JPH08169822A - In vivo radical scavenging agent - Google Patents

Abstract

PURPOSE: To obtain a radical scavenging agent and antitumor agent having a long action time and high safety in vivo, capable of being orally administered without forming hydrogen peroxide and other radicals in radical scavenging action, by using cystathionine. CONSTITUTION: This in vivo radical scavenging agent and antitumor agent comprises cystathionine. Or, the in vivo radical scavenging agent and antitumor agent contain cystathionine as an active ingredient. Cystathionine as an active ingredient is mixed with any of a superoxide dismutase preparation, a vitamin C analog compound, a vitamin E analog compound, a cystine analog compound, a reduced type glutathione, a peroxidase preparation and a crude medicine to give the objective in vivo radical scavenging agent. Cystathionine shows radical scavenging and ischemia-reperfusion damage protecting effects and can protect diseases caused by free radicals. The in vivo radical scavenging agent and the antitumor agent can orally be administered relatively in a large amount and stable in vivo by taking advantage of the cystathionine as an intravital substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a radical scavenger and an anti-ulcer agent consisting of cystathionine which is effective in vivo.

[0002]

2. Description of the Related Art A radical has one unpaired electron.
A molecule or atom having one or more atoms, such as nitric oxide, nitrogen dioxide, or active oxygen. Active oxygen is generated in the process of energy metabolism in living cells,
Superoxide (O ₂ ^-), hydrogen peroxide (H ₂ O ₂₎
Alternatively, there are hydroxy radicals (.OH) and the like. These active oxygens are essential for the bactericidal mechanism of phagocytes and play an important role in eliminating viruses and cancer cells. However, excessive production beyond the control of the place, amount, and time that the living body possesses attacks the in-vivo molecules that make up the in-vivo membranes and tissues,
Induces various diseases. At present, disease states and diseases that are considered to involve radicals include arteriosclerosis, cerebral infarction, cerebral ischemia-reperfusion injury, digestive system ischemia-reperfusion injury, ulcer, malignant tumor,
Chemical carcinogenesis, paraquat poisoning, diabetes, epilepsy, aging, etc. have been reported.

The first radical produced in the living body is superoxide. Other radicals such as OH radicals are generated from this superoxide through the Fenton reaction and the like. Therefore, SOD, a specific enzyme that eliminates this superoxide in the living body
(Superoxide dismutase) is present. Therefore, clinical applications of SOD preparations and related substances (Japanese Patent Application No. 2-75805) have been studied as radical scavengers. In addition, vitamin E analog compounds (Japanese Patent Application No. 3-128)
96), a vitamin C analog compound (Japanese Patent Application No. 2-13419)
9) Development of radical scavengers such as cysteine analogs, reduced glutathione peroxidase preparations, Wakhan medicine and the like (Japanese Patent Application No. 1-8694) has been reported.

[0004]

However, in SOD preparations, there is a possibility of anaphylactic shock in SOD preparations derived from different animals, it cannot be orally administered as it is because it is a protein, and in vivo half-life is reduced by intravenous administration. There was a drawback of being a few minutes.

In addition, the elimination of superoxide by SOD or SOD-related preparations always involves the generation of hydrogen peroxide. Although it is decomposed by catalase in the healthy body, the activity of catalase is reduced due to ischemia, etc., and OH radicals are generated as shown in formulas (1) and (2) by iron ions released from hemoglobin.
These can cause new tissue damage.

[0006]

Embedded image On the other hand, low molecular weight compounds such as vitamin E and C analogs have a short half-life in vivo and need to be ingested constantly. Since cysteine and glutathione have free SH groups, they can bind to metal species in the body if administered in large amounts. There was a problem of toxicity such as easy to do.

Therefore, there has been a demand for a radical scavenging and anti-ulcer agent which is orally administrable without fear of anaphylactic shock and the like, acts without generating other radicals at the time of radical scavenging, and has a long half-life in vivo.

[0008]

It is considered that cystathionine, which is an in-vivo substance, plays an important role in vivo as a precursor of cysteine, but its action on radicals has not been studied. The inventors of the present invention invented a liver damage inhibitor for cystathionine (Japanese Patent Application No.
-284639) is further advanced, and it was found that cystathionine has an effect on radical scavenging and protection against ischemia-reperfusion injury, and the present invention provides a radical scavenger and an anti-ulcer agent comprising cystathionine.

