JPH0121808B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anti-inflammatory agent containing 3,4-dihydroxybenzaldehyde as an active ingredient. In the course of studying the anti-inflammatory properties of various substances, the present inventors discovered that 3,4-dihydroxybenzaldehyde (hereinafter referred to as the "substance") has excellent anti-inflammatory properties. I've reached it. This substance itself is a known substance that exists in various edible plants, and has been proposed as an anticancer agent (Japanese Patent Application Laid-Open No.
51018) has been done. However, its mechanism of action is largely unclear, and no report has been found so far regarding the anti-inflammatory effect of this substance. The present inventors investigated the anti-inflammatory effect of this substance using various methods, and found that it has a granuloma growth suppressing effect, an adjuvant arthritis suppressing effect, a leukocyte migration suppressing effect, and a platelet aggregation suppressing effect, and is also low in toxicity. Therefore, the present invention was obtained based on the knowledge that the compound has suitability as an anti-inflammatory and anti-rheumatic agent. The toxicological and pharmacological properties of this substance will be explained below. (1) Acute toxicity Acute toxicity was investigated by oral and intraperitoneal administration routes using female JCL-ICR mice. For oral administration, the drug was dissolved and dispersed in 0.2% CMC, and for intraperitoneal administration, the drug was dissolved in physiological saline and was adjusted to a predetermined amount using a stomach probe or syringe. After administration, poisoning symptoms were continued to be observed, and the mortality rate over time was determined for up to 7 days, and the LD ₅₀ value was determined using the Litchfield-Wilcoxon chart calculation method.

[Table] (2) Mutagenicity It was investigated using the following method. Recombinant repair-deficient strain (Bacillus subtilis M45)
B-agar medium (10 g of meat extract, 10 g of polypeptone, 5 g of NaCl, 15 g of agar) was added to a small pipette.
(g, distilled water 1000 ml, pH 7.0) was streaked so that the starting points did not touch. This substance was dissolved in DMSO, and 0.05 ml of the solution was soaked into a circular paper with a diameter of 8 mm, placed so as to cover the starting point of the streak, and after culturing overnight at 37°C, the length of the growth inhibition zone was measured. Kanamycin was used as a negative control, and mitomycin was used as a positive control. The results are shown in the table below, and the results showed that this substance did not exhibit mutagenicity.

[Table] (3) Platelet aggregation inhibitory effect Platelets are collected from the ear vein of rabbits (domestic rabbit native species) in the presence of 3.8% titrate (green cross), centrifuged and converted into platelet-rich plasma (PRP).
and platelet poor plasma (PPP) were obtained, and during aggregation reaction, PRP was diluted with PPP to reach a platelet count of 300,000/
The one adjusted to μ was used. Arachidonic acid used as an aggregating agent was Na salt (manufactured by Sigma), dissolved in physiological saline, and added to platelets. This substance was dissolved in physiological saline and added in a predetermined amount to platelets. In addition, naproxen was used as a control drug, but because it is insoluble in water,
It was dissolved in physiological saline as Na salt and added in a predetermined amount to platelets. For the aggregation reaction, platelet aggregation tracer PAT manufactured by Niko Bioscience was used.
-Measured using 4A. The IC ₁₀₀ of this substance was 75 μM, and the IC ₁₀₀ of the control drug naproxen was 140 μM. From this result, it is clear that this substance has the effect of suppressing platelet aggregation. (4) Polymorphonuclear leukocyte migration inhibitory effect (in vitro) This was carried out using rat polymorphonuclear leukocytes according to the Boyden method ["Nippon Clinical" Vol. 27, No. 9, p. 172 (1969)]. That is, Escherichia coli (E.
Coli) culture medium and serum in a 10:1 ratio.
After incubation at 37°C for 1 hour and diluting it 5 times with physiological saline, the test drug was pre-incubated with a polynuclear leukocyte suspension for 10 minutes, and the migration of polynuclear leukocytes was measured. This substance dissolves in physiological saline and enters the white blood cell layer.
It was added at a concentration of 100 μM. In addition, since the control drugs naproxen and indomethacin are insoluble in water,
It was added as Na salt so that the concentration in the system was 100 μM. At a drug dose of 100 μM, the inhibition rates of polymorphonuclear leukocyte migration by this substance, naproxen, and indomethacin were 49%, 0, and 26%, respectively, indicating that the migration-inhibiting activity of this substance is quite strong. (5) Inhibitory effect on polymorphonuclear leukocyte migration (in vivo) Experiments were conducted using 5-week-old Donryu (male).
An exudation test of polymorphonuclear leukocytes into the inflamed site was performed according to the pouch method (Ishikawa et al., Yakushu 88 1472, 1968). This substance and the control drug indomethacin are
It was dispersed in 0.2% CMC and a prescribed amount was given orally. The results of measuring the number of polymorphonuclear leukocytes in the exudate at 3, 6, and 24 hours after CMC injection were as follows, and it was found that this substance suppressed the exudation of polymorphonuclear leukocytes into the inflamed site.

[Table] (6) Granuloma inhibitory effect The experiment was conducted using 5-week-old Donryu (〓) according to the method of Fujimura et al. [Applied Pharmacology 19 (3), 329 (1980)]. Paper disk is 8mmφ×0.26mm(d)
paper with 2% CMC solution (dihydroxystreptomycin, 0.1 million units of penicillin)
mg/ml) was used. These two discs were implanted subcutaneously on the back of a rat under ether anesthesia. The test drug was dispersed in a 0.3% CMC solution and administered for 10 days, and granulation was removed after 11 days.
The weight of the granuloma was measured.

