JPS6325600B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel physiologically active substance (hereinafter referred to as NSQ), its production method, and a pharmaceutical containing the substance as an active ingredient. The present inventor inoculated various poxviruses,
As a result of research on medicinal active substances extracted by processing various animal tissues (excluding humans) that have been infected with pox, cultured cells or cultured tissues (hereinafter simply referred to as pox tissues), it has been found that they contain high concentrations of specific active ingredients. We succeeded in extracting and purifying the new physiologically active substance NSQ. NSQ includes various types of stress, such as biological stress such as viruses, bacteria, protozoa, and Rickettsia, oxygen deprivation, chemical stress such as drugs (ACTH, cortisone, etc.), physical stress such as cold, noise, radiation, fear, It acts on the body's homeostasis maintenance mechanism, which regulates and repairs distortions in the nervous system, secretory system, immune system, etc. that occur when the body's organic damage is caused by mental stress such as anxiety and mania. It has the effect of increasing natural healing power and normalizing biological functions. As described below, NSQ is not only useful as an analgesic, sedative, anti-stress, anti-ulcer, anti-hypertensive, anti-allergic, immunomodulating, anti-tumor drug, but also widely used in various diseases associated with various types of stress and autonomic nervous imbalance. In addition, when used in combination with other drugs, the side effects of the drugs can be reduced and a synergistic effect can be expected. According to the invention, NSQ grinds the pox tissue and
After adding an extraction solvent to this to remove protein, the extract is brought into contact with an adsorbent under acidic conditions, then the active ingredients are eluted using water or an organic solvent, and the insoluble materials are removed by dissolving this in water. It is produced by contacting it with an adsorbent under acidic conditions and then eluting it with a weakly basic organic solvent or a water mixture thereof. The method of the present invention will be described in more detail. In the present invention, variola tissue refers to virus-infected cultured tissue, cultured cells, and virus-infected inflamed tissue of various animals obtained by various inoculation methods or culture methods of poxviruses such as vaccinia virus, cowpox virus, and ectromelia virus. Or the chorioallantoic membrane of hatched chicken eggs. The pox tissue is collected aseptically and ground, and 1 to 5 times the amount of extraction solvent is added to make it milky. Distilled water, physiological saline, weakly acidic or weakly basic buffer solutions, or glycerin aqueous solutions can be used as the extraction solvent, and sterilization and sterilization may be performed as appropriate.
Phenol etc. may be added for preservative purposes. or,
At this time, the cell tissue can be destroyed by treatments such as freezing and thawing, ultrasound, cell membrane lytic enzymes, surfactants, etc. to make extraction easier. The resulting milky extract is filtered or centrifuged to remove tissue fragments and protein removal. Protein removal operation can be carried out by known physicochemical methods, such as heating, ultrasound, treatment with protein denaturants such as acids, bases, urea, guanidine, organic solvents, surfactants, etc., isoelectric precipitation, salting out, etc. method can be applied. Next, asbestos, Celite, Seitz filter plate, ultrafiltration,
Remove insoluble proteins using ion exchange resin, centrifugation, etc. The thus obtained extract containing the active ingredient is made weakly acidic using hydrochloric acid, sulfuric acid, hydrobromic acid, etc., and then adsorbed onto a suitable adsorbent. Kaolin, activated carbon, or ion exchange resin can be used as the adsorbent, and the active ingredients can be removed by passing the extract through a column packed with the adsorbent or by adding the adsorbent to the extract and stirring. Adsorption can be achieved. The adsorbent is water, methanol, ethanol,
