WO2019188950A1 - Medicinal composition - Google Patents

Abstract

A medicinal composition for treating a central neurodegenerative disease, said medicinal composition comprising at least one member selected from the group consisting of nicotine, nobiletin, sinensetin and Nchinpi, characterized in that the central neurodegenerative disease is associated with mitochondrial dysfunction.

Description

Pharmaceutical composition

The present invention relates to a pharmaceutical composition for treatment of a central neurodegenerative disease accompanied by mitochondrial dysfunction, comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi.

Maintenance of mitochondrial function, which is a stable ATP supply system, is essential for cell survival. Therefore, failure of the stable supply system of ATP in many eukaryotic cells, that is, mitochondrial dysfunction, can be a decisive cause of cell death. For example, Alzheimer's disease (AD) and Parkinson's disease (PD), known as the two major central neurodegenerative diseases, are known to be neuronal cell death as a cause of the onset.

Here, the actin cytoskeleton is indispensable for nerve cells to maintain the morphological structures such as nerve axons and dendrites, and the ATP consumption of the actin depolymerization-polymerization cycle is the total energy consumption of the brain. It is estimated that it is 1 to 50% (Non-Patent Documents 1 and 2). Therefore, there is a possibility that a stable supply system of ATP is incorporated in the nerve cells of the brain so that the balance of ATP balance in the brain does not break.

On the other hand, V-1 (also referred to as Myotrophin) protein is an ankyrin repeat protein, and human V-1 consists of 118 amino acids. V-1 binds to actin capping proteins (CP, CapZ) via intramolecular ankyrin repeats. CP binds to the end of the actin filament and blocks it to block the polymerization of actin. V-1 is known to inhibit its actin capping activity and thereby promote the formation of the actin cytoskeleton (Non-patent Document 3).

However, the function of V-1 on mitochondria was not known.

Synensetin and nobiletin are polymethoxyflavonoids contained in the skin of the genus Citrus, and chimp is a mature skin of Satsuma mandarin or mandarin orange. Nobiletin and sinensetin are known to have an antidementia effect. Chimpi has long been used as a crude drug. However, the function for any mitochondria was not known.
Nicotine is a kind of alkaloid contained mainly in the leaves of tobacco Nicotiana tabacum. A systematic review that smoking reduces the risk of developing Parkinson's disease is known (Non-Patent Document 4). However, the function of nicotine on mitochondria was not known.

Berstein, B.W. & Bamburg, J.R., J. Neurosci. 2003, 23: 1-6 Engl, E. & Attwell, D., J. Physiol. 2015, 593.16: 3417-3429) Takeda, S., Minakata, S., Koike, R., Kawahata, I., Narita, A., Kitazawa, M., Ota, M., Yamakuni, T., Maeda, Y. & Nitanai, Y. Two distinct mechanisms for actin capping protein regulation-steric and allosteric inhibition. PLoS Biology, e1000416, 2010. Allam, M.F. et al., Smoking and Parkinson ’s Disease: Systematic Review of Prospective Studies, Movement Disorders, 19, 6, 2004, 614-621

An object of the present invention is to provide a pharmaceutical composition for the treatment of a central neurodegenerative disease accompanied by mitochondrial dysfunction, comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin and chimpi. .

In the central nervous system culture system, the present inventor has shown that the complex of V-1 protein and actin capping protein up-regulates the mRNA level of COX I, nicotine, nobiletin, cinecetin and chimpi are V-1 protein and actin capping protein. Has been found to promote the formation of the complex and up-regulate COX I mRNA levels.
The present invention includes the following aspects.
[1] A pharmaceutical composition for treatment of a central neurodegenerative disease, comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi, wherein the central neurodegenerative disease is a mitochondrial dysfunction A pharmaceutical composition comprising:
[2] The pharmaceutical composition of [1], wherein the central neurodegenerative disease is Alzheimer's disease with mitochondrial dysfunction, Parkinson's disease with mitochondrial dysfunction, or cerebrovascular dementia with mitochondrial dysfunction,
[3] The pharmaceutical composition of [2], wherein the treatment is promotion of formation of a complex of V-1 protein and actin capping protein,
[4] at least one compound selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi for use in the treatment of mitochondrial dysfunction;
[5] The compound according to [4], wherein the mitochondrial dysfunction is Alzheimer's disease with mitochondrial dysfunction, Parkinson's disease with mitochondrial dysfunction, or cerebrovascular dementia with mitochondrial dysfunction.
[6] The compound of [5], wherein the treatment is promotion of formation of a complex of V-1 protein and actin capping protein.
[7] contacting a test substance with a nerve cell;
Determining the level of the complex of V-1 protein and actin capping protein and / or the expression level of COX I in the nerve cells contacted with the test substance,
When the determined level and / or expression level is higher than the level of the complex of V-1 protein and actin capping protein and / or the expression level of COX I in a nerve cell not in contact with the test substance, Selecting the test substance as a candidate substance useful for maintaining and strengthening mitochondrial homeostasis,
A method for screening candidate substances useful for maintaining and / or enhancing mitochondrial homeostasis.

