Uses of Creosote

Abstract

[Subject] The present invention aims to explore and provide a novel acetylcholinesterase inhibitor. [Solution] Provide an acetylcholinesterase inhibitor containing creosote as an active ingredient.

Description

Technical Field

[0001] The present invention relates to acetylcholinesterase inhibitors containing creosote and various uses thereof.

[0002] Prior Art

[0003] Acetylcholine is a neurotransmitter that is released from the terminals of parasympathetic nerves or motor nerves and transmits nerve stimulation. Acetylcholine is synthesized by choline acetyltransferase (ChAT) and decomposed by acetylcholinesterase (AChE), maintaining a constant concentration in the body.

[0004] If the concentration of acetylcholine is low in a part of the body, the cholinergic neurotransmission is insufficient. For example, such a state in the brain is considered to be one of the causes of reduced learning function or the onset of Alzheimer's disease. Donepezil (trade name: Aricept), a cholinesterase inhibitor, is used as a therapeutic drug for Alzheimer's disease.

[0005] Other drugs are used for therapeutic purposes, such as distigmine bromide for urinary disorders, distigmine bromide or ambenonium chloride for myasthenia gravis, and neostigmine or edrophonium as antagonists of muscle relaxants during general anesthesia. These drugs are drugs that aim to improve pathological effects by inhibiting acetylcholinesterase and increasing the concentration of acetylcholine at nerve terminals.

[0006] However, these acetylcholinesterase inhibitors have common side effects such as gastrointestinal symptoms such as vomiting, nausea, and diarrhea, and studies have been conducted on drugs with fewer side effects (eg, Patent Document 1).

[0007] [Prior art literature]

[0008] [Patent Document]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-204274. Summary of the invention

[0010] (Problems to be solved by the invention)

[0011] The present invention aims to explore and provide novel acetylcholinesterase inhibitors.

[0012] (Methods to solve the problem)

[0013] As a result of intensive research, the present inventors unexpectedly discovered that creosote, which is known to have antidiarrheal effects, has an acetylcholinesterase inhibitory effect, and thus completed the present invention.

[0014] That is, the present invention relates to an acetylcholinesterase inhibitor containing creosote as an effective ingredient.

[0015] One embodiment of the present invention is a pharmaceutical composition containing an acetylcholinesterase inhibitor containing creosote as an active ingredient.

[0016] One embodiment of the present invention is a composition prepared for oral administration or rectal administration, wherein the dosage of creosote as an active ingredient is 0.1 to 500 mg per day per kg of body weight.

[0017] One embodiment of the present invention is a composition prepared for parenteral administration, wherein the dosage of creosote as an active ingredient is 0.2 to 300 mg per day per kg of body weight.

[0018] Another embodiment of the present invention relates to a pharmaceutical composition for treating, improving or inhibiting the progression of a disease or condition associated with decreased acetylcholine activity.

[0019] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of myasthenia gravis, comprising creosote as an active ingredient.

[0020] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of glaucoma, comprising creosote as an active ingredient.

[0021] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of Alzheimer's type dementia, comprising creosote as an active ingredient.

[0022] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of dementia with Lewy bodies, comprising creosote as an active ingredient.

[0023] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of Parkinson's disease, comprising creosote as an active ingredient.

[0024] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of dysuria, comprising creosote as an active ingredient.

[0025] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of constipation, comprising creosote as an active ingredient.

[0026] Another embodiment of the present invention relates to a pharmaceutical composition for treating or suppressing the progression of functional dyspepsia, comprising creosote as an active ingredient.

[0027] Another embodiment of the present invention relates to a pharmaceutical composition for detoxification of anticholinergic drug poisoning, which contains creosote as an active ingredient.

[0028] Another embodiment of the present invention relates to a pharmaceutical composition for suppressing (eliminating the residual muscle relaxant effect) a non-depolarizing muscle relaxant containing creosote as an active ingredient.

[0029] Another embodiment of the present invention relates to a pharmaceutical composition for increasing the incidence of lucid dreams (increasing the rapid eye movement sleep ratio) comprising creosote as an active ingredient.

[0030] Another embodiment of the present invention relates to a pharmaceutical composition for treating cognitive impairment in schizophrenia patients, comprising creosote as an active ingredient.