Extract with an organic solvent such as isopropanol. At this time, when a weakly basic organic solvent or an aqueous mixture thereof, which will be described later, is used as the eluent, the following steps are not necessarily necessary. The obtained eluate is suitably neutralized and then evaporated to dryness or freeze-dried under reduced pressure to obtain an active ingredient-containing substance. Furthermore, 5 to 15 times the amount of water is added to the substance to remove water-insoluble substances, and then the substance is adsorbed onto an adsorbent under acidic conditions as described above. Next, it is eluted with a weakly basic organic solvent or a water mixture thereof. Particularly preferred weakly basic organic solvents include pyridine and lutidine. If desired, the obtained purified substance may be further purified by fractionation with water, methanol, acetonitrile, etc. by column chromatography using silica gel, microcrystalline cellulose, etc. NSQ extracted and purified by the method of the present invention is substantially free of inorganic substances, contains water-soluble organic active ingredients, and has the following physicochemical properties. Properties: Pale yellow-brown amorphous hygroscopic powder Solubility: Soluble in water and methanol Insoluble in benzene and ether PH: 6.5-7.5 Ultraviolet absorption: λ _nax 265-275 nm Elemental analysis: C 41.4-47.0% H 5.4- 7.8% O 32.1-37.4% N 11.4-15.6% Ninhydrin reaction: positive Orsine-hydrochloric acid reaction: positive Color reaction by arsenic molybdate method: positive Various protein detection reactions: negative Next, an example of manufacturing NSQ will be shown. Example 1 The skin of a healthy adult rabbit was inoculated with vaccinia virus, the infected skin was aseptically removed and cut into small pieces, and then phenol-added glycerin water was added and ground with a homogenizer to form an emulsion. Obtained. This was then centrifugally filtered, and the resulting filtrate was adjusted to pH 4.8-5.5 with hydrochloric acid, heated to 100°C with flowing steam, and filtered. The filtrate was further filtered using a Seitz filter plate, adjusted to pH 9.2 with sodium hydroxide, heated to 100°C, and filtered. The filtrate was adjusted to pH 4.5 with hydrochloric acid, 1.5% activated carbon was added, stirred for 1 to 5 hours, filtered, water was added to the activated carbon, and adjusted to pH 9.4-10 with sodium hydroxide for 3 to 5 hours.
After stirring for an hour, it was filtered. PH7.0− of the filtrate with hydrochloric acid
7.2 and dried under reduced pressure. Water was added thereto, dissolved, and insoluble matter was filtered. The filtrate was adjusted to pH 4.0 with hydrochloric acid and adsorbed onto an activated carbon column. After washing with 0.01N hydrochloric acid, it was eluted with 10% pyridine-water and the eluate was dried. . 0.8-1.5 g of NSQ was obtained from 1 kg of pox skin. Elemental analysis values: C 42.3% H 5.8% O 32.9% N 14.8% Furthermore, this was subjected to microcrystalline cellulose column chromatography, and after flowing a mixed solvent of methanol, isopropanol, benzene, and water, the active fraction was separated with water. It was purified by elution. Elemental analysis values: C 46.3% H 6.4% O 34.0% N 12.1% Example 2 Adsorption activated carbon obtained in the same manner as Example 1 was mixed with 10%
Eluted with lutidine-water. This was subjected to silica gel column chromatography to obtain an active fraction purified with acetonitrile-water. 1.0 to 1.6 g of NSQ was obtained from 1 kg of infected skin. Elemental analysis values: C 42.7% H 7.2% O 36.8% N 11.9% The pharmacological action of the NSQ of the present invention is shown below. Acute toxicity test An acute toxicity test of NSQ was conducted using 10 mice per group. The results are shown in Table 1.

