JP2727431B2 - In vivo radical scavenger - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an in vivo effective radical scavenger and an anti-ulcer agent comprising cystathionine.

[0002]

2. Description of the Related Art A radical is one unpaired electron.
One or more molecules or atoms, such as nitric oxide, nitrogen dioxide, and active oxygen. Reactive oxygen is generated in the process of energy metabolism in living cells,
Superoxide (O ₂ ^-), hydrogen peroxide (H ₂ O ₂₎
Alternatively, there is a hydroxyl radical (.OH). These active oxygens are essential for the phagocytic cell bactericidal mechanism and play an important role in removing viruses and cancer cells. However, excessive production that exceeds the control of the field, amount, and time of the living body attacks the in vivo molecules that constitute the membranes and tissues in the living body,
Induces various diseases. At present, pathological conditions and diseases that are thought to involve radicals include arteriosclerosis, cerebral infarction, cerebral ischemia-reperfusion injury, gastrointestinal ischemia-reperfusion injury, ulcer, malignant tumor,
Chemical carcinogenesis, paraquat poisoning, diabetes, epilepsy, aging, etc. have been reported.

[0003] In vivo, the first radical generated is superoxide. Other radicals such as OH radicals are generated from this superoxide through a Fenton reaction or the like. Therefore, in living organisms, SOD, a specific enzyme that eliminates this superoxide,
(Superoxide dismutase). Therefore, clinical applications of SOD preparations and related substances (Japanese Patent Application No. 2-75805) have been studied as radical scavengers. Vitamin E related compounds (Japanese Patent Application No. 3-128)
96), a compound related to vitamin C (Japanese Patent Application No. 2-13419).
9) Development of radical scavengers such as cysteine-related compounds, reduced glutathione peroxidase preparations, and Kampo medicines (Japanese Patent Application No. 1-8694) has been reported.

[0004]

However, among SOD preparations, the possibility of anaphylactic shock in a SOD preparation derived from a different animal, that it cannot be administered orally as it is as a protein, and that the half-life in vivo by intravenous administration is low. There was the disadvantage of being a few minutes.

[0005] Elimination of superoxide by SOD or SOD-related preparations always involves generation of hydrogen peroxide. It is decomposed by catalase in a healthy subject, but the activity of catalase is reduced due to ischemia or the like, and OH radicals are generated as in formulas (1) and (2) by iron ions released from hemoglobin, etc.
These can cause new tissue damage.

[0006]

Embedded image On the other hand, low-molecular compounds such as vitamin E and C-related compounds have a short half-life in vivo and must be taken at all times, and cysteine and glutathione have free SH groups. There was a problem of toxicity, for example, it was easy to do.

[0007] Therefore, there has been a demand for a radical scavenger and antiulcer agent which can be administered orally without fear of anaphylactic shock and the like, and which acts without generating other radicals during radical scavenging and has a long half-life in vivo.

[0008]

Means for Solving the Problems Cystathionine, a substance in the body, is thought to play an important role in the body as a precursor of cysteine, but its action on radicals has hardly been studied. The present inventors have invented a hepatic disorder inhibitor with respect to cystathionine (Japanese Patent Application No. Sho 63)
Further, the present inventors have found that cystathionine has an effect on radical scavenging and protection against ischemia-reperfusion injury, and provide a radical scavenger comprising cystathionine and an anti-ulcer agent in the present invention.

[0009] Ischemia-reperfusion injury is a new cell injury that occurs upon reperfusion after hemostasis. The mechanism is that oxygen re-supplied from the interruption of blood flow becomes active oxygen (radical), causing chain peroxidation of the phospholipids present in the cell membrane to cause cell damage. The injury causes erosion and ulceration of the mucous membrane.

[0010] Cystathionine eliminates the radicals that cause these injuries and protects against diseases caused by the radicals.

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

[0012]

Embedded image The generated hydrogen peroxide generates 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 operation. Focusing on the fact that cystathionine does not generate other radicals in the radical scavenging reaction with superoxide due to its -S- group as shown in formula (4), radical scavenging that does not generate other radicals during radical scavenging action The agent is provided.

[0013]

Embedded image For effective radical scavenging, relatively large doses are required. Utilizing that cystathionine is a substance in the body, it is possible to administer a relatively large amount orally,
In addition, it is possible to provide a radical scavenger that is stable in a living body.

The cystathionine which is a radical scavenger according to the present invention has almost no toxicity and the results of an acute toxicity test using mice are as follows.

