JPH0930975A - Inhibitor for secondary damage caused by leucocyte followed by recovery of blood circulation to tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor containing a specific phosphoric diester compound, suppressing tissue damage caused by leucocyte such as myocardinal infarction, lung infarction, etc., in natural recovery of blood circulation after temporary disorder of blood circulation, surgical infusion, etc., having low toxicity. SOLUTION: This inhibitor for secondary damage caused by leucocyte followed by recovery of blood circulation to tissue comprises a phosphoric diester compound of the formula (R1 and R2 are each H or methyl) or its salt. The potassium salt of the diester of L-ascorbic acid, L-α-tocopherol and phosphoric acid may be cited as the compound of the formula I or its salt. A dose of the compound of the formula is preferably 2-50mg per adult daily in the case of an injection. The dose is preferably 20-500mg per adult daily in the case of internal medicine. The inhibitor may contain a component having the same kind of another medical action and/or a component having a different kind of another medical action as long as it is not against the purpose.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a drug for suppressing the secondary damage of a tissue caused by leukocytes accompanying the restoration of blood flow to various tissues with impaired blood flow.

[0002]

BACKGROUND OF THE INVENTION Many mechanisms of tissue damage after impaired blood flow have been reported. For example, neutrophils include (a) superoxide anion (O ₂
^-1 ) production (Fantone JC et al., The American Journa
l of Pathology, 107: 397-418 (1982)), (b) Release of lysosomal enzymes (Weisman G et al., The New Engl.
and Journal of Medicine, 303: 27-34 (1980)), and (c) Release of lipoxygenase product (Sasaki K et al.,
Cardiovascular Research, 22: 142-48 (1988)) is said to be involved in ischemic tissue damage. Permanent ligation of the left coronary artery in rats also increased leukotriene B ₄ (LTB ₄ ) levels, which was 8 post-ligation prior to the peak of polymorphonuclear leukocyte infiltration into heart tissue.
It has been reported that a peak was reached at time (Sasaki K et al., Cardiovascular Research, 22:
142-48 (1988))). The peak value of leukotriene B ₄ was 8 times that of the hearts that underwent sham surgery.
Furthermore, activation of phospholipase A ₂ induces lysosomal membrane rupture and releases lysosomal enzymes (Schror K et al., The American Journal of Patholo.
gy, 238: H87-H92 (1980)) and swelling mitochondria (Broekemeier KM et al., The Journal of Bioche
mistry, 260: 105-113 (1985)), is said to bring about the release of arachidonic acid. The release of arachidonic acid leads to the formation of leukotrienes and thromboxanes which have been shown to strongly exacerbate ischemic damage.

Α-Tocopherol is known to be a radical scavenger (McCay PB, Ann Re.
v Nutr, 5: 323-340 (1985)), also reported to reduce myocardial infarct size in dogs (Tanabe M et.
al., The Japanese Journalof Pharmacology, 50: 467-
476 (1989)). However, this drug has not been used as a therapeutic agent for myocardial infarction due to its low water solubility.

Ascorbic acid is also known as a radical scavenger (Nishikimi M et al., Bi
ochemical and Biophysical Research Communications,
63: 463-468 (1975) and Bodannes RS et al., FEBS Le.
tters, 105: 195-196 (1979)), under some conditions its oxidant nature becomes central (Heikkila RE et al., Journal of Neurochemistr.
y, 41: 1384-1392 (1983)).

On the other hand, the following formula in which ascorbic acid and tocopherols are bound by a phosphodiester bond

[0006]

Embedded image

[In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a methyl group. ] And its salts are known to have pharmacological profiles different from those of mere mixtures of ascorbic acid and tocopherols.

That is, these compounds are anticataract agents, agents for treating / preventing climacteric disorders, cosmetics having a skin beautifying effect (Japanese Patent Publication No. 2-44478), anti-inflammatory agents (Japanese Patent Publication 1-2.
7044), an anti-ulcer agent (JP-A-63-270626).
No.), a preventive / therapeutic agent for ischemic organ injury (JP-A-2-111)
722) and various other uses are known.

[0009] Although the compound has a stronger antioxidant effect on rat brain homogenate than α-tocopherol, when observed by electron spin resonance spectroscopy, the compound is strong against the hydroxy radical (.OH). It has also been reported that it exhibits excellent scavenging activity (Mo
ri A et al., Neurosciences, 15: 371-76 (1989)).

It has also been shown that the compounds alleviate tissue acidosis and energy damage during and after cerebral ischemia in mongolian gerbillus (Ku).
ribayashi Y et al., The Japanese Journal of Pharma
Biology, 49: supp. 130P (1989)).

