococcus thermophilus . In the first paragraph, The above intestinal beneficial bacteria are B. animalis subsp . lactis BL03, S. thermophilus BT01, B. animalis A composition comprising L. subsp . lactis BI04, L. plantarum BP06, L. acidophilus BA05, L. paracasei BP07 , L. helveticus BD08, and B. breve BB02. In the first paragraph, A composition according to claim 1, wherein the neuropathic pain is selected from the group consisting of peripheral neuropathic pain, diabetic neuropathic pain, post-herpetic neuralgia, neuropathic pain due to spinal cord injury, pain syndrome after spinal surgery, trigeminal neuralgia, complex regional pain syndrome (CRPS), phantom pain, terminal pain, cancer neuropathic pain, and central pain after stroke. In the first paragraph, The above neuropathic pain is pain caused by damage to the central nervous system, the composition. In the first paragraph, The above intestinal beneficial bacteria are a composition for analgesic use in neuropathic pain. In the first paragraph, A composition wherein the above intestinal beneficial bacteria have the ability to inhibit the expression of TNF-α. In the first paragraph, The above composition is a composition for oral administration. A health functional food composition for preventing or improving neuropathic pain, comprising intestinal beneficial bacteria such as lactobacillus bacteria, bifidobacteria, and streptococci. A method for preventing, improving, or treating neuropathic pain, comprising administering to a subject in need thereof lactobacillus bacteria, bifidobacteria, and streptococci, which are beneficial intestinal bacteria.

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