JPH0665092A - Anti-withdrawal symptomatic agent containing lps and anti-withdrawal symptomatic agent for animal - Google Patents

Abstract

PURPOSE:To provide the subject new agents administrable through any of the following routes: peroral, percutaneous, injection and intraperitoneal routes. CONSTITUTION:The objective agents containing the LPSs represented by the following three kinds thereof: (1) LPS-1: (a) primary molecular weight: 5000+ or -1000 (SDS-PAGE method), (b) phosphorus number: 2+ or -1/molecular weight 5000, (c) hexosamine number: 9+ or -1/molecular weight 5000, (d) KDO number: 2+ or -1/molecular weight 5000; (2) LPS-2: (a) primary molecular weight: 6500+ or -2500 (SDS-PAGE method), (b) phosphorus number: 1-2/molecular weight 5000, (c) hexosamine number: 7+ or -1/molecular weight 5000, (d) KDO number: 1-2/molecular weight 5000; (3) LPS-3: (a) primary molecular weight: 6500+ or -2500 (SDS-PAGE method), (b) phosphorus number, 2+ or -1/molecular weight 5000, (c) hexosamine number: 5+ or -1/molecular weight 5000, (d) KDO number: 2+ or -1/molecular weight 5000.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel anti-withdrawal agent and anti-withdrawal agent for animals. More specifically, the present invention relates to a novel anti-withdrawal agent containing LPS and a novel anti-withdrawal agent for animals.

[0002]

2. Description of the Related Art It is generally well known that if the use of alcohol, morphine narcotics, nicotine, etc. becomes addictive and the intake thereof is suddenly prohibited, so-called withdrawal symptoms occur. Therefore, it is a well-known fact that rehabilitation of addicts such as alcohol is hindered, and clinical use of narcotics is restricted. Methadone (methado) is currently used as a drug for suppressing such withdrawal symptoms.
ne), clonidine, dizocilpine, etc. are known.
However, methadone is said to cause drug addiction by itself. [Pee. Ah. Doherty (PR
Doughherty) et al., Neuropharmacology, 26 , 15
95-1600, 1987]. Also, clonidine is
It has been reported that intraperitoneal administration of 0.16 mg / kg suppresses swaying. [Stuart Fielding (S
The Field of Pharmacology and Experimental Therapeutics (THE JOURNAL OF P)
HARMACOLOGY AND EXPERIMET
AL THERAPEUTICS), Vol. 207,
No. 7, 899-905, 1978] However,
Even when the present inventors intravenously administered 0.1 to 10 mg / kg, there was no effect of suppressing the jump phenomenon, which is a more severe withdrawal symptom. Furthermore, 10 mg / kg of intravenous administration caused spasticity. Disocilpine lacks safety because the difference between the toxicity value and the effective value is extremely small. [Keith A. et al., Science (Science)
e), 251 , 85-87, 1991].

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention Currently used anti-convulsant drugs have the drawbacks as described above, and no satisfactory anti-continence drug has been provided yet.

SUMMARY OF THE INVENTION The present invention has as its technical subject the provision of a novel anti-withdrawal symptom agent in which the above-mentioned drawbacks of the prior art have been solved and a novel anti-withdrawal symptom agent for animals.

[0005]

[Means for Solving the Problems] The above technical problems have a high anti-withdrawal symptom effect, a high chemotherapeutic index, and can be used for a long period of time, and any route of oral, transdermal, injection and intraperitoneal routes can be used. However, it is achieved by providing a novel anti-continence symptom agent and animal anti-continence symptom agent which can be administered, can be produced at low cost, and can be supplied in a large amount.

Bacterial Separation Source The LPS-derived bacterium used in the present invention has been isolated from wheat examined by the present inventors regardless of its origin and type. Therefore, it is presumed that it is separated from wheat and wheat products of all production areas and types. The origins and types of wheat flour that the present inventors have confirmed to be able to separate these bacteria are as follows. Nominal Origin of Oat Flour Dark Northern Springs USA 1. Canadian Wheat Canada Hard Red Winter Semi-Hard USA Australian Standard Wheat Australia Horosiri Japan

Separation of PS In order to separate the LPS that can be used in the present invention from the above-mentioned bacteria, Westphal et al. Described “Methods in Carbohydrate Chemistry (Met).
hods in Carbohydrate Chem
(Istry) vol. V [published by Academic Press, Inc., New York, USA, 1965], page 83, using the hot phenol method and further purification with an anion exchange resin.

That is, after suspending the cells in distilled water, the cells are stirred with distilled water and an equal volume of hot phenol, and then the aqueous layer is recovered by centrifugation, and the aqueous layer is dialyzed to remove phenol. After removal and concentration by ultrafiltration, a crude LPS fraction was obtained, and this fraction was subjected to a conventional method, for example, using a Pharmacia FPLC system, Mono Q-Sepharose manufactured by Pharmacia, or Q-Sepharose (manufactured by Pharmacia). Sepharose) may be used for anion exchange chromatography for purification, and desalting may be performed according to a conventional method. By the above operation, a purified sample with a purity of 96% or more can be obtained.

