JPH07126176A - Active oxygen inhibitor - Google Patents

Abstract

PURPOSE:To provide an active oxygen inhibitor having high superoxide dismutase(SOD)-and catalase(CAT)-like activities and useful for prevention and therapy of various kinds of disorders or diseases caused by active oxygen. CONSTITUTION:This active oxygen inhibitor is characterized by its active component composed of manganese(III)-2,6-pyridinecarboxylate complex.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an active oxygen inhibitor.

[0002]

BACKGROUND OF THE INVENTION AND SUMMARY OF THE INVENTION neutrophils in leukocytes, migration response to enemy penetrated into the body, phagocytosis, active oxygen O ₂ ^- production, by the release of lysosomal enzymes Since it has a bactericidal action, it is considered to play an important role in biological defense. On the other hand, along with the biological defense reaction, active oxygen released by neutrophils or other tissues destroys cells during ischemia of each tissue and subsequent reperfusion of blood or during acute inflammation.
It has been reported to impair organizational function [BRL
ucchesi, Annual Review of Pharmacology and Toxicology (Ann. Rev. Pharmacol. To
xicol.), 26, 201 (1986); BA Freeman et al., Laboratory Inves
tigation), 47, 419 (1982); E. Braunwald., RA Kl
oner, Journal of Clinical Investigation (J. Clin. Invest.), 76, 1713 (1985); J.
L. Romson et al., Circulation
n), 67, 1016 (1983).

In order to protect the living body from such excess active oxygen, enzymes such as superoxide dismutase (hereinafter referred to as SOD) and catalase (hereinafter referred to as CAT) are present in the living body, and excess active oxygen is present. To prevent illness. However, when such a protective action does not function sufficiently or when a large amount of active oxygen is generated, such a protective action alone cannot sufficiently protect the living body. Such cases include, for example, excessive stress, ingestion of a large amount of alcohol, peroxides and other drugs, excessive exercise, and the like.

Therefore, there has been a demand for the provision of a drug having an activity similar to that of the above-mentioned enzymes such as SOD and CAT. The object of the present invention is to solve the above technical problems,
An object is to provide an active oxygen inhibitor having SOD-like activity or CAT-like activity.

[0005]

Means and Actions for Solving the Problems
In view of the fact that the main cause of the above-mentioned cytotoxicity, particularly heart, brain, kidney, lung, and post-ischemic reperfusion injury in the digestive system is active oxygen released from neutrophils,
As a result of earnest studies to provide a new drug having an activity of suppressing the release of active oxygen species or removing active oxygen species, manganese (III) -2,6-pyridinedicarboxylate complex was found to be extremely active. The present inventors have completed the present invention by finding the fact that they have strong SOD-like activity and CAT-like activity.

That is, the active oxygen suppressor of the present invention is characterized by containing a manganese (III) -2,6-pyridinedicarboxylate complex as an active ingredient. This complex has the formula:

[0007]

[Chemical 1]

The compound represented by the formula (1) is obtained, for example, by reacting manganese (III) acetate with 2,6-pyridinedicarboxylic acid. This compound has the activity of suppressing the release of reactive oxygen species from neutrophils or removing the released reactive oxygen species. Therefore, it exerts an action of preventing the production of lipid peroxide in the living body or an action of lowering lipid peroxide. Accordingly, it is useful as a preventive and therapeutic agent for various disorders or diseases caused by excessive generation of reactive oxygen species, bioaccumulation of lipid peroxide, or lack of defense mechanism against these. More specifically, the active oxygen depressant of the present invention is a drug that protects various tissue cells from damage associated with ischemia and recanalization of blood, for example, a therapeutic agent for cardiac ischemic diseases such as myocardial infarction and arrhythmia; Hepatic and renal function improving agents for disorders such as transplantation and microcirculatory insufficiency; therapeutic agents for gastrointestinal ulcers such as gastric ulcers; therapeutic agents for cerebral hemorrhage, cerebral infarction, transient cerebral ischemic disease; or for causes other than ischemia Drugs that suppress various cellular disorders that are thought to be caused by abnormally generated active oxygen, such as skin diseases such as cutaneous vasculitis, psoriasis, erythema multiforme, Behcet's disease, chickenpox dermatitis, and cement dermatitis; Arteriosclerosis; Diabetes; Eye diseases such as retinopathy of prematurity, eyeball disease, retinitis, pigmentation; emphysema; adult respiratory distress syndrome; arthritis; rheumatoid malignancy; ulcerative colitis; Crohn's disease; Reino In addition, such as Mr. disease, age spots, freckles and pigmentation prevention, burns, trauma, they are useful as therapeutic agents, such as fatigue.

