JP2000026295A - Therapeutic agent for ischemic reperfusion injury and therapeutic agent for cell dysfunction including cardiac insufficiency due to arrhythmia and cardiac infarction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a therapeutic agent useful for ischemic reperfusion injury and cell dysfuntion of mammals by including pyridoxal phosphate, pyridoxine, pyridoxal, etc. SOLUTION: This therapeutic agent is obtained by including a compound selected from (A) pyridoxal-5'-phosphate represented by the formula, (B) pyridoxine, (C) pyridoxal and (D) pyridoxane. The therapeutic agent is preferably administered in a daily dose of about 1-50 mg based on 1 kg body weight of a patient. The ingredients A to D and a pharmaceutically acceptable carrier (a physiological saline solution, a Ringer's solution, etc.), are contained to afford a pharmaceutical composition. The composition is preferably enterally or parenterally administered. Great effects on treatment of ischemic reperfusion injury and cell dysfunction including cardiac insufficiency due to arrhythmia and cardiac infarction can be produced by properly administering the ingredients A to D.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a therapeutic agent for ischemia-reperfusion injury in various organs and a therapeutic agent for cell dysfunction including arrhythmia and heart failure due to myocardial infarction.

[0002]

BACKGROUND OF THE INVENTION Ischemia is the inability of an organ or part of the body to receive an adequate blood supply. Organs not supplied with blood are said to be hypoxic. The organs also become hypoxic when there is a temporary loss of blood supply, such as during surgery or temporary arterial occlusion. After a brief stop, blood begins to flow again into the organ, which is known as ischemia reperfusion of the organ.
Ischemic reperfusion of an organ causes structural and functional abnormalities in the tissue of the organ and can cause damage to the organ. Symptoms observed with ischemia-reperfusion injury include neutrophil infiltration, bleeding, edema and necrosis.

[0003] One example of ischemia-reperfusion injury is myocardial infarction. Myocardial infarction results from the interruption of blood supply to the myocardium (muscle wall of the heart). Due to the interruption of the blood supply, the infarct portion, which is a region of the myocardium where there is no blood supply, spreads, and eventually results in a functional loss of myocardial infarction or heart failure.

[0004] When blood is reperfused into the ischemic organs, excessive oxidative free radicals are produced by the blood and cell storage of adenosine triphosphate (ATP) is impaired, resulting in ischemia reperfusion injury. It is considered. For example, myocardial infarction has been linked to impairment in ATP and the occurrence of intracellular calcium overload.

[0005]

Heretofore, there has not been proposed a therapeutic agent for ischemia-reperfusion injury and a therapeutic agent for cell dysfunction including heart failure due to arrhythmia and myocardial infarction, which have excellent drug efficacy. An object of the present invention is to provide a therapeutic agent for ischemia-reperfusion injury in various organs and a therapeutic agent for cell dysfunction including heart failure due to arrhythmia and myocardial infarction.

[0006]

According to the present invention, there is provided a therapeutic agent for mammals according to the present invention, which is a therapeutic agent for ischemia-reperfusion injury and cell dysfunction, comprising pyridoxal-5 '.
-It is characterized by containing a compound selected from the group consisting of phosphoric acid, pyridoxine, pyridoxal and pyridoxamine.

[0007] Preferably, the amount of the therapeutic agent ranges from about 1 to 50 mg / kg of the patient's body weight. The amount of the therapeutic agent is about 5 to 25 m / kg of the patient's weight.
It is preferably in the range of g. Preferably, the compound is a therapeutic for enteral or parenteral administration. Preferably, the compound is pyridoxal-5'-phosphate.

Next, the therapeutic agent of the present invention is a therapeutic agent for ischemia-reperfusion injury in mammals, which comprises a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. Features. Next, the therapeutic agent of the present invention is a therapeutic agent for mammalian cell dysfunction, characterized by containing a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine.

Next, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and a therapeutic amount of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. I do. Preferably, the compound is pyridoxal-5'-phosphate. The pharmaceutical composition is preferably in a form suitable for enteral or parenteral administration.