According to the present invention, it is possible to provide a pharmaceutical composition for the treatment of a central neurodegenerative disease accompanied by mitochondrial dysfunction, comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi. .

In the midbrain culture system, the V-1 / CP complex upregulates COX I mRNA levels. D44R is an artificial mutant V-1 (dominant negative V-1 mutant). Like wild type V-1 (WT), it was introduced into neurons using a lentiviral vector VSV-G (KatoＧet al., Hum.Gene.Ther. 2011, 22: 1511-1523). Wild-type V-1 (WT), artificial mutant V-1 (D44R), mutant SRF (Ishikawa et al., J. Neurosci. 2010, 285: 32734-32743) , C3 enzyme (RhoA inhibitor), or Y-27632 (ROCK inhibitor). Numerical values are mean ± SEM (n = 3). For control, **** is p <0.0001. It is the effect on the V-1 / CP assembly (complex formation) of donepezil, memantine, nobiletin, sinensetin, N-thinpi or a mixture of nobiletin and sinensetin in the central nervous system. Cultured cranial neurons or cultured hippocampal neurons were each treated with 10 μM donepezil, 10 nM or 10 μM memantine, 30 μM nobiletin, 30 μM synencetin, 100 μg / ml N-thinpi, or a mixture of 30 μM nobiletin and 3 μM sinencetin. It is the effect on the mRNA level of COX I (cytochrome C oxidase subunit I) of donepezil, memantine, nobiletin, sinensetin, N-thinpi or a mixture of nobiletin and nobiletin in the central nervous system. Numerical values are mean ± SEM (n = 3). Tukey 対 test after one-way ANOVA was performed for all pairs. For controls, * is p <0.05, ** is p <0.01, *** is p <0.001, and **** is p <0. .0001, for a nobiletin and cinecetin mixture, # is p <0.05. The concentration is the same as in FIG. FIG. 5 is an effect on mRNA levels of glutathione synthetase and glutamine-cysteine ligase catalytic subunit (GCLC) of wild type (WT) or artificial mutant (D44R) V-1 in a midbrain nerve culture system. Numerical values are mean ± SEM (n = 3). Tukey 対 test after one-way ANOVA was performed for all pairs. For GFP control, *** is p <0.001, for V-1 (D44R), ### is p <0.001, and #### is p <0.001 0.0001. Formation of V-1 / CP complex is promoted by 10 μM NMDA and 10 μM nicotine in the mesencephalic nerve culture system (cultured mesencephalic neurons or cultured hippocampal neurons). FIG. 4 shows the effects of 30 μM nobiletin and 300 μg / ml N chipi on V-1 / CP protein complex formation in cultured hippocampal neurons. Cells were treated with test samples for 24 hours. The cell lysate is subjected to immunoprecipitation with anti-V-1 antibody, and CP co-immunoprecipitated with V-1 is detected with mouse anti-CPα antibody, and then this immunoprecipitate is re-reacted with rabbit anti-V-1 antibody. Labeled. Independently, each input sample was assayed with anti-total ERK antibody. FIG. 6 shows the effect on ATP production of wild type (WT) or artificial mutant (D44R) V-1 and 30 μM nobiletin, 30 μM synencetin, 100 μg / ml N chipi, or a mixture of 30 μM nobiletin and 3 μM sinencetin in PC12D cells. ATP production of wild-type (WT) or artificial mutant (D44R) V-1 in hippocampal neurons and 30 μM nobiletin, 30 μM sinensetin, 100 μg / ml N chimpi, a mixture of 10 μM nobiletin and 1 μM sinencetin or a mixture of 30 μM nobiletin and 3 μM sinensetin. Show the impact on. For GFP controls, ** is p <0.01 and **** is p <0.0001. The change of the mRNA level of the mitochondrial associated protein COXI with time is shown. Numerical values are mean ± SEM (n = 3). For P14 mice, ** is p <0.01 and *** is p <0.001. For P49 mice, #### is p <0.0001. The expression change of V-1 and TH accompanying the postnatal development and aging of a midbrain dopamine nerve is shown. Midbrain dopamine neurons were double-stained with bispecific antibodies against V-1 and TH. Scale: 20 μm. The broken rectangle in the upper panel shows the dark substantia of the midbrain, and the lower two panels show the magnified image. FIG. 6 shows the interaction between V-1 and CP in substantia nigra TH positive neurons (substantia nigra dopamine nerve) in mice. Scale: 20 μm. The broken rectangle in the upper panel shows the dark substantia of the midbrain, and the lower two panels show the magnified image. Note that the complex of V-1 and CP is not observed in the TH positive nerve in the substantia nigra of P14 mice. N skin for the expression of each subunit protein of mitochondrial electron transport complex I to V in the middle brain of aged (17 months old) male C57BL / 6N mice (oral administration for 2 weeks at a dose of 0.5 g / kg body weight / day) Of the mitochondrial electron transport complex I to V in the midbrain of the NMDA receptor subunit NR2D-deficient C57BL / 6N mice (NR2D knockout mice, male adult) (Panel right). Panel left: In old mice, N-Chan enhanced the expression of each constituent subunit protein of mitochondrial electron transport complex I to V in the midbrain. Panel right: In the midbrain of NR2D-deficient mice, a decrease in the expression level of each of the constituent subunits of the above complexes I to V was observed compared to wild-type (WT) mice. These findings suggest that the mechanism of mitochondrial homeostasis in the midbrain dopamine neurons may be dependent on the NMDA receptor subunit NR2D.