[0031] Another embodiment of the present invention relates to a herbicide comprising creosote as an active ingredient.

[0032] Another embodiment of the present invention relates to an insecticide (insecticide for insects or nematodes) containing creosote as an active ingredient.

[0033] Another embodiment of the present invention relates to the use of creosote for the manufacture of a medicine for treating, ameliorating or inhibiting the progression of a disease or condition associated with decreased acetylcholine activity.

[0034] Other embodiments of the present invention relate to methods for treating or inhibiting the progression of a disease or condition associated with decreased acetylcholine activity in a subject,

[0035] A method comprising the step of applying to said subject an effective amount of creosote.

[0036] In one embodiment of the present invention, the disease or condition associated with decreased acetylcholine activity is one or more diseases or conditions selected from the following groups:

[0037] -Myasthenia gravis,

[0038] -glaucoma,

[0039] - Alzheimer's dementia,

[0040] -Dementia with Lewy bodies,

[0041] -Parkinson's syndrome,

[0042] - Difficulty urinating,

[0043] - Difficulty in defecation,

[0044] - Functional dyspepsia,

[0045] - Anticholinergic poisoning,

[0046] - Residual muscle relaxation via non-depolarizing muscle relaxants,

[0047] - Reduced incidence of lucid dreams and

[0048] -Cognitive impairment in patients with schizophrenia.

[0049] In one embodiment of the present invention, creosote is administered orally or intrarectally to a subject, and the dosage of creosote is 0.1 to 500 mg per kg of body weight per day.

[0050] In one embodiment of the present invention, the creosote is administered parenterally to the subject, and the dosage of the creosote is 0.2 to 300 mg per kg of body weight per day.

[0051] Inventions in which one or more of the above-listed features of the present invention are arbitrarily combined are also included in the scope of the present invention.

[0052] (Effects of the Invention)

[0053] The acetylcholinesterase inhibitor of the present invention not only has the drug effect based on the acetylcholinesterase inhibitory activity, but also can reduce the side effects (digestive symptoms) of the existing acetylcholinesterase inhibitors through the antidiarrheal effect of creosote. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A graph showing the acetylcholinesterase inhibitory activity of creosote plotted at each concentration.

[0055] Figure 2 A graph showing the acetylcholinesterase inhibitory activity of guaiacol plotted at each concentration. DETAILED DESCRIPTION

[0056] The present invention relates to an acetylcholinesterase inhibitor using creosote as an effective ingredient and various uses thereof.

[0057] Creosote

[0058] The wood creosote, which is an effective ingredient of the present invention, is known in the art and is listed in the Japanese Pharmacopoeia, the National Formulary of the United States, etc. The wood creosote, which is an effective ingredient of the present invention, is obtained by distilling wood tar obtained from trees such as beech, elm, maple, pine, etc., especially broad-leaved trees, and collecting the fraction at about 200 to 230° C. (760 mmHg) by fractionation. It is clearly distinguished from creosote obtained from coal tar (N. Ogata and T. Baba, Res. Commun. Chem. Pahthol. Pharmacol, 66, 411-423, 1989).

[0059] Creosote oil, which is an effective ingredient of the present invention, is a colorless to light yellow liquid including guaiacol, creosol, phenol, p-cresol, 4-ethylguaiacol, o-cresol, and the like.

[0060] Preparation (form)

[0061] In the present invention, the creosote is used in the form of a common pharmaceutical composition (e.g., a medical agent). That is, it can be prepared in various forms according to the therapeutic purpose using pharmaceutically acceptable excipients or diluents and prepared according to conventional methods. Representative forms thereof include tablets, pills, powders, capsules, soft capsules, granules, oral liquids, etc., injections, etc., for intravascular administration, intramuscular administration, subcutaneous administration or intradermal administration, and suppositories, etc., for rectal administration.