[0009] Ischemia-reperfusion injury is a cell injury that newly occurs when blood flow is reopened after hemostasis. The mechanism is that oxygen re-supplied from the interruption of blood flow becomes active oxygen (radical), which in turn causes peroxidation reaction of phospholipids existing in the cell membrane to cause damage to cells. This injury causes erosion and ulceration on the mucous membrane.

Cystathionine eliminates radicals causing these injuries and protects from diseases caused by radicals.

SOD and SOD-related substances also have a radical scavenging action, but the reaction produces H ₂ O ₂ when scavenging radicals as shown in formula (3).

[0012]

Embedded image The generated hydrogen peroxide produces new OH radicals and the like as in the above reactions (1) and (2), which further cause tissue damage. Therefore, it is desirable that the radical scavenger does not generate other radicals during its action. Due to its -S- group, cystathionine does not generate other radicals in the radical scavenging reaction with superoxide as shown in formula (4), and radical scavenging does not generate other radicals during the radical scavenging action. The agent is provided.

[0013]

Embedded image A relatively large dose is required for effective radical scavenging. Utilizing the fact that cystathionine is an in-vivo substance, it is possible to administer a relatively large amount orally,
In addition, it has become possible to provide a radical scavenger that is stable in vivo.

The radical scavenger of the present invention, cystathionine, has almost no toxicity, and the results of the acute toxicity test using mice are as follows.

Results of acute toxicity test Route of administration LD50 Oral 10 mmol / kg (2200 mg / k
g) or more Intraperitoneal 10 mmol / kg (1250 mg / k
g) As above, this acute toxicity test was conducted for oral administration and intraperitoneal administration using DDY male mice (body weight: 35 to 40 g).
LD50 mmo is observed for 14 days (112H) after administration.
l / kg (mg / kg) was calculated. Although described as LD50 value here, none of the mice actually died during the test could be confirmed.

The radical scavenger according to the present invention is a mixture of liquid or solid pharmaceutical auxiliary components such as excipients, binders, diluents and the like, and powders, granules, tablets,
It can be orally or parenterally administered in any dosage form such as capsules and injections.

It is also possible to mix with other drugs if necessary. The dose may be adjusted according to age, body weight, symptoms, etc., but usually, for adults, it is usually 10 mg to 1 a day.
0 g is desirable.

[0018]

EXAMPLES The radical scavenging action and antiulcer action of cystathionine according to the present invention will be explained in detail by the following experimental examples.

Experiment 1 Elimination of superoxide derived from xanthine-xanthine oxidase (X-XOD) system (Cyprinus luciferin derivative (MCL
Method A)) When xanthine (X) is added to xanthine oxidase (X-XOD), superoxide is produced in proportion to the concentration of xanthine. The generated superoxide is MCL which is a superoxide reaction fluorescent reagent.
A (2-methyl-6 [p-methoxyphe
nyl] -3,7-dihydroimidazo
[1,2-α] pyrazin-3-one hydr
It can be expressed as chemifluorescence intensity. Cystathionine and cysteine were added to this system, and their effects were comparatively examined. Cysteine has already been shown to have a radical scavenging action, and experiments were conducted in parallel to compare the effects. In this experiment, xanthine oxidase (X-XOD) 10 mU / ml
Xanthine (X) (30 μM) was added thereto. The relationship between the amount of each added cystathionine and the remaining amount of superoxide is shown in FIG.

Experiment 2 Eliminating action on human washed leukocyte-producing superoxide. After washing leukocytes were prepared from normal healthy subjects and suspended in a buffer, PMA (phor) which activates leukocytes was prepared.
bol myristate acetate) was added to produce superoxide, and the effects of cystathionine and cysteine on this were examined.
The superoxide produced was measured in the same manner as in Experiment 1. The relationship between the amount of each added cystathionine and the remaining amount of superoxide is shown in FIG.

As can be seen from FIGS. 1 and 2, cystathionine is xanthine-xanthine oxidase (X-XO).
D) System-derived superoxide and human washed leukocyte-produced superoxide were suppressed in a concentration-dependent manner.

