JP2006176428A - Neural network reconstructive agent and method for reconstructing neural network - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop drug(s) in view of the fact that neurodegenerative diseases including Alzheimer's disease, senile dementia, cerebrovascular dementia and Parkinson's disease means symptoms presenting disorders in memory and/or cognizance by neural network failure with various causes, and for these diseases with no effective curative methods, such treatments as to enable neurological functions to be brought to conditions close to normal state have been demanded even from conditions being already in progression of neural network disorders. <P>SOLUTION: In cultured neurocytes, such conditions as to present the atrophy in neurites and dendrites and decrease in presynapse number and postsynapse number are created. The objective drug(s) capable of restoring neurocyte dendrites and synapse to normal conditions by treating neurocytes in the above-mentioned conditions is(are) retrieved from among components in traditional drugs, and withanosides and sominone as a metabolite thereof are identified. Furthermore, it is found that the above metabolite and/or an astragaloside metabolite is(are) useful as neural network reconstructive agent(s). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a neural network restructuring agent comprising a herbal medicine-derived compound effective for the treatment of cognitive dysfunction caused by the breakdown of a neural network in the brain as an active ingredient, and a neural network restructuring method using the same. Is.

Alzheimer's disease, cerebrovascular dementia, senile dementia, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Huntington's chorea, and the like are known as diseases that impair memory and cognition. These are diseases caused by neurite atrophy and dysfunction due to synapse deficiency, although the etiology is different.In addition, amyotrophic lateral sclerosis, cerebral hemorrhage, cerebral infarction, brain tumor, brain trauma, spinal cord injury, etc. are also caused by neurite atrophy. It is a disease caused by dysfunction due to synapse loss.

Cholinesterase inhibitors that are clinically used as anti-dementia drugs do not treat the disease, although they delay the progression of the disease, and are less likely to be effective in patients with moderate dementia. Drugs classified as neurotrophic factor-like substances are also being studied as anti-dementia drug candidates, but all of them are mainly neuroprotective, and the neurite outgrowth and synapse formation in a neurodegenerative environment are clear. Not shown.

On the other hand, herbal components, for example, a component of ginseng ginsenosides (ginsenoside), among others, for the ginsenoside Rb _1, are known to be used as brain cells or nerve cell protecting agent (Patent Document 1).
Cognition Enhancer for withanolide glycosides and withanolide aglycones, components of Ashwagandha, used as an anti-dementia drug in Ayurvedic medicine, a traditional Indian medicine Is known for its use. (Patent Document 2). According to the literature, the recognition enhancement of the Ashwagandha component is attributed to the promotion of muscarinic acetylcholine transmission.
In addition, with regard to vitanolide A, vitanoside IV, and vitanoside VI, which are components of Ashbaganda, the present inventors have clarified that they are compounds having both a neurite extension action and a synapse remodeling action (Non-patent Document 1). ).
Moreover, it is known that steroid saponin or sapogenin is useful for diseases (Alzheimer's disease or Alzheimer type senile dementia) characterized by a deficiency in the number of postsynaptic membrane-bound receptors or function (Patent Document 3). And this document mentions astragaloside (astragaloside) which is a component such as jaundice as one of steroid sapogenins.

International Publication WO00 / 37481 Special table 2004-500151 JP2004-217666 Abstracts of the 51st Annual Meeting of the Japanese Biopharmaceutical Society, page 151, 2004

The above-mentioned neurodegenerative diseases such as Alzheimer's disease, senile dementia, cerebrovascular dementia, and Parkinson's disease have different etiologies, but all indicate symptoms that impair memory and cognition due to the breakdown of the neural network. For these diseases for which there is no effective therapeutic method, there is a real need for a treatment that can bring the nerve function closer to normal even when the nerve network has already been impaired. Moreover, considering the patient's standpoint, treatment with less burden is more desirable than nerve cell transplantation and gene therapy that require surgery. Therefore, it is necessary to develop a drug that regenerates the neural network even when the nerve cells are damaged or after the damage.

Amyotrophic lateral sclerosis is an intractable disease in which the limbs of the cerebral cortex motor area, the spinal cord, and the brain stem lose their limbs, and there is no effective treatment. Furthermore, motor neurons in the cerebral cortex motor cortex are also damaged by cerebral hemorrhage, cerebral infarction, brain tumor, brain trauma, etc., leading to paralysis of the body. Traumatic spinal cord injury also results in limb paralysis due to damage to spinal motor neurons. In either case, it is a dysfunction due to the breakdown of the neural network, but if the surviving nerve cells are activated to form a neural network again, recovery of the function can be expected.