[0062] When preparing tablets, granules, and powders, a wide range of known carriers can be used, such as excipients such as lactose, white sugar, glucose, starch, and crystalline cellulose, binders such as hydroxypropylcellulose, methylcellulose, gelatin, tragacanth gum, gum arabic, and sodium alginate, disintegrants such as starch, carboxymethylcellulose, and calcium carbonate, and lubricants such as magnesium stearate, talc, and stearic acid. For tablets, a conventional coating can be applied if necessary, and it can be made into, for example, sugar-coated tablets, film-coated tablets, etc., and can also be made into double-layer tablets or multi-layer tablets. In addition, granules or powders can also be applied with a conventional coating.

[0063] The same is true when preparing the pill form. Various carriers that have been known in the art can be used, such as excipients such as licorice powder, glucose, wheat flour, binders such as glycerin, water syrup, gum arabic, tragacanth gum, gelatin, disintegrants such as medicinal yeast, kudzu root, kelp powder, etc.

[0064] When preparing the capsule form, various carriers conventionally known in the art, for example, excipients such as lactose, olive oil, and soybean oil can be used.

[0065] Internal liquid preparations may also be aqueous or oily suspensions, solutions, syrups or other forms. These liquid preparations may use commonly used additives, such as suspending agents, such as sorbitol syrup, methylcellulose, gelatin, carboxymethylcellulose, emulsifiers, such as lecithin, sorbitan monoolefin esters, gum arabic, etc.

[0066] When preparing an injection, the composition may be in the form of a suspension, a solution, or an emulsion in an oily or aqueous medium, and may contain a prescribing agent such as a suspending agent, a stabilizer, and a dispersing agent.

[0067] When preparing the suppository form, a wide range of known carriers can be used. For example, bases such as cocoa butter, glycerin gelatin, and polyethylene glycol can be used. Emulsifiers and suspending agents can be used for suppositories as necessary. In addition, colorants, flavoring agents, etc. can be added to the medicament of the present invention as necessary.

[0068] The amount of creosote contained as an active ingredient in the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected according to the form of the preparation, and is generally preferably about 0.2 to 60% of the total weight of the preparation.

[0069] The dosage of the pharmaceutical composition of the present invention is appropriately selected depending on the patient's sex, age, body weight, symptom severity, etc. Generally, for oral administration and rectal administration, the dosage of creosote as an active ingredient is about 0.1 to 500 mg per day per kg of body weight for adults, preferably about 2 to 100 mg, and more preferably about 2 to 25 mg. For parenteral administration as an injection, similarly, for adults, the dosage of creosote as an active ingredient is about 0.2 to 300 mg per day per kg of body weight, preferably about 0.2 to 50 mg, and more preferably about 0.5 to 5 mg. In addition, these can be divided into about 2 to 4 doses per day.

[0070] Specific uses

[0071] The acetylcholinesterase inhibitors of the present invention can be used for various specific purposes. For example, the acetylcholinesterase inhibitors of the present invention can be used to treat myasthenia gravis, glaucoma, Alzheimer's dementia, Lewy body dementia, Parkinson's syndrome, urination and defecation difficulties, functional dyspepsia, etc., which have been confirmed to be treated by acetylcholinesterase inhibitors. In addition, the acetylcholinesterase inhibitors of the present invention can also be used for the detoxification of anticholinergic poisoning, the inhibition of non-depolarizing muscle relaxants, the increase in the incidence of lucid dreams, the treatment of cognitive impairment in schizophrenia patients, etc. In addition, the acetylcholinesterase inhibitors of the present invention can also be used as herbicides or insecticides (e.g., insecticides for repelling insects) based on their mechanism of action.

[0072] The terms used in this specification are used to describe specific implementations, and the present invention is not limited thereto.

[0073] In addition, the term "comprising" used in this specification intends the existence of the matters (parts, steps, elements or numbers, etc.) recorded, except for cases where there is an obvious different understanding in the words, and does not exclude the existence of matters (parts, steps, elements or numbers, etc.) other than these.

[0074] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those fully understood by those skilled in the art. If no different definition is given, the terms used herein shall be interpreted as integrating the meaning of this specification with the relevant technical field, and shall not be interpreted as idealized or excessive formal meaning.

[0075] Embodiments of the present invention will be described with reference to schematic drawings. However, in the schematic drawings, some drawings may be exaggerated for the purpose of clear description.

[0076] In the present specification, when expressed as "1 to 10 w / w %", for example, a skilled person can understand that the expression specifically indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 w / w %.