The physical properties of LPS, which can be used in the present invention as described in detail in the production examples below
The physical properties of 3 kinds of S (96% or more pure sample) were as follows. (SDS-PAGE method is defined in Production Example 1) LPS1 Main molecular weight: 5,000 ± 1,000 (S
DS-PAGE method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 9 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 LPS2 Main molecular weight: 6,500 ± 2,500 ( S
DS-PAGE method) Phosphorus number: 1-2 / molecular weight 5,000 Hexosamine number: 7 ± 1 / molecular weight 5,000 KDO number: 1-2 / molecular weight 5,000 LPS3 Main molecular weight: 6,500 ± 2,500 ( S
DS-PAGE method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000

Measurement of anti-withdrawal symptom effect The anti-withdrawal symptom effect of the LPS of the present invention is determined by the effect of naloxone, which is known as a morphine antagonist on morphine-addicted mice [The Journal of Pharmacology and Experimental Therapy, above].
901 page. In addition, it was confirmed that the number of occurrences of the "jumping" phenomenon, which is considered to be the most serious of the withdrawal symptoms induced by the administration of Endlabs, Inc. of the United States (available from Endo Labs. Inc.), was used as an index. .

Further, the effect on the psychological withdrawal symptoms is evaluated by the place preference conditioned reflex test (Conditione).
d Place preference) [Japanese Journal of Psychopharmacology published in 1992 (Japanese
J. Psychopharmacology), 1
2 , pp. 25-30, Suzuki Tsutomu et al., 5-HT ₃ receptor antagonists block place preference induced by cocaine and methamphetamine ”(5-HT
₃ receptor antagonists bl
ock cocaine- and method
etamine-induced place pre
ference)]]. The test is a method of measuring the dependence on a drug by empirically learning the relationship between a drug and the place where it is administered to experimental animals, and then measuring the preference for the same place. As well established in the industry.

The LPS which can be used in the present invention can be used individually, and as long as its intended use is not impaired, two or more kinds of them can be arbitrarily combined or further combined with any other substance. Can be used. It can also be used as one component of quasi drugs, foods, functional foods, beverages, feeds and the like. LP of the present invention
Anti-withdrawal drug containing S can be administered as an oral drug, an intravenous drug, an intramuscular drug, by an ordinary pharmaceutical technique or a conventional method for producing an animal drug.
As a transdermal drug, an intraperitoneal drug alone or as a mixture with other drugs, powders, granules, pills, tablets, troches, capsules, liquids, patches, ointments, liniments, lotions, It can be provided in the form of suppositories, injections and the like. Further, for animals, it can be further prepared as a feed additive, a premix preparation, and a drinking water additive. When used as a feed additive, it is preferably a powder or granules. The premix formulation refers to a product diluted with a feed component such as starch to facilitate mixing with a feed. The feed additive containing the LPS of the present invention and the feed to which the premix preparation can be added may be any commercially available feed.
It may also be a feed containing feed additives such as minerals, vitamins and amino acids.

If desired, additives such as excipients, preservatives, buffers and the like may be added to these preparations in order to maintain storability and homogeneity. Further, a corrigent, a flavoring agent, and a coloring agent can be included. As the excipient, for example, lactose or starch can be used. As the preservative, for example, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate and propyl paraoxybenzoate, sodium dehydroacetate, phenol, methylparaben, ethylparaben, propylparaben and the like can be used. As the buffer, for example, citrate, acetate, phosphate and the like can be used.

The present invention will be described in more detail with reference to production examples, examples and experimental examples. The "Escherichia coli LPS" used in them is O128: B8 manufactured by Difco, USA.

Production Example 1 1.04 g of hard wheat flour (Canadian Wheat from Canada) containing 1.09% ash was weighed and put into a 50 ml Corning tube, and 20 ml of distilled water was added to 50 mg /. ml of flour solution was prepared.

This solution was cultivated with shaking in a water bath at 37 ° C., and the elapsed time was 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours.
0 o'clock, 10 o'clock, 12 o'clock, 20 o'clock, 24 o'clock, 45 o'clock each.
Collect 5 ml, dilute 10 ° to 10 ⁵ times, and add 1 to standard agar medium (Nissui Pharmaceutical Co., Ltd. medium having the following composition).
A total of 00 μl was seeded, and the viable cell count was measured and colonies were observed. Standard agar medium (Nissui Pharmaceutical Co., Ltd. code number: 05618) In 1 liter Yeast extract 2.5 g Peptone 5.0 g Glucose 1.0 g Agar 15.0 g pH 7.1 ± 0.1

Yellow to cream opaque colonies (colony 1), cream opaque colonies (colony 2), and yellow semi-transparent colonies, which were considered to be of different types, were observed at 8 hours and 10 hours of the elapsed culture time. The colony 3), the milky white opaque colony (colony 4), and the small white opaque colony (colony 5) are spread on another standard agar medium of the same species as described above and subcultured, while colonies 1 to
Gram stainability and limulus activity of 5 bacteria were examined.

The term "limulus activity" as used herein means to be positive in a limulus test, which is an endotoxin quantification method using a horseshoe crab blood cell extract and a chromogenic synthetic substrate, which was invented by Levin in 1968. This limulus test is known as the LPS detection method,
For example, it can be carried out using a reagent set commercially available from Seikagaku Corporation under the name Toxicolor System.

Of the above colonies, the limus activity of colonies 4 and 5 (both Gram stain +) was extremely lower than that of colonies 1 to 3 (both Gram stain −). The morphology and biochemical properties of colonies 1 to 3 were observed using a medium manufactured by Sui Pharmaceutical Co., Ltd. and an ID test EB-20. The following results were obtained.

Bacteria forming colony 1 (identification number: 9
(0081-1) (Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Microbial Technology)
Deposited in Japan as Microbiology Research Institute Contribution No. 11664 from August 17, 2013, and Microconservation Research Article Contribution No. 350 from August 12, 1991.
No. 9 was transferred to international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, this bacterium is presumed to belong to the genus Serratia of the family Enterobacteriaceae.