The compound further comprises serum phospholipase A ₂
It has also been confirmed to inhibit (PLA ₂ ) activity (Ku
ribayashi Y et al., Arzneimittel Forschung / Drug Re
search, 42 (II), 9: 1072-1074 (1992)).

The present inventor has conducted further studies based on these backgrounds, and as a result, as shown below, the compound suppresses the secondary damage to the tissue caused by leukocytes accompanying the restoration of blood flow to the tissue. The present invention has been completed and the present invention has been completed.

[0013]

That is, the present invention provides the following formula:

[0014]

Embedded image

[In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a methyl group. ] The phosphoric acid diester compound shown by or its pharmaceutically acceptable salt (henceforth a "this compound" is collectively called), It comprises.
It is an inhibitor of secondary tissue damage caused by leukocytes accompanying recovery of blood flow to the tissue.

As shown in the section of the pharmacological effect test described below, this compound suppresses the establishment of myocardial infarction in rats, suppresses the production of superoxide anion in rat polymorphonuclear leukocytes, and acid phosphatase which is a lysosomal enzyme. It has been found according to the invention to inhibit the activity. In light of these findings, the effect of the present compound on the tissue damage associated with blood flow recovery is as follows: the activity to remove oxygen-derived radicals such as superoxide anion produced by leukocytes such as neutrophils and the acid phosphatase that is a lysosomal enzyme. It is considered to be based on the suppression of the secondary damage caused by leukocytes by the combined action of the inhibitory action. Therefore, the drug of the present invention is useful for suppressing secondary damage caused by leukocytes such as neutrophils, which accompanies restoration of blood flow in various tissues. The most common examples of such tissue damage are myocardial infarction, which is myocardial necrosis resulting from coronary artery occlusion, and cerebral infarction, and the agent of the present invention is effective for transient blood flow in these tissues and other tissues. It can be used to suppress the tissue damage caused by leukocytes such as neutrophils in natural blood flow recovery after surgery, surgical reperfusion and the like.

The present compound is, for example, Japanese Patent Publication No. 2-44478.
It can be synthesized according to the method described in JP-A No. 62-205091.

In the drug of the present invention, the present compound can be used in the free form shown in the above formula, or in the form of a pharmaceutically acceptable salt. Examples of such pharmaceutically acceptable salts include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and other salts. However, it may be used as long as it is pharmaceutically acceptable.

The drug of the present invention may contain a combination of two or more compounds included in the present compound.

The compound has very low toxicity and is excellent in safety. For example, a potassium salt of a diester of L-ascorbic acid and DL-α-tocopherol, which is one of the present compounds, and phosphoric acid (R ₁ = R ₂ = methyl in the formula (I)) (hereinafter abbreviated as EPC-K). .) LD ₅₀ is 5 g /
kg (rat, oral) or more, and 100 mg / kg (rat, intravenous). This very low toxicity is a great advantage in using this compound for the purposes of the present invention.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The drug of the present invention can be appropriately used orally or parenterally (eg, intravenous injection, subcutaneous injection, intramuscular injection, drip infusion, etc.). As the formulation form, for example, any of solid formulations such as tablets, granules, powders and capsules, and liquid formulations such as injections can be formulated by a conventional method. These formulations include commonly used excipients, binders, disintegrants, dispersants, absorption enhancers, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers. Various additives such as a pH adjuster and the like can be appropriately used.

The dose of the present compound depends on the patient's age, body weight,
Although it may vary depending on sex, symptoms, etc., for example, in the case of an injection, it is usually about 0.5 to 200 mg, preferably about 2 to 50 mg per day for an adult. ~ 2000 mg, preferably about 20-500 mg.

The drug of the present invention may contain other components having the same kind of medicinal effect and / or components having another kind of medicinal effect, as long as the object of the present invention is not violated.

Hereinafter, the present invention will be described in more detail with reference to drug efficacy test examples and examples, but the present invention is not limited thereto. The EPC-K used is
It was produced by the known method. Statistical analysis
Differences were considered significant when p <0.05, using t's-t test.