Here, cystathionine cannot act as cysteine. This is because there is no metabolic enzyme from cysteine to cystathionine in the X-XOD system and the leukocyte system. In addition, the scavenging action of superoxide is due to its active free S in cysteine.
Due to the H group, the same reaction mechanism cannot be adopted depending on the -S- of cystathionine. Therefore, although cystathionine is a precursor of cysteine, it shows a completely different action / effect in radical scavenging.

Further, the radical scavenger composed of cystathionine does not produce other radicals when it reacts with superoxide, so that even if it is used in combination with other existing radical scavengers, it affects their action. Never. Therefore, the radical scavenger can be administered in any combination with the existing radical scavenger, and a radical scavenger more effective for the target disease can be provided.

Next, an experiment was carried out on the protective action of cystathionine in rat acute gastric mucosal injury due to ischemia-reperfusion. As a sample, the abdominal cavity of a Wistar male rat (10-week-old, 230 to 250 g) fasted for 18 hours is clamped, ischemic for 30 minutes, and then the clamp is removed for reperfusion. 60 minutes later, after exsanguination, the stomach was removed and
A rat acute gastric mucosal lesion model was prepared. The ischemic state for 30 minutes was confirmed by adhering a laser blood flow meter (BRL-100) manufactured by Bio Research Center to the serosa surface of the stomach and measuring the blood flow. Changes in blood flow before and after clamping are shown in FIGS. 3 and 4. In this rat acute gastric mucosal lesion model, the tissue damage of the gastric mucosa was visually observed and the lipid peroxide in the stomach was quantified. As shown in FIG. 3, the blood flow was reduced by clamping and the blood flow was recovered by removing the clamp. Therefore, the ischemia-reperfusion state was reliably reproduced. As shown in FIG. 4, there was almost no difference in the blood flow rate as a reference from the case of administration of cystathionine, and it is considered that the two suffered from ischemia-reperfusion injury to the same extent.

Experiment 3 Protective action of cystathionine on gastric mucosal injury After a predetermined amount of cystathionine was orally administered to each mouse group in advance, the acute gastric mucosal lesion model was prepared. The relationship between each dose of cystathionine and the macroscopically observed gastric mucosal tissue injury area is shown in FIG. Furthermore, the examination criteria for gastric mucosal tissue damage at the time of ischemia-reperfusion and the areas of gastric mucosal tissue damage when cystathionine, cysteine, SOD and allopurinol were administered were observed macroscopically and shown in FIG. FIG. 7 shows a comparison of tissue damage between the case of intraperitoneal administration of cystathionine and the case of oral administration.

As shown in FIG. 5, the gastric mucosal tissue injury area was decreased with an increase in the dose of cystathionine, and as shown in FIG. 6, compared with cysteine, SOD, etc., which are said to have a gastric mucosa protective action. Moreover, cystathionine showed remarkable protective effect on gastric mucosal tissue injury. Although cystathionine has a gastric mucosal tissue injury protective action both in the intraperitoneal and oral administrations, intraperitoneal administration is more effective. Cystathionine effectively protects against various mucosal injuries caused by radicals such as ischemia-reperfusion during surgery and stress-induced chronic gastric ulcer, and this effect is considered to have a similar effect other than gastric mucosa. To be

Further, the inhibitory effect of cystathionine on the production of lipid peroxide in the stomach will be described. When polyunsaturated fatty acids localized in the lipids that make up the cell membrane are attacked by radicals, lipid radicals are generated and chain lipid peroxides are produced. It is believed that this lipid peroxide causes, directly or indirectly, a decrease in membrane function and eventually erosion and ulcer. This experiment clarifies the antiulcer action of cystathionine by quantifying the amount of lipid peroxide produced.

Experiment 4 Inhibitory action of cystathionine on the production of lipid peroxide in the stomach After homogenizing gastric mucosal tissue with KCl, the amount of lipid peroxide was measured by the Aust method to determine the amount of thiobarbituric acid (T
hiobarbituric acid react
e substances (TBA-RS) value
Display as e).