(A) Morphology Short rod-like shape No mobility Gram stainability:-

(B) Growth state Approximate semi-agar medium: Yellow to cream-colored, round, opaque colonies are formed. SS agar: forming white, translucent colonies. [SS agar medium: Nissui Pharmaceutical Co., Ltd. code number: 05031] In 1 liter composition, meat extract 5.0 g bile salt 9.0 g peptone 7.5 g lactose 10.0 g sodium citrate 8.5 g sodium thiosulfate 5.5 g citric acid Ferric acid 1.0 g Neutral red 0.025 g Brilliant gulin 0.033 Agar 13.5 g pH: 7.1 ± 0.1 TSI agar medium: No change on the slope, but yellow on the upper part. Produces gas. [TSI agar medium: Nissui Pharmaceutical Co. code number: 0510
3] In a composition of 1 liter Meat extract 5.0 g NaCl 5.0 g Peptone 15.0 g Lactose 10.0 g Sucrose 10.0 g Glucose 1.0 g Ferric citrate 0.2 g Sodium thiosulfate 0.2 g Phenol red 02g agar 15.0g pH: 7.6 ± 0.1

(C) Physiological properties Forges-Broscauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Carbon source Availability of lactose: + Adnit: -Rhamnose: + Mannitol: + Esculin: + Inosit: -Sorbit: + Arabinose: + Raffinose: + ▲ 10 ▼ Sucrose: +

(E) Others Decarboxylation reaction of lysine: -Use of malonic acid: -Decomposition of arginine: -Deamination reaction of phenylalanine: -Decarboxylation reaction of ornithine:-

Bacteria forming colony 2 (identification number: 9
(Headed to the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology, Heisei 2)
Deposited in Japan as Microbiology Research Institute No. 11665 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 0 was transferred to the international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, the bacterium is presumed to belong to the genus Enterobacter of the Enterobacteriaceae family.

(A) Morphology Short rod shape No ageability Gram stainability:-

(B) Growth state Standard agar medium: A cream-colored and opaque colony is formed. SS agar: forms red, opaque colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas.

(C) Physiological Properties Forges-Proscher's reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: +

(D) Utilization of carbon source Lactose: + Adonite: -Rhamnose: + Mannitol: + Esculin: + Inosit: -Sorbit: + Arabinose: + Raffinose: + (10) Sucrose: +

(E) Others Decarboxylation reaction of lysine: -Use of malonic acid: + Decomposition of arginine: + Deamination reaction of phenylalanine:-Decarboxylation reaction of ornithine: +

Bacteria forming colony 3 (identification number: 9
(Heisei 2 in the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Science and Technology)
Deposited in Japan as Microbiology Research Institute No. 11666 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 1 was transferred to an international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, this bacterium is presumed to belong to the genus Pantoea of the family Enterobacteriaceae.

(A) Morphology Short rod-like shape No mobility Gram stainability:-

(B) Growth state Standard agar medium: yellow, round translucent colonies are formed. SS agar: does not form colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Does not generate gas.

(C) Physiological properties Forges-Proskerwell reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: +

(D) Utilization of carbon source Lactose: + Adonite: -Rhamnose: + Mannitol: + Esculin: + Inosit: -Solpit: + Arabinose: + Raffinose:-(10) Sucrose: +

(E) Others Decarboxylation reaction of lysine: -Use of malonic acid: + Decomposition of arginine: -Deamination reaction of phenylalanine: -Decarboxylation reaction of ornithine:-

Each of the colonies 1, 2, and 3 was transferred to 1 liter of L-broth medium and shaken at 37 ° C. overnight.
The cells were collected by centrifugation at 000 G for 20 minutes at 4 ° C. The L-broth medium was prepared by adding 10 g of polypeptone manufactured by Difco, 5 g of yeast extract manufactured by Difco, and special grade NaCl (5 g) manufactured by Wako Pure Chemical Industries, Ltd. to distilled water, adjusted to pH 7.5 with NaOH, and autoclaved. Separately, prepare a 40% solution of special grade glucose from Wako Pure Chemical Industries, which has been prepared in advance.
It was prepared by adding it after diluting it by 00 times.

Each bacterial cell was suspended in 50 ml of distilled water, and 50 ml of 90% hot phenol was added to the suspension to give 6 cells.
Stir for 20 minutes at 5 to 70 ° C, and after cooling, 10,000
G was centrifuged at 4 ° C. for 20 minutes to collect an aqueous layer. The phenol layer was subjected to the same operation as above twice more. The three water layers were combined and dialyzed overnight to remove phenol, and the internal solution was advantech toyo (ADVANTEC).
The product was subjected to ultrafiltration using UK-200 manufactured by TOYO) and concentrated by a molecular weight cut-off of 200,000 (N.
₂ pressure: 2 atm).

This concentrated sample was used as Q-Sepharose Fast Flow (Q-Seph) manufactured by Pharmacia.
Arose Fast Flow) and subjected to anion exchange chromatography. That is, after applying the sample to the column with a buffer solution containing 10 mM Tris-HCl (pH 7.5) and 10 mM NaCl, 400 mM NaCl /
The limulus active fraction was eluted with 10 mM Tris-HCl (pH 7.5). This eluate is subjected to ultrafiltration under the same conditions as above to desalinize and concentrate to obtain LPS with a purity of 96% or more.
Got The nucleic acid is 1M NaCl / 10 mM Tris-
Elute with HCl (pH 7.5).

