JPH0859473A - Agent for protecting cranial nerve cell - Google Patents

Abstract

PURPOSE: To provide a cranial nerve cell protecting agent containing a specific compound as an active component, effective for remarkably suppressing the death of nerve cell induced by glutamic acid, having cranial nerve cell protecting action and useful for the prevention and treatment of cerebral thrombosis, cerebral infarction, dementia, etc. CONSTITUTION: This protecting agent contains a compound of formula I [R1 to R5 are each H or OH; R6 is an (OH-substituted)alkyl], e.g. 20-hydroxyecdyson of formula II as an active component. The compound of formula I, especially a natural ecdysteroid can be produced, e.g. by extracting a plant such as paffia or anthotaxis with water or a hydrophilic solvent, adjusting the extracted liquid to alkaline state, treating with a reversed phase adsorbent, washing the reversed phase adsorbent with an alkaline washing liquid to remove impurities, eluting the component adsorbed to the reversed phase adsorbent and crystallizing from the eluate. The daily administration dose of the compound of formula I for adult is preferably l mg to 1 g for oral administration and 0.1mg to 1g for injection.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cerebral nerve cell protective agent. More specifically, the present invention relates to a cerebral nerve cell protective agent which is effective and highly safe for the prevention or treatment of ischemic brain injury and the like.

[0002]

Prior art and problems to be solved by the invention Cerebral thrombosis, cerebral embolism, cerebral vasospasm, rapid blood pressure decrease, cerebral infarction due to hypoperfusion vasculopathy, cerebral thrombosis, cerebral embolism, cerebral hemorrhage, subarachnoid hemorrhage, Stroke due to transient cerebral ischemic attack, hypertensive encephalopathy, etc., as well as brain damage and cardiac arrest, hypoxia,
Hypoglycemia causes ischemic changes in cerebral nerve cells, which in turn degenerate and necrotize cerebral nerve cells. In addition, secondary degeneration and necrosis of cerebral nerve cells gradually occurring after primary infarction such as cerebral infarction cause dementia, psychiatric disorder, and behavioral disorder as aftereffects of cerebrovascular disorder.

As methods for suppressing such cerebral nerve cell damage, anticoagulants (edetic acid, sodium citrate, heparin, coumarin derivatives, indandione derivatives) and thrombolytic agents against cerebral thrombosis and embolism have hitherto been used. (Urokinase, streptokinase), fibrinolytic inhibitors (plasminogen activator inhibitors such as tranexamic acid) for cerebral hemorrhage and subarachnoid hemorrhage, and glucose-permeable diuretic (electrolyte) for cerebral edema. , Urea, mannitol), etc.
In addition, brain function improving agents (vinca alkaloids, ergot alkaloids, xanthine derivatives) have been mainly used as symptomatic therapy for the purpose of improving circulation and metabolism at the lesion site. On the other hand, focusing on the mechanism of degeneration and necrosis of brain neurons, various drugs such as glutamate receptor antagonists, calcium influx inhibitors, and free radical scavengers are being actively developed.

However, there are still many unknown parts in the mechanism, and in spite of many search efforts, it is the current situation that a satisfactory drug having an effective and high safety cannot be found. Therefore, an object of the present invention is to search for a compound that inhibits glutamic acid-induced cerebral nerve cell death, and to create a cerebral nerve cell protective agent effective in the prevention or treatment of ischemic encephalopathy and the like.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that compounds represented by the following formula (I), particularly natural polyhydroxy steroids generally called ecdysteroids The inventors have found that these derivatives significantly suppress glutamate-induced neuronal cell death and have a cerebral neuronal cell protective action, and completed the present invention. That is, the present invention provides a cerebral nerve cell protecting agent, which comprises a compound represented by the following formula (I) as an active ingredient.

[0006]

Embedded image

(In the formula, R _{1 to} R ₅ each independently represent a hydrogen atom or a hydroxyl group, and R ₆ represents an alkyl group which may be substituted with a hydroxyl group.) In the above general formula (I) of the present invention, In the represented compounds,
R ₆ has 2 to 8 carbon atoms which may be substituted with a hydroxyl group.
Alkyl groups of 10 are preferred, moreover, R ⁶ is of the formula —C (CH ₃ ) XC
A group represented by HXCHXCHXC (CH ₃ ) (CH ₂ X) X (in the formula, X represents a hydrogen atom or a hydroxyl group, and 6 Xs may be the same or different) is preferable.