[Table] Pharmacological test The SART stressed animals used in the following animal experiments were prepared using the method of Kita et al. [Japan Pharmacological Journal, 71 , 195-210]
(1970)]. SART-stressed animals raised using this method exhibit severe stress, exhibiting weight loss, increased heart rate, and prolonged QRS duration, similar to human autonomic imbalance caused by environmental changes. It can be seen as an animal model that represents the state. (1) Suppressive effect on weight loss in stressed animals Groups of 15 mice were raised under SART stress conditions, and changes in body weight were compared between the NSQ 5mg/Kg/day administration group and the non-administration group. At the same time, changes in body weight of mice raised under normal environmental conditions were also measured. The results are shown in Table 2.

[Table] (2) Effects on increased respiration rate, heart rate, and prolongation of QRS time in stressed animals A group of 10 mice was subjected to SART stress for 5 days, and the effects on respiration rate, heart rate, and QRS time were
We investigated the effects of NSQ. As shown in Table 3, intraperitoneal administration of NSQ at 15 mg/Kg/day significantly suppressed the increases in respiratory rate, heart rate, and QRS duration caused by SART stress.

[Table] (3) Acetylcholine (ACh) in isolated intestinal tract
Sensitivity recovery effect on the sensitivity of ACh (10 ^-7 g/ml) in the intestinal tract of mice subjected to SART stress for 5 days
We investigated the effects of NSQ. As shown in Table 4,
By administering 20mg/Kg/day of NSQ, ACh
Sensitivity returned to almost normal values.

[Table] (4) Sedative effect (4.1) Locomotor activity suppression effect NSQ was intraperitoneally administered to mice, and 50 minutes later, locomotor activity was measured for 15 minutes. The control group received 25 ml/Kg of physiological saline. (4.2) Inhibitory effect on exploratory behavior In the same manner as above, 50 minutes after intraperitoneal administration of NSQ.
The amount of exploratory behavior was measured for 15 minutes. These results are shown in Table 5.

[Table] (5) Analgesic effect The SART stress mice used in the following analgesic test were subjected to stress for 4 days. (5.1) Acetic acid method 0.1ml/10g of 0.7% acetic acid-added saline to a mouse
The drug was administered intraperitoneally, and the number of writhing events occurring for 15 minutes from 15 minutes after administration was measured. Effect judgment is NSQ
An effect was defined as the number of writhing mice subcutaneously administered was reduced to 50% or less of that of the control group, and the results are expressed as a percentage of the mice. (5.2) Phenylquinone method A 0.075% phenylquinone solution dissolved in a 5% ethanol solution was intraperitoneally administered to a mouse at 0.1 ml/10 g, and the number of writhing events occurring in 20 minutes from 5 minutes after administration was measured. The effectiveness was evaluated in the same manner as in the acetic acid method. These results are shown in Table 6.

[Table] (5.3) D'Amour-Smith
Method: We irradiated the ridge of a mouse with a heating wire for a maximum of 15 seconds, and measured the time it took for it to show an escape response.
Efficacy was evaluated at 30, 60, 90 and 30 days after intraperitoneal administration of NSQ.
When the average reaction time at 120 minutes was extended to more than twice the value before administration, it was considered effective and expressed as a percentage of mice. The results are shown in Table 7.

[Table] (5.4) Tail pressure method Stimulation was applied to the mouse ridge using a Randall-Selitto pressure stimulation measuring device, and the pressure was increased until the mouse exhibited an escape response.
The average value of the pressurized weight at 30, 60, 90, and 120 minutes after intraperitoneal administration of NSQ was divided by the value before administration, and this value was expressed as an analgesic coefficient. The results are shown in Table 8. ED _1.5 of _NSQ for SART stress mice
It was 1.5mg/Kg.

[Table] (6) Stress ulcer suppressive effect After fasting male rats for 24 hours, restraint water immersion stress (25°C, 20 hours) was applied and the degree of ulcers formed in the stomach body was observed. NSQ was administered intraperitoneally 10 times in total before the start of fasting and before stress loading. The control group received 3 ml/Kg/times of physiological saline. The results are shown in Table 9. The stress-induced ulcer showed severe hemorrhagic erosion and mucosal defects throughout the gastric body. In contrast, with NSQ administration, almost no bleeding was observed, and defects and erosions were significantly reduced.

[Table] (7) Blood pressure normalization effect Intraperitoneal administration of NSQ90 using Wistar rats and spontaneously hypertensive rats (SHR)
The blood pressure after minutes was compared with that before administration. As is clear from Table 10, NSQ has no effect on normal blood pressure, but lowers hypertension to a normal level.