The results of each bacterial cell are shown in Tables 1 to 3 below. In addition, the amount of LPS is E. coli L
PS conversion value, sugar is phenol-sulfuric acid method [M. Authored by M. Dubois, Analytical
Chemistry (Analytical Chemistr
y), Vol. 28, p. 350, 1956], the protein is the Lowry method [oh, h. Raleigh (OH Low
ry) et al., Journal of Biological Chemistry (Journal of Biological
Chemistry)], vol.193, p. 65, 1
951]. In addition, the amount of nucleic acid is OD (260n
Based on the measured value in m) (1 OD = 50 μg), the purity (%) was calculated based on the following formula.

[Equation 1]

[0044]

[Table 1]

[Table 2]

[Table 3]

Molecular weight : LPS obtained from each bacterial cell was dissolved in distilled water to obtain 2 m.
A g / ml solution was prepared and 10 μl thereof was placed in a 1.5 ml plastic tube. Separately, 180μ
l 10% (w / v) SDS, 45 μl 5% β-mercaptoethanol, 90 μl CBB dye solution, 11
2.5 μl of 0.5 M Tris-HCl (pH 6.8) and 2
SDS treatment solution 10 prepared by adding 2.5 μl of distilled water
μl was added and mixed well, then immersed in a boiling water bath for 5 minutes. Immediately after this heating, it was immersed in ice water and rapidly cooled.

10 ml of 10% (w / v) SDS, 1
The migration buffer prepared by dissolving 7.9 g of tricine and 3.03 g of Tris in 1 liter of distilled water was placed in a slab gel electrophoresis tank manufactured by Marisol. A 20% polyacrylamide gel was fixed in an electrophoretic bath, a sample was put in a sample groove, the voltage was fixed at 50 v for 1 hour, and then fixed at 150 v, and the electrophoresis was continued until the dye was eluted from the gel (herein). This electrophoresis method is called SDS-pAGE method). After electrophoresis, Bio-Rad's silver staining kit 161-04
Silver staining was carried out at room temperature using No. 43 to confirm the behavior. Simultaneous protein molecular weight markers [LMw kit E manufactured by Pharmacia: phosphorylase b (94k), albumin (67k), ovalbumin (43k), carbonic anhydrase (30k), trypsin inhibitor (20k), α- Lactalbumin (14k)],
Peptide molecular weight marker [1860 manufactured by Pharmacia
-101 molecular weight marker: myoglobin (16.9
k), myoglobin I & II (14.4k), myoglobin I (8.2k), myoglobin II (6.0k),
When the molecular weight of LpS of the present invention was calculated from the migration position of [myoglobin Iv (2.5k)], it was 5,000 ± 1,000.
0 (LPS1 derived from bacterial cell 900814-1), 6,
500 ± 2,500 (LPS2 derived from bacterial cell 900814-2 and LPS derived from bacterial cell 900814-3
It was 3). The molecular weight of Escherichia coli LPS (Escherichia coli 0127: B8LPS manufactured by Difco) measured in the same manner was 30,000 ± 20,000.

LPS1, LPS2 in the above silver staining,
The stained band of LPS3 is shown in FIG. In FIG. 1, number 1
Is LPS1, number 2 is LPS2, number 3 is LPS3
It is a dyed belt. As shown in FIG. 1, LPS1 also showed a slightly aggregated staining band even at a molecular weight of around 30,000. LP
S2 has a dyeing zone between 30,000 and 43,000, but it is presumed that the number of macromolecules is extremely small in comparison with the dyeing degree of the dyeing zone of 14,000 or less.
LPS2 can be judged from the amount of sugar and hexosamine described below.
Has the lowest sugar content, followed by LPS3 and LPS1 in that order, which is considered to be consistent with the pattern observed by electrophoresis. Also, the ratio of LPS amount / total dry yield is LPS.
2, LPS3, LPS1 in this order. From the above observation results, it is estimated that LPS2 has a relatively large amount of low-molecular-weight LPS, and then the ratio of LPS3 and LPS1 decreases in that order.

Phosphorus Content Chen-Toribara Method [Chien et al., "Analytical Chemistry (Ana
(Lytical Chemistry), vol. Two
8, 1756-1758 (1956). LPS1, LPS2 and LPS3 were separately dissolved in distilled water to give 31.6 μg and 57.
A 20 μl solution containing 6 μg, 103.6 μg LPS was prepared and placed in a small test tube. 20 μl of 50 v / v%
Sulfuric acid was added and heated at 160 ° C. for 2 hours. Then 2
After adding 0 μl of 10 v / v% perchloric acid, the mixture was heated with a gas burner for 1 minute to incinerate. Then 0.5 ml
Distilled water, then 0.5 ml of reaction reagent (1 ml of 6N
Sulfuric acid, 2 ml of distilled water, 2 ml of 2.5 v / w% ammonium molybdate and 1 ml of 10 v / w% ascorbic acid were mixed and prepared, 0.5 ml of which was used) and added for 30 minutes at room temperature. After standing, the absorbance OD at 820 nm (820 nm) was measured. In addition, as a sample for preparing a calibration curve, potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and phosphoric acid weights of 2.5 μg, 1 μg, 0.25 μg, and 0 μg were included, respectively.
A 5 ml solution was prepared and used. In addition, 1 g of phosphorus corresponds to 4.39 g of potassium dihydrogen phosphate. The results are shown in Table 4 below. In the table, the numerical value indicating the absorbance is a value obtained by subtracting the data of the control not subjected to the heat treatment in order to avoid an error due to the incorporation of inorganic phosphorus (for example, derived from the phosphate buffer solution). The phosphorus amount (μg) is a value calculated from the absorbance. The phosphorus amount (% by weight) was calculated by the following formula. In the formula, “0.67” indicates the OD value of 1 μg of standard phosphorus, and the sample concentration indicates the concentration (mg / ml) of each LPS dissolved in distilled water.