The complex according to the present invention is SOD.
Since it exhibits CAT-like activity of erasing active oxygen that cannot be erased depending on the situation, that is, H ₂ O ₂ , it has an advantage of further increasing the effect. The active oxygen suppressor of the present invention is usually
It is used in the form of a general pharmaceutical preparation. The preparation is prepared using diluents or excipients such as fillers, fillers, binders, moisturizers, disintegrants, surfactants and lubricants which are usually used. Various forms of this pharmaceutical preparation can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules,
In addition to suppositories, injections (solutions, suspensions, etc.), external preparations such as lotions, creams, ointments, etc. can be used. In the case of molding in the form of tablets, as the carrier, any of those conventionally widely used in this field can be used, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin,
Excipients such as crystalline cellulose, silicic acid, water, ethanol,
Propanol, simple syrup, glucose solution, starch solution,
Gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dry starch, sodium alginate, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, lauryl sulfate Disintegrators such as sodium, stearic acid monoglyceride, starch, lactose, etc., sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium base, absorption promoters such as sodium lauryl sulfate, glycerin, starch, etc. Examples thereof include humectants, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearates, boric acid powders and lubricants such as polyethylene glycol. Further, the tablet may be a tablet which is coated with a usual coating, if necessary, such as a sugar coating, a gelatin-coated tablet, an enteric coated tablet, a film-coated tablet, a double tablet or a multilayer tablet.

In the case of molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin and talc. , Gum arabic powder,
Examples include tragacanth powder, gelatin, binders such as ethanol, disintegrators such as laminaran and agar. In the case of molding in the form of suppositories, conventionally known carriers can be widely used, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semisynthetic glycerides. You can Furthermore, when prepared as an injection, the solution, emulsion and suspension are preferably sterilized and isotonic with blood, and when molded into the form of these solution, emulsion and suspension, Any of those conventionally used in this field as a diluent can be used, and examples thereof include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. it can. In this case, a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be further added. Coloring agents, preservatives, fragrances, flavors, if necessary,
Sweeteners and other pharmaceuticals may be included in the therapeutic agent. In the case of molding into a paste, cream or gel, white petrolatum, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicone, bentonite and the like can be used as a diluent.

The amount of the complex contained in the active oxygen suppressor of the present invention is not particularly limited and may be selected in a wide range, but it is usually 1 to 70% by weight based on the total composition. The administration method of the active oxygen suppressor of the present invention is not particularly limited, and the active oxygen inhibitor is administered according to various preparation forms, patient age, sex and other conditions, and degree of disease. For example, tablets, pills, liquids,
Orally in the case of suspensions, emulsions, granules and capsules.

[0012] In the case of an injection, it is administered alone or mixed with an ordinary replenishing solution such as glucose or amino acid, and is intravenously administered. Further, if necessary, it is intramuscularly, intradermally, subcutaneously or intraperitoneally administered. It In the case of suppositories, it will be administered rectally. Furthermore, in the case of external preparations such as lotions, creams and ointments, they are applied and administered. The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on the usage, age of the patient, sex and other conditions, degree of disease, etc., but the amount of the compound of the present invention is usually 1 to 10 mg per 1 kg of body weight per day, preferably 2 to 5 mg. It is preferable that the dose be administered once or twice a day.