[0010] The present invention provides a method for treating ischemia-reperfusion injury in organs, wherein pyridoxal-5'-phosphate (also called PLP and P-5P) treats ischemic reperfusion injury, resulting in heart failure following arrhythmia and myocardial infarction. Based on the finding that it can be used for the treatment of

Pyridoxal-5'-phosphate (PLP)
Is chemically represented by the following chemical formula (Chemical Formula 1).
-Hydroxy-methyl-5-[(phosphonoxy) methyl]
—4-pyridine-carboxaldehyde.

Embedded image

PLP is a derivative of vitamin B ₆ (pyridoxine hydrochloride) and has strong B ₆ activity. Mammal generates PLP by phosphorylating and oxidized vitamin B _6. Vitamin B ₆ phosphorylation is associated with pyridoxal kinase. PLPs can be chemically synthesized in various ways, for example by the action of ATP on pyridoxal, the action of phosphate chloride on pyridoxal in aqueous solution and the phosphorylation of pyridoxamine with concentrated phosphoric acid, followed by oxidation. .

[0013] The biological role of PLP is to act as accessory enzymes and antagonists. PLP is an auxiliary enzyme in glycogen phosphorylase levels (glycogen degradation) and transamination levels in the malate-aspartate shuttle (glycolysis and glycogenolysis). Furthermore, PLP affects ATP binding as it is a purinergic receptor antagonist. Until now,
PLP has been used therapeutically as an enzyme cofactor vitamin.

The present invention includes a therapeutic agent for ischemia-related conditions and a composition thereof. In one aspect, the invention includes a method of treating ischemia reperfusion injury and cell dysfunction in a mammal, wherein the method is selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine. Administering a therapeutic amount of the compound to a mammal.

[0015] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutic amount of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine. The present invention relates to a pharmaceutical composition for treating ischemia-reperfusion injury and cytotoxicity.

[0016]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions and methods for the treatment of ischemia-related conditions such as ischemia-reperfusion injury and cell dysfunction. The present invention generally relates to the administration of the pharmaceutical composition comprising therapeutic amount at least one vitamin B ₆ from the compound.

According to the present invention, PLP can be used for the treatment of ischemia-reperfusion injury and cell dysfunction. Cell dysfunction is, for example, arrhythmia and myocardial infarction followed by heart failure. “Treatment” and “treating” as used herein include the prevention, inhibition, alleviation and treatment of ischemia-related conditions or symptoms affecting mammalian organs and tissues. For example, administration of a composition of the present invention prior to ischemia can prevent, inhibit or prevent ischemia-reperfusion injury and cell dysfunction of organs and tissues. Alternatively, by administering the composition of the present invention during or after ischemia (including during or after reperfusion), ischemic reperfusion injury and cell dysfunction of organs and tissues is reduced.
Can be reduced or treated.

Vitamin B ₆ suitable for treating ischemia-related symptoms
Other drug compounds of origin include pyridoxine, pyridoxal, and pyridoxamine. Those skilled in the art will recognize that these derivatives have almost the same effect as PLP after adjusting for metabolic and molecular weight differences.

In one aspect, the invention includes a method of treating ischemia-reperfusion injury and cell dysfunction in a mammal, the method comprising pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. Administering to a mammal a therapeutic amount of a compound selected from: Cell dysfunction may include cardiac arrhythmias or heart failure from myocardial infarction. As used herein, “therapeutic amount” includes a prophylactic amount, eg, an amount effective for preventing or preventing ischemia-related symptoms and an amount effective for reducing and treating ischemia-related symptoms.

Preferably, the therapeutic amount of the compound for the treatment of ischemia reperfusion injury and cell dysfunction is in the range of about 1 to 50 mg / kg of the patient's body weight per day,
More preferably, about 5 to 25 m / kg of patient weight
g. The compounds may be administered for short and long periods of time. Depending on the circumstances of the individual, it is preferable to administer a short-term dose of more than 25 mg / kg of the patient's body weight over a long period. For example, as described in the Examples, compounds can be administered for a short period of time, for example, 21 days.
There are no noticeable side effects when doses of up to 50 mg / kg of patient weight are administered. If long-term (months or years) administration is required for this vein, the patient's body weight should be 1 kg
A dose of 25 mg or less per dose is preferred.