[Equation 2] The phosphorus number is a conversion number per 5,000 molecular weight calculated by the following formula.

[Equation 3]

[0049]

[Table 4]

Hexosamine Content Elson-Morgan Method (Tokyo Kagaku Dojin Shuppan "Biochemistry Experiment Course" No. 37)
7 to 379) and performed as follows. Dissolve LPS in distilled water to give 1.58 mg (LPS1), 2.88
A solution of mg (LPS2), 5.18 mg (LPS3) / ml was prepared, and 100 μl thereof was put into a Spitz (manufactured by Iwaki Glass Co., Ltd.) with a screw cap, and 100
μl 8N HCl was added and heated at 110 ° C. for 16 hours. About 200 μl of 4N NaOH was added to adjust the pH to 7. A 100 μl portion thereof was taken, placed in another Spitz equipped with a screw cap, and after adding 200 μl of the following reagent A, the mixture was heated at 105 ° C. for 1.5 hours and then cooled with running water. Then 100 μl was taken and 670 μl of 96
% Ethanol was added, and 67 μl of the following reagent B was added, and the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 535 nm. 0.20 to 200μ as a sample for preparing a calibration curve
g / ml N-acetylglucosamine (manufactured by Wako Pure Chemical Industries, Ltd.) was used. (Reagent A) It was prepared by mixing 75 μl of acetylacetone and 2.5 ml of 1.25N sodium carbonate. (Reagent B) It was prepared by mixing 1.6 g of p-dimethylbenzaldehyde, 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol. As a result, the number of hexosamines in LPS1, LPS2, and LPS3 was 9 ± 1 / molecular weight 5,000 and 7 ± 1 / molecular weight 5, respectively.
It was 5,000, 5 ± 1 / molecular weight 5,000.

KDO Content KDO (2-keto-3-deoxyoctonate) content was measured by the diphenylamine method [Shaby (Shaby
R. ) Et al., Analytical Biochem (Analy
mechanical Biochem. ), 58 (1), 123
~ 129 (1974)].

A KDO detection sample was prepared by combining 500 mg of diphenylamine, 5 ml of ethanol, 45 ml of glacial acetic acid, and 50 ml of concentrated hydrochloric acid (all manufactured by Wako Pure Chemical Industries, Ltd.). In 500 µl thereof, (1) 0.505 mg / ml of LPS1 was added in 250 µl distilled water solution; (2) 0.57
250 μl distilled aqueous solution containing 6 mg / ml LPS2;
(3) 250μ containing 0.518 mg / ml LPS3
1 distilled water solution; and heated in a boiling water bath at 100 ° C. for 33 minutes and then cooled in cold water (24.5 ° C.) for 30 minutes, and then using a Hitachi spectrophotometer 320, 420,
Ultraviolet absorption at 470, 630 and 650 nm was measured (measurement values were A420, A470, A630 and A65, respectively).
0). As a standard sample, 250 μl of distilled water containing 0.5 μmol / ml KDO ammonium salt [manufactured by Sigma, USA] was used.

The value of the following equation was determined for each of the specimen sample and the standard sample. S = A420-A470 + A630-A650

The value (S _T ) of the sample specimen is 0.1 for LPS1.
09, LPS2 was 0.078, and LPS3 was 0.099. The value of the standard sample _{(S S)} is 0.246, the value of the distilled water alone was 0.005.

By comparing these values, LPS1 has a value of 2 ± 1.
/ Molecular weight 5,000, 1-2 for LPS2 / Molecular weight 5,
KD of 2 ± 1 / molecular weight of 5,000 for LPS3 and 5,000
It was estimated that O was included.

Note that these values are calculated as follows, taking LPS1 as an example. When the concentration of KDD contained in the solution is x (μmol / ml),

[Equation 4] From the above equation, x = 0.221. Therefore, LPS1
If y is the number of moles of KDD contained in 1 mole (assumed to be 5,000) of, then y = 2.19 from the following equation.

[Equation 5]

Production Example 2 (Production of Bordetella pertussis LPS) A pertussis solution for test (2.
0 × 10 ¹⁰ cells / ml) were used as dead cells.

25 mg (dry weight) / ml of the dead cells
Was suspended in sterilized water. To this, an equal amount of 90% hot phenol solution (68 to 70 ° C) was added, and extraction was performed while shaking at 68 ° C for 1 hour. The aqueous layer was separated by centrifugation at 8,000 G and 4 ° C. for 20 minutes. To the remaining phenol layer, the same amount of sterilized water as the above aqueous layer was added, and the same extraction was performed. The obtained aqueous layer was combined with the previous aqueous layer, dialyzed in running water overnight, and then concentrated to 1/10 with a rotary evaporator. This was centrifuged at 8,000 G and 4 ° C. for 20 minutes. Separate the supernatant and add a small amount of sodium acetate.
Cold ethanol at 6 ° C was added in an amount of 6 times, and the mixture was left at -20 ° C overnight. The precipitate recovered by centrifugation at 4,000 G for 30 minutes at 4 ° C. was washed twice with ethanol, then once with acetone, and dried with an aspirator.