[Table] (8) Immunomodulatory effect (8.1) Hemolytic plaque-forming cell (HPFC) production test Mice that had been intraperitoneally administered with cyclophosphamide (CP) at 25 mg/Kg for 2 days were immunized with sheep red blood cells, and then NSQ was administered intraperitoneally. Administer intravenously and by CP
The recovery effect against suppression of HPFC production was investigated. As shown in FIG. 1, NSQ significantly restored the antibody production ability decreased by CP. (8.2) Effect on IgE production Rats were immunized with DNP-As, which is an extract of Ascaris suum bound to a dinitrophenyl group, and pertussis vaccine, and NSQ or CP was administered intraperitoneally for 5 days, followed by intraperitoneal administration for 9 days. Increases the amount of IgE in the eye serum by 48
Time was measured by homologous PCA reaction. As shown in Table 11, it was found that NSQ had no effect on IgE production.

[Table] (8.3) Immune ability recovery effect of SHR Using SHR and WKA rats as a control, the method of Takeichi et al. [Igaku no Ayumi, Vol. 111, No. 6,
376-378 (1979)], a rosette formation test, a hemolytic plaque formation test, and a lymphocyte blastogenesis reaction were performed. NSQ and commercially available antineoplastic protein polysaccharides are
10 mg/Kg and 300 mg/Kg were administered intraperitoneally for one week, respectively. Table 12 shows the radioactivity of ³ H-thymidine, which incorporates the rosette formation rate, the number of hemolytic plaque-forming cells in ¹⁰⁶ spleen cells, and the rejuvenation reaction to phytohemagglutinin (PHA) and concanavalin A (Con A). Shown as counts. NSQ significantly restored the immunocompetence of SHR, especially the decline in T cell function.

[Table] (8.4) Antitumor effect Methylcholanthrene-induced SHR tumor cells 5x
10 ^Five cells were implanted subcutaneously into a group of 10 SHRs, and then 6
Daily NSQ, said protein polysaccharide, glucan or WKA
Rat thymus extract was administered. As shown in Table 13, the number of deaths was significantly reduced in the NSQ administration group, and the survival days of the dead rats were also prolonged.

[Table] (8.5) Radiation protection effect A group of 10 mice was irradiated with 900R radiation.
The subsequent mortality rate of the mice was examined. Each test drug was administered intraperitoneally for 7 days. As shown in Table 14, NSQ restored and enhanced the immune function decreased by radiation and significantly reduced the mortality rate.

[Table] (9) Antiallergic effect (9.1) PCA reaction suppression effect Homologous (homologous) immunization of rats with DNP-As and pertussis vaccine
-logous) effects on PCA were investigated. That is,
Anti-DNP-As serum was injected intradermally into the back of rats.
After hours, antigen DNP-As and Evans blue
(Evans blue) was injected intravenously to induce the response. When NSQ was administered intraperitoneally 30 minutes before response induction,
It significantly suppressed the PCA reaction. (Table 15)

[Table] (9.2) Degranulation inhibitory effect Rat anti-DNP-As serum was intraperitoneally injected into rats to passively sensitize them, and 48 hours later, the mesentery was removed. The cells were incubated in Tyrode's solution, NSQ was added, and then the antigen DNP-As was added, and after 15 minutes, the degranulation rate was measured by staining with toluidine blue. As shown in Table 16, NSQ significantly inhibited degranulation.

[Table] (9.3) Effect of suppressing histamine release Anti-benzylpenicilloyl bovine gamma globulin (BPO-BGG) serum was injected into the hearts of guinea pigs to passively sensitize them, and 24 hours later, the lungs were removed and cut into small pieces. . NSQ and benzylpenicilloyl bovine serum albumin (BPO-BSA) were added and incubated in Tyrode's solution, and the amount of histamine in the supernatant was measured. The results are shown in Table 17.

[Table] (9.4) Experimental asthma suppressive effect Anti-BPO-BGG serum was injected into the hearts of guinea pigs to passively sensitize them. After 48 hours, the trachea was incised and connected to a transducer, and the respiratory flow rate and respiration were measured. The respiratory rate and the ratio of respiration time to inspiratory time were measured from the flow rate. 20 mg/Kg of NSQ was administered intraperitoneally in four divided doses prior to intrajugular administration of BPO-BSA. trigger 18
After a few minutes, the asthmatic state was most evident, and even at this point, NSQ significantly suppressed the symptoms. In Table 18, the respiratory rate, respiratory flow rate, and exhalation/inhalation are each shown with the pre-induction values set at 100.