A “neuron cell” is a cell that constitutes the nervous system, and has dendrites and axons in a form specific to the nerve extending from the cell body. These are the places where chemical signals are received and electrical signals are generated and transmitted, and are therefore indispensable structures for neurons. Nerve cells have excitability to generate electrical signals and simultaneously synthesize and release neurotransmitters. Excitatory synapses release excitatory neurotransmitters as chemical signals from axon terminals, while inhibitory synapses release inhibitory neurotransmitters. These excitatory or inhibitory neurotransmitters regulate the generation of electrical signals and neurotransmitter release in the target cells. In this way, the nerve cells that construct the neural network have a built-in mechanism for generating electrical signals. That is, in nerve cells, a resting membrane potential is formed by the action of an ATP-consuming Na ⁺ , K ⁺ -ATPase (sodium ion pump), and the preparation for electric signal generation is constantly arranged. Furthermore, dendrites and axons that are characteristic of nerves are maintained in the form of actin fibrils and microtubule cytoskeletons, but these cytoskeletons all maintain their structure and consume ATP. To do. Therefore, neurons need to have a mitochondrial ATP supply system not only in their cell bodies but also in dendrites and axons. For this reason, the vulnerability of the ATP supply system in nerve cells is directly linked to the functional vulnerability of the cranial nervous system. Therefore, the discovery of drugs that contribute to “maintenance and strengthening of mitochondrial homeostasis” is extremely important.

“V-1 protein” is an acidic protein discovered in the rat brain by Yamakuni et al. In the early 1990s. Subsequently, genes were also found in mice and humans, and their expression in living bodies was also confirmed. V-1 mRNA encodes a 118 amino acid protein, but in the brain it exists as a 117 amino acid protein with the amino-terminal methionine removed. This protein is expressed in almost all peripheral and central nervous system neurons and adrenal medulla chromaffin cells, as well as in skeletal muscle, cardiomyocytes, and smooth muscle. The molecule is the same protein as myotrophin found in the myocardium. In catecholamine-producing cells such as substantia nigra dopamine neurons and adrenal medulla cells, the function of the protein that promotes transcription of catecholamine synthase gene and enhances catecholamine production is known. In addition, physiological functions that promote the formation of actin cytoskeleton by binding to actin capping protein and inhibiting its activity have been clarified.

“Actin capping protein (CP)” is a regulator of intracellular actin skeleton formation. Such a name was given because it has the activity of binding to the positive end of actin fibers and suppressing its elongation. It is also called CapZ because it is a protein that constitutes the Z band of skeletal muscle cells. It binds to the positive end of actin filaments as a heterodimeric molecule composed of one molecule each of α-subunit and β-subunit. It has been demonstrated that free actin capping protein present in the cytoplasm loses actin capping activity when bound to V-1.

“Maintenance / enhancement of mitochondrial homeostasis” refers to maintenance / enhancement of stable ATP production ability of mitochondria. In eukaryotic organisms, it is indispensable for all cells constituting organs and tissues having various functions to stably produce and supply ATP themselves in order to ensure their survival. Therefore, mitochondrial dysfunction, the main source of ATP production in cells, that is, the cessation of ATP supply, not only causes the end of cell life in humans but also decisive causes of individual death. Become. The failure of the “maintenance and strengthening of mitochondrial homeostasis” system has been pointed out as a mechanism for the development of central neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and cerebrovascular dementia (Michel, P. et al., Neuron 90, 2016, 675-691; Ittner, LM and Goets, J., Nature Reviews Neuroscience 12, 2011, 67-21). In this connection, there is also evidence that aging causes deterioration of mitochondrial ATP production function at specific brain sites. Therefore, "maintenance and strengthening of mitochondrial homeostasis" is indispensable for maintaining brain health.

“COXI” is cytochrome c oxidase subunit I (cytochrome c oxidase subunit I, COX I), which is an important component of complex IV that catalyzes the final step of the mitochondrial electron transport system (respiratory chain) One of the subunits. In the complex IV, the electrons received from each cytochrome c via the complex III are passed to oxygen to generate two molecules of water. COXI is encoded by mitochondrial DNA. Therefore, it is used as a molecular marker for mitochondria.

As the method for determining the protein level, any conventionally known method can be used. For example, there is a Western blot analysis using an antibody. In addition, immunoprecipitation can be used for quantification of the V-1 / CP complex level.

As the method for determining the mRNA expression level, any conventionally known method can be used. For example, a real-time RT-qPCR method can be used.