The residue was suspended in distilled water so as to have a concentration of 20 mg / ml, and subjected to ultrasonic treatment (output control 5, 15 minutes, room temperature) with Sonifia Model 185 manufactured by Branson, USA. Then 2,500G, 4
Centrifugation was performed at 10 ° C. for 10 minutes to collect the supernatant.

This supernatant was added at 4 ° C. to Sigma (USA).
a) Nucleolytic enzymes DNase I and RNase A manufactured by the company
Treatment for 15-16 hours (finally 10 μg / ml
DNase I and 20 μg / ml RNase A
It was used). Add the same concentration of nucleolytic enzyme and add 3
Heated at 7 ° C. for 2 hours. Then, at 2,500 G and 4 ° C, 1
The mixture was centrifuged for 0 minutes, and the supernatant was collected.

This supernatant was used in Gelman, USA
It was filtered with a pore size of 0.2 μm using an Acrodisc of the company. The filtrate was passed through a molecular sieve [Resin: Sepharose 6B manufactured by Pharmacia, USA, column size = inner diameter 5].
cm × length 100 cm, buffer = 10 mM Tris-H
Cl, 10 mM NaCl (pH 7.5), flow rate = about 3
ml / cm ² / hour), the limus activity positive fractions were examined and combined using a LS-1 kit manufactured by Seikagaku Corporation, and filtered with the acrodisc manufactured by Gelman Co. at a pore size of 0.2 μm. The filtrate is subjected to ion exchange [apparatus: FPLC manufactured by Pharmacia, USA, resin: Mono Q HR10 / 10 manufactured by Pharmacia, USA, buffer = 1
0 mM Tris-HCl + 10 mM NaCl (pH
7.5) for 15 minutes, then adjust the amount of NaCl to 165
wash to 30 mM for 30 minutes, then for 20 minutes,
Washing while increasing the amount of NaCl so that the amount of NaCl becomes a concentration gradient from 165 mM to 1 M, and then washing with 1 M amount of NaCl for 30 times, flow rate = 2 ml / min], LS-1 kit manufactured by Seikagaku Corporation Fractions positive for limulus activity were examined by using and were combined. The combined fractions were desalted on a column [resin: Sephadex G-25 fine manufactured by Pharmacia, USA, column size = inner diameter 2 cm × length 25 cm, eluent = distilled water], and then freeze-dried. did.

The substance most likely to be mixed in this freeze-dried preparation (4.50 mg) is nucleic acid. Therefore,
Take an ultraviolet absorption curve (200-400 nm), 260n
The absorbance at m was determined. Nucleic acid concentration is 5 when absorbance is 1.
It was 1% or less when the nucleic acid concentration was calculated from the above absorbance using 0 μg / ml. Also, SDS-PAG
No protein was clearly detected by method E. Therefore,
Considering the detection sensitivity, the protein mixed in the freeze-dried preparation is estimated to be 0 to 3% at most. Therefore, the purity of the freeze-dried preparation was estimated to be 96% or higher.

The physical properties of this B. pertussis LPS measured in the same manner as in Production Example 1 were as follows. Physical properties of B. pertussis LPS Main molecular weight = 6,000 ± 1,000 (SDS-pAG
E method) Phosphorus number = 4 / molecular weight 6,000 Hexosamine number = 12 / molecular weight 6,000 Fatty acid number = 4 / molecular weight 6,000 KDO number = 2 ± 1 / molecular weight 6,000

The physical properties of Escherichia coli LPS measured in the same manner were as follows. Physical properties of Escherichia coli LPS Main molecular weight = 40,000 ± 10,000 8,000 ± 4,000 (by SDS-PAGE method) Phosphorus number = 12 / molecular weight 30,000 Hexosamine number = 45 ± 6 / molecular weight 30,000 Fatty acid number = 18 / Molecular weight 30,000 Number of KDO = 5 ± 1 / Molecular weight 30,000

The following is a formulation example of the preparation containing the LPS of the present invention. The LPS amounts in Examples 1 to 4 are E. coli LPS-equivalent amounts by the limulus test.

Example 1 (tablets) LPS1 0.04 g 6% HPC lactose 178 g talc stearate 8 g potato starch 14 g The above ingredients were mixed and compressed to form 400 0.5 g tablets containing 0.1 mg LPS1. Prepared.

Experimental Example 1 (Effect of anti-withdrawal symptom-1) 6 to 12 4 to 5 week old ddY mice (body weight: 20)
˜24 g), 12.7 mg of morphine pellets (prepared by impregnating molecular sieves with morphine hydrochloride morphine obtained from Takeda Pharmaceutical Co., Ltd. of Japan; and so on) subcutaneously just below the back neck. 2 days after transplantation, 50 μg / kg body weight E. coli LPS (6 animals),
LPS3 (7 animals) or B. pertussis L produced in Production Example 1
PS (6 animals) was dissolved in physiological saline and intravenously injected. The control group received only physiological saline. 1 hour later 1
Naloxone (0 mg / kg body weight) was intraperitoneally administered, and immediately after that, the number of "jumps" in the mice was counted for 40 minutes to determine the jumping-suppressing effect. The results are shown in Table 5. In Table 5,
Numerical values represent the number of relevant mice. The jumping suppression effect was determined as follows. The average number of jumps / animal in the physiological saline administration group (12 animals) as a control group was 62.7 ±.
Since it was 25.5, it was determined from 62.5-25.5 = 37 that there was no effect when the number of jumps was 37 or more, and there was an effect when the number of jumps was less than 37.