[Table] (10) Hepatic damage inhibitory effect (10.1) Liver cirrhosis inhibitory effect Using a group of 10 rats, carbon tetrachloride was mixed with the same amount of olive oil at 10ml/kg for 15 weeks.
A total of 30 subcutaneous administrations were performed to treat induced liver cirrhosis.
We investigated the inhibitory effect of NSQ. NSQ at the same time as carbon tetrachloride to 20mg/Kg, 5 times a week
When administered intraperitoneally, no signs of liver cirrhosis were observed in the control group. (10.2) Hepatocellular carcinoma inhibitory effect The effect of NSQ on hepatocellular carcinoma induced by 3'-methyl-diethylaminoazobenzene (DAB) was investigated using a group of 10 rats. DAB as feed
NSQ was administered intraperitoneally 5 times a week. Experimental animals were divided into the following groups. The results are shown in Table 19. Group A: Administer DAB only Group B: DAB for 12 weeks, NSQ 20mg/Kg for 13-26 weeks
Weekly administration Group C: DAB for 12 weeks, then DAB and NSQ 20mg/
Co-administration of Kg Group D: Co-administration of DAB and NSQ 2.5mg/Kg until 26 weeks

[Table] As is clear from the results of the above pharmacological tests, the NSQ of the present invention has low toxicity and a wide range of pharmacological effects, particularly regulating and repairing abnormalities in biological functions caused by various stresses, and maintaining biological homeostasis. It is a useful biodefense regulator. Therefore, NSQ is not only useful as a drug for analgesia, sedation, anti-ulcer, normalization of blood pressure, immune regulation, anti-tumor, and anti-allergy, but also widely used as an anti-stress agent, autonomic nerve regulator, etc. It can be applied as a time repair agent. Examples of diseases for which NSQ is applicable are as follows. Neuralgia, low back pain, essential hypertension, hypotension, gastritis, peptic ulcer, stress ulcer, menstrual abnormality, obesity, diabetes, nocturnal enuresis, skin pruritus, stress-induced diseases such as Meniere's syndrome, cardiac neuropathy, gastrointestinal Neurosis, respiratory neurosis, headache, dizziness, fatigue, insomnia, trembling, cold extremities, palpitations, shortness of breath, anorexia, constipation, autonomic dysfunction such as diarrhea, agammaglobulinemia, hypogammaglobulinemia , Wiskott-Aldrich syndrome, Louis-Bar syndrome, Gitlin syndrome, Nezelof
syndrome, DiGeorge syndrome,
Lymphoma, Hodgkin's disease, leukemia, immunodeficiency syndromes such as immunodeficiency caused by steroids and anticancer drugs, various malignant tumors, bronchial asthma, hay fever, allergic rhinitis, enteritis, ulcerative colitis, periarteritis nodosa,
Allergic diseases such as urticaria, eczema, contact dermatitis, allergic conjunctivitis, rheumatoid arthritis, and lupus erythematosus, as well as influenza, dysentery,
Tetanus, sepsis, various other infectious diseases, and radiation damage. NSQ can be used alone or in combination with other drugs as a prophylactic agent, therapeutic agent, or therapeutic adjuvant for these diseases. moreover,
When NSQ is used in combination with other drugs, it can be expected to reduce the side effects of other drugs, reduce dosage, and increase efficacy. Pharmaceutical compositions containing the NSQ of the present invention as an active ingredient can be formulated into tablets, capsules, granules, liquids, injections, ointments, suppositories, aerosols, and other commonly used dosage forms. . In addition to excipients such as starch, lactose, and mannitrate, formulations such as tablets, capsules, and granules may contain, as appropriate,
Binder such as crystalline cellulose, gum arabic, corn starch, gelatin, polyethylene, polyvinyl alcohol, polyvinylpyrrolidone, disintegrant such as carboxymethyl cellulose, polyethylene glycol, potato starch, talc,
Lubricating agents such as magnesium stearate, wetting agents such as glycerol, other coating agents, coloring agents, and flavoring agents can be used. For the preparation of injections, aqueous or non-aqueous solutions or suspensions sterilized using solvents such as distilled water for injection, physiological saline, glucose injection, vegetable oil for injection, propylene glycol, polyethylene glycol, etc. It can be done. In addition, sodium pyrosulfite, sodium hydrogen sulfide, 1-ascorbic acid, thioglycolic acid, citrate,
Stabilizers such as acetates and phosphates, procaine hydrochloride,
A soothing agent such as glucose, other tonicity agents, solubilizing agents, preservatives, suspending agents, emulsifying agents, etc. can be added. This type of preparation may be in the form of a sterilized solid composition that is dissolved before use. Ointments contain fatty oils, paraffin, lanolin,
Suppositories can be formulated using petrolatum, glycols, glycerin, higher alcohols, and other suitable bases, and suppositories can be formulated using bases such as cacao butter, macrogol, glycerogelatin, lanolin fat, etc., as appropriate. Surfactants and preservatives may be added. Furthermore, it can be made into an aerosol in the form of a liquid or fine powder together with a suitable propellant, and if desired, auxiliary agents such as wetting agents and dispersants may be added. The dosage of the NSQ of the present invention varies depending on the subject and the method of administration, but in order to obtain the desired effect, it is generally preferable to administer 1 μg to 10 mg/Kg per day, and it can be changed as appropriate depending on the disease and symptoms. Examples of formulations of pharmaceutical compositions containing the NSQ of the present invention are shown below. Tablet NSQ 50mg Lactose 125〃 Crystalline cellulose 20〃 Magnesium stearate 5〃 Total 200mg Capsule NSQ 10mg Lactose 160〃 Potato starch 75〃 Magnesium stearate 5〃 Total 250mg Injection NSQ 1mg Sodium chloride Appropriate amount Distilled water for injection Appropriate amount Suppository NSQ 10mg Cocoa butter 1990〃Total 2000mg Ointment NSQ 1g Emulsified wax 24〃 White petrolatum 60〃 Liquid paraffin 15〃Total 100g Aerosol NSQ 0.1% by weight Lecithin 0.9 〃 Dichlorodifluoromethane 39 〃 Dichlorotetra Fluoroethane 60 〃 100 Weight %