Results of acute toxicity test Administration route LD50 Oral 10 mmol / kg (2200 mg / k
g) or more Intraperitoneal 10 mmol / kg (1250 mg / k)
g) or more The present acute toxicity test was performed for oral administration and intraperitoneal administration using DDY male mice (body weight 35 to 40 grams).
Observation was performed for 14 days (112H) after administration, and LD50 mmo was observed.
l / kg (mg / kg) was determined. Although described as the LD50 value here, none of the mice actually died during the test.

The radical scavenger according to the present invention is obtained by mixing with liquid or solid pharmaceutical auxiliary components such as excipients, binders, diluents, etc.
It can be administered orally or parenterally in any dosage form such as capsules and injections.

[0017] It is also possible to mix with other drugs as required. The dose may be appropriately increased or decreased depending on the age, body weight, symptoms, and the like.
1.0 g is desirable.

[0018]

EXAMPLES The radical scavenging action and the anti-ulcer action of cystathionine according to the present invention will be described in detail with reference to the following experimental examples.

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

Experiment 2 Elimination action on human washed leukocyte-producing superoxide Washed leukocytes were prepared from a normal healthy person, suspended in a buffer, and then activated with PMA (phor).
(Bol myristate acetate) was added to generate superoxide, and the effects of cystathionine and cysteine on the superoxide were examined.
The generated superoxide was measured in the same manner as in Experiment 1. FIG. 2 shows the relationship between the amount of each added cystathionine and the remaining amount of superoxide.

As can be seen from FIGS. 1 and 2, cystathionine is a xanthine-xanthine oxidase (X-XO).
D) Superoxide derived from the system and superoxide producing human washed leukocytes 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 leukocyte system. The elimination action of superoxide is due to its active free S
Due to the H group, the same reaction mechanism cannot be taken depending on -S- of cystathionine. Therefore, cystathionine is a precursor of cysteine, but has a completely different action and effect on radical scavenging.

Further, the radical scavenger composed of cystathionine does not produce other radicals when reacting with superoxide. Therefore, even when used in combination with other existing radical scavengers, the effect thereof is affected. Nothing. Therefore, it can be arbitrarily combined with an existing radical scavenger and administered, and a radical scavenger more effective for a target disease can be provided.

Next, an experiment was conducted on the protective effect of cystathionine on acute gastric mucosal injury in rats caused by ischemia-reperfusion. Specimens are clamped in the celiac artery of a Wistar male rat (10-week-old, 230-250 g) fasted for 18 hours, left in an ischemic state for 30 minutes, and then unclamped and reperfused. 60 minutes later, after bleeding and lethality, the stomach is removed,
A rat gastric mucosal lesion model was prepared. The ischemic state for 30 minutes was confirmed by attaching a laser blood flow meter (BRL-100, manufactured by BioResearch Center) to the serosal surface of the stomach and measuring the blood flow. Changes in blood flow before and after clamping are shown in FIGS. For this rat acute gastric mucosal lesion model, macroscopic observation of tissue damage of the gastric mucosa and quantification of lipid peroxide in the stomach were performed. As shown in FIG. 3, the blood flow was reduced by clamping, and the blood flow was restored by removing the clamp. Thus, the ischemia-reperfusion state was reliably reproduced. As shown in FIG. 4, there was almost no difference in blood flow between the case where cystathionine was administered and the control standard, and it is considered that both were injured by ischemia-reperfusion to the same extent.

Experiment 3 Protective Effect of Cystathionine on Gastric Mucosal Injury The above-mentioned acute gastric mucosal lesion model was prepared after oral administration of a certain amount of cystathionine to each mouse group. FIG. 5 shows the relationship between the dose of each cystathionine and the visually observed area of gastric mucosal tissue injury. Further, gastric mucosal tissue damage during ischemia-reperfusion was checked visually, and each gastric mucosal tissue damage area when cystathionine, cysteine, SOD and allopurinol were administered was visually observed, and is shown in FIG. FIG. 7 shows a comparison of tissue damage between cystathionine administered intraperitoneally and oral administration.