[Table 5]

As is clear from Table 5, the incidence of anti-withdrawal symptoms was only about 8% in the control group, 50% in the E. coli LPS administration group, and about 67% in the B. pertussis LPS (Production Example 2) administration group.
%, And 100% in the LPS3 administration group.

Experimental Example 2 (Anti-withdrawal symptom effect-2) In order to examine the dose dependence of the anti-withdrawal symptom effect of LPS of the present invention upon intravenous administration, 4-5 week old ddY male mice (average weight 20 g) , 12.7 mg of morphine pellet was subcutaneously transplanted just below the neck of the back, and 2 days later, 0.5 μg / kg body weight (6 animals), 5 μg / kg body weight
(6), 15 μg / kg body weight (9), 50 μg / kg body weight (12), or 500 μg / kg body weight (6)
LPS3 was dissolved in physiological saline and administered intravenously.
The control group (10 animals) was administered with physiological saline only. Two hours after that, 10 mg / kg body weight of naloxone was intraperitoneally administered, and immediately after that, the number of "jumps" in the mice was counted for 40 minutes. The results are shown in Table 6 as an average per animal of each group.

[Table 6]

FIG. 2 is a graph of the results shown in Table 6.

Experimental Example 3 (Anti-withdrawal symptom effect-3) Experimental example 2 was repeated in order to investigate the dose dependence of the anti-withdrawal symptom effect of LPS of the present invention upon intradermal administration. However, the amount of LPS3 is 50 μg / kg body weight (7 animals) or 5
The control group (physiological saline administration group) was set to 8 animals, and the amount was set to 00 μg / kg body weight (5 animals). The results are shown in Table 7 as an average per animal in each group.

[Table 7]

FIG. 4 is a graph of the results shown in Table 7. It is clear from FIGS. 2 and 3 that the anti-withdrawal effect of LPS of the present invention is dose-dependent.

Experimental Example 4 (Effect of anti-withdrawal symptom-4) In order to examine the administration time dependency of the effect of anti-withdrawal symptom of the LPS of the present invention, 5-9 ddY male mice (5-9 mice in each group) ( In a body weight of 20 to 24 g, 12.7 mg of morphine pellet was subcutaneously transplanted just below the neck of the back, and 2 days later, 10 mg / body dish kg of naloxone was intraperitoneally administered,
However, 5 hours before administration of naloxone (7 animals), 3 hours (8 animals), 8 hours (6 animals), and 18 hours (5 animals)
LPS3 at 0 μg / kg was intravenously administered, and the number of “jumps” in the mice was counted for 40 minutes immediately after the administration of naloxone. The LPS3 non-administration group was 9 animals. The results are shown in Table 8 as an average per animal in each group.

[Table 8]

FIG. 4 is a graph of the results shown in Table 8. From FIG. 4, it is estimated that the LPS of the present invention has a withdrawal symptom preventive effect, and that the preventive effect is maximized when it is administered immediately before the onset of withdrawal symptom.

Experimental Example 5 (Anti-withdrawal symptom effect-5) Sprague-Da of 4 animals per group
LPS to 6-week saponified rats (average weight 165 g)
3 (5 or 15 μg / kg body weight) in physiological saline (1 c
c / kg body weight) or physiological saline alone (1 cc / kg body weight) was intraperitoneally administered. One hour later, cocaine (commercially available from Sankyo Co., Ltd., 4
(mg / kg of body weight) was intraperitoneally administered, and then 50 in black or white in a box (KN-80 type Cpp device manufactured by Natsume Seisakusho) partitioned by a partition plate into two rooms colored in white and black. I put it in for a minute. The next day, physiological saline (1 cc / body weight k
After the administration of g) alone, it was placed in a room of the opposite color for 50 minutes. The above steps were repeated twice (total of 4 days).

On the next day, the partition plate of the same box was removed, a platform was set up at the boundary between both rooms, and the rat was allowed to move freely, and the staying time of the rat in the white or black room was measured for 15 minutes thereafter. did. The result is the value obtained by subtracting the staying time (second) in the room of the opposite color from the staying time (second) in the room of the same color as the room first put in after the drug administration by the device installed in the same box. (Sec) was automatically measured. This value approaches "0" if LPS3 or the like has the effect of suppressing the dependence on cocaine, and increases as the effect of increasing the dependence increases. The result
Table 9 shows the average of each group.

FIG. 5 is a graph of the results shown in Table 9.

In the above experiment, cocaine hydrochloride was converted to morphine hydrochloride (commercially available from Sankyo Co., Ltd.) (4 mg / kg body weight), or methamphetamine hydrochloride (commercially available from Dainippon Pharmaceutical Co., Ltd., 2 mg / body weight kg). LPS3 (45 μg / kg body weight) showed a time difference of 150 seconds and 130 seconds (average of 3 animals each) in the case of morphine hydrochloride and methamphetamine hydrochloride, respectively, compared with the physiological saline administration group. It has been proved to have a strong anti-withdrawal effect.

[Table 9]

Dose, Dosage Interval, Toxicity Value Dose of the anti-withdrawal agent of the present invention, the anti-withdrawal agent for animals,
The dosing interval is, of course, individually determined under the strict control of the attending physician or veterinarian, taking into consideration the age, symptoms, weight, and administration effect of the subject, but it is an oral administration for a human adult (60 kg). 1 μg to 100 mg, 10 ng to 10 by intravenous administration
mg, 100 ng to 1 mg by percutaneous administration is a temporary guideline for the once-daily dose. For animals, for large animals such as cows and horses, use 1/60 of the above amount as a guideline for the amount per 1 kg of body weight, and for medium-sized and small animals such as pigs, dogs and cats, double the amount. The dose per kg can be used as a standard, and for birds such as chickens, double the dose can be administered as a guide for the amount per kg of body weight.