[0077] In this specification, numerical values ​​used to express component contents or numerical ranges are interpreted as including the meaning of the term "about" unless otherwise specified. For example, "10 times" is understood to mean "about 10 times" unless otherwise specified.

[0078] All the disclosures of the documents cited in this specification are cited in this specification. The technician can refer to the text of this specification and understand that the relevant disclosures in these prior art documents are part of this specification without departing from the spirit and scope of the present invention.

[0079] [Example]

[0080] Example 1: Determination of Acetylcholinesterase Activity of Creosote

[0081] <Experimental Materials>

[0082] 1. Tested substances

[0083] 1.1 Tested substance 1

[0084] Name: Creosote (hereinafter referred to as WC)

[0085] Molecular weight: Since it is a mixture, the molecular weight is not indicated.

[0086] Purchaser: Taiko Pharmaceutical Co., Ltd.

[0087] Storage conditions: Refrigerate (4°C), keep away from light

[0088] 1.2 Test substance 2 (positive control)

[0089] Name: Galantamine Hydrobromide (hereinafter referred to as GAL)

[0090] Molecular weight: 368.3

[0091] Product number: G1660-2MG

[0092] Purchaser: Sigma-Aldrich

[0093] Storage conditions: Frozen (-20℃)

[0094] * Galantamine is known as a substance having an acetylcholinesterase inhibitory effect and is used for the treatment of mild to moderate Alzheimer's type dementia or memory impairment.

[0095] 2. Drug testing

[0096] 2.1AchE enzyme

[0097] Name: Acetylcholinesterase from Electrophorus electricus (electric eel) Type VI-S

[0098] Product number: C3389-500UN

[0099] Purchaser: Sigma-Aldrich

[0100] Storage conditions: Frozen (-20℃)

[0101] 2.2AchE activity assay kit

[0102] Name: Acetylcholinesterase Activity Assay Kit

[0103] Product number: MAK119

[0104] Purchaser: Sigma-Aldrich

[0105] Storage conditions: room temperature

[0106] 2.3 Solvent

[0107] Name: UltraPure 1M Tris-HCl pH7.5

[0108] Product number: 15567-027

[0109] Purchased from: Invitrogen

[0110] Storage conditions: Refrigerate (4°C)

[0111] 2.4 Water

[0112] Use water purified by the ultrapure water production device UL-Pure (hereinafter referred to as ultrapure water)

[0113] 3. Use of machines and equipment

[0114] 3.1 Electronic Scale

[0115] Type: AUX120

[0116] Manufacturer: SHIMADZU

[0117] 3.2 Straw

[0118] Type: pipetman P series

[0119] Manufacturer: GILSON

[0120] 3.2 Continuous Dispenser (Multi-channel Pipette)

[0121] Type: pipetman Neo series

[0122] Manufacturer: GILSON

[0123] 3.3 Constant temperature water tank

[0124] Type: BF400

[0125] Manufacturer: Yamato Scientific Co., Ltd

[0126] 3.4 Refrigerated Storage

[0127] Model:GR-38ND

[0128] Manufacturer: TOSHIBA

[0129] 3.5 Microplate Reader

[0130] Type: SH-9000Lab

[0131] Manufacturer: CORONA ELECTRIC

[0132] 3.6 96-well microplate

[0133] Type: 353072

[0134] Manufacturer: FALCON

[0135] <Test method>

[0136] 1. Prepare the test substance 1 solution

[0137] WC was weighed and dissolved in ultrapure water to prepare a concentration of 5 mg / mL as the test substance 1 solution.

[0138] 2. Prepare the test substance 2 solution

[0139] GAL was weighed and dissolved in ultrapure water to a concentration of 1 mg / mL, and then diluted 1.25 times with ultrapure water to a concentration of 800 μg / mL to prepare the test substance 2 solution.

[0140] 3. Prepare AchE enzyme solution

[0141] 2 mg (500 units) of AchE enzyme (freeze-dried powder) was dissolved in 2 mL of Tris-HCl buffer pH 7.5 adjusted to a concentration of 20 mM, and then diluted 30 times with ultrapure water to a concentration of 33.3 μg / mL.