On the other hand, saponins that are components of herbal medicines, for example, protopanaxadiol saponins contained in ginseng crude drugs, are produced by intestinal bacteria by 20-O-β-D-glucopyranosyl- (20S) -protopanaxadiol (M1). It is known to be metabolized. It is also known that M1 itself exhibits the same pharmacological action as protopanaxadiol.

  The present inventors treated an active partial sequence of amyloid beta (Aβ (25-35)), which is a causative agent of Alzheimer's disease, in primary cultured cells of rat cerebral cortical nerves as a cell model of neural network failure. We prepared a state that exhibited atrophy of axons and dendrites, a decrease in the number of pre-synapses and post-synapses. By treating nerve cells in this state, it is possible to search for drugs that can return the neurites and synapses of the nerve cells to a normal state.

As an animal model of neural network failure, the present inventors have administered a single dose of Aβ (25-35) into the lateral ventricle of a mouse, and spatial memory impairment that begins to appear one week later, and axons in the brain. And dendritic atrophy, presynaptic and postsynaptic numbers decreased. Oral administration of a drug with a neurite outgrowth effect in a cell model to mice in this state brings about recovery of spatial memory, and at the same time all of the axons, dendrites, and presynapses in the brain are in a normal state Confirm that the expression level is restored.

In the search method using the cell model and the animal model described above, for example, various world traditional drugs are selected as test drugs, extracts are extracted, and extracts having a neurite extension action are first identified. Next, a method of isolating and identifying an active compound from an extract exhibiting activity and further confirming the action of each compound to regenerate a neural network can be mentioned.

In the present invention, the metabolite of witanoside and the metabolite of astragaloside means a compound in which witanoside and astragaloside are produced in vivo by metabolic enzymes or enteric bacteria. In addition, Ashbaganda (Withania somnifera), Withania coagulans, Withania frutescens, Acnistus arborescens, Datura fastuosa, Datura fastaosa, Datura metel Datura stramonium, Deprea orinocensis, Deprea subtriflora, Discopodium penninervium, Dunalia australis bra, Dunalia atrabria , Dunalia solanacea, Hyosyamus niger, Iochroma fuchsioides, Jaborosa bergii Lycium chinensis, Nicandra physaloides, Physalis angulata, Physalis chenopodifolia, Physalis minima, Physalis minima var. ), Physalis ixocarpa, Cape gooseberry (Physalis peruviana), Phisalis viscosa (Physalis viscosa), Solanum ciliatum, black cat (Tacca chantrieri), and Astragalus membrangalus m Also included are compounds having the same chemical structure obtained from, for example, Meliotus officinalis, Astragalus spinosus, Trifolium plants.
One of the metabolites of withanoside is sominone.

Isolation and identification of the active compound may be carried out by generally known methods, and examples thereof include the following methods for the components of the present invention and related compounds.
(1) Isolation of withanolide A (formula 1), isolation of withanoside IV (formula 2), withanoside VI (withanoside VI) (formula 3) Withania somnifera Dunal root). Specifically, the method described in Chem. Pharm. Bull., 50, 760-765 (2002) may be used.

(2) Isolation of sominone (formula 4) Sominone is a metabolite of withanoside IV in the body. This can also be obtained by treating Witanoside IV with an enzyme, for example, naringinase (pH 5.25, 37 ° C., 3 days), and separating and purifying it by partition high performance liquid chromatography (HPLC). Can do. In addition, sominone can be isolated from Ashbaganda, and specifically, the method described in Heterocycles, 34, 689-698 (1992) may be used.

(3) Isolation of Astragaloside I (Chemical Formula 5), Astragaloside II (Chemical Formula 6), Astragaloside IV (Chemical Formula 7) These three components are yellow It can be done from moths (roots of Astragalus membranaceus Bunge or Astragalus mongholicus Bunge). Specifically, the method described in Chem. Pharm. Bull., 31, 698-708 (1983) may be used.

When the metabolite of witanoside and / or the metabolite of astragaloside is used as a pharmaceutical product, various pharmaceutical preparations such as solutions, suspensions, syrups, An elixir, extract, powder, granule, fine granule, tablet, capsule or the like may be used.
In addition, in the case of administration as a pharmaceutical, the administration method, dosage, and frequency of administration can be appropriately selected depending on the age, weight and symptoms of the patient, but in the case of oral administration, as metabolites of withanoside and / or astragaloside What is necessary is just 0.01-100 micromol / kg.