As shown in FIG. 5, as the dose of cystathionine increases, the area of gastric mucosal tissue injury decreases, and as shown in FIG. 6, compared with cysteine, SOD, etc., which are said to have gastric mucosal protective action. Cystathionine also showed a remarkable protective effect on gastric mucosal tissue injury. Cystathionine has a protective effect on gastric mucosal tissue injury in both intraperitoneal and oral administration, but intraperitoneal administration works more effectively. Cystathionine effectively protects against various mucosal injuries caused by radicals such as ischemia-reperfusion during surgery and chronic gastric ulcers with stress, and this effect is considered to have a similar effect other than in gastric mucosa. Can be

Further, the effect of cystathionine on the production of lipid peroxide in the stomach will be described. When highly unsaturated fatty acids localized in lipids constituting a cell membrane are attacked by radicals, lipid radicals are generated, and lipid peroxides are generated in a chain. It is believed that this lipid peroxide directly or indirectly causes a decrease in membrane function, and thus erosion and ulceration. This experiment clarifies the anti-ulcer effect of cystathionine by quantifying the amount of lipid peroxide produced.

Experiment 4 Inhibitory effect of cystathionine on the production of lipid peroxide in the stomach After homogenizing the gastric mucosal tissue with KCl, the amount of lipid peroxide was measured by the Aust method, and the amount of thiobarbituric acid (T
hiobarbituric acid reactive
esubstances (TBA-RS) value
e).

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

FIG. 10 shows the control criteria and cystathionine,
Cysteine and SOD were administered. 4 is a graph showing the relationship between the amount of lipid peroxide produced in the stomach and the area of gastric mucosal tissue injury during ischemia-reperfusion in each case. The amount of lipid peroxide produced in the stomach and gastric mucosal tissue injury had a positive correlation, and the control standard and cysteine administration showed a linear y =-
The distribution is generally around 44.980 + 3.2814x, whereas cystathionine administration is clustered near the origin. This indicates that cystathionine can suppress and prevent both production of lipid peroxide in the stomach and damage to gastric mucosal tissue. This is because cystathionine has an action of eliminating radicals that cause generation of lipid peroxide in the stomach and damage to gastric mucosal tissue.

[0031]

As described above, according to the radical scavenger according to the present invention, the radical scavenging action can be exerted without generating other radicals at the time of radical scavenging, thereby effectively preventing and suppressing ulcer formation. it can. This is useful for healthy subjects, but is more effective when catalase activity is reduced in ischemia and the like.

Cystathionine also suppresses ischemia-reperfusion injury of cell membranes and suppresses ulcer formation (gastric peroxidation) even when compared with conventional radical scavengers and SOD, which is a gastric lipid peroxide production inhibitor. It has a remarkable effect on suppressing lipid production and protecting against gastric mucosal tissue injury. This is because cystathionine can eliminate these causative radicals without generating other radicals. Therefore, cystathionine effectively protects against various mucosal injuries caused by radicals, and this action can be applied to other than gastric mucosa.

Furthermore, since cystathionine is a substance in the living body, there is no need to worry about toxicity, anaphylactic shock, etc., it has a long half-life in the living body, it can be administered orally, and it can be used with any other radical scavenger. This has an advantageous effect as compared with conventional radical scavengers and antitumor agents that can be combined.

[Brief description of the drawings]

FIG. 1. Xanthine-xanthine oxidase (XS-
XOD) Elimination action on superoxide derived from the system (Seafly luciferin derivative (MCLA) method)

FIG. 2: Elimination effect on human washed leukocyte producing superoxide

FIG. 3. Blood flow over time before and after ischemia-reperfusion

[FIG. 4] Reference criteria and blood flow of cystathionine-administered specimens during ischemia and reperfusion

FIG. 5 shows the protective effect of cystathionine on gastric mucosal injury.

FIG. 6 Comparison of protective effects against gastric mucosal injury

FIG. 7: Study on administration route of cystathionine in protective action against gastric mucosal injury

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

FIG. 9: Comparison of the inhibitory action on production of lipid peroxide in the stomach

FIG. 10: Relationship between gastric mucosal injury protective action and gastric lipid peroxide production inhibitory action

Claims (6)

(57) [Claims] 1. An in vivo radical scavenger comprising cystathionine. 2. An in vivo radical scavenger comprising cystathionine as an active ingredient. 3. A composition comprising cystathionine as an active ingredient, a superoxide dismutase preparation, a vitamin C-related compound,
In vivo radical scavenger, wherein one or more of vitamin E analogs, cysteine analogs, reduced glutathione, peroxidase preparations, and crude drugs are added. 4. An anti-ulcer agent comprising cystathionine 5. An anti-ulcer agent comprising cystathionine as an active ingredient. 6. A super oxid dismutase preparation, a vitamin C-related compound comprising cystathionine as an active ingredient,
An anti-ulcer agent, wherein one or more of vitamin E analogs, cysteine analogs, reduced glutathione, peroxidase preparations, and crude drugs are added.