Of the compounds represented by the formula (I) of the present invention,
Examples of naturally-occurring compounds in which R ₆ has 8 carbon atoms include abutasterone, ajugasterone C, 22-deoxy-20,26-dihydroxyecdysone, 22,25-dideoxyecdysone, 2-deoxyecdysone and 14-deoxyecdysone. , 22-deoxyecdysone, 25-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, 14-deoxy-20-hydroxyecdysone, 22-deoxy-26-hydroxyecdysone, 22-deoxyinocosterone, deoxybiperidone, 2 , 22-deoxyecdysone, 2, 22-dideoxy-20-hydroxyecdysone, 20, 26-dihydroxyecdysone, ecdysone,
Geraldiasterone, 5α-20-hydroxyecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 26-hydroxy-polypodin B, inocosterone, muristerone A, 5α-polypodin B, polypodin B, ponasterone A, ponasterone C, pterosterone, Taxisterone, 2, 14, 22, 25-tetradeoxyecdysone, 2, 22, 25-trideoxyecdysone, turquesterone, ajugasterone D and the like can be mentioned.

Further, among the compounds represented by the formula (I), R ₆
A compound having 9 or 10 carbon atoms and obtained from nature include, for example, ajugalactone, ajugasterone B, amalasterone A, amalasterone B, capitasterone,
Calsamosterone, Siasterone, Dacristerone, 24
(28) -dehydromaxisterone A, 24,25-dehydropreciasterone, 20-deoxymaxasterone A, 2-deoxymaxasterone A, episiasterone, 24-epi-
Makisterone A, 23-hydroxy-siasterone, isocyanasterone, maxisterone A, maxisterone B, maxisterone C, maxisterone D, 29-norciasterone,
29-norsengosterone, 22-oxo-siasterone,
Paxylosterone, presiasterone, lapisterone,
Examples include sengosterone, cidasterone A, cidasterone B and the like. In addition, examples of the compound obtained from nature with R ₆ having 2 carbon atoms include dihydropostosterone, poststerone and the like.

And these natural ecdysteroids are
From plants and animals containing them, for example, JP-A-4-279528
It can be obtained by extraction and purification by a known production method as described in No. That is, plants containing ecdysteroids, such as Paphia, Inokozuchi, Kiranso, Inumasaki, and Vitex are extracted with water, a hydrophilic solvent or a mixed solvent thereof, the extract is adjusted to be alkaline, and the alkaline extract is Treated with a reversed-phase adsorbent, washed the reversed-phase adsorbent with an alkaline cleaning solution to remove impurities, then elute the components adsorbed on the reversed-phase adsorbent with a solvent and crystallize ecdysteroid from the eluate. The purified ecdysteroid can be obtained by precipitation.

Among the compounds represented by the formula (I) of the present invention, the chemically synthesized compounds include, for example, 2β,
3β, 14, 20, 22, 25-hexahydroxy-27-propyl-5β-cholest-7-en-6-one, 2β, 3
β, 14, 20, 22, 25-hexahydroxy-27- (3-hydroxy-propyl) -5β-cholest-7-ene-6
-On (Tetrahedron, 47 (2
3), 3753-3772 (1991)], 5α-2-deoxy-20-hydroxyecdysone, 24-epi-2-deoxymaxisterone A, 5, 24-epi-2-deoxymaxisterone A, 5 -Epi-2-deoxymaxterone A [The above is the method described in Journal of Organic Synthetic Chemistry, 48 (1), 43-55 (1990) and its references], 22-deoxy-5α-ecdysone, 22-deoxy- 20-hydroxyecdysone, 14-deoxymuristerone A, 22-deoxy-24-nor-5α
-Ecdysone, 2,25-dideoxyecdysone, 22,25
-Dideoxy-5α-ecdysone, 22,25-dideoxy-5β-hydroxyecdysone, 22,25-dideoxy-
20-hydroxyecdysone, 22,25-dideoxy-24-
Methylecdysone, 22,25-dideoxy-27-nor-ecdysone, 2,22-dideoxy-24-nor-ecdysone, 22,25-dideoxy-5α-hydroxyecdysone, 5α-ecdysone, 2, 14, 22, 25- Tetradeoxy-5α-ecdysone, 2, 14, 22, 25-tetradeoxy-5α-hydroxyecdysone, 2, 22, 25-trideoxy-5α-ecdysone, 14, 20, 25-trideoxy-5α-hydroxyecdysone, 2, 22, 25-Trideoxy-5α-hydroxyecdysone, 2,22,25-Trideoxy-5β-hydroxyecdysone [The above are Comprehensive Insect Physiology Biochemistry and Pharmacology (Comprehensive In
sect Physiology Biochemistry and Pharmacology), Vo
l.7, p185, Pergamon Press (19
86) and its references, etc.] and the like. Then, these synthetic ecdysteroids can be obtained by, for example, the known production method described in the above [].