Incidentally, Behrence Kelver (Behre
ns K

[Outer 1] rber) method, 7-week-old average body weight 22 g
, LPS1, LPS2 in C3H / He male mice
LDS of LPS3 is 150, 180, 180 respectively
μg / animal, and E. coli LPS value of 300 μg / animal 6
It was 0% or less. Also, E. coli LPS, B. pertussis LPS
Toxicity value LD ₅₀ of (Production Example 2) (2 male BALs per group)
The average values in B / C mice, average body weight 45 g) were 3.4 and 11 mg / kg by intravenous administration and 16 and 32 mg / kg by intradermal administration, respectively.

[0081]

INDUSTRIAL APPLICABILITY According to the present invention, it has a high anti-withdrawal effect, a high chemotherapeutic index, long-term use, and can be administered by any of the oral, transdermal, injection and intraperitoneal routes. , A novel anti-continence drug and veterinary anti-contraindication drug that can be produced in a large amount at low production cost are provided.

[Brief description of drawings]

FIG. 1 is an SDS-PAG of LPS that can be used in the present invention.
It is a figure which shows the pattern in E method.

FIG. 2 is a graph showing the dose dependence of the anti-withdrawal effect of the anti-withdrawal agent of the present invention in intravenous administration.

FIG. 3 is a graph showing the dose dependence of the anti-withdrawal effect of the anti-withdrawal agent of the present invention in intradermal administration.

FIG. 4 is a graph showing the administration time dependency of the anti-withdrawal symptom prevention effect of the anti-withdrawal symptom agent of the present invention.

FIG. 5 is a graph showing the effect of the anti-withdrawal drug of the present invention on mental drug dependence.

[Explanation of symbols]

In FIG. 1, 1 is LPS1, 2 is LPS2, and 3 is LPS.
3 shows the pattern.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Daisuke Tsukioka 1-21-17 Kasuga, Chuo-ku, Chiba City, Chiba Prefecture (72) Denichi Mizuno 18 Okamoto, Kamakura City, Kanagawa Prefecture (72) Inventor Haruyuki Oshima Hachioji, Tokyo 1097 Ichidatemachi Ichigaoka housing complex 2-1 −513

Claims (8)

[Claims] 1. An anti-withdrawal agent containing LPS having the following physical properties. Main molecular weight: 5,000 ± 1,000 (SDS-PAG
E method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 9 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 2. The anti-withdrawal agent according to claim 1, wherein the LPS is derived from Gram-negative bacilli having the following properties. (A) Morphology Short rod-like no motility Gram stainability :-( b) Growth state Standard agar medium: Yellow to cream colored, round, opaque colonies are formed. SS agar: forming white, translucent colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (C) Physiological properties Forges-Proscher's reaction: + Formation of indole:-Formation of hydrogen sulfide:-Utilization of citric acid: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbite: + Arabinose: + Raffinose: + ▲ 10 ▼ Sucrose: + (e) Others Decarboxylation reaction of lysine: Use of malonic acid :-Decomposition of arginine:-Deamination reaction of phenylalanine:-Decarboxylation reaction of ornithine:- 3. An anti-withdrawal agent containing LPS having the following physical properties. Main molecular weight: 6,500 ± 2,500 (SDS-PAG
E method) Phosphorus number: 1-2 / molecular weight 5,000 Hexosamine number: 7 ± 1 / molecular weight 5,000 KDO number: 1-2 / molecular weight 5,000 4. The anti-withdrawal agent according to claim 3, wherein the LPS is derived from Gram-negative bacilli having the following properties. (A) Morphology Short rod shape No motility Gram stainability :-( b) Growth state Standard agar medium: A cream-colored and opaque colony is formed. SS agar: forms red, opaque colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (C) Physiological properties Forges-Proscher's reaction: + Production of indole:-Production of hydrogen sulfide:-Utilization of citric acid: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbit: + Arabinose: + Raffinose: + ▲ 10 ▼ Sucrose: + (e) Others Decarboxylation of lysine:-Utilization of malonic acid : + Decomposition of arginine: + Deamination reaction of phenylalanine:-Decarboxylation reaction of ornithine: + 5. An anti-withdrawal agent containing LPS having the following physical properties. Main molecular weight: 6,500 ± 2,500 (SDS-PAG
E method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 6. The anti-withdrawal agent according to claim 5, wherein the LPS is derived from Gram-negative bacilli having the following properties. (A) Morphology Short rod shape No mobility Gram stainability :-( b) Growth state Standard agar medium: Yellow, round, translucent colonies are formed. SS agar: does not form colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Does not generate gas. (C) Physiological properties Forges-Proscher's reaction: + Formation of indole:-Formation of hydrogen sulfide:-Utilization of citric acid: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbite: + Arabinose: + Raffinose:-▲ 10 ▼ Sucrose: + (e) Others Decarboxylation of lysine:-Utilization of malonic acid + Decomposition of arginine: -Deamination reaction of phenylalanine: -Decarboxylation reaction of ornithine: -Anti-withdrawal agent containing LPS selected from the group consisting of compounds. 7. The L according to any one of claims 1 to 6.
An anti-withdrawal agent containing a mixture of PS. 8. The L according to any one of claims 1 to 6.
LP selected from the group consisting of PS and mixtures thereof
Anti-withdrawal agent for animals containing S.