JP2900580B2 - Reactive oxygen disorder protective agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an agent for preventing and treating a disease based on cell damage caused by active oxygen.

(Prior Art) There are a number of active oxygen scavenging systems in a living body, which protect the living body from oxidative stress. It has been clarified that active oxygen resulting from the disturbance of these defense systems is involved in the development of various diseases. In particular, in recent years, attention has been paid to the fact that damage to vascular endothelial cells by active oxygen is deeply involved in the pathogenesis of arteriosclerosis.

When vascular endothelial cells are damaged by reactive oxygen, the basement membrane is exposed, platelets in the blood stream are activated, and an agglutination reaction occurs. Platelet-derived vascular smooth muscle cell growth factor is released from the aggregated platelets, and smooth muscle cells that exist in the media and play a role in relaxing and contracting blood vessels migrate to the intima, where smooth muscle cells are deposited in the intima layer. Proliferate, intimal thickening occurs, and arteriosclerosis develops. When arteriosclerosis develops, endothelial cells produce endothelial cell-derived relaxing factors and prostacyclin, which regulate blood pressure and blood flow.

Taking the above-mentioned arteriosclerosis as an example, cell damage due to active oxygen is considered to be a part of the causes of various diseases, and up to now, drugs for eliminating these active oxygen in vivo have been studied. Antioxidant compound having an effect such as degradation of the erase and lipid peroxide active oxygen have been developed by the approach from many fields as active oxygen eliminator (Kenji Fukuzawa: radical scavenger, Mebio, 5 90～94,198
8). Water-soluble vitamin C and the like are known to effectively eliminate active oxygen in the water-soluble fraction such as the cytoplasm, but α-lipid is effective as a fat-soluble substance that exerts an effect in a hydrophobic region such as a cell membrane. Tocopherol derivatives, β-carotene,
Like estrogens are known (Toshio Futaki: vivo antioxidant, 37, 893 screwed 897,1988). In order to make vitamin C exhibiting strong erasing activity in an aqueous solution work in a hydrophobic region, this fat solubilized product is also being studied as an erasing agent (Kane
yoshi Kato et al .: Studies on scavengers of activeoxygen
species, J. Med. Chem., 31 , 793-798, 1988).

(Problems to be Solved by the Invention) Recent research has revealed that active oxygen generation sites during disease are located inside and outside cells. In the former, electrons leak randomly from the electron transport system in intracellular organelles, particularly mitochondria, and active oxygen appears in the cytoplasm and damages the cell. In this case, the radical scavenger must pass through the cell membrane and exhibit scavenging activity. However, superoxide dismutase and catalase, which have been studied to date, have a large molecular weight and do not enter tissues in a negative manner. There were problems with the administration method, such as a short half-life and a short duration in the blood.

In addition, as an example of cytotoxicity due to extracellular reactive oxygen, protection from vascular endothelial cell damage not only regulates blood pressure but also prevents arteriosclerosis, thus suppressing vascular endothelial cell damage The development of drugs is expected.

Compounds such as the α-tocopherol derivative, β-carotene, estrogens, and fat-solubilized vitamin C often act on the immune system or affect the circulatory system such as blood pressure and pulse rate, and are toxic. If the potential is induced to be too high in order to enhance the activity, there is a problem that toxicity is exhibited again. Many of these substances show sufficient erase activity at the in vitro level,
It is also known that it has no therapeutic effect at the in vivo level. Moreover, these fat-solubilized derivatives of antioxidants in vivo change the deformability of erythrocytes and make it easier to cause hemolysis.

An object of the present invention is to solve these problems and to provide an agent for treating or preventing a disease caused by cell damage caused by active oxygen.

(Means for Solving the Problems) The present invention is a 9-cis-octadecenoic acid or 9-trans-octadecenoic acid as an active ingredient, and a cytotoxicity preventive agent based on active oxygen containing these as a main component.

In general, active oxygen species containing lipid peroxide are known as substances that damage cells, for example, vascular endothelial cells, in vivo. The present inventors have created an experimental system for examining endothelial cell damage caused by activated leukocytes after culturing vascular endothelial cells by radioactive chromium release reaction, and using various fatty acids as drugs to demonstrate the effect of preventing vascular endothelial cell damage. As a result of the investigation, it was found that 9-cis-octadecenoic acid or 9-trans-octadecenoic acid had a strong protective effect, and the present invention was completed.

These fatty acids can be of natural or synthetic origin, and any commercially available high-purity fatty acid is sufficient.

When the active ingredient of the present invention is used as a protective agent for active oxygen damage, the active ingredient is mixed with a pharmacologically acceptable carrier, excipient, diluent or the like known per se, and a pharmaceutical composition is prepared according to a known method. For example, they can be administered orally or parenterally as tablets, capsules, solutions, suppositories, and injections.

In addition, since the active ingredient of the present invention is a fat component, the following dosage forms can be mentioned for parenteral administration. For injection and infusion, there are oil-based preparations such as water-soluble suspensions, liposome preparations and lipid microsphere preparations. Topical dosage forms include intraocular eye drops and ointments, and rectum into lipid surfactant mixed micelle suppositories.

The dose varies depending on the administration subject, administration route, symptoms, etc., but when administered orally, the active substance is usually administered in a dose of about 1 mg / kg to 100 mg / kg body weight, preferably about 5 mg / kg to 100 mg / kg body weight. Administer 50 mg / kg body weight about 1 to 3 times a day.