On the other hand, the protective effect on the cerebral nerve cells described in the present invention means cerebral thrombosis, cerebral embolism, cerebral vasospasm, rapid decrease in blood pressure,
Cerebral infarction due to hypoperfusion vasculopathy, cerebral thrombosis, cerebral embolism,
Stroke due to cerebral hemorrhage, subarachnoid hemorrhage, transient cerebral ischemic attack, hypertensive encephalopathy, etc.
It means suppressing degeneration or necrosis of cerebral nerve cells associated with hypoxia, hypoglycemia and the like. This also means suppressing secondary degeneration and necrosis of cerebral nerve cells that gradually occur after primary infarction such as cerebral infarction and cause dementia, mental disorders, behavioral disorders, and the like.

The more specific brain neuronal cell protective action is based on the following working hypothesis. The mechanism of cerebral nerve cell damage due to ischemic injury, etc., in particular, the delayed nerve cell death of the cerebral cortex system has been studied as a mechanism of glutamate neurotoxicity, and the "glutamate / calcium / nitric oxide" hypothesis has been proposed. This is because nitric oxide synthase existing in neurons is activated by the calcium ion influxed by stimulation of NMDA (N-methyl di aspartate) receptor, which is a subtype of glutamate receptor, to generate nitric oxide. It is the theory that the generated nitric oxide reacts with active oxygen to generate radicals, which causes damage to the cell membrane and ultimately leads to cell death.

Therefore, the novel agent for protecting cerebral nerve cells of the present invention could be found by searching for a compound having an inhibitory effect on glutamate neurotoxicity and clarifying its mechanism of action. More specifically, the active ingredient of the present invention could be found by conducting the following experiment, for example.

Cerebral cortex cells isolated and isolated from rat fetuses are cultured to remove non-neuronal cells such as glial cells expressed in the culture. This is glutamic acid or NM
The cells are treated with excitatory amino acids such as DA, and then incubated in Eagle's solution containing no excitatory amino acids for 1 or 24 hours, and the viability of cells is determined by the trypan blue staining method. The survival rate of the cultured cells is calculated by counting at least 200 cells in one sample and determining the ratio of the number of viable cells to the total number of counted cells.
In such experiments, cell viability was 40% of the untreated group.
Since it decreases to the following level, it is possible to evaluate the protective effect of the test substance against nerve cell death by administering the test substance to the same experimental system. The protection effect is calculated as the protection rate according to the following formula. Protection rate (%) = [(D−C) / (A−C)] × 100 (where, A is the survival rate of the untreated group (control group), C is the survival of the excitatory amino acid administration group) Rate, D
Means the survival rate of the cell group to which the drug and excitatory amino acid were administered. ) In the case of chronic administration, the drug is administered until just before the excitatory amino acid is administered, and the drug is not added to the incubation solution during and after the excitatory amino acid treatment, while in the case of acute administration, it is added to the normal solution. Using the cultured cells maintained as above, the drug is added to the excitatory amino acid-containing solution and the subsequent incubating solution.

In this experimental system, glutamic acid was administered,
Then, by culturing in a glutamic acid-free liquid containing the compound represented by the formula (I) of the present invention, a dose-dependent cerebral nerve cell protective action is observed. On the other hand, many of the mammalian steroid hormones (for example, adrenocortical hormones such as corticosterone and aldosterone) that are not included in the compound represented by the formula (I) do not show a brain neuronal cell protective action in this experimental system, and The invention is the first example of a group of steroid compounds having a protective effect on brain neurons.

Further, many of the compounds (MK-801, etc.) which have been known to have a neuroprotective effect on the brain are NMD.
While the compound of formula (I) of the present invention acts on the A receptor to suppress glutamate cytotoxicity, it does not act directly on the receptor and suppresses the process after receptor stimulation. Therefore, it is considered that the mechanism of action is different from the conventional one.