[0142] 4. Preparation of Working Solution for AchE Activity Assay

[0143] 2 mg of the reagent contained in the AchE activity assay kit was weighed and dissolved in 200 mL of assay buffer to prepare a working solution.

[0144] 5. Determination steps

[0145] 5.1 Determination of AchE activity of test substance 1 (WC)

[0146] The measurement was carried out according to the following procedure.

[0147] 1) AchE enzyme solution (33.3 μg / mL), test substance 1 solution (5 mg / mL), and ultrapure water were mixed according to the following Table 1.

[0148]

Table 1

[0149] Table 1 Composition of reaction solution

[0150]

[0151] 2) The reaction was carried out in a constant temperature water bath at 37°C for 10 minutes.

[0152] 3) Add 200 μL of ultrapure water to each of the 4 wells (n=4) of a 96-well microtiter plate (blank well)

[0153] 4) Add 200 μL of the calibrator included in the AchE activity assay kit to each of the other 4 wells (n=4) in step 3). (Calibrator well)

[0154] 5) Add 190 μL of the working solution to each of the other 24 wells (6 samples x 4). (Sample well)

[0155] 6) 10 μL of the post-reaction solutions No. 1 to 6 obtained in step 2) were added to each of the 4 wells (n=4) to which the working solution was added.

[0156] 7) The plate was tapped to mix, and the reaction was allowed to proceed at room temperature for 2 minutes, and then the absorbance at 412 nm was measured using a microplate reader.

[0157] 8) After the reaction was allowed to proceed for another 8 minutes at room temperature (10 minutes in total), the absorbance at 412 nm was measured.

[0158] 5.2 Determination of AchE activity of test substance 2 (GAL)

[0159] The measurement was carried out according to the following procedure.

[0160] 1) AchE enzyme solution (33.3 μg / mL), test substance 2 solution (800 μg / mL), and ultrapure water were mixed according to the following Table 2.

[0161]

Table 2

[0162] Table 2 Composition of reaction solution

[0163]

[0164] 2) The reaction was carried out in a constant temperature water bath at 37°C for 10 minutes.

[0165] 3) Add 200 μL of ultrapure water to each of the 4 wells (n=4) of a 96-well microtiter plate (blank well)

[0166] 4) Add 200 μL of the calibrator included in the AchE activity assay kit to each of the other 4 wells (n=4) in step 3). (Calibrator well)

[0167] 5) Add 190 μL of the working solution to each of the other 24 wells (6 samples x 4). (Sample well)

[0168] 6) 10 μL of the post-reaction solutions No. 1 to 6 obtained in step 2) were added to each of the 4 wells (n=4) to which the working solution was added.

[0169] 7) The plate was tapped to mix, and the reaction was allowed to proceed at room temperature for 2 minutes, and then the absorbance at 412 nm was measured using a microplate reader.

[0170] 8) After the reaction was allowed to proceed for another 8 minutes at room temperature (10 minutes in total), the absorbance at 412 nm was measured.

[0171] 6. Data processing

[0172] The AchE activity (units / mL) was calculated by the following formula using the obtained absorbance values, and the average value and standard deviation of the AchE activity (units / mL) at each concentration of each test substance were calculated.

[0173]

Number 1

[0174]

[0175] (A 412 ) final : Absorbance at 412 nm after 10 minutes of reaction

[0176] (A 412 ) initial : Absorbance at 412 nm after 2 minutes of reaction

[0177] n: dilution factor (n = 120)

[0178] 0.2: coefficient by calibrator

[0179] In addition, the AchE activity at each concentration of the test substance was used to obtain a one-phase exponential decay curve using GraphPad Prism, and the IC 50 Value (mg / mL).

[0180] <Test results>

[0181] 1. Acetylcholinesterase activity of wood creosote (WC)

[0182] The average value and standard deviation of the AchE activity (units / mL) obtained in each well of the test substance 1 (WC) and the test substance 2 (GAL) are shown below.