[Table] Number of rats: 5 per group As can be seen from the above results, this substance suppresses proliferative granulomas without suppressing body weight. In addition, after the test, autopsies were performed to observe the gastric mucosa and to measure the weight of the thymus gland. As a result, bleeding and ulcer formation in the gastric mucosa were observed in the indomethacin and prednisolone-administered group, and significant thymus atrophy was observed in the prednisolone-administered group. It was seen. This substance administration group
No abnormalities were observed compared to the control group. (7) Adjuvant arthritis inhibitory effect The preventive effect of adjuvant arthritis was evaluated using Fujihira's method (Applied Pharmacology 5(2), 169,
(1971). That is, Freund's complete adjuvant (0.6 mg/0.1 ml) was inoculated into the tail of a rat anesthetized with ether. Two weeks after vaccination, the test drug was orally administered once a day for 20 consecutive days. The results are shown in Figure 1. As is clear from the results, this substance showed a therapeutic effect on adjuvant arthritis. In addition, in the group treated with the control drug prednisolone, body weight was significantly suppressed and significant atrophy of the weight of the thymus gland removed by autopsy was observed, but in the group treated with this substance, no weight suppression or thymus atrophy was observed, and there were few side effects. It turned out that. Therefore, this substance is effective as a therapeutic agent for chronic inflammation such as rheumatoid arthritis. From the above results, it can be understood that this substance has excellent granuloma growth inhibitory activity, adjuvant arthritis inhibitory activity, leukocyte migration inhibitory effect, and platelet aggregation inhibitory effect, and has low toxicity. Therefore, this substance has extremely useful uses as an anti-inflammatory agent and an anti-rheumatic agent for chronic rheumatoid arthritis, systemic lupus erythematosus (SLE), and the like. Next, formulation for applying this substance as the above-mentioned therapeutic agent will be explained. This substance can be applied in various forms. The substances can also be used alone or in admixture with pharmaceutically acceptable diluents and other agents. At this time, in order to improve the irritation of this substance, it is possible to make this substance into an clathrate compound using a compound with inclusion ability such as cyclodextrin, or to form a mixture or condensate with various amines, amino acids, or sugars. It is also effective to use it after. The substances can also be applied orally or parenterally and may take any form suitable for their administration. Additionally, the substance can be presented in dosage unit form, such as powders, granules, tablets, dragees, capsules, suppositories, suspensions, solutions, emulsions, ampoules, injection solutions, etc., provided they contain an effective dosage. It can take various forms. It should therefore be understood that the medicament of the present invention can be prepared by applying any conventionally known formulation means. The content of the substance (active ingredient) in the drug of the present invention can be adjusted within a wide range of 0.01 to 100%, preferably 0.1 to 70% (by weight). As mentioned above, the drug of the present invention is administered orally or parenterally to humans and animals, with oral administration being particularly preferred. In this case, oral administration also includes sublingual administration, and parenteral administration includes subcutaneous, intramuscular, intravenous, etc. injections and infusions. The dose of the drug of the present invention depends on whether the subject is an animal or a human, and is influenced by age, individual differences, medical conditions, etc., and in some cases doses outside the range shown below may be administered, but in general, the drug is administered to humans. In this case, the oral dosage of this substance is 0.1 to 500 per kg body weight per day.
mg, preferably 0.5 to 200 mg, and the parenteral dosage is 0.01 to 200 mg, preferably 0.1 to 100 mg, administered in 1 to 4 divided doses per 1 kg of body weight per day. Specific examples of formulation of the drug of the present invention are shown below as examples. Parts in the examples indicate weight unless otherwise specified. Example 1 10 parts of this substance, 15 parts of heavy magnesium oxide and 75 parts of lactose are uniformly mixed to form a powder or granules, and this powder is placed in a capsule container to form capsules. Example 2 45 parts of this substance Starch 15〃 Lactose 16〃 Crystalline cellulose 21〃 Polyvinyl alcohol 3〃 Water 30〃 were uniformly mixed and simmered, then crushed, granulated, and dried.
It is sieved and made into granules. Example 3 4 parts of calcium stearate was added to 96 parts of the granules obtained in Example 2, and the mixture was compressed to form tablets with a diameter of 10 mm. Example 4 Granules were prepared in the same manner as in Example 2 using 94 parts of this substance, 6 parts of polyvinyl alcohol, and 30 parts of water. 10 parts of crystalline cellulose is added to 90 parts of the obtained granules and compressed to form tablets with a diameter of 8 mm, and syrup gelatin precipitated calcium carbonate is added to the tablets to form sugar-coated tablets. Example 5 10 parts of this substance, 3 parts of benzyl alcohol, 3 parts of physiological saline, and 87 parts of physiological saline are added and mixed under heating, followed by sterilization to prepare an injection.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the experimental results of the anti-onset effect against adjuvant arthritis.

Claims (1)

[Claims] 1. An anti-inflammatory agent containing 3,4-dihydroxybenzaldehyde as a main component. 2. The anti-inflammatory agent according to claim 1, wherein the inflammation is chronic inflammation. 3. The anti-inflammatory agent according to claim 1 or 2, wherein the chronic inflammation is rheumatism.