[Pharmaceutical efficacy test 1] Effect of EPC-K on superoxide anion production of polymorphonuclear leukocytes (Test method) Preparation of polymorphonuclear leukocytes: 1% sodium caseinate (Wako Pure Chemical Industries) in sterile saline Male Spra weighing 200-400 g
gue-Dawley rats were injected intraperitoneally. After 6 hours the animals were sacrificed and the peritoneal exudates were collected in plastic tubes and centrifuged at 180 xg for 5 minutes at 4 ° C. To lyse contaminated red blood cells, cells were suspended in Tris-HCl buffer (pH 7.65) containing 150 mM ammonium chloride (Carpenter DL et al., The Journal of Pharmacology.
and Experimental Therapeutics, 251: 983-991 (198
9)). Hank's equilibrium buffer solution containing no calcium ions (H
Polymorphonuclear leukocytes were washed twice with BSS), then cells 2 ×
The cells were resuspended at a rate of 10 ⁷ cells / ml.

[0026] O ₂ ^- production assay: O ₂ ^- production was measured by the reduction of ferricytochrome C (Goldstei
n IM et al., The Journal of Clinical Investigatio
n, 56: 1155-1163 (1975) and Markert M et al., Metho.
ds in Enzymology, 105: 358-65 (1984)). That is, HBSS or EPC-K, suspension of polymorphonuclear leukocytes (4 × 10 ⁶ cells / ml), ferricytochrome C (type III, derived from horse heart: Sigma Chem. Co., St. Loui
s, MO, USA) (2.5 mg / ml) and phorbol-1
2-myristic acid-13-acetic acid (PMA, Wako Pure Chemical) (2
μg / ml) were sequentially added to plastic test tubes.
Incubate the tubes at 37 ° C, and after 5 minutes,
After centrifuging at 8000 xg for 30 seconds, the absorbance of the supernatant was measured at 550 nm using a spectrophotometer (150-20, Hitachi). The amount of O ₂ ⁻ produced can be calculated according to the following formula. ^{_{O 2 - (nmol) = 47.7}} × Absorbance

(Results) The results are shown in Table 1. Each data, O ₂ by induced rat polymorphonuclear leukocytes by PMA ^- on the production, shows the effect of EPC-K at each concentration. Values are expressed as mean ± mean deviation (n = 3), and the numbers in parentheses represent the inhibition rate (%) with respect to the O ₂ ⁻ amount in the control.

[0028]

[Table 1]

[0029] Table 1 than As is apparent, EPC-K is, O ₂ by polymorphonuclear leukocytes ^- production level was dose-dependently inhibited,
Its IC ₅₀ was 4.2 × 10 ^-5 M.

[Pharmaceutical efficacy test 2] Effect of EPC-K on polymorphonuclear leukocyte lysosomal enzyme (Test method) A final concentration of 0.2% was added to a suspension of polymorphonuclear leukocytes prepared in the same manner as in the above-mentioned efficacy test 1. So that
Samples were prepared by adding Triton X-100. Triton X-100 is supposed to release all leukocyte enzyme activity into the suspension medium (Traynor JR et al.,
Biochimica et Biophysica Acta, 665: 571-77 (198
1)). After centrifugation at 600 xg for 5 minutes, the resulting supernatant was used for enzyme activity assay. The assay of acid phosphatase activity is performed by the method described by Bessey et al.
A et al., The Journal of Biological Chemistry, 16
4: 321-29 (1946)) using p-nitrophenyl phosphate as a substrate. The β-glucuronidase activity can be determined by the method described by Kato et al. (Chemical & Pha
rmaceuticalBulletin, 8: 239-42 (1960))
-Measured using nitrophenyl glucuronide as substrate. Myeloperoxidase activity was measured by o-dianisidine oxidation (Jandle RC et al., The Journal of Clinical
Investigation, 61: 1176-1185 (1978)). One unit of enzyme activity was defined as the enzyme activity that cleaves 1 μmol of substrate in 1 minute under the conditions used.

(Test Results) Table 2 shows the effect of each concentration of EPC-K on the acid phosphatase activity. Each value represents the mean ± mean deviation (n = 3), and the numbers in parentheses indicate the inhibition rate (%) with respect to the control activity.

[0032]

[Table 2]

As seen in Table 2, EPC-K inhibited acid phosphatase activity in a concentration-dependent manner, and IC ₅₀ was
It was 2.4 × 10 ^-5 M. However, it had no effect on β-glucuronidase and myeloperoxidase (data not shown).

[Pharmaceutical efficacy test 3] Suppression of rat myocardial infarction (Test method) Induction of myocardial infarction: Selye et al. (Angiology, 2: 398-407)
(1960)) and Sasaki et al. (Cardiovascular Research, 22:
142-48 (1988)) to induce myocardial ischemia.
That is, a male Sprague-Dawley rat weighing 240-280 g was anesthetized with ether and the chest was opened. The heart was quickly abducted from the thoracic cavity and the left main coronary artery was ligated with a suture 1-2 mm below its origin. The heart was returned to its original position in the chest cavity and the chest was immediately closed. EPC-K was dissolved in physiological saline and intravenously administered 5 minutes before ligation (5 mg / k).
g).