[0183]

Table 3

[0184] Table 3 Mean values ​​and standard deviations of AchE activity (units / mL) (WC) (n=4)

[0185]

[0186]

Table 4

[0187] Table 4 Mean values ​​and standard deviations of AchE activity (units / mL) (GAL) (n=4)

[0188]

[0189] 2. Calculate IC 50

[0190] Nonlinear regression curve and IC obtained using the AchE activity values ​​of the test substance 1 at each concentration 50 The value is shown in Figure 1 .like Figure 1 As shown, IC 50 The value is 0.27 mg / mL. In addition, the average molecular weight of the components contained in creosote is 129. When the unit is converted to mM, IC 50 The value became 2.1 mM.

[0191] The above results confirmed the inhibitory effect of wood creosote (WC) on acetylcholinesterase. In addition, wood creosote was shown to have an acetylcholinesterase inhibitory activity comparable to that of galantamine (GAL), which is known to be a practical acetylcholinesterase inhibitor.

[0192] <Investigation>

[0193] The results of this study show that creosote has an excellent acetylcholinesterase inhibitory effect. This result suggests that creosote can be used to treat or improve various diseases or conditions caused by acetylcholinesterase. In addition, creosote is known to be a substance widely used as an antidiarrheal drug, and the acetylcholinesterase inhibitor of the present invention is expected to reduce digestive symptoms that are common side effects of existing acetylcholinesterase inhibitors.

[0194] Example 2: Comparison of the Acetylcholinesterase Activity of Creosote and Guaiacol

[0195] <Experimental Materials>

[0196] 1. Tested substances

[0197] Tested substance 3

[0198] Name: Guaiacol (hereinafter referred to as GUA)

[0199] Molecular weight: 124.14

[0200] Purchaser: Tokyo Chemical Industry Co., Ltd.

[0201] Storage conditions: room temperature, dark

[0202] 2. Drug testing

[0203] 2.1AchE enzyme

[0204] Name: Acetylcholinesterase Type VI-S from electric eel

[0205] Product number: C3389-500UN

[0206] Purchaser: Sigma-Aldrich

[0207] Storage conditions: Frozen (-20℃)

[0208] 2.2AchE activity assay kit

[0209] Name: Acetylcholinesterase Activity Assay Kit

[0210] Product number: MAK119

[0211] Purchaser: Sigma-Aldrich

[0212] Storage conditions: room temperature

[0213] 2.3 Water

[0214] Use water purified by the ultrapure water production device UL-Pure (hereinafter referred to as ultrapure water) 3. Use of machines and equipment

[0215] 3.1 Electronic Scale

[0216] Type: AUX120

[0217] Manufacturer: SHIMADZU

[0218] 3.2 Straw

[0219] Type: pipetman P series

[0220] Manufacturer: GILSON

[0221] 3.2 Continuous Dispenser (Multi-channel Pipette)

[0222] Type: pipetman Neo series

[0223] Manufacturer: GILSON

[0224] 3.3 Constant temperature water tank

[0225] Type: BF400

[0226] Manufacturer: Yamato Scientific Co., Ltd.

[0227] 3.4 Refrigerated Storage

[0228] Model:GR-38ND

[0229] Manufacturer: TOSHIBA

[0230] 3.5 Micro-disc analyzer

[0231] Type: SH-9000Lab

[0232] Manufacturer: CORONA ELECTRIC

[0233] 3.6 96-well microplate

[0234] Type: 353072

[0235] Manufacturer: FALCON

[0236] <Test method>

[0237] 1. Prepare the test substance 3 solution

[0238] GUA was weighed and dissolved in ultrapure water to prepare a concentration of 40 mM to prepare a test substance 3 solution.

[0239] 2. Prepare AchE enzyme solution

[0240] 2 mg (500 units) of AchE enzyme (freeze-dried powder) was dissolved in 20 mM Tris-HCl buffer pH 7.52 mL, and then diluted 30 times with ultrapure water to a concentration of 33.3 μg / mL.

[0241] 3. Preparation of AchE Activity Assay Solution

[0242] 2 mg of the reagent contained in the AchE activity assay kit was weighed and dissolved in 200 mL of the assay buffer to prepare a working solution.

[0243] 4. Determination steps

[0244] 4.1 Determination of AchE activity of test substance 3

[0245] The measurement was carried out according to the following procedure.