[0013]

EXAMPLES The active oxygen suppressor of the present invention will be described below with reference to Reference Examples and Examples. Reference Example (Production of Manganese (III) -2,6-Pyridinedicarboxylate Complex) 5 ml of acetic acid was placed in a round-bottomed flask, and manganese acetate Mn (CH ₃
0.180 g of COO) ₃ .2H ₂ O was added. When manganese acetate was completely dissolved, 0.200 g of 2,6-pyridinedicarboxylic acid was gradually added. While stirring with a stirrer, the mixture was subsequently heated in a water bath at 70 ° C. After reacting for about 1 hour, the mixture was left standing and cooled at room temperature. The product was filtered and thoroughly washed with acetic acid to obtain the target compound. (1) Electron Spin Resonance The aqueous solution of the obtained product was measured by electron spin resonance at 77K. As a result, since no Mn ²⁺ signal was observed, it was confirmed that manganese in the product was present as Mn (III). (2) Elemental Analysis Mn (III) · _{[C 5 H 3 N (COOH)} 2 ] ₂ · CH ₃
COO ^- · at 1/2 H ₂ O Calculated (%) C 42.22 H2.66 N6.15 Found (%) C 42.07 H2.38 N5.85 ( 3) Infrared absorption spectrum tablets (KBr) Method The infrared absorption spectrum was measured.
As a result, tertiary amine (-N =) and carboxylic acid (-
Peak due to CH ₂ COOH) appeared respectively 1313Cm ^-1 and 1690 cm ^-1. (4) Mass spectrometry (fast-atom-bombardment-mass spectrome
try) JEOL JMS-D300 (manufactured by JEOL Ltd.) Device: FAB Gun High Voltage: 6 kV Filament current: 1.5 A Emission current: 10 mA Gas: Xe The results are shown in FIG. Formulation Example 1 A tablet having the following composition in one tablet was produced according to a conventional method.

(Component) (Compounding amount) Manganese (III) -2,6-pyridinedicarboxylate complex 5 mg Starch 132 mg Magnesium stearate 18 mg Lactose 45 mg Total 200 mg Formulation Example 2 (Component) (Compounding amount) Manganese (III) ) -2,6-Pyridine dicarboxylate complex 500 mg Polyethylene glycol (molecular weight 4000) 300 mg Sodium chloride 900 mg Polyoxyethylene sorbitan monooleate 400 mg Sodium metabisulfite 100 mg Methylparaben 180 mg Propylparaben 20 mg Distilled water for injection 100 ml Parabens and metaheavy Sodium sulfite and sodium chloride were dissolved in a part of the above distilled water for injection at 80 ° C. with stirring. The resulting solution is cooled to 40 ° C,
Manganese (III) -2,6-pyridinedicarboxylate complex, polyethylene glycol and polyoxyethylene sorbitan monooleate were sequentially dissolved. Then,
The remaining distilled water for injection was added to the solution to adjust the final volume, sterile filtration was performed using an appropriate filter paper, and 1 ml each was dispensed into ampoules to prepare an injection solution. Test Example 1 (Measurement of SOD-like activity) 1. Preparation of Reagents ・ Solution A (50 mM phosphate buffer) NaHPO ₄ 8.9
The pH was adjusted to 7.80 by adding 100 ml of an aqueous solution of 0.68 g of KH ₂ PO _{4 to} 500 ml of an aqueous solution of 5 g. Solution B (1 mM cytochrome C solution oxidized with K ₃ Fe (CN) ₆ ) Cytochrome C 30.96 mg was added to the solution A
Was dissolved in 1 mM cytochrome C solution to a total volume of 2.5 ml, and 100 mM K ₃ Fe (CN) ₆ aqueous solution 59 was added.
After adding μl and reacting for 1 hour, dialysis was performed with purified water for 1 day. -Solution C (0.5 mM xanthine sodium solution) 1.74 mg of sodium xanthine in solution A in a total volume of 20 ml
I chose Solution D (xanthine oxidase solution) Xanthine oxidase (5 units, 0.3 ml, 41 mg protein / ml, 0.42 unit / mg protein = 17.22 units / ml or 5 units, 0.3 ml, 38 mg
Protein / ml, 0.47 unit / mg protein = 17.
(86 units / ml) was used after being diluted 100-fold with the solution A. 2. Measuring instrument JASCO U best 50 under the condition of 25 ℃ as electronic spectrum
(Manufactured by JASCO Corporation) was used. 3. Measurement method The following operations were performed with all temperatures kept at 25 ° C.

(1) Solution A 3 ml and solution B5 in the cell
0 μl was added and stirred well. 550 n of the obtained solution
The absorbance A ₁ at m was measured. To this solution, a spatula of Na ₂ S ₂ O ₄ (reducing agent) was added to reduce cytochrome C, and the absorbance A of the solution at 550 nm was measured.
₂ was measured. Absorbance difference ΔA (= A ₂ −A ₁ ) is 0.2
The addition amount of the solution B was adjusted so as to be 1.

