JP2019094304A - Autophagy derivative - Google Patents

Abstract

To provide a new autophagy derivative and a new Parkinson disease preventive and therapeutic agent.SOLUTION: An autophagy derivative contains a component selected from memantine, clemastine and a salt thereof as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to an agent for inducing autophagy and a therapeutic agent for preventing and treating Parkinson's disease.

  Parkinson's disease (hereinafter referred to as PD) is the second most common neurodegenerative disease in Japan, and in addition to progressive movement disorder, non-exercise symptoms (cognitive decline, psychiatric symptoms, autonomic nervous symptoms) etc progress over time and develop As they become bedridden after 15-20 years, they cause serious damage to social assets due to the large burden of medical and nursing care. Although early detection and early treatment intervention is considered important to realize the radical treatment of this disease, it has already been noted that 30% or more of the substantia nigra neurons have dropped out when motor symptoms appear, and it is 5-10 About 70% of the substantia nigra neurons drop out after 1 year, so a radical treatment such as prevention of the onset and suppression of progression will be carried out by performing medium- to long-term treatment intervention before the onset or at the very early onset to about 5-10 years. Is considered necessary.

  Analysis of familial PD causative gene product functions has been vigorously advanced since 1997 as a PD onset mechanism, and eight of 20 responsible genes identified by November 2016, sporadic PD risk gene products About 50% of functions are also considered to be involved in the broad autophagy lysosome system. In autophagy, isolated membranes generated from rough endoplasmic reticulum / mitochondria contact sites in cells surround a substrate to form a double membrane structure called autophagosomes, which fuse with lysosomes (including an internal hydrolytic enzyme) It becomes an autolysosome with a single membrane structure, and refers to a series of flows in which the inner substrate is degraded. Since α-synuclein, which is a cause of neurodegeneration in Parkinson's disease and the like, is included as one of the substrates, certain expectations have been drawn for PD therapeutic drug development by autophagy promotion (Non-patent Document 1).

J. Neurol. Neurosurg. Psychiatry 83, 430-436, doi: 10.1136 / jnnp-2011-301205 (2012)

  An object of the present invention is to provide a novel autophagy inducing agent and an agent for preventing and treating PD by autophagy induction.

  Therefore, the inventor of the present invention has studied to find an ingredient having autophagy-inducing effect among ingredients (557 species) already used as a medicine and effective for preventing and treating Parkinson's disease. The present inventors have found that memantine used for suppressing the progression of cognitive symptoms of Alzheimer's disease and clemastine used as an antihistamine have excellent autophagy inducing action and Parkinson's disease preventing and treating action based on the above, and completed the present invention.

  That is, the present invention provides the following [1] to [8].

[1] An autophagy inducer comprising a component selected from memantine, clemastine and salts thereof as an active ingredient.
[2] An agent for preventing and treating Parkinson's disease, which comprises a component selected from memantine, clemastine and salts thereof as an active ingredient.
[3] Use of a component selected from memantine, clemastine and their salts for producing an autophagy inducer.
[4] Use of a component selected from memantine, clemastine and salts thereof for the preparation of a preventive and therapeutic agent for Parkinson's disease.
[5] A compound selected from memantine, clemastine and their salts for use in induction of autophagy.
[6] A compound selected from memantine, clemastine and their salts for use in the preventive treatment of Parkinson's disease.
[7] A method of inducing autophagy comprising administering an effective amount of a component selected from memantine, clemastine and salts thereof.
[8] A method for preventing and treating Parkinson's disease, which comprises administering an effective amount of a component selected from memantine, clemastine and salts thereof.

  According to the present invention, autophagy is induced by administering a safety-established component which has been used in clinical practice, and autophagy in dopamine neurons derived from Parkinson's disease patients who are deeply involved in Parkinson's disease. It can also be strongly induced to provide a preventive and therapeutic agent for Parkinson's disease.