The therapeutic amount of the compound for the treatment of ischemia-related conditions may be administered before, during or after ischemia (including during or after reperfusion), or before or after ischemia. It may be continuously administered after blood. For example, the compound
It may be administered prior to cardiac procedures such as bypass surgery, thrombolysis, angioplasty, and other procedures that require the blood flow to be stopped or resumed. In addition, the compound
It may be administered periodically to prevent cell dysfunction resulting from arrhythmias and heart failure.

The administration of a pharmaceutical composition containing PLP to humans will be described as an example. When a human undergoes a cardiac procedure such as, for example, bypass surgery, thrombolysis, angioplasty, or a procedure that requires cessation of blood flow, an aqueous solution containing a therapeutic amount of PLP immediately prior to surgery and during hospitalization of the patient. May be administered intravenously. Alternatively, one immediately before and after surgery
An aqueous solution containing a therapeutic amount of PLP may be administered continuously for up to a week. After hospitalization, the human may receive intestinal administration for a period of time that the physician determines is appropriate for PLP, typically for a period not exceeding, for example, 8 to 12 months.

Similarly, a human may receive PLP enterally throughout the surgical procedure from the onset of symptoms of ischemia-related symptoms. In addition, humans at risk for arrhythmia or heart failure may receive regular enteral administration of PLP to prevent cell dysfunction.

In a preferred aspect of the present invention, the method for treating ischemia-reperfusion injury and cell dysfunction in a mammal comprises a PLP for treating ischemia-reperfusion injury and cell dysfunction.
To a mammal in a therapeutic amount. In another aspect, the compound administered may be pyridoxine, pyridoxal or pyridoxamine.

In yet another aspect of the present invention, a method for preventing or treating certain cellular dysfunctions known as mammalian cardiac arrhythmias comprises a pyridoxal-5'-phosphate, pyridoxine for treating cardiac arrhythmias. Administering to the mammal a therapeutic amount of a compound selected from the group consisting of pyridoxal or pyridoxamine. In yet another aspect of the invention, the cell dysfunction treated is heart failure from myocardial infarction.

The pharmaceutical composition of the present invention relates to a composition suitable for treating ischemia-reperfusion injury and cell dysfunction. Cell dysfunction may include cardiac arrhythmias or heart failure from myocardial infarction. The pharmaceutical composition of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine. Pharmaceutically acceptable carriers include, but are not limited to, for example, saline, Ringer's solution, phosphate buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, antioxidants, coloring agents and diluents. Pharmaceutically acceptable carriers and excipients are selected so that side effects from the drug compound are minimized and the performance of the compound is not lost or impaired and therapeutic efficacy is not lost. Preferably, the compound selected is PLP.

The pharmaceutical compositions may be administered enterally and parenterally. Parenteral administration is by subcutaneous, intramuscular, intradermal, intramammary, intravenous and other methods of administration known in the art. Enteral administration includes solutions, tablets, sustained release capsules,
Includes enteric coated capsules and syrup. When administered, the pharmaceutical composition may be at or near body temperature.

[0028] Methods of preparing pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutic compound selected from the group consisting of PLP, pyridoxine, pyridoxal, and pyridoxamine are known to those skilled in the art. A method for preparing a pharmaceutical composition containing PLP will be described as an example.

Generally, the PLP solution is a mixture of PLP and a pharmaceutically acceptable carrier, such as buffered saline at an acid or alkaline pH, because PLP is essentially insoluble in water, alcohols and ethers. And) are prepared by simply mixing in a sterile condition at least at room temperature. PLP
Preferably, the solution is prepared immediately before administration to mammals. However, if the PLP solution is prepared some time before administration to mammals, the preparation should be stored in a sterilized and refrigerated state. Further, because PLP is photosensitive, the PLP solution may be stored in a container suitable for blocking light from the PLP, such as an amber vial or bottle.

Although the present invention is not limited to a particular mechanism of action or theory, examples will now be described to enhance the understanding of the invention as a whole.