“Nobiletin” is a polymethoxyflavone compound that is a kind of flavonoids characteristic of the genus Citrus. It exhibits various biological activities such as antidementia effect, anti-Alzheimer's disease effect, antihypertensive effect, and anti-inflammatory effect.

“Sinecetin” is a kind of polymethoxyflavone compound of flavonoids characteristic of the genus Citrus similar to nobiletin. Anti-dementia effects have been reported in animal experiments.

“Chimpi” is the mature pericarp of Citrus unshiu Markovich or C. reticulate Blanco, and is a herbal medicine used as an aromatic bitterness stomachic medicine, antitussive and expectorant in Chinese medicine. “N Chinpi” refers to a nobiletin or synencetin-rich skin. It is known to exhibit a stronger antidementia effect than nobiletin alone.

The mitochondrial homeostasis maintenance / enhancement agent may be added with pharmaceutically acceptable materials such as preservatives and stabilizers. “Pharmaceutically acceptable” may be a material itself having the above-mentioned action or a material not having the above-mentioned maintenance action, and can be administered together with the mitochondrial homeostasis maintenance / enhancement agent. Means a pharmaceutically acceptable material. Moreover, it is a material which does not have a maintenance effect | action and has a synergistic effect or an additive stabilization effect by using together with a maintenance agent.

Examples of materials that are acceptable for formulation include sterilized water, physiological saline, stabilizers, excipients, buffers, preservatives, binders, and the like.

Buffers include phosphate, citrate buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, etc. Organic acids, etc., or carbonate buffer, Tris buffer, histidine buffer, imidazole buffer, and the like.

Alternatively, a solution formulation may be prepared by dissolving in an aqueous buffer known in the field of solution formulation. The concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM.

The mitochondrial homeostasis maintenance / enhancement agent may contain other low molecular weight polypeptides, serum albumin, proteins such as gelatin and immunoglobulins, saccharides such as amino acids, polysaccharides and monosaccharides, carbohydrates, and sugar alcohols. Good.

Examples of saccharides and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

In the case of an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. Adjuvants such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. may be used in combination.
If desired, it may further contain a diluent, a solubilizer, a pH adjuster, a soothing agent, a sulfur-containing reducing agent, an antioxidant and the like.

In addition, if necessary, it can be enclosed in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (See "Remington's Pharmaceutical Science 16th edition", Oslo Ed., 1980, etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982, 12: 98-105; US Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP 133,988).
The pharmaceutically acceptable carrier to be used is selected appropriately or in combination from the above depending on the dosage form, but is not limited thereto.

When mitochondrial homeostasis maintenance / enhancement agent is used as a medicine for humans or other animals, in addition to administering these substances themselves to patients, they should be formulated by known pharmaceutical methods. Is also possible. In the case of formulation, the above-described formulation-acceptable materials may be added.

The mitochondrial homeostasis maintenance / enhancement agent can be administered in the form of pharmaceuticals, and can be administered systemically or locally orally or parenterally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient Can do. The effective dose is selected in the range of 0.001 mg to 100 mg per kg of body weight per time. Alternatively, a dose of 0.1 to 1000 mg, preferably 0.1 to 50 mg per patient can be selected. For example, in the case of an anti-Arid5A antibody, an effective dose is an effective dose such that an amount of free antibody is present in the blood. As a specific example, one month (4 Weekly) 0.1 mg to 40 mg, preferably 1 mg to 20 mg divided into 1 to several times, for example, 2 times / week, 1 time / week, 1 time / 2 weeks, 1 time / 4 weeks, etc. In the method of administration, such as intravenous injection such as infusion or subcutaneous injection. The administration schedule is 2 times / week or once / week to once / 2 weeks, once / 3 weeks, once / 4 weeks, etc. while observing the state after administration and observing the trend of blood test values It is also possible to adjust such as extending the administration interval.

The present invention relates to a central neurodegenerative disease associated with mitochondrial dysfunction, comprising administering to a subject in need thereof a pharmaceutical composition comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi Related to treatment.
The central neurodegenerative disease is preferably Alzheimer's disease with mitochondrial dysfunction, Parkinson's disease with mitochondrial dysfunction, or cerebrovascular dementia with mitochondrial dysfunction. The treatment is preferably promotion of formation of a complex of V-1 protein and actin capping protein.

The present invention relates to a screening method for candidate substances useful for maintaining and strengthening mitochondrial homeostasis. The screening method detects the influence of a test substance on the formation of a complex of a VI protein and an actin capping protein, and uses a test substance that has a detected effect different from that when a test substance is not used as a candidate substance. Including selecting.

The biological species from which V-1 is used in the screening method is not limited to a specific biological species. Examples include humans, monkeys, mice, rats, guinea pigs, pigs, cows, chickens and insects.

The “test substance” is not particularly limited. For example, a natural substance, an organic compound, an inorganic compound, a single substance such as a nucleic acid, a protein, and a peptide, and a compound library, nucleic acid library, peptide library, gene live Examples include rally expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts. The test substance can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label. Further, in addition to the test substance, a mixture obtained by mixing a plurality of these test substances is also included.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.