(2) The cell was charged with 2.7 ml of the solution A, 300 μl of the solution B and the solution C in the amounts determined in the above (1). Solution D was added to this to start the reaction, and active oxygen was generated. At this time, the increase in absorbance at 550 nm is 0.0
Solution D to be 250absorbance / min (± 10%)
The amount added was adjusted. (3) As in (2) above, add 2.7 ml of solution A to the cell,
After adding 300 μl of the solution B and the solution C in the amounts determined in (1), manganese (III) -2,6-pyridinedicarboxylate complex was added. Then, the amount of solution D obtained in (2) above was added to this, and the increase in absorbance at 550 nm was measured, and the amount at which the generation of active oxygen was suppressed by 50% was obtained as IC ₅₀ . 4. Measurement result The amount of the solution B added in (1) above was set to 45 μl. At this time, the absorbance was A ₁ = 0.295, A ₂ = 0.200, and ΔA = 0.200 absorption.

Further, the addition amount of the solution D in the above (2) was set to 42 μl. The increase in absorbance at this time is 0.0 on average.
It was 2550 absorption / min. Manganese (III) −
The absorbance increase and the active oxygen generation suppression rate when the concentration of the 2,6-pyridinedicarboxylate complex was changed and added were measured. The results are shown in Table 1.

[0018]

[Table 1]

Manganese (II) calculated from the results shown in the table
The IC ₅₀ for the addition of I) -2,6-pyridinedicarboxylate complex was 0.17 μM. The same operation was repeated 6 times in total, and the average value and standard deviation were obtained. As a result, IC ₅₀ (μM) was 0.16 ± 0.067. Test Example 2 (Measurement of CAT-like activity) 1. Measurement principle CAT is known to catalyze the oxidation reaction of methanol, which is important in clinical chemistry analysis. The reaction formula is shown below.

[0020]

[Chemical 2]

The CAT-like activity of the complex was measured by quantifying the formaldehyde produced in this reaction.
Formaldehyde was quantified using a formaldehyde test reagent (manufactured by Wako Pure Chemical Industries, Ltd.). This method uses a test reagent, 4-amino-3-hydrazino-5.
-Mercapto-1,2,4-triazole is alkaline and binds to formaldehyde and is further oxidized to give 5
Red-purple 6-mercapto-S with maximum absorption at 50 nm
-Triazolo [4,3-b] -S-tetrazine is used, and the absorbance of the reaction solution is 55
The amount of formaldehyde can be measured by measuring at 0 nm. 2. Measurement method and measurement results Manganese (III) -2,6-pyridinedicarboxylate complex was dissolved in distilled water or a phosphate buffer (pH 7.00) to prepare a 0.005 M solution.

4 ml of this solution was added to a mixed solution of 2 ml of methanol and 0.5 ml of hydrogen peroxide (150 μg / ml), and the mixture was shaken well and left for 10 minutes. To 2 ml of the obtained reaction solution, 2 ml of an aqueous solution of sodium hydroxide as an alkali and 2 ml of an aqueous solution of 4-amino-3hydrazino-5-mercapto-1,2,4-triazole as a test reagent were added, and the mixture was shaken until foaming was completed. . Then, the absorbance at 550 nm was measured. The measured absorbance is based on the calibration curve of formaldehyde (graph showing the relationship between the concentration of formaldehyde (ppm) and the absorbance) obtained in advance, and when the pH is 3.5, the amount of formaldehyde produced per 1 mM of the manganese complex is 0.0
It was found to be 69 μM.

[0023]

INDUSTRIAL APPLICABILITY As described above, the active oxygen suppressor of the present invention has high SOD-like activity and CAT-like activity, and thus is useful as a prophylactic and therapeutic drug for various disorders or diseases caused by active oxygen. There is.

[Brief description of drawings]

FIG. 1 shows the manganese (III) -2,6-obtained in the examples.
It is a graph which shows the mass spectrometry result of a pyridine dicarboxylate complex.

Claims (1)

[Claims] 1. An active oxygen depressant comprising a manganese (III) -2,6-pyridinedicarboxylate complex as an active ingredient.