[0031] According to vitamin B ₆ homocysteine theory, the relative deficiency of vitamin B _6, homocysteine accumulates. Homocysteine is an atherogenic amino acid. Homocysteine accumulation causes vascular endothelial damage, platelet dysfunction and atherosclerosis. Vitamin B ₆
Is known to act to prevent the adverse effects of homocysteine accumulation. PLP is because the auxiliary enzymes required for the catabolism of homocysteine, therapeutic potential of vitamin B ₆ has been suggested to be related to an increase in the formation of PLP. Thus, the effect of PLP in treating ischemia-related symptoms results from the catabolism of homocysteine by PLP administered according to the present invention.

As an alternative to the vitamin B ₆ homocysteine theory, there is the ATP theory. ATP becomes excessive due to ischemia. Since ATP affects the contraction of cardiomyocytes and vascular smooth muscle, extracellular ATP greatly affects cardiovascular function. For example, ATP regulates ionic flux, calcium homeostasis, and excitation-contraction coupling in atrial and ventricular myocytes. PLP has been shown to suppress ATP-induced increase in intracellular calcium. Thus, PLP acts as an endogenous antagonist of the ATP receptor (P _2x ) and may buffer the action of externally released ATP on cardiomyocytes and vascular myocytes.

According to the present invention, PLP, pyridoxine,
Appropriate administration of pyridoxal and pyridoxamine has been shown to have a very unexpected and profound effect on the treatment of mammalian ischemia-reperfusion injury and cardiac dysfunction following coronary artery occlusion. The effects of PLP administration will be described in detail with reference to the following specific examples.

[0034]

EXAMPLES Example 1 In Vitro—Ischemia Reperfusion and Left Ventricular Developing Pressure Measurements in Isolated Rat Hearts Sprague-Dawley
The male rat head (200-250 g) was dissected, the heart was quickly removed and, according to the Langendorff method, 95%
Krebs, which is oxidized with the O ₂ and 5% CO ₂ -
Perfusion was carried out at a constant flow rate of 10 ml / min using Henselite buffer (KH buffer) pH 7.4. After equilibration, KH
PLP using a Langerdorf perfusion apparatus with buffer
The effect of isotope on ischemia-reperfusion injury was investigated.

After equilibration for 15 minutes, global ischemia was induced by stopping perfusion for 30 minutes while maintaining the heart at constant humidity and a temperature of 37 ° C. In the ischemic reperfused heart, 30 minutes after total ischemia, perfusion with normal KH buffer was performed again for 60 minutes. The duration is 1.times.2 times the threshold voltage.
The heart was electrically stimulated with a 5 millisecond square wave at 300 beats / minute (Hipps and Bird stimulator). Left ventricular development pressure (LVDP), rate of change in development pressure (+ dP / dt) and change in relaxation (-dP / dt) were measured by inserting a latex balloon filled with water into the left ventricle. At the beginning of each experiment, the volume of the balloon was adjusted with a left ventricular diastolic pressure (LVEDP) of 10 mmHg and the balloon was connected to a pressure transducer (Model 1050BP-Biopack Systems). Data was recorded online via an analog-digital interface (MP100, Biopack Systems) and stored and processed in "Windows Acknowledge 3.01". In experiments examining the effect of pyridoxal-5'-phosphate (PLP), the heart was perfused with PLP (15 μM) + KH buffer for 10 minutes before ischemia occurred. Transmission of PLP (15 μM) in KH buffer was performed during reperfusion in these experiments.

The left ventricular developed pressure (LVDP) reflects the contractile activity of the heart. After ischemia, when the heart is perfused, the heart tends to become less rhythmic. The heart has time to settle to a normal rhythm.

The results of these experiments are shown in Table 1 below. The control group was 13 animals and the PLP treated grape was 6
It is an animal. All values in Table 1 are percentages from pre-ischemic values.

[0038] Global ischemia resulted in a decrease in left ventricular developed pressure (LVDP). Recovery of LVDP changes due to reperfusion of the ischemic heart was slow. These parameters are 6
0 min reperfusion showed about 40% recovery.

On the other hand, when the heart was perfused with PLP for 10 minutes before ischemia, about a 80% recovery of the LVDP reduction was seen. Also, the time to 50% recovery (time to half of maximal contractile recovery at reperfusion) was reduced in treated hearts.