(Culture of primary rat hippocampal and midbrain neurons)
Pregnant Sprague-Dawley (SD) rats were bred by feeding and watering in a 12 hour light / dark cycle. The uterus was aseptically removed from the rat on day 18 of gestation (E18) by a midline abdominal incision under isoflurane deep anesthesia. Under a stereomicroscope, the fetal hippocampus was removed in ice-cold phosphate buffered saline (PBS), and the tissue was dispersed by treatment for 30 minutes at 37 ° C. in a nerve cell dispersion (Sumitomo Bakelite). After centrifugation, the supernatant was removed. Next, the cell pellet is dispersed in a dispersion liquid (Sumitomo Bakelite), and then the removal liquid (Sumitomo Bakelite) is added to the cells sufficiently dispersed by pipetting, followed by centrifugation at 900 rpm for 5 minutes, and then the supernatant is removed. did. Next, the pellet was suspended using Neurobasal (Neurobasal® Medium 500 ml / Phenol Red free, 50 × B27 supplement 10 ml, 0.5 mM L-glutamine, 0.005% penicillin-streptomycin) -Seeded on plastic dishes or plates coated with L-lysine. After 1 day of culture, the medium was changed, and then half of the medium was changed every 3 to 4 days. The medium was cultured in a medium containing 10 μM Ara-C at 37 ° C. in a 5% CO ₂ incubator for 14 days. As a test medium for drug treatment experiments, Ara-C and antibiotic-free neurobasal medium were used.
Rat midbrain primary neurons were collected from Wistar rats on day E16 according to a previously reported method (Wakita et al. 2010), dispersed using a nerve culture dispersion (Sumitomo Bakelite), and then seeded. . As the medium, EMEM medium containing 10% FBS was used. The primary cultured neurons of the midbrain were subjected to various analyzes 48 hours after introduction of various expression vectors or treatment with crude drug extracts and various compounds on the 5th day after the seeding and half-day medium exchange every other day thereafter. It was used for.

(Immunoprecipitation and Western blot analysis)
In immunoprecipitation experiments and Western blot analysis of V-1 / CP complex formation changes by various samples or samples, 1 × 10 ⁶ cells of hippocampal neurons or 2 × 10 ⁶ cells of midbrain dopamine neurons were analyzed in a 35 mm dish. Cells containing the cells were seeded, and after the above-described culture, a medium containing various compounds or herbal extracts and natural foods was added and treated for 48 hours. In the immunoprecipitation experiment, the above-mentioned cells were washed twice with PBS (−) and centrifuged at 1000 rpm, and the cells were collected, frozen once, and then 300 μl of cell lysate mammalian lysis buffer (Promega) was added, and left on ice for 30 minutes. Thereafter, the cell lysate was sucked 10 times with a 27 gauge needle and centrifuged at 4000 rpm, and the supernatant was collected as a cell extract. Thereafter, the cell extract containing 400 μg of protein is transferred to a 2 ml tube, mixed with Dynabeads Protein G (Veritas / Invitrogen) to which anti-V-1 antibody is bound, and mixed by inverting at 4 ° C. for 16 hours. I let you. Thereafter, Dynabeads are collected with a magnet, washed 3 times with an ice-cooled washing buffer (containing PBS / 0.01% Tween-20), and then anti-V-1 antibody with an elution buffer (50 mM glycine-HCl, pH 2.8). And bound protein was eluted and collected. The immunoprecipitate was analyzed by Western blot using an anti-CPα antibody. First, a 12% polyacrylamide gel was used as a separation gel for SDS-PAGE. After electrophoresis at a constant voltage of 100 V, the protein was transferred from the gel to a PVDF membrane, and a TBS-T buffer containing 5% skim milk (10 mM Tris-HCl (pH 7) was used. 4), 100 mM NaCl, 0.05% Tween 20; hereinafter referred to as blocking buffer), and blocked at room temperature for 1 hour. Incubation was carried out at 4 ° C. for 16 hours with anti-CPα antibody diluted 1000 times with blocking buffer. Further, the membrane was washed with TBS-T, incubated with HRP-labeled IgG antibody (CST) diluted 1000 times with blocking buffer at room temperature for 1 hour, and washed 3 times with TBS-T. Antibody positive bands were detected by ECL method using Immobilon Western chemiluminescence HRP substrate (Millipore).

(Quantitative analysis of cell viability using MTT method)
In the quantitative analysis using the MTT method of cell viability change due to various natural compounds, crude drugs and V-1 gene, cells containing midbrain dopamine neurons were seeded in a 48-well plate at 1.6 × 10 ⁵ cells / well. After the above culture, various expression vectors were introduced for 48 hours or drug treatment was performed for 24 hours. Thereafter, MTT reagent (Dojindo, 0.01 g / 2 mL) dissolved in PBS was prepared, 20 μL / well was added to each well, and incubated at 37 ° C. for 4 hours in a CO ₂ incubator. Thereafter, the medium was aspirated, 100 μl of DMSO was added to each well and shaken for 10 minutes. After confirming that the MTT reagent was completely dissolved, the absorbance at 590 nm was measured with an absorptiometer. The rate of change was calculated.