The autophagy induction effect (LC3-II rise) of memantine is shown. Autophagy induction can be confirmed by the additive effect on LC3-II elevation by the combined use of lysosomal inhibitors. It shows the production promoting effect of memantine on autolysosomes. The time course of the autophagy induction effect in each concentration of memantine is shown. It shows that memantine has no mTORC1 inhibitory action. The effect of mitochondrial autophagy in iPS cell-derived neurons of memantine is shown. Figure 7 shows the autophagy-inducing effect of clemastine. Fig. 6 shows the autolysosomal production promoting effect of clemastine.

  The active ingredients of the autophagy inducer and the agent for preventing and treating Parkinson's disease of the present invention are components (compounds) selected from memantine, clemastine and salts thereof.

  Memantine is the chemical name 1-amino-3,5-dimethyladamantane. Memantine has an NMDA (N-methyl-D-aspartate) receptor channel inhibitory action that is a subtype of glutamate receptor and is used to suppress the progression of cognitive symptoms in moderate and severe Alzheimer's disease . However, the action of memantine on autophagy and the action on Parkinson's disease are not known.

Clemastine is the chemical name (2R) -2- {2-[(1R) -1- (4-chlorophenyl) -1-phenylethoxy] ethyl} -1-methylpyrrolidine. Clemastine is an antihistamine agent having an H ₁ receptor antagonistic action, and is used as a therapeutic drug for allergic skin diseases, allergic rhinitis, sneezing, nasal discharge and the like. However, the action of clemastine on autophagy and Parkinson's disease is not known.

  Examples of salts of memantine and clemastine include mineral acid salts such as hydrochlorides, sulfates and nitrates, and organic acid salts such as acetates, fumarates, citrates and tartrates. Among these, hydrochloride is preferable for memantine and fumarate is preferable for clemastine.

As shown in the examples below, memantine and clemastine increase the expression level of LC3-II, which is a marker for autophagy, and show an additive effect in the presence of E64d and pepstatin A to induce autophagy. In addition, immunohistochemistry shows an increase in LC3-positive granules (autophagosomes or autolysosomes) and increases co-localization pressure, thereby increasing autophagy flux. Furthermore, memantine and clemastine show mitochondrial autophagy (mitophage) inducing effects in dopamine neurons derived from Parkinson's disease patients, and are useful as preventive and therapeutic agents for Parkinson's disease.
The agent for preventing and treating Parkinson's disease according to the present invention is a symptom of Parkinson's disease, such as autonomic nervous disorders such as tremor at rest, muscle rigidity, peristalsis / kinesis, postural reflex disorder, constipation, and neurological symptoms such as depression. Control the occurrence of and improve these symptoms.

  When memantine, clemastine or a salt thereof is used as a medicine, it can be administered orally or parenterally. Memantine, clemastine or a salt thereof can be made into a pharmaceutical composition by being combined with a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers, known ones such as excipients, binders, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, preservatives and the like can be used. It can be formulated by the usual methods.

  Examples of preparations for oral administration include tablets (including coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like.

  The preparation for oral administration can be prepared according to a known method by incorporating additives commonly used in the field of preparation. Examples of such additives include excipients such as lactose, mannitol and anhydrous calcium hydrogen phosphate; binders such as hydroxypropyl cellulose, methyl cellulose and polyvinyl pyrrolidone; disintegrants such as starch and carboxymethyl cellulose; magnesium stearate, Lubricants such as talc and the like can be mentioned. Parenteral administration can be administered as an injection, a preparation for rectal administration, a topical administration and the like, and among them, an injection is preferred.

  Injections include, for example, sterile solutions or suspensions. These injections are produced, for example, by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in water for injection by Japan Post. If necessary, an isotonicity agent such as sodium chloride; a buffer such as sodium dihydrogen phosphate or sodium monohydrogen phosphate; a solubilizing agent etc. may be blended. In addition, it can be used as an injectable preparation in a soluble form (powder filling, lyophilization), and in this case, it can be manufactured by an ordinary method by adding an excipient such as mannitol or lactose.

  Suppository etc. are mentioned as a preparation for rectal administration. The suppository is produced, for example, by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in a base such as cocoa butter or macrogol, and pouring it into a mold for molding. Alternatively, the liquid or cream may be placed in a container for injection to give a preparation for rectal administration.