[0040]

[Table 1]

Example 2 Isolation of Membrane Fraction and Quantification of Adenylyl Cyclase Activity At the end of each perfusion / reperfusion, the heart was removed from the cannula and was subjected to the procedure described by Sethi et al. J. Cardiac Failure 1 (5) (1995) and Seshi et al. (Sethi
et al.) Am. J. Raw membranes were prepared by methods previously used in Physiol., 272 (1997).
Briefly, the heart is minced and then PT-2
0 polytron (Brinkman Instruments, Westbury, New York, USA), 50 mM Tris-HCl, pH 7.5 (1 g tissue)
(15 ml per 15 ml), twice for 20 seconds each, at a setting of 5. The resulting homogenate was centrifuged at 1000 × g for 10 minutes and the pellet was discarded. Supernatant 304 for 25 minutes
Centrifuged at 8000 × g. The resulting pellet was resuspended and centrifuged twice with the same buffer at the same speed.
The final pellet was resuspended in 50 mM Tris-HCl, pH 7.4 and used for various biochemical assays.

As adenylyl cyclase is increased during ischemic perfusion, cAMP levels increase and arrhythmias and damage to the myocardium occur due to increased calcium. PLP
Treatment partially suppresses this increase in enzyme activity and regulates the level.

As described in Ceshi et al.
Adenylyl cyclase activity was quantified by measuring the formation of [α- ³² P] cAMP [α- ³² P] ATP.
Unless otherwise indicated, the incubation assay medium was
50 mM glycylglycine (pH 7.5), 0.5 mM M
gATP, [α- ³² P] ATP (1-1.5 × 106 cp
m) 5 mM MgCl ₂ (above ATP concentration), 100 mM
NaCl, 0.5 mM cATP, 0.1 mM EGTA,
0.5 mM 3-isobutyl-1-methylxanthine, 1
It contained 0 U / ml adenosine deaminase, and an ATP regeneration system containing 2 mM creatine phosphate and 0.1 mg creatine kinase per ml in a final volume of 200 μl. Incubation was started by adding membrane (30-70 ug) to the reaction mixture equilibrated at 37 ° C. for 3 minutes. Incubation time is 3
0.5 mM unlabeled cAM for 10 minutes at 7 ° C
The reaction was terminated by adding 0.6 ml of 120 mM zinc acetate containing P. 0.5 ml, 144 mM Na ₂
Other nucleotide concomitant precipitation reaction in NaCO ₃ by addition of CO _3, by chromatography next to it, to quantify the [α- ³² P] cAMP formed during the reaction. Unlabeled c
AMP is measured by measuring the adsorptivity at 259 nm.
Helped monitor the recovery of [α- ³² P] cAMP. Under the assay conditions used, adenylyl cyclase activity was linear with protein concentration and incubation time.

The membrane fraction prepared as described in Example 2 of Control Group C had a stable KH after a 20 minute stabilization period.
From hearts perfused with buffer or normal KH buffer plus PLP for 90 minutes. After a 20 minute stabilization period, the membrane fraction of the group indicated by IR (ischemic perfusion) was 3
0 minutes of ischemia, then 6 minutes with normal KH buffer.
It was from a 0 minute reperfused heart. The fractions of the group marked PR show 15 minutes after a 20 minute stabilization period.
The heart was perfused with μm PLP plus normal KH buffer for 10 minutes to cause 30 minutes of ischemia, then normal KH
From heart reperfused with buffer for 60 minutes. PLP was present during the reperfusion period.

Table 2 shows the results. n = 6 experiments.

[0046]

[Table 2]

Example 3-In Vivo-Coronary Artery Ligation As described in Cessi et al., Supra, a male Sprague Dawley rat (20
Myocardial infarction occurred at 0-250 g). 100% O ₂
Rats were anaesthetized with 1-5% isoflurane at pH 5.
An incision is made in the skin along the border of the left sternum and a fourth
The ribs were cut and a retractor was inserted. The pericardial sac was opened and the heart was outside. The coronary artery descending to the left ventricle was ligated with 6-0 silk suture about 2 mm from the coronary artery above the aorta. The heart was then returned to the chest again and the incision was closed with a purse string suture. A rat treated similarly except that the artery was ligated was used as a test rat. Surgical mortality was less than 1%. Unless otherwise indicated in the present specification, in this test, an experimental animal showing an infarct size of the left ventricle> 30% was used. All animals were allowed to recover, food and water were provided ad libitum, and electrocardiogram (ECG), hemodynamic and histological assessments were performed for 21 days.