(Quantitative analysis of mRNA level using PCR method)
In quantitative analysis of mRNA level changes by various natural compounds, crude drugs and V-1 gene using PCR method, cells containing isolated hippocampal neurons or midbrain dopamine neurons are each 1 × 10 ⁶ cells in a 35 mm dish or 2 × 10 ⁶ cells were seeded, and after the aforementioned culture, RNA was collected and purified from the cells using RNeasy mini kit (Qiagen). Template cDNA synthesis was performed using RverTra Ace qPCR RT Kit (Toyobo). A quantitative PCR method was used for analysis of changes in various mRNA levels, and analysis was performed using the following primers:
Rat COX I
Forward primer: 5'-TCACAGCCCATGCATTCGTA-3 '
Reverse primer: 5'-AGATAGAAGACACCCCGGCT-3 ')
Rat β-actin forward primer: 5′-TGTGTTGTCCCTGTATGCCT-3 ′
Reverse primer: 5'-AATGTCACGCACGATTTCCC-3 '.

Real-time quantitative PCR analysis device LightCycler Nano (Roche) was used for PCR, and quantitative analysis was performed by two-step PCR (95 ° C./15 seconds, 60 ° C./30 seconds 50 cycles) using the CyberGreen method. The amount of COX I mRNA was corrected by the amount of β-actin mRNA of the reference gene product, and the increase or decrease rate was calculated.

(Quantitative analysis of intracellular ATP content)
In quantitative analysis of changes in intracellular ATP amount by various natural compounds, crude drugs and V-1 gene, PC12D cells or cultured hippocampal neurons were seeded at 48 × ^{4 4} cells / well in a 48-well plate. The cultured hippocampal neurons were cultured for 48 hours after introduction of various expression vectors or treated with drugs for 24 hours, and the whole medium was extracted, and an ATP extraction reagent Intracellular ATP measurement kit (Toyo Benet) was added. After extracting ATP from the cells by standing for 5 minutes, transfer 100 μL of the sample to the cuvette, mix with 100 μl of the L / L reagent, which is the fermentation reagent in the kit, and shake the cuvette three times to mix. Measurement was performed for each specimen. For the obtained data, the rate of change was calculated with the light emission amount of the control being 1.

(Changes in mRNA level over time of the mitochondrial protein COX I with age)
COX I mRNA levels in the mouse midbrain became maximal in adulthood and decreased with aging. C57BL6 mouse midbrain tissue was collected, mRNA was extracted with QIAGEN RNeasy kit, reverse-transcribed to cDNA using Toyobo RiverTra Ace, and the cDNA was analyzed by quantitative PCR. 5′-CGGAGCCCCAGATATAGCAT-3 ′ was used as the forward primer, and 5′-ATGGGCTAGATTTCCCGCTA-3 ′ was used as the reverse primer.

V-1 / CP complex upregulates COX I mRNA levels in mesencephalic nerve culture system As shown in FIG. 1, in the mesencephalic nerve culture system, the complex of wild type V-1 and CP becomes COX I mRNA level. Was up-regulated. In contrast, artificial mutant V-1 (D44R) down-regulated COXI mRNA levels. These results indicate that V-1 binds to actin capping protein and upregulates COX I RNA levels in the midbrain nerve. Since COX I is encoded by mitochondrial DNA, it is used as a molecular marker for mitochondria. Thus, V-1 has been shown to promote mitochondrial function. The expression of COX I mRNA in the mouse midbrain increased about 1.7 times at 49 days of age and decreased to about 0.4 at 512 days of age compared to 14 days of age. It is understood that mitochondrial function decreases with aging (FIG. 10).

Effect of V-1 on cell survival rate of mesencephalic nerve culture system In the above-mentioned mesencephalon nerve culture system, wild type V-1 increased cell survival rate, whereas artificial mutant V-1 (D44R) Reduced cell viability (FIG. 2). It is also an SRF (serum response factor) deletion mutant involved in Rho signal activation, a botulinum C3 enzyme that specifically inactivates Rho, and a selective and potent ROCK (Rho binding kinase) inhibitor. Y-27632 reduced cell viability. Since mitochondrial dysfunction, ie, ATP depletion, results in cell death, V-1 has been shown to increase cell viability through mitochondrial function enhancement.

Effect of V-1 on COXI expression in neuronal culture system In cultured mesencephalic or cultured hippocampal neurons, nobiletin, sinensetin, N-thinpi or a mixture of nobiletin and sinensetin promoted V-1 / CP assembly (complex formation) Promoted the expression of COXI (FIGS. 3 and 4). In contrast, commercially available Alzheimer's disease drugs such as donepezil (cholinesterase inhibitor) and memantine (NMDA receptor antagonist) did not show such effects. Existing therapeutic agents for Alzheimer's disease did not show the effect of promoting mitochondrial function.