Evaluation of infarcted area: The infarcted area of the heart was examined at 12, 24 and 48 hours after ligation by the method of Sasaki et al. (Cardio
Vascular Research, 22: 142-48 (1988)). That is, the heart is rapidly removed, and it is 2 to 2.5 mm.
Sliced into four transverse rings (base to rings A, B, C and D) of width. 1 for 3 transverse rings (rings B, C and D)
% Triphenyltetrazolium chloride (TTC) solution
Incubated at 37 ° C for 20 minutes. TTC is reduced to red formazan by accepting an electron.
This reaction does not occur in mitochondria or in regions where the oxidative system is impaired. Therefore, the lack of staining
Distinguish areas with disabilities from areas without disabilities (Klei
n H. et al., Virchows Archiv A. Pathological Anato
my and Histology, 393: 287-297 (1981)). A drawing tube (SZH) in which the stained area and the unstained area of the bottom surface of each ring are attached to an optical microscope (SZH-ILLD, Olympus Optical Co., Ltd.) on paper.
-DA, Olympus Optical Co., Ltd.).
The area of the unstained area (ie, the impaired area) was expressed as a percentage of the total cross-sectional area of the left ventricle (% LV).

(Test Results) FIG. 1 shows the time course of the area of the TTC unstained region of the heart after ligation of the left main coronary artery. Each point on the graph represents mean ± mean deviation. Ligation of the coronary artery resulted in an infarct region that was not stained with TTC.
It reached a plateau at 12 hours in the untreated group as seen in. The infarct areas at 12 hours, 24 hours, and 48 hours after ligation were 50.1 ± 1.7 and 45.7 ± 2.2, respectively.
And 45.5 ± 5.5% LV. EPC 5 minutes before ligation
Intravenous injection of -K significantly reduced the extent of infarction at 24 and 48 hours. The inhibition rates were 28.5, 54.0 and 52.3% at 12, 24 and 48 hours, respectively.

Interestingly, in the EPC-K-treated animals, the unstained areas at 24 and 48 hours showed
It was smaller than that of 12 hours. TTC can in fact reliably delineate well-established necrosis, but it even detects reversibly impaired tissue. That is, the above
Klein et al. (Virchows Archiv A. Pathological Anatomy
and Histology, 393: 287-297 (1981)), tetrazolium staining showed NAD and / or NAD in the early phase of myocardial ischemia.
Or, it has been found that the activity of tissue dehydrogenase is not necessarily diminished although a large infarct region is delineated due to the loss of NADH. In the rat model we used in this study, blood flow from surrounding normal tissue to the ischemic region gradually recovers within 12 hours after ligation. However, in the absence of drug treatment, the area that was once in the ischemic state becomes necrotic despite the restoration of blood supply (Sasaki et al., Cardiovascular.
Research, 22: 142-48 (1988)). Combining the previous findings, the observation results of this test, and the results of the above-mentioned drug efficacy tests 1, 2, etc., pretreatment with EPC-K before ligation was
It is believed that the tissue that had been reversibly impaired was restored by blocking the secondary tissue damage caused by infiltrating neutrophils as the blood flow was restored.

[0038]

【Example】

[Example 1] Oral tablet EPC-K 100 mg Lactose 75 mg Starch 20 mg Polyethylene glycol 6000 5 mg The above ingredients are mixed by a conventional method to give 1 tablet. Sugar coating may be applied as necessary.

Example 2 Injection EPC-K 200 mg Mannitol 5.0 mg 1N sodium hydroxide Appropriate amount distilled water Total amount 100 ml The above components are mixed by a conventional method and sterile filtered. The filtrate is aseptically filled in glass ampoules in an amount of 5 ml each and the mixture is sealed to obtain an injection.

[Brief description of drawings]

FIG. 1. Heart TTC after ligation of the left main coronary artery of the rat.
Time-dependent change in area of unstained area (displayed as mean ± mean deviation).

Claims (2)

[Claims] 1. The following formula: [In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a methyl group. ] The inhibitor of the secondary damage of the tissue by the leukocytes accompanying the restoration of blood flow to the tissue, which comprises the phosphodiester compound represented by the above or a pharmaceutically acceptable salt thereof. 2. The inhibitor according to claim 1, wherein the secondary damage to the tissue is necrosis of myocardial tissue.