Occlusion of the rat coronary arteries has been shown to cause cardiomyocyte damage, resulting in left ventricular wound formation and cardiac dysfunction. It was reported that the wound healed within 3 weeks after coronary occlusion, but mild, moderate and severe symptoms of congestive heart failure appeared 4, 8, and 16 weeks after ligation. Therefore, the contractile dysfunction in rats seen 3 weeks after coronary occlusion is due to acute ischemic changes.

Rats were placed in a transparent cage in a temperature and humidity controlled room, and the light-dark cycle was 12 hours. Food and water were provided ad libitum. Any rats were divided into five groups. Apparent treatment, coronary artery ligation without PLP treatment, apparent treatment with PLP treatment, PLP for 2 days before surgery
Coronary artery ligation with treatment (body weight orally by gastrometer)
25 mg / kg), coronary artery ligation with PLP treatment 1 hour after surgery (25 mg / kg body weight). These animals were used for all of the following tests. The ECG test used these animals as controls before surgery, so take an ECG before operating on these animals,
It was used as a control for the same animals after surgery.

Example 4 Hemodynamic Changes Animals prepared as described in Example 3 were treated with ketamine hydrochloride (60 mg / kg) and xylazine (10 mg / k).
The mixture of g) was injected and anesthetized. The right carotid artery is exposed and a microchip pressure transducer (Model P
R-249, Miller Instruments, Houston,
(Texas). A catheter was carefully inserted into the lumen of the carotid artery until the tip of the transducer entered the left ventricle. The catheter was secured around the artery with a ligature. Left ventricular systolic pressure (LVLSP),
Left ventricular end diastolic pressure (LVEDP), systolic rate (+ dP / d
Hemodynamic parameters such as t), relaxation rate (-dP / dt) were recorded on a computer system (Acknowledge 3.1 Harvard, Montreal, Canada).

Due to myocardial infarction for 3 weeks, left ventricular diastolic pressure (LVEDP) increased progressively and neither heart rate of left ventricular systolic pressure (LVSP) changed. Further, the contraction power factor (+ dP / dt) and the relaxation power factor (−dP
/ Dt) were significantly reduced in infarcted animals.
By treating infarcted animals with PLP for 3 weeks, L
Increase in VEDP and + dP / dt and -dP / d
The decrease in t was partially prevented.

The results are shown in Tables 3 and 4 below. Data are expressed as mean ± SE of 10 animals. All measurements were performed using a mirror microcatheter. The catheter was inserted into the left ventricle by inserting the catheter into the right carotid artery. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; + dP / dt, systolic rate; -dP / dt,
Relaxation rate. Animals were arbitrarily divided into four groups. Apparent treatment, apparent treatment plus drug treatment, coronary artery ligation group (MI) 2 days before ligation, no more than 21 days of drug treatment initiation (PrD). PLP (25m / kg body weight)
g) was given to the treatment group orally by gastric instrument once a day.

[0053]

[Table 3]

[0054]

[Table 4]

There were three groups of rats. 20 each. (MI) Untreated coronary ligation, (PP1) PLP oral treatment once daily, starting 1 hour after ligation, (P1
P2) PLP oral treatment once daily, starting 2 days before ligation.