Effects of V-1 on glutathione synthetase and GCLC expression in neuronal culture systems In midbrain neurons, wild-type V-1 acts to eliminate reactive oxygen species responsible for mitochondrial dysfunction and glutamine-cysteine The mRNA level of ligase catalytic subunit (GCLC) was up-regulated (FIG. 5). In contrast, the artificial mutant V-1 (D44R) down-regulated the expression of both genes.

Various substances (NMDA, nicotine, donepezil, memantine, nobiletin, synencetin, N-thinpi, and a mixture of nobiletin and cinecetin) in cultured midbrain neurons or cultured hippocampal neurons that promote the formation of V-1 / CP complex The effect on the formation of the V-1 / CP complex was investigated. NMDA is an agonist of NMDA-type glutamate receptors involved in memory and learning, donepezil and memantine are therapeutic agents for Alzheimer's disease, and nobiletin, sinensetin and N-thinpi are substances or herbal medicines derived from citrus fruits. NMDA, nicotine, nobiletin, sinensetin, and N-thinpi promoted the formation of the V-1 / CP complex (FIG. 6). Moreover, 30 μM nobiletin showed the same effect as 300 μg / ml N chimney (FIG. 7).

PC12D cells are an undifferentiated nerve model derived from adrenal medullary pheochromocytoma. V-1 proteins, nobiletin, sinensetin, and N-thinpi increased ATP production in PC12D cells and hippocampal neurons, indicating that they have mitochondrial function-promoting effects (FIGS. 8 and 9).

Changes in V-1 and TH expression during postnatal development and aging of midbrain dopamine neurons Immunochemical analysis of V-1 and TH Tyrosine, the rate-limiting enzyme of V-1 and dopamine biosynthesis with age in the mouse midbrain In order to analyze changes in the expression level of hydroxylase (hereinafter referred to as TH) at the cellular level, immunochemical analysis was performed. First, a 4% paraformaldehyde-fixed mouse mesencephalon section prepared at a thickness of 50 μm was treated with PBS containing 0.1% Triton-X for 1 hour and then subjected to blocking treatment with PBS containing 5% goat serum for 2 hours. . Thereafter, mouse monoclonal anti-TH antibody (200-fold dilution) and rabbit polyclonal anti-V-1 antibody (500-fold dilution) diluted with PBS containing 1% goat serum were added dropwise and treated at 4 ° C. for 16 hours. Next, after washing 3 times with PBS for 5 minutes, anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546 diluted 500-fold with PBS containing 2% goat serum were added dropwise, treated for 1 hour at room temperature, and then treated with PBS for 10 hours. Washing was performed 3 times per minute. Finally, it was sealed with a mounting agent for preventing fading (Agilent Technology), allowed to stand for 15 minutes, and then photographed using a fluorescence microscope (Keyence).
Expressions of tyrosine hydroxylase (TH) and V-1 which are rate-limiting enzymes for dopamine synthesis were examined at each stage after birth, that is, 14 days old, 49 days old, and 512 days old. It was revealed that the expression of V-1 and TH decreased remarkably with aging (FIG. 11).

Interaction between V-1 and CP in substantia nigra TH positive neurons (ie, substantia nigra dopamine neurons) in mice The interaction between V-1 and CP was determined using PLA (Proximity Ligation Assay). To detect endogenous proteins in fixed cells, secondary antibody specificity was combined with complementary oligonucleotide probe rolling circle amplification (RCA). A pair of oligonucleotide-labeled antibodies (PLA probes) produced an amplified signal only when the probes were in close proximity (within 40 nm).

Visualization analysis of V-1 / CP complex (Dulink (registered trademark) PLA method)
A Duolink In Situ kit (Sigma-Aldrich) was used for visualization analysis of the V-1 / CP complex accompanying aging in mouse midbrain tissue. First, 4% paraformaldehyde-fixed mouse mesencephalon sections prepared at a thickness of 50 μm were treated with a blocking solution (included in the kit) at room temperature for 2 hours. Thereafter, mouse monoclonal anti-CPα antibody (200-fold dilution) and rabbit polyclonal anti-V-1 antibody (500-fold dilution) diluted with PBS containing 1% goat serum were added dropwise and treated at 4 ° C. for 16 hours. In order to perform the proximity ligation assay (PLA) treatment on the anti-CPα antibody and anti-V-1 antibody bound to the antigen, 5-fold diluted anti-mouse PLA probe and anti-rabbit PLA probe (included in the kit) were added dropwise at 37 ° C. After treatment for 60 minutes, washing was performed twice for 5 minutes with washing solution A (included in the kit). Next, in order to ligate the added PLA probe, a 40-fold diluted ligase (included in the kit) was added dropwise, treated at 37 ° C. for 30 minutes, and then washed twice with washing solution A (included in the kit) for 2 minutes went. Finally, in order to label the ligated PLA probe, a 80-fold diluted polymerase (included in the kit) is dropped, treated at 37 ° C. for 100 minutes, and then washed twice with Washing Solution B (included in the kit) for 10 minutes. After washing, washing was further performed with washing solution B diluted 100 times for 1 minute. Thereafter, the mixture was allowed to stand for 15 minutes after encapsulating in an antifade encapsulant (included in the kit), and a Duolink positive spot was observed and photographed using a fluorescence microscope (Keyence).
As a result, the interaction between V-1 and CP was hardly detected in the dopamine nerve at 14 days after birth, but was clearly detected at 49 days of age, and a marked increase was observed. However, it became clear after that that it decreased remarkably with aging (512 days of age) (FIG. 12).