  The topical administration preparations include solutions, eye drops, creams, ointments, gel preparations, sprays, powders and the like. The liquid preparation may be produced by adding the compound of the present invention or a pharmaceutically acceptable salt thereof to water, and adding a stabilizer, a solubilizer, a thickener, a dispersant, a suspending agent, etc. as necessary. it can. As this thickener, gelatin, sodium hyaluronate, high molecular weight dextran, sodium alginate, sodium chondroitin sulfate and the like can be used. Eyedrops can be produced by adding a preservative, in addition to a buffer, pH adjuster, tonicity agent. Creams and ointments can be prepared using aqueous or oily bases such as water, liquid paraffin, vegetable oils (peanut oil, castor oil, etc.), macrogol and the like. The gel preparation may be gelatin, pectin, carrageenan, agar, tragacanth, alginate, cellulose ether (methylcellulose, sodium carboxymethylcellulose etc.), pectin derivative, polyacrylate, polymethacrylate, polyvinyl alcohol, polyvinyl pyrrolidone etc. Can be manufactured. A spray can be prepared by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in water or the like, and then placing it in a spray container. In the case of a powder, the compound of the present invention or a pharmaceutically acceptable salt thereof can be used as it is, but can be produced by mixing it with a suitable excipient.

  The dose of memantine, clemastine or a salt thereof is appropriately determined according to individual cases in consideration of the target disease or condition, the age, body weight, sex and the like of the administration subject. In general, when orally administered, the dose of memantine, clemastine or a salt thereof per day for an adult (about 60 kg body weight) is 0.01 to 100 mg, preferably 0.01 to 30 mg, more preferably 0.1 to 10 mg. It is administered once or in 2-4 divided doses. In the case of intravenous administration, the daily dose for an adult is usually 0.03 to 3000 μg, preferably 0.03 to 300 μg, more preferably 0.03 to 30 μg per kg body weight, and 1 day 1 It is divided into multiple doses and administered.

  The present invention will now be described in more detail by way of examples.

Example 1
A compound library (hereinafter referred to as existing drug library) which has already been clinically applied consists of about 1,200 species of compounds independently collected by the inventor Hideyuki Satani. The autophagy-inducing effects of the same compounds were examined sequentially for 557 species. GFP (tag that emits green fluorescence) -LC3 (protein that serves as an indicator of autophagy) stably expressing HeLa cells (see Autophagy 7: 176-187, 2011) were seeded at 0.1 × 10 ⁵ / well in 96-well plates . Drugs of the existing drug library (10 μM each) were added to the cells, and after 24 hours, GFP color development was observed with a fluorescence microscope using Floid (manufactured by Life Technologies). Torin1 (5 μM) as a positive control (showing GFP-LC3 positive granules increase by inducing autophagy), negative control (block the final step of autophagy, and inhibit the degradation of GFP-LC3 positive granules) BafA1 (100 nM) was used as positive granules are increased). LC3 dot positive cells (the number of cells in which the number of LC3 dots increases to 5 or more among 100 GFP positive cells) is determined to be 30% or more as the primary hit compound (standard: J Biol Chem 282: 564 1, 2007 )did. Add E64d (10 μM) + pepA (10 μM) together with each primary hit compound to the presence or absence of the autophagy induction effect from the primary hit compounds group for which the autophagy induction effect has not been reported in literature (69 types) Set up conditions / conditions not added, collect cells after 24 hours, extract protein using RIPA buffer, evaluate expression of LC3-II and actin by Western blotting, confirm autophagy induction effect As a secondary hit compound.
Secondary hit compounds were clomiphene hydrochloride, memantine hydrochloride and clemastine fumarate.