Example 5 Electrocardiogram (ECG) Recording All groups (apparent treatment, coronary artery ligation (M
I), apparent treatment with drug treatment, coronary artery ligation with drug treatment 2 days before ligation, coronary artery ligation with drug treatment within 1 hour after ligation) rats before coronary artery ligation and 1, 3 after occlusion , 7, 14, and 21, 6 reads (I, II, III, aVr, aVf, aVI)
ECG recording was performed. Surface ECG was recorded under isoflurane anesthesia using a model EC-60 heart and respiratory monitor (Cilogic International Limited, UK). An ST segment abnormality was defined as a decrease or increase of at least 1 mm from the baseline, which lasted for 1 minute or more.
ST at 60 ms after J point in all six leads
The magnitude of the segment shift was measured. The QT interval is measured on a standard basis, then the Bazette equation (QT _c = Q
The square root of the T / RR interval) was used to correct for heart rate. For all leads, the QT interval from the start of the longest Q wave to the end of the T wave was measured. If no Q wave exists, the starting point of the R wave was used. The RR interval immediately before the QT interval measurement was used for correction in heart rate. Prior to the R-wave, a negative deflection with an amplitude of at least 25 uV
Waves.

ST segment changes ST segments that reflect subendocardial hypoperfusion are most often used in the ECG to represent ischemia, and ST segment deviation is used as a clear marker of cardiac perfusion status. be able to. Electrodes located above the lesion record the rise of the ST segment, while electrodes in the opposite region of the torso detect a corresponding fall in the ST segment.
In this study, from day 1 to day 21 after coronary artery ligation in untreated rats, a decrease in ST segment was recorded in lead I and an increase in ST segment in leads II and III. PLP treatment attenuated the extent of ST segment elevation / decline after occlusion and promoted ST segment recovery. The results are shown in Tables 5 and 6 below. In Table 5, MI
And in all three leads of the PrD group,
The ST segment deviation value recorded before occlusion (control) was
0.01, 0.02, 0.01 and 0.0 respectively
15, 0.02 and 0.01. In Table 6, M
In all three reads of the I and PrD groups, the ST segment deviation values recorded before occlusion (control) were 0.02, 0.02, 0.01 and 0.01, 0.009, 0. 015.

[0058]

[Table 5]

[0059]

[Table 6]

[QT interval and mortality due to myocardial infarction]
The QT interval on a surface electrocardiogram is an indirect measure of the potential duration of ventricular action, and its prolongation is often associated with the occurrence of a patient's malignant ventricular arrhythmia.
Long QT intervals in the ECG have been associated with an increased risk of sudden cardiac death following myocardial infarction. Data is QT
Prolonged intervals occurred on day 1 and then showed that post-ligation mortality of untreated rats gradually decreased on days 3 to 21, the highest period. PLP treatment attenuated QT prolongation and hastened the recovery time of the QT interval after coronary occlusion. The results are shown in Tables 7, 8 and 9 below.

Myocardial infarction was caused by coronary ligation. All animals remaining in the following weeks were used for ECG evaluation. Values are means ± SE. Treated animals received PLP (25 mg / kg) orally once or twice daily. The value before the occurrence of myocardial infarction was set as a control value.

[0062]

[Table 7] There were two groups of rats. 20 each.
(MI) Untreated coronary ligation, (PrD) PL once daily
P oral treatment.

[0063]

[Table 8] There were three groups of rats. 20 each.
(MI) Untreated coronary ligature, (PP1) PL once daily
P oral treatment, (PP2) PLP oral treatment twice daily, n
= 20, values are means ± SE. The value before the occurrence of myocardial infarction was set as a control value.

[0064]

[Table 9] There were three groups of rats. 20 each.
(MI) Untreated coronary ligature, (PP1) PL once daily
P oral treatment, (PP2) PLP oral treatment twice daily, n
= 20, values are means ± SE. The value before the occurrence of myocardial infarction was set as a control value. Therefore, mortality was also significantly lower in the PLP treated group.

Mortality The earliest deaths after a myocardial infarction occur within a few hours and are primarily due to fatal ventricular arrhythmias. The mortality was highest in this study 48 hours after coronary ligation for both treatment and non-treatment. However, mortality was significantly lower in the treated animals. With such a decrease in mortality, ECG results improved, suggesting an anti-arrhythmic effect of PLP (reducing the incidence of pathological Q-waves and PVCS).

The test rats were arbitrarily divided into four groups. 20 each. Apparent treatment, apparent treatment plus drug treatment, drug treatment onset 2 days before ligation (MI + drug) not exceeding 21 days, and arterial ligation group (MI). The apparent treatment group and the apparent treatment plus drug treatment group were considered as one group because there was no difference in mortality and other hemodynamic changes. The results are shown in Tables 10, 11, 12, and 13 below.

