JP2000136137A - Ischemic reperfusative encephalopathia reducer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an ischemic reperfusative encephalopathia reducer reducing encephalopathia caused from acute cerebral blood reduction such as cerebral hemorrhage. SOLUTION: This ischemic reperfusative encephalopathia reducer is suppressible nerve cell death generated with acute ischemic reperfusative encephalopathia by previously administrating phosphatidyl-L-serine or its salt. A transformed-type phosphatidyl-L-serine in which a constitutive aliphatic acid of the phosphatidyl-L- serine is derived from soybean lecithin is effective.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition having an effect of reducing cerebral nerve cell death due to sudden ischemia, and more particularly, to a method for reducing cerebral damage caused by an acute decrease in cerebral blood flow such as a stroke. And a drug for reducing ischemia-reperfusion encephalopathy.

[0002]

A. Bruni et al. Reported that injection of phosphatidylserine extracted from bovine brain into the tail vein of mice increased brain glucose concentration by about four times compared to the control group. (Nature, vol. 260, p. 331, 1976). In addition, it has been reported that oral administration of phosphatidylserine extracted from the bovine brain for 12 weeks improves the behavior of aged rats showing a decrease in memory.
(A. Zanotti et al., Psychopharmacology Berl., Vol. 99,
p316,1989).

Further, in clinical studies in humans, it has been reported that bovine brain phosphatidylserine is effective for Alzheimer's disease and senile memory impairment in a double-blind-placebo study (PJ Delwaide et al., Acta Neurol). .Scand., Vol.7
3, p.136,1986; RREngel et al., Eur. Neuropsychophar
macol., vol. 2, p. 123, 1993).

On the other hand, Japanese Patent Application Laid-Open No. Hei 6-279313 discloses that phosphatidylserine having a specific fatty acid composition obtained by a synthetic method is used for treating senile dementia based on the activating action of protein kinase C-isozyme. Although promising, its efficacy is not clear in vivo.

[0005] In addition, German Published Patent Application DE 4117629.
And EP 0396080 also disclose that phosphatidylserine obtained by a synthesis method has an effect of increasing brain glucose and the like.

[0006] In addition, Applicants have disclosed that phosphatidylserine from the brain of animals other than cattle ameliorates scopolamine-induced memory impairment in rodents.
No. 754 discloses that soybean lecithin, rapeseed lecithin, and yolk lecithin-derived transferable phosphatidylserine have a cerebral glucose-increasing effect and a scopolamine-induced memory impairment effect in rodents.
No. 133984.

[0007] As described above, it has been reported that the administration of phosphatidylserine is effective for Alzheimer's disease, senile memory impairment, and the like. However, the effect on cerebral nerve cell death due to an acute decrease in cerebral blood flow such as a stroke. Has not been reported yet.

[0008]

Problems such as cerebral ischemia, cerebral hemorrhage and subarachnoid hemorrhage occupy most of the acute brain damage, and once the brain is damaged, treatment is difficult and fundamental. There is no cure. For this reason, these acute brain disorders are reduced
Drugs that can be prevented are expected.

As a result of intensive studies, the present inventors have found that pre-administration of phosphatidyl-L-serine can reduce or prevent ischemia-reperfusion encephalopathy caused by ischemia, leading to the present invention.

[0010] That is, the present invention is an agent for reducing ischemic reperfusion encephalopathy which reduces cerebral nerve cell death due to sudden ischemia, and more specifically, cerebral injury due to acute cerebral blood flow decrease such as stroke. The aim is to obtain an agent for reducing ischemia-reperfusion encephalopathy. Another object of the present invention is to provide an agent for reducing ischemia-reperfusion encephalopathy, which is not greatly restricted in terms of cost and supply, as the agent for reducing brain injury.

[0011]

The agent for reducing ischemia-reperfusion encephalopathy according to the first aspect of the present invention comprises phosphatidyl-L-serine or a salt thereof as an active ingredient.

[0012] The ischemia-reperfusion performance disorder reducing agent according to the second aspect of the present invention is effective for transfer type phosphatidyl-L-serine or a salt thereof in which the constituent fatty acid of phosphatidyl-L-serine is derived from soybean lecithin. Component.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the agent for reducing ischemia-reperfusion encephalopathy of the present invention, phosphatidyl-L-serine or a salt thereof is administered to a subject in advance to cause sudden ischemia-reperfusion encephalopathy. Nerve cell death can be suppressed. Therefore, cerebral damage due to ischemia of the administered subject can be reduced or prevented.

The salt of phosphatidyl-L-serine may be used in the form of a pharmaceutically acceptable salt. Specifically, there are a sodium salt, a potassium salt, a magnesium salt, an ammonium salt and the like, and a sodium salt and a potassium salt are preferred.

The administration of phosphatidyl-L-serine or a salt thereof was carried out orally in the examples described below.
Intravenous administration is also effective. Further, other excipients such as lipids, sugars and proteins may be mixed and processed into capsules or granules having improved ease of handling and storage. Furthermore, since there is no problem in terms of safety, it can be incorporated into foods and drinks that are taken daily and can be sufficiently taken, which is effective in reducing cerebral dysfunction due to sudden ischemia-reperfusion encephalopathy. It is.