Example 2
The effect of inducing autophagy was examined for memantine. As shown in FIG. 1, memantine elevates the expression level of LC3-II at 10 μM to 40 μM, and at each concentration, an additive effect is observed in the LC3-II expression level in the presence of E64d and pepstatin A. It was thought to induce. Furthermore, memantine was added to HeLa cells in a state where GFP-RFP-LC3 plasmid was transiently expressed by lipofection. The increase in both yellow autophagosome and red autolysosome was confirmed by starvation as a positive control, and it was confirmed that only yellow autophagosome accumulated by addition of Bafilomycin A1 for 4 hours as a negative control. Under the conditions of this experiment, it was considered that autophagy flux was increased because memantine (100 μM) increased the LC3 positive granules (autophagosomes or autolysosomes) in yellow and red (FIG. 2).

  Next, the time required to induce autophagy was examined. As shown in FIG. 3, memantine was added at a concentration of 1 to 100 μM, and SH-SY5Y cells were recovered after 4 hours, 8 hours and 24 hours, and when LC3-II elevation was examined by western blotting, it was at least 8 hours The increase effect of LC3-II was recognized by the addition of 3 μM memantine. At 8 hours and 24 hours, autophagy induction was observed in a dose-dependent manner.

  Furthermore, when the involvement with the PI3K / Akt / mTORC1 pathway was examined, it was thought that autophagy was induced by a mechanism different from the pathway, since no particular change was observed in p-p70S6K or p-S6K. ).

  Furthermore, in order to confirm the mitochondrial autophagy (mitophage) induction effect in dopamine neurons, dopamine neurons induced to differentiate from iPS cells derived from hereditary Parkinson's disease (PARK2, PARK6) were used. In this cell line, PINK1 / parkin-mediated mitophagy can not be induced because each lacks expression of parkin and PINK1 proteins (Imaizumi Y, Okada Y, Akamatsu W, Koike M, Kuzumaki N, Hayakawa H , Nihira T, Kobayashi T, Ohyama M, Sato S, Takanashi M, Funayama M, Hirayama A, Soga T, Hishiki T, Suematsu M, Yagi T, Ito D, Kosakai A, Hayashi K, Shouji M, Nakanishi A, Suzuki N, Mizuno Y, Mizushima N, Amagai M, Uchiyama Y, Mochizuki H, Hattori N, Okano H. Mitochondrial Dysfunction Associated with Incremental Oxidative Stress and s Synuclein Accumulation in PARK 2 iPSC-derived Neuron Brain Muscle Membranes 5: 35 (2012), Shiba-Fukushima K, Ishikawa KI, Inoshita T, Izawa N, Takanoshi M, Sato S, Onodera O, Akamatsu W, Okano H, Imai Y, Hattori N. Evidence that phosphorylated ubiquitin signaling is involved in Hum Mol Genet. 26: 3172-3185 (2 The etiology of Parkinson's disease. 017)). iPS cells are induced to dopamine neurons, and 30 μ MCCCP (mitochondrial uncoupling agent) is added to induce mitophagy 10 days after nerve induction, fixed 48 hours later, β-III-tubulin (neural marker) and Complex III- Immunostaining with core 1 (mitochondrial marker) was performed to evaluate mitophagy by quantifying mitochondrial area in neurons. When observation was made 48 hours after adding 10 μM memantine together with CCCP to each cell, the mitochondrial area (total area of the portion stained with the antibody showing the inner mitochondrial membrane) decreased, and therefore, the memantine involved PINK1 / parkin Not, it was confirmed to induce PINK1 / parkin non-mediated mitophagy (FIG. 5). These results demonstrate that memantine induces autophagy in cultured neurons and iPS cell-derived neurons.

Example 3
Next, an additive effect was observed for the expression level of LC3-II in the presence of E64d and pepstatin A also for clemustine 10 μM (Fig. 6), and endogenous LC3-positive granules (autophagosomes or autolysosomes) were increased by immunocytostaining. Since co-localization of endogenous LC3 positive granules and LAMP2 positive granules (lysosome or autolysosome) was increased (FIG. 7), it was confirmed that autophagy flux was promoted.

Claims (2)

  An autophagy inducer comprising a component selected from memantine, clemastine and their salts as an active ingredient.   An agent for preventing and treating Parkinson's disease, which comprises as an active ingredient a component selected from memantine, clemastine and salts thereof.