When administered parenterally, for example, about 5 mg / kg to 20 mg / kg body weight of the present active substance is administered once or twice a day as a suppository. About 0.1 mg / kg of this active substance as an oily injection
It is preferred to administer 2020 mg / kg body weight once or twice a day.

In addition, since the active ingredient of the present invention has a carboxyl group in a free state, it can be easily processed into a stable oily preparation by using a surfactant because it has high water solubility compared to general neutral lipids.

(Example) Bovine vascular endothelial cell culture method Bovine carotid artery 5 to 10 cm of blood vessel was removed and PBS supplemented with antibiotics (penicillin, streptomycin, etc.)
(Phosphate buffer solution) and wash with ME.
The cells were immersed in M (Eagle's medium, minimal medium), cooled on ice, and brought back to the culture room. Blood vessels also contain antibiotics
Washed several times with MEM medium. Thereafter, the fat adhering to the blood vessel was removed neatly, the bifurcation was cut off with scissors, and the blood vessel was cut vertically open through the bifurcation. The blood vessels were pinned in an intimal surface on a flat fixation surface and pulled. Using a scalpel # 11, endothelial cells were detached by gently touching the intimal surface. At that time, remove the scalpel in advance with 20% FBS
Moistened MEM medium (containing antibiotics) containing (fetal calf serum) smoothed the female movement and prevented smooth muscle cell invasion. The endothelial cells attached to the scalpel were dispersed in 10 ml of the MEM medium and centrifuged at 800 rpm for 10 minutes. Thereafter, the above-mentioned MEM was added to the sediment, dispersed by a pipette until it became a rice ear with dozens of endothelial cells collected, and seeded and cultured on a plastic petri dish.

Reactive Oxygen Protection Experiment Using Vascular Endothelial Cells Bovine carotid artery-derived endothelial cells isolated and cultured by the above-described method were confluent in 24-well multiwells. Various test drugs were added therein at 5 μg / ml, and cultured for 2 days to be taken up into endothelial cells. Then ⁵¹ Cr- and sodium chromate was added per well 2 .mu.Ci, and cultured for another 18 hours, in the cell ⁵¹ Cr-
Sodium chromate was incorporated. Thereafter, the cells were washed twice with Hanks' solution, and 10 times the amount of endothelial cells of leukocytes (neutrophils separated from human peripheral blood with Ficoll (trade name, manufactured by Pharmacia)) and 12-O-tetradecanoyl-phorbol 10 ng / ml of -13-acetate was added. (This substance stimulates the NADPH-dependent pentasaccharide phosphate cycle acting on the leukocyte membrane to promote the production of active oxygen and damage endothelial cells. At this time, the cells radiated from the cells damaged by active oxygen The radioactivity released into the culture medium after 5 to 6 hours is measured with a γ-scintillation counter,
The release amount was in the state of uptake of the test drug. The total amount of ⁵¹ Cr incorporated into the endothelial cells was measured by adding 0.1% Triton X-100 and dissolving the cell membrane, and measuring the radioactivity released into the culture solution. did.

The amount of radioactivity when no leukocytes and 12-O-tetradecanoyl-phorbol-13-acetate were added was defined as the amount released without stimulation.

The amount of ⁵¹ Cr released is Y: Calculated by [No stimulation release amount].

^The active oxygen defense effect can be determined from the amount of ⁵¹ Cr released.

Example 1 In the above-described active oxygen protection experiment using vascular endothelial cells, the amount of radioactive release was measured using 9-cis-octadecenoic acid and 9-transoctadecenoic acid as test drugs.

 The results are shown in Table 1.

Comparative Example 1 In the vascular endothelial cell experiment, 9-cis-tetradecenoic acid 9-cis-hexadecenoic acid 9-cis-icosenoic acid 9,12,15-all-cis-octadecatrienoic acid 5,8 was used as a test drug. , 11,14-all-cis-eicosatetraenoic acid 4,7,10,13,16,19-all-cis-docosahexaenoic acid 6-cis-octadecenoic acid 11-cis-octadecenoic acid 5,8,11, 14,17-all-cis-icosapentaenoic acid was used. In addition, an experiment was performed without using a test drug as a control.

 The results are shown in Table 1.

Example 2 Test for Inhibition of Injury Due to Concentration Dependence of Cis-Octadecenoic Acid
Performed at 1,1,5 and 10 μg / ml. The results are shown in Table 2.

Comparative Example 2 In a vascular endothelial cell experiment, after culturing without adding a test substance, ascorbic acid palmitate, superoxide dismutase, mannitol and vitamin E were added to endothelial cells at the time of adding leukocytes, and thereafter radioactivity was determined according to the above-described procedure. It was measured. The results are shown in Table 3.

Conventional active oxygen scavengers for scavenging active oxygen such as hydroxyl radicals and superoxide have not been found to have the effect of suppressing cell damage due to active oxygen derived from leukocytes.

Example 3 A tablet was produced by the usual means using the following components. The composition per tablet is as follows.

Administer 2 to 6 tablets daily after meals per adult.

(Effects of the Invention) Since the active ingredient of the present invention is an in-vivo component, it has a remarkably low toxicity, exhibits an active oxygen damage inhibitory effect at a physiologically effective concentration in a living body, and is smoothly incorporated into a biological membrane, so that tissue transfer is achieved. It is an active oxygen disorder protective agent with excellent properties.

Claims (1)

(57) [Claims] 1. An active oxygen damage protective agent comprising an active ingredient containing 9-cis-octadecenoic acid or 9-trans-octadecenoic acid as a main component.