Furthermore, the relationship between the fatty acid composition of the agent for reducing ischemia-reperfusion encephalopathy according to the present invention and the effect of reducing ischemia-reperfusion encephalopathy is not always clear, but at least the effect is demonstrated in Examples described later. The confirmed transfer type phosphatidyl-L-serine derived from soybean lecithin was confirmed to be effective.

The phosphatidyl-L-serine or a salt thereof according to the present invention is a transfer-type phosphatidyl-L-serine obtained by a transfer reaction using lecithin as a raw material, and is usually obtained from various lecithins such as soybean lecithin as a raw material. Phosphatidyl-L-serine can be produced by a purification operation using an organic solvent according to the method.

The soybean lecithin and the like used as raw materials are all phosphatidyl-isolated and purified from animal brain.
It can be provided in a much larger amount and at a lower cost than L-serine, and there are few quantitative problems in supply. Furthermore, there is no risk of infection such as Creutzfeldt-Jakob disease and mad cow disease.

It is desirable that transfer-type phosphatidyl-L-serine derived from soybean lecithin and the like is subjected to an appropriate purification treatment step to remove impurities, but there are problems with administration and problems that hinder the effect. As long as it is not used, it may be used while containing impurities from the raw materials and the production process.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The hippocampus is one of the brain tissues that plays an important role in learning and memory, and is susceptible to damage by ischemia and oxygen deprivation, resulting in memory impairment in humans, And various reports in experimental animals.
Recently, a cerebral ischemia animal model has been widely used as a screening for anti-amnesic drugs for cerebrovascular dementia because it can produce ischemic foci in the forebrain with good reproducibility. In this,
Pulsinelli and Brierley reported a transient cerebral ischemia model in rats with bilateral vertebral and common carotid artery occlusion (Kirino Takaaki; hippocampus and delayed neuronal cell death: Clin. Neurosci., 12,6)
1-65 (1994)). Furthermore, in order to save the labor of cauterizing the vertebral carotid artery, a model of cerebral ischemia in a rat with dysplasia in the ring of the Willis artery (Kirino, T., Tamura, A. and Sano, K .; Selecti
ve vulnerability of the hippocampus to ischemia-re
versible and irreversible types of ischemic cell d
amage .: Brain Research. 63, 39-58 (1985)).

As part of the evaluation of the efficacy of soybean-transferred phosphatidylserine (hereinafter referred to as “SB · tPS”) in animals, cerebral ischemia was performed using sand rats, which are widely used as an ischemic animal model. The effects on associated hippocampal pyramidal cell damage were examined. That is, using a sand rat cerebral ischemia model, and using as an index the delayed neuronal cell death in the hippocampal CA1 region occurring after 4 or 5 days after ischemia,
The effects of SB / tPS oral administration daily were investigated.

1. Preparation The used animals were 14-week-old male sand rats purchased from Japan SLC Co., Ltd., room temperature 24 ± 2 ° C., humidity 55 ± 1.
The animals were preliminarily reared for one week under the conditions of 0%, 12-hour light / dark cycle (8:30 to 20:30 light period). Sand rats were kept 4 to 5 animals in a stainless steel cage, and were allowed to freely take feed for feeding MF (manufactured by Oriental Yeast) and water.

The sample used (soy-transferred phosphatidylserine (SB.tPS)) was prepared as follows. That is, phospholipase D is allowed to act on a mixture of soybean lecithin and L-serine, and the solution after the reaction is subjected to solvent fractionation, whereby phosphatidyl-L having a purity of 95% is obtained.
-Serine was obtained. In addition, at the time of administration, a substance having a purity of 95% was suspended in distilled water for injection and then subjected to ultrasonic treatment before use.

The experimental procedure is as follows. First, the following operation was performed to cause cerebral ischemia in an animal model. Under 2% halothane anesthesia, sand rats were fixed in a dorsal position.
The bilateral carotid arteries were dissected from surrounding tissue, and a thread was hooked on each. Then, under 1% halothane anesthesia, Sugita-type clips were attached to both carotid arteries while gently lifting the thread. After stopping the bleeding for 5 minutes or 15 minutes on a 37 ° C. heat retaining mat, the clip was removed and the blood flow was resumed. After suturing the wound site, the animals were returned to their home cage.

2. Oral administration of SB / tPS Sand rats (33 animals) were divided into two groups, which were a PS administration group and a distilled water administration group (control group). In the administration group, SB · tPS (240 mg / kg) was orally administered once a day for 5 days before cerebral ischemia treatment. Then, the rat was subjected to cerebral ischemia-reperfusion according to the method described above. From the next day, behavioral observations, changes in body weight, and confirmation of life and death were performed, and on the 10th day, surviving animals were sacrificed urgently. In the control group, cerebral ischemia-reperfusion treatment was performed in the same manner, and the surviving animals were sacrificed on the 10th day.