Effect on expression of complex of electron transport system (oxidative phosphorylation) in mitochondria in mouse midbrain 17-month-old male C57BL / 6N mice orally treated with N-skin, or NMDA reception in previous report (Yamamoto et al. 2016) Somatic NR2D subunit knockout homozygous C57BL / 6N mice (approximately 2 months old male adult) were used. In the above 17-month-old mice, N-skin was orally administered (0.5 g / kg / day) for 2 weeks. Thereafter, the midbrain region was removed from each animal used, homogenized in sample buffer (50 mM Tris-HCl, pH 7.5, 100 mM DTT, 2% SDS, 10% Glycerol), and 95 minutes at 95 ° C. After boiling for 15 minutes (for mitochondrial blot analysis) at 50 ° C. (for Western blot analysis of total ERK), ultrasonic disruption (Bioraptor) was performed. After centrifugation (13 krpm, 10 minutes, room temperature), the obtained supernatant (protein amount 20 μg / well) was separated by SDS-PAGE (12% separation gel) and subjected to Western blot analysis.
Total OXPHOS Rodent WB antibody Cocktail (Abcam, ab110413) was used for detection of each complex constituent subunit of mitochondria. After separating the midbrain tissue protein by SDS-electrophoresis as described above, the protein was transferred from the gel to the PVDF membrane, and the blot was blocked at room temperature for 1 hour while shaking in PBS (-) containing 1% skim milk. Thereafter, the mixture was reacted overnight at 4 ° C. with the same antibody diluted to 250 μg / mL with PBS (−) containing 1% skim milk (250 times). Subsequently, after reaction with an anti-mouse HRP-labeled secondary antibody (CST, 1000-fold dilution), detection was performed by the ECL method. In addition, total ERK was used as protein loading control. After reacting overnight with an anti-p44 / 42 (Erk1 / 2) antibody (CST # 9102) diluted 1000-fold at 4 ° C, it was reacted with an anti-rabbit HRP-labeled secondary antibody (CST, diluted 1000-fold) and the ECL method described above The total amount of ERK was detected. About said experimental result, experiment was repeated at least twice and the reproducibility was confirmed.
As a result of examining the expression of each constituent subunit protein of complex I to V of mitochondrial electron transport system in the middle brain of aged mice by Western blotting, oral administration of N-Chan increases all the constituent subunit protein levels, As a result, it recovered to the expression level of the adult (FIG. 13, panel left). Furthermore, in adult mice lacking the NMDA receptor subunit NR2D (NR2D knockout mice), which is expressed in the midbrain dopamine neurons of adult mice, the above complex I to V is extracted using the midbrain tissue extract. A decrease in the expression level of each component subunit was observed (FIG. 13, right panel). This suggests that mitochondrial homeostasis in the midbrain dopamine neurons is dependent on NMDA receptor subunit NR2D.

According to the present invention, a pharmaceutical composition for the treatment of central neurodegenerative diseases is provided. In addition, the present invention can provide a screening method for candidate substances useful for maintaining and / or enhancing mitochondrial homeostasis.

Claims (7)

A pharmaceutical composition for treatment of a central neurodegenerative disease, comprising at least one selected from the group consisting of nicotine, nobiletin, cinecetin, and chimpi, wherein the central neurodegenerative disease is accompanied by mitochondrial dysfunction The said pharmaceutical composition characterized by these. The pharmaceutical composition according to claim 1, wherein the central neurodegenerative disease is Alzheimer's disease associated with mitochondrial dysfunction, Parkinson's disease associated with mitochondrial dysfunction, or cerebrovascular dementia associated with mitochondrial dysfunction. The pharmaceutical composition according to claim 2, wherein the treatment is promotion of formation of a complex of V-1 protein and actin capping protein. At least one compound selected from the group consisting of nicotine, nobiletin, cinecetin and chimpi for use in the treatment of mitochondrial dysfunction. The compound according to claim 4, wherein the mitochondrial dysfunction is Alzheimer's disease associated with mitochondrial dysfunction, Parkinson's disease associated with mitochondrial dysfunction, or cerebrovascular dementia associated with mitochondrial dysfunction. The compound according to claim 5, wherein the treatment is promotion of formation of a complex of V-1 protein and actin capping protein. Contacting a test substance with a nerve cell;
Determining the level of the complex of V-1 protein and actin capping protein and / or the expression level of COX I in the nerve cells contacted with the test substance,
When the determined level and / or expression level is higher than the level of the complex of V-1 protein and actin capping protein and / or the expression level of COX I in a nerve cell not in contact with the test substance, Selecting the test substance as a candidate substance useful for maintaining and strengthening mitochondrial homeostasis,
A method for screening candidate substances useful for maintaining and / or enhancing mitochondrial homeostasis.

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