When the compound represented by the formula (I) of the present invention is used for the prevention or treatment of a disease requiring protection of cerebral nerve cells, it may be used as a drug substance, but usually as a pharmaceutical preparation not particularly limited, It is administered orally or parenterally. For example, as an oral agent, tablets, capsules, troches,
It can be used as solid preparations such as granules and powders, or as liquid preparations such as water preparations and syrup preparations. These various preparations can be produced by a known method using a conventional inorganic or organic or solid or liquid pharmaceutical preparation carrier.

The dose of the compound represented by the formula (I) of the present invention varies depending on the kind and symptom of the target disease, but in the case of general adults, it is 1 mg to 1 g per day for oral administration.
It is preferably 10 to 500 mg, and 0.1 to 1 g, preferably 1 to 500 mg per day for injection administration.

[0020]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited to these examples.

Example 1 Protective action of 20-hydroxyecdysone represented by the following formula (II) on cerebral nerve cells

[0022]

[Chemical 3]

Cerebral cortex cells were extracted and isolated from a rat fetus with embryonic day 18 to 20 and cultured in Eagle medium for 10 to 14 days. However, cytosine and arabinoside 10 days after 6 to 7 days of culture
Non-neuronal cells such as glial cells expressed in culture were removed by adding μM. Life and death of cells after excitatory amino acid treatment and drug treatment
It was judged by the blue staining method. The viability of cultured cells was calculated by counting at least 200 cells or more in one sample and determining the ratio of the number of viable cells to the total number of counted cells. Using the data counted from one cover slip as one example, the average value and standard error were obtained from the data of 5 cases per group. Glutamic acid was used as the excitatory amino acid. The cultured cells were treated with a solution containing glutamic acid (1 mM) for 10 minutes, and then cultured in an Eagle medium containing no glutamic acid for 24 hours. In contrast, in the administration group,
20-Hydroxyecdysone (0.1-100 μM) was added to the culture medium after administration of glutamic acid, and the mixture was cultured for 24 hours. The protective effect of 20-hydroxyecdysone was calculated by the following formula. Protection rate (%) = [(D−C) / (A−C)] × 100 In this formula, A is the survival rate of the untreated group (control group), C
Indicates the survival rate of the glutamic acid-administered group, and D indicates the survival rate of the cell group administered with 20-hydroxyecdysone and glutamic acid. As a result, as shown in Table 1, a dose-dependent protective effect on cerebral nerve cells was observed.

[0024]

[Table 1]

Note) *: p <0.5 **: p <0.01 (test the difference from the group administered with glutamic acid alone by Dunnett's method) Example 2: Protective action of various steroids on cerebral nerve cells In the same manner as in Example 1. , Each of the mammalian steroid hormones shown in Table 2, prednisolone, aldosterone, and corticosterone (100 μM) was administered,
A comparison was made with the action of 20-hydroxyecdysone. Table 2 shows the results.

[0026]

[Table 2]

Note) *: p <0.01 (Dunnett's method is used to test the difference from the glutamic acid alone administration group)

[0028]

The active ingredient represented by the above formula (I) of the present invention exhibits a cerebral nerve cell protecting action. Therefore, the active ingredient represented by the formula (I) of the present invention is a drug containing an effective and non-toxic amount of cerebral thrombosis, cerebral embolism, cerebral vasospasm,
Sudden decrease in blood pressure, hypoperfusion vasculopathy, etc. and cerebral infarction due to them, cerebral thrombosis, cerebral embolism, cerebral hemorrhage, subarachnoid hemorrhage, transient cerebral ischemic attack, hypertensive encephalopathy, etc., cerebral stroke, and further brain damage It can also be used for the prevention or treatment of heartbeat pause, hypoxia, hypoglycemia, dementia due to secondary degeneration and necrosis of cerebral nerve cells, mental disorders, and behavioral disorders.

Claims (3)

[Claims] 1. A cerebral nerve cell protective agent, which comprises a compound represented by the following formula (I) as an active ingredient. Embedded image (In the formula, R _{1 to} R ₅ each independently represent a hydrogen atom or a hydroxyl group, and R ₆ represents an alkyl group which may be substituted with a hydroxyl group.) 2. The R _{6 of the} above formula (I) is an alkyl group having 2 to 10 carbon atoms which may be substituted with a hydroxyl group.
The described brain nerve cell protective agent. 3. R ^{6 in} formula (I) above is of the formula —C (CH ₃ ) XCHXCH.
_{XCHXC (CH 3) (CH 2} X) X ( wherein, X represents a hydrogen atom or a hydroxyl group, the six X may be the same or different.) Of claim 1, wherein a group represented by Cerebral nerve cell protective agent.