[0067]

[Table 10] ## At day # 21, three animals in the MI group became severe and were unlikely to survive for another week.

[0068]

[Table 11] (MI) Untreated coronary ligature, (PP1) PL once daily
P oral treatment, (PP2) PLP oral treatment twice daily. In a second similar test, the test rats were arbitrarily divided into four groups. 20 each. Apparent treatment, apparent treatment plus drug treatment, one hour after ligation, drug treatment started (Pr
D) Period not exceeding 7 days, and arterial ligation group (M
I). The apparent treatment group and the apparent treatment plus drug treatment group were considered as one group because there was no difference in mortality and other hemodynamic changes. The results are shown in Table 12 below.

[0069]

[Table 12]

[0070]

[Table 13] There were three groups of rats. (MI) 20 animals. Untreated coronary ligation, (PP1) 25 animals. PLP once a day
Oral treatment, (PP2) 25 animals. PLP oral treatment twice daily.

[Arrhythmia Prevention Effect of PLP Expressed in ECG] Some of the results of ECG of animals in the above-mentioned mortality test showed the arrhythmia prevention effect of PLP. In one of them, the occurrence of pathological Q waves was reduced. Tables 14 and 15 show these results.

[0072]

[Table 14] There were three groups of rats. 20 each.
(MI) Untreated coronary ligature, (PP1) PL once daily
P oral treatment, (PP2) PLP oral treatment twice daily, n
= 20, values are mean ± SE. The apparently treated group received saline.

[0073]

[Table 15] There were three groups of rats. 20 each.
(MI) Untreated coronary ligature, (PP1) PL once daily
P injection, (PP2) PLP injection twice daily, n = 20,
Values are means ± SE. The apparently treated group received saline.

In another such result, the occurrence of anterior ventricular contraction (PVC) after coronary artery ligation was reduced. Tables 16 and 17 show these results.

[0075]

[Table 16]

[0076]

[Table 17]

Those of skill in the art will understand the description of these preferred details, and will understand that the compounds and methods may be substantially modified, without affecting or altering the function of the described embodiments. It will be appreciated that modifications, changes and substitutions are possible. While embodiments of the present invention have been described above, various modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the spirit, nature and scope of the claimed and described invention. is there.

[0078]

As described above, according to the present invention,
A therapeutic agent for ischemia-reperfusion injury in various organs and a therapeutic agent for cell dysfunction including heart failure due to arrhythmia and myocardial infarction can be provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C07D 213/67 C07D 213/67 (72) Inventor Setti, Raja Canada, Manitoba, Winnipeg, Sorb Crescent 68 (72) Inventor Duck Sinamuti, Krishnamouti Canada, Manitoba, Winnipeg, Crestview Park Drive 934

Claims (10)

[Claims] 1. A therapeutic agent for mammalian ischemia-reperfusion injury and cell dysfunction, comprising a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. 2. The therapeutic agent according to claim 1, wherein the amount of the therapeutic agent ranges from about 1 to 50 mg / kg of the patient's body weight. 3. The method of claim 1, wherein the amount of the therapeutic agent ranges from about 5 to 25 mg / kg of the patient's body weight. 4. The therapeutic agent according to claim 1, wherein the compound is a therapeutic agent for enteral or parenteral administration. 5. The method according to claim 5, wherein the compound is pyridoxal-5′-.
The therapeutic agent according to claim 1, which is phosphoric acid. 6. A therapeutic agent for treating ischemia-reperfusion injury in mammals, comprising a compound selected from the group consisting of pyridoxal-5′-phosphate, pyridoxine, pyridoxal and pyridoxamine. 7. A therapeutic agent for a mammalian cell dysfunction, comprising a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. 8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutic amount of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine. 9. The method according to claim 9, wherein the compound is pyridoxal-5′-.
The pharmaceutical composition according to claim 8, which is phosphoric acid. 10. The pharmaceutical composition according to claim 8, wherein said pharmaceutical composition is in a form suitable for enteral or parenteral administration.