[0246] 1) AchE enzyme solution (33.3 μg / mL), test substance 3 solution (40 mM), and ultrapure water were mixed according to the following Table 5.

[0247]

Table 5

[0248] Table 5 Composition of reaction solution

[0249] sample GUA1 GUA2 GUA3 GUA4 GUA5 GUA6 AchE enzyme solution (33.3μg / mL) 25μL 25μL 25μL 25μL 25μL 25μL Ultrapure water 75μL 74.5μL 73.3μL 70μL 57.5μL 25μL Test substance 3 solution (40 mM) 0μL 0.5μL 1.75μL 5μL 17.5μL 50μL Total 100μL 100μL 100μL 100μL 100μL 100μL GUA concentration 0mM 0.2mM 0.7mM 2mM 7mM 20mM

[0250] 2) The reaction was carried out in a constant temperature water bath at 37°C for 10 minutes.

[0251] 3) Add 200 μL of ultrapure water to each of the three wells (n=3) of a 96-well microtiter plate (blank well)

[0252] 4) Add 200 μL of the calibrator included in the AchE activity assay kit to each of the other 3 wells (n=3) in step 3). (Calibrator well)

[0253] 5) Add 190 μL of the working solution to each of the other 18 wells (6 samples x 3). (Sample well)

[0254] 6) 10 μL of the post-reaction solutions No. 1 to 6 obtained in step 2) were added to each of the three wells (n=3) to which the working solution was added.

[0255] 7) The plate was tapped to mix, and the reaction was allowed to proceed at room temperature for 2 minutes, and then the absorbance at 412 nm was measured using a microplate reader.

[0256] 8) After the reaction was allowed to proceed for another 8 minutes at room temperature (10 minutes in total), the absorbance at 412 nm was measured.

[0257] 5. Data processing

[0258] The AchE activity (units / mL) was calculated by the following formula using the obtained absorbance values, and the average value and standard deviation of the AchE activity (units / mL) at each concentration of each test substance were calculated.

[0259]

Number 2

[0260]

[0261] (A 412 ) final: Absorbance at 412 nm after 10 minutes of reaction

[0262] (A 412 ) initial : Absorbance at 412 nm after 2 minutes of reaction

[0263] n: dilution factor (n = 120)

[0264] 0.2: coefficient through the calibrator

[0265] In addition, the AchE activity obtained at each concentration of the test substance was used to find a regression curve using GraphPad Prism to calculate the IC 50 Value (mM).

[0266] <Test results>

[0267] 1. Test results of test substance 3

[0268] The average value and standard deviation of the AchE activity (units / mL) obtained in each well of the test substance 3 (GUA) are shown below.

[0269]

Table 6

[0270] Table 6 Mean values ​​and standard deviations of AchE activity (units / mL)

[0271]

[0272] 2. Calculate IC 50

[0273] Nonlinear regression curve and IC obtained using the AchE activity values ​​of each concentration of the test substance 3 50 The value is shown in Figure 2 .like Figure 2 As shown, IC 50 The value is 17mg / mL.

[0274] <Investigation>

[0275] In the acetylcholinesterase inhibition test of Example 1, the IC 50 for 2.1mM In Example 2, the acetylcholinesterase inhibition test of guaiacol was carried out under the same conditions as in Example 1. 50 for 17mM That is, it is shown that wood creosote has an acetylcholinesterase inhibitory activity that is about 8 to 9 times higher than that of guaiacol as a single component.

Claims

1. Use of creosote for the manufacture of a pharmaceutical composition for treating a disease or condition that is treated by acetylcholinesterase inhibition, wherein: The disease or condition is one or more diseases or conditions selected from the group consisting of myasthenia gravis, glaucoma, Alzheimer's type dementia, and residual muscle relaxation by non-depolarizing muscle relaxants.

2. The use according to claim 1, wherein: The pharmaceutical composition is for oral administration or rectal administration, and is prepared so that the dosage of the creosote to a subject is 0.1 to 500 mg per kg of body weight per day.

3. The use according to claim 1, wherein: The pharmaceutical composition is for parenteral administration and is prepared so that the dosage of the creosote to a subject is 0.2 to 300 mg per kg of body weight per day.

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