In contrast to the axonal and dendritic atrophy induced by the active partial sequence of amyloid beta (Aβ (25-35)) and the decrease of presynaptic and postsynaptic, sominone is associated with vitaminide A, withanoside IV and withanoside VI. Similarly, it has a remarkable improvement effect.
In addition, with Witanolide A, Vitanoside IV, and Vitanoside VI, normal levels of spatial memory impairment and axonal / dendritic / presynaptic reduction induced in mice by intracerebroventricular administration of Aβ (25-35). In the case of oranoside IV administered orally, the active substance in the body was sominone.
From these, the compound in the present invention has a novel mode of action not found in existing drugs, Alzheimer's disease, senile dementia, cerebrovascular dementia, frontotemporal dementia, Lewy body dementia, Parkinson's disease, It is useful for the treatment of various neurodegenerative diseases such as Huntington's disease, as well as dysfunction of motor neurons due to amyotrophic lateral sclerosis requiring axon formation, cerebral hemorrhage, cerebral infarction, brain tumor, brain trauma, spinal cord injury, etc. It is also useful for treatment.

Example 1
Neural network formation (method) Cerebral cortical neurons of embryonic day 18 SD rats were dispersedly cultured. Nerve cells were treated with 10 μM Aβ (25-35) to cause injury. After treatment of nerve cells after induction of injury, phosphorylated NF-H (axon marker), MAP2 (dendritic marker), synaptophysin (pre-synaptic marker), PSD-95 (post-synaptic marker) Immunostaining was performed using an antibody against and the axon per cell, dendrite length, and synaptic density per dendrite unit length were measured.

(Results) When 10 μM Aβ (25-35) was treated 1 day after the start of culture of rat cerebral cortical neurons, atrophy of axons and dendrites was observed 4 days later. When sominone, withanolide A, withanoside IV and withanoside VI were each treated with 1 μM, 4 days later, both axons and dendrites were significantly extended as compared with the solvent-treated group. Next, after culturing rat cerebral cortical neurons for 3 weeks to sufficiently form synapses, treatment with 10 μM Aβ (25-35) for 4 days significantly reduced both pre-synapse and post-synapse. When Sominone, Withanolide A, Withanoside IV and Withanoside VI were each treated with 1 μM, the density of pre-synapse and post-synapse increased significantly compared to the solvent-administered group 7 days later.

Example 2
It shows the effect of improving spatial memory impairment.
(Method) 25 nmol Aβ (25-35) was administered to the right ventricle of a 6-week-old ddY mouse (male). After 7 days, the drug (10 μmol / kg) was orally administered once a day and continued for 13 days. Seven days after the start of drug administration, a spatial memory acquisition test was conducted in the Morris water maze for 6 days. On the last day of the acquisition test, drug administration was discontinued, and 7 days later, a spatial memory retention test was conducted in the water maze. After the behavioral experiment, immunostaining using antibodies against phosphorylated NF-H (axon marker), MAP2 (dendritic marker), synaptophysin (pre-synaptic marker), PSD-95 (post-synaptic marker), The density of axons, dendrites, presynapses, and postsynapses was measured.

(Results) Intraventricular administration of 25 nmol Aβ (25-35) significantly reduced the memory retention ability of mice. 7 days after administration of Aβ (25-35), memory impairment started, and axons, dendrites, and decreased synaptic density were markedly observed in the cerebral cortex and hippocampus. After confirming, consecutive administration of oritanolide A, withanoside IV and withanoside VI was performed at 10 μmol / kg. All four compounds restored memory retention to normal mouse levels, and axons, dendrites, and presynaptic densities in the cerebral cortex and hippocampus to normal mouse levels.

Witanoside metabolites and astragaloside metabolites show neuronal network formation and spatial memory impairment, and are useful as drugs to treat cognitive dysfunction caused by neural network breakdown in the brain .

Chemical structure of withanolide A (chemical formula 1) Chemical structure of Witanoside IV (withanoside IV) Chemical structure of Witanoside VI (withanoside VI) Chemical structure of sominone (Chemical formula 4) Chemical structure of Astragaloside I (astragaloside I), Astragaloside II (astragaloside II), Astragaloside IV (astragaloside IV) Remodeling of presynapse and postsynapse by sominone, withanolide A, and withanoside IV

Claims (4)

A neural network restructuring agent comprising as an active ingredient at least one selected from a metabolite of withanoside or an astragaloside metabolite. The neural network restructuring agent according to claim 1, wherein the metabolite of withanoside is sominone. A method for reconstructing a neural network using a composition containing as an active ingredient at least one selected from a metabolite of withanoside or a metabolite of astragaloside (astragaloside). 4. The method for reconstructing a neural network according to claim 3, wherein the metabolite of withanoside is sominone.

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