The above-mentioned acute gastric mucosal lesion model was prepared after previously administering each amount of cystathionine to each mouse group. The relationship between each dose of cystathionine and the production amount of gastric lipid peroxide is shown in FIG. Further, FIG. 9 shows the inspection criteria for the production of lipid peroxide in the stomach during ischemia-reperfusion and the amount of each lipid peroxide in the stomach when cystathionine, cysteine and SOD were administered. As shown in FIG. 8, cystathionine decreases the amount of gastric lipid peroxide produced with an increase in its dose, and as shown in FIG. 9, cysteine, which is said to have a gastric lipid peroxide production inhibitory action, Even when compared with SOD and the like, it showed a remarkable inhibitory effect on the production of lipid peroxide in the stomach.

FIG. 10 shows the inspection criteria and cystathionine,
Cysteine and SOD were administered. FIG. 2 is a graph showing the relationship between the amount of gastric lipid peroxide produced in each case and the gastric mucosal tissue injury area during ischemia-reperfusion. There is a positive correlation between gastric lipid peroxide production and gastric mucosal tissue injury, and the linear y =-for the reference and cysteine administration.
It is distributed around 44.980 + 3.2814x as a whole, whereas cystathionine administration is concentrated near the origin. This shows that cystathionine can suppress and protect both the production of gastric lipid peroxide and gastric mucosal tissue damage. This is because cystathionine has an action of scavenging radicals that cause the production of gastric lipid peroxide and gastric mucosal tissue damage.

[0031]

As described above, according to the radical scavenger according to the present invention, the radical scavenging action is exerted without effectively generating other radicals at the time of radical scavenging, and the ulcer is effectively prevented or suppressed. it can. This is also useful for healthy people, but more effective when catalase activity is reduced, such as in ischemia.

In addition, cystathionine inhibits ischemia-reperfusion injury of cell membrane and ulcer generation (stomach peroxidation) even when compared with conventional radical scavengers and SOD, which is a gastric lipid peroxide production inhibitor. It has a remarkable effect on suppression of lipid production and protection against gastric mucosal tissue damage. This is because cystathionine can eliminate these causative radicals without generating other radicals. Therefore, cystathionine effectively protects against various mucosal damages caused by radicals, and this action can be applied to other than gastric mucosa.

Furthermore, since cystathionine is a substance in vivo, there is no fear of toxicity, anaphylactic shock, etc., its half-life in vivo is long, it can be orally administered, and it may be combined with any other radical scavenger. It has an advantageous effect as compared with the conventional radical scavenger and antitumor agent that can be combined.

[Brief description of drawings]

FIG. 1: Xanthine-xanthine oxidase (XS-
XOD) -based superoxide scavenging action (cypridina luciferin derivative (MCLA) method)

[Fig. 2] Extinction effect on human washed leukocyte-producing superoxide

[FIG. 3] Time course blood flow before and after ischemia-reperfusion

[Fig. 4] Reference criteria for ischemia and reperfusion and blood flow of cystathionine-administered sample

FIG. 5: Protective action of cystathionine on gastric mucosal injury

FIG. 6 Comparison of gastric mucosal injury protective action

[Fig. 7] Examination of administration route in gastric mucosal injury protective effect of cystathionine.

FIG. 8: Inhibitory effect of cystathionine on lipid peroxide production in the stomach

FIG. 9: Comparison of gastric lipid peroxide production inhibitory effect

FIG. 10: Relationship between gastric mucosal injury defense effect and gastric lipid peroxide production inhibitory effect

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area A61K 38/45 45/06

Claims (6)

[Claims] 1. An in vivo radical scavenger comprising cystathionine. 2. An in-vivo radical scavenger containing cystathionine as an active ingredient. 3. A superoxide dismutase preparation, a vitamin C analog compound, which contains cystathionine as an active ingredient,
In vivo radical scavenger, characterized in that one or more kinds of vitamin E analogs, cysteine analogs, reduced glutathione, peroxidase preparations, and crude drugs are added. 4. An antiulcer agent comprising cystathionine 5. An anti-ulcer agent characterized by containing cystathionine as an active ingredient. 6. A superoxide dismutase preparation, a vitamin C analog compound, which contains cystathionine as an active ingredient,
Anti-ulcer agent characterized in that one or more kinds of vitamin E analogs, cysteine analogs, reduced glutathione, peroxidase preparations, and crude drugs are added