[Brief explanation of the drawing]

FIG. 1 is a graph showing the immune function recovery effect of the substance of the present invention, NSQ.

Claims (1)

[Claims] 1. After grinding the pox tissue and adding an extraction solvent to it to remove protein, the extract is brought into contact with an adsorbent under acidic conditions, and then the active ingredient is extracted using water or an organic solvent. The following properties can be obtained by eluting this, dissolving it in water to remove insoluble matter, contacting it with an adsorbent under acidic conditions, and then eluting it with a weakly basic organic solvent or its water mixture. NSQ, a new physiologically active substance with Properties: Pale yellow-brown amorphous hygroscopic powder Solubility: Soluble in water and methanol Insoluble in benzene and ether PH: 6.5-7.5 Ultraviolet absorption: λ _nax 265-275 nm Elemental analysis: C 41.4-47.0% H 5.4- 7.8% O 32.1-37.4% N 11.4-15.6% Ninhydrin reaction: positive Orsine-hydrochloric acid reaction: positive Color reaction by arsenic molybdate method: positive Various protein detection reactions: negative 2 Grind the pox tissue and extract it After removing protein by adding a solvent, the extract is brought into contact with an adsorbent under acidic conditions, then the active ingredients are eluted using water or an organic solvent, and the insoluble matter is removed by dissolving this in water. A method for producing a novel physiologically active substance NSQ, which comprises contacting with an adsorbent under the following conditions and then eluting it using a weakly basic organic solvent or an aqueous mixture thereof. 3.An anti-stress and autonomic nerve regulator containing NSQ as an active ingredient. 4 Analgesic containing NSQ as an active ingredient,
Sedatives. 5. An immunomodulating and anti-allergic agent containing NSQ as an active ingredient. 6. An anti-ulcer agent containing NSQ as an active ingredient. 7. A blood pressure regulator containing NSQ as an active ingredient. 8. An antitumor agent containing NSQ as an active ingredient.