3. Histopathological examination After the observation period, sand rats were opened under ether anesthesia,
After thoracotomy, 2% paraformaldehyde and 2.
Perfusion fixation was carried out with a 5% glutaraldehyde mixed fixative solution. After excision of the brain, it was fixed with a 10% buffered formalin solution and embedded in paraffin to prepare a hematoxylin-eosin stained specimen.

Observing the obtained sample under an optical microscope,
The degree of pyramidal cell damage in the hippocampal CA1 region was evaluated by scoring the following five levels. Score 0: No obstacle at all. Score 1: slight cell necrosis. Score 2: Small area necrosis of cells. Score 3: Extensive cell necrosis. Score 4: Loss of all cells.

4. Statistical processing The obtained data was subjected to one-way analysis of variance (ANOVA), and comparison of the effects between the PS administration group and the control group was performed by Dunn.
Statistical processing was performed by the ett type multiple comparison test, and the
% Or less was determined to have a significant difference.

5. Setting of ischemic time Under halothane anesthesia, the degree of damage to pyramidal cells in the hippocampal CA1 region due to ischemia was compared between 5 minutes and 15 minutes. As a result, in 5 of the 20 minutes of ischemia, histological images of incomplete ischemia such as only one-sided injury or mild injury with a score of about 1 or 2 were observed in 8 of 20 cases. On the other hand, in 15 minutes ischemia, histological images that seem to be incomplete ischemia were observed in 4 of 22 cases, and in the remaining 18 cases, almost complete loss of pyramidal cells was observed. Admitted. From this, 15 minutes of ischemia was performed to verify the effect of SB · tPS on cone cell damage.

6. Effect of SB / tPS administration on hippocampal pyramidal cell damage due to cerebral ischemia In the sham operation group, no damage was observed. Among the groups to which only distilled water was administered (16 cases), during the observation period of 10 days after ischemia, death cases (4 cases) and incomplete ischemia cases (2 cases)
The average of the cytotoxicity scores of 10 cases excluding ex.) Was 3.9.
0 ± 0.32 (SD). In contrast, SB
-Among the tPS administration groups (17 cases), the cytotoxicity score of 15 cases excluding death cases (1 case) and incomplete ischemia cases (1 case) during the observation period 10 days after ischemia Is 3.2 on average
7 ± 0.70 (SD), indicating that the cell damage was significantly reduced as compared with the distilled water administration group (Table 1). In addition, there was no difference between the two groups in general behavior and body weight change for 10 days after resumption of blood flow.

[0031]

[Table 1]

7. Discussion As indicated above, SB · tPS (240 mg
/ Kg / day) for 5 days.
It has been shown to reduce the degree of cone cell damage. This mechanism cannot be explained at this time due to the lack of basic data on animals. However, in experiments using mice, it has been reported that the administration of SB / tPS increases glucose in the brain, and one of the mechanisms is the impaired production of high-energy phosphate compounds such as ATP caused by ischemia. May have a protective effect. However, details are unknown.

On the other hand, after transient cerebral ischemia, an early phenomenon caused by reperfusion of blood flow includes an increase in extracellular glutamate and binding to its receptor → an increase in intracellular calcium ion → lipid peroxidation and monoxide. Increasing nitrogen (NO) → generating radicals. Therefore, the inhibitory effect of SB / tPS on delayed neuronal death will be examined in the future.
It is necessary to consider the two phenomena in detail.

As described above, the effect of oral administration of SB.tPS on hippocampal pyramidal cell damage was examined using the murine cerebral ischemia model. As a result, SB.tPS (2
(40 mg / kg) every day to give hippocampal CA
It has been shown to reduce the degree of damage to one pyramidal cell. This suggests that SB.tPS may be effective in improving learning and memory disorders associated with cerebrovascular disorders.

[0035]

As described above, the present invention relates to an agent for reducing ischemia-reperfusion encephalopathy which reduces cerebral nerve cell death due to sudden ischemia, and more particularly, to a brain caused by acute cerebral blood flow decrease such as stroke. An agent for reducing ischemia / reperfusion encephalopathy which reduces injury can be obtained. The present invention has an effect that an agent for reducing ischemia-reperfusion encephalopathy, which is not greatly limited in terms of cost and supply, can be obtained as the agent for reducing brain injury.

Continued on the front page (72) Inventor Masahisa Takahashi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Masashi Sakai 1-1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo Stock Company Yakult Honsha (72) Inventor Satoshi Kudo 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo F-term (reference) 4C086 AA01 DA41 MA04 NA14 ZA36 4H050 AB21

Claims (2)

[Claims] 1. An agent for reducing ischemia-reperfusion encephalopathy comprising phosphatidyl-L-serine or a salt thereof as an active ingredient. 2. A transferable phosphatidyl-L in which the constituent fatty acid of phosphatidyl-L-serine is derived from soybean lecithin.
-An agent for reducing ischemia-reperfusion encephalopathy comprising serine or a salt thereof as an active ingredient.