JP6912072B2 - Pharmaceuticals for the prevention or treatment of frontotemporal dementia - Google Patents

Description

The present invention relates to a tau protein aggregation inhibitor containing phorbol ester as an active ingredient, and a pharmaceutical for preventing or treating neurodegenerative diseases caused by tau protein aggregation containing such a tau protein aggregation inhibitor.

The increase in dementia patients is a global medical problem centered on developed countries. The number of dementia patients is expected to increase rapidly in Japan as well, and there are concerns that the number will reach 3.5 million within 20 years and that the prevalence of people aged 65 and over will rise to 10% or more. Approximately 70% of patients with this dementia are patients with Alzheimer's disease.

Tau protein and beta-amyloid protein are known as proteins involved in Alzheimer's disease. Tau protein is a 55-60 kD protein that resides in nerve cells and binds to microtubules. Abnormal phosphorylation of such tau protein results in the formation of tau protein aggregates, resulting in lesions called neurofibrillary tangles. On the other hand, beta-amyloid protein exists outside nerve cells and aggregates to cause lesions called amyloid plaques.

Since the beta-amyloid theory has been the mainstream from the basic research so far, vaccine therapy based on the establishment of antibodies against beta-amyloid (AN-1792) and inhibitors of beta-amyloid-producing enzymes (gamma-secretase inhibitor: Semagacestat) Etc. have been developed. However, despite the reduction or disappearance of amyloid plaque, the former vaccine therapy resulted in progressive impairment of cognitive function, and the latter enzyme inhibitor resulted in further decline in cognitive function.

In addition to the above, various therapeutic agents have been developed for neurodegenerative diseases, but all of them actually used for treatment are symptomatic treatments, and development of therapeutic agents that lead to curative treatment. Has not been successful. Almost all neurodegenerative diseases are unmet medical needs, and in particular, in Alzheimer's disease, both treatment satisfaction and drug contribution to treatment are recognized as the worst 1 diseases (see Non-Patent Document 1). ). Therefore, in recent years, the development of dementia preventive drugs targeting tau protein has been further promoted.

By the way, phorbol ester is a natural plant-derived organic compound of the diterpene Tiglium family, and hydrolyzate production of croton oil derived from the seed of Croton tiglium, which is a foliar shrub of the family Croton tiglium native to Southeast Asia. It was first isolated in 1934 as a thing. Among the phorbols, phorbol-12-millistate-13-acetate (PMA) is considered to be a inducer of differentiation and / or apoptosis in multiple cell lines and primary cells, including malignancies, leukemia, HIV, and Parkinson. It is disclosed that it is used as a therapeutic agent for diseases and the like (see Patent Documents 1 and 2). However, the relationship between such phorbol ester and tau protein aggregation has not been known so far.

Special Table 2015-513315 Japanese Unexamined Patent Publication No. 2016-056192

JPMA New Letter No.140 (2010/2011)

As mentioned above, various therapeutic agents have been developed for neurodegenerative diseases, but all of them are actually treated as symptomatic treatments, and development of therapeutic agents that lead to curative treatment. Has not been successful. Therefore, an object of the present invention is to provide an agent that suppresses the aggregation of tau protein that causes Alzheimer's disease and frontotemporal dementia.

The present inventor first found that the heat shock transcription factor Heat Shock Transcription Factor 1 (HSF1) is involved in the aggregation of tau protein in the course of research on cognitive dysfunction. Therefore, we proceeded with further research and comprehensively investigated the substances involved in the activation of HSF1 based on the experience and intuition of the inventors. The present invention was completed.

That is, the present invention is as shown below.
(1) A tau protein aggregation inhibitor containing phorbol ester as an active ingredient.
(2) The tau protein aggregation inhibitor according to (1) above, wherein the phorbol ester is phorbol 12-millistart 13-acetate.
(3) A drug for preventing or treating a neurodegenerative disease caused by aggregation of tau protein containing the tau protein aggregation inhibitor according to (1) or (2) above.
(4) The prophylactic or therapeutic drug according to (3) above, wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease.

INDUSTRIAL APPLICABILITY According to the present invention, aggregation of tau protein can be suppressed. Further, by suppressing the aggregation of tau protein, it becomes possible to prevent or treat the neurodegenerative disease caused by the aggregation of tau protein.

It is a figure which shows the fluorescence micrograph of the mutant tau protein-introduced cell. FIG. 2A is a diagram showing the proportion of cells that produced aggregates when treated at each concentration of active HSF1. FIG. 2B is a diagram showing the results of Western blot analysis of cells treated at each concentration of active HSF1. It is a figure which shows the ratio of the cell which formed the aggregate when PMA was added to Neuro-2a culture medium at the same time as the addition of pEGFP-N1-tauR406W adenovirus vector. It is a figure which shows the ratio of the cell which formed the aggregate when PMA was added to the Neuro-2a culture medium 6 hours after the addition of pEGFP-N1-tauR406W adenovirus vector.

The tau protein aggregation inhibitor of the present invention is not particularly limited as long as it contains phorbol ester as an active ingredient. Here, tau protein exists in nerve cells and binds to microtubules55. It is a protein of ~ 60 kD. Abnormal phosphorylation of such tau protein forms aggregates of tau protein.

Other aspects of the present invention include (1) a method for suppressing tau protein aggregation, which comprises administering phorbol ester to a subject, and (2) phorbol for use as a tau protein aggregation inhibitor. Examples thereof include the use of esters and (3) phorbol esters in the preparation of tau protein aggregation inhibitors.

In the present invention, the phorbol ester is not particularly limited as long as it is an acid ester of phorbol, and horbol 12-millimeter start 13-acetate (PMA), horbol 12,13-dibutyrate, horbol 12,13-didecanoate, horbol. 13-Buchilate, Phorbol 12-decanoate, Phorbol 13-decanoate, Phorbol 12,13-Diacetate, Phorbol 13,20-Diacetate, Phorbol 12,13-Dibenzoate, Phorbol 12,13-Dihexanoate, Phorbol 12,13-dipropionate, Phorbol 12-Millistate, Phorbol 13-Millistate, Phorbol 12, 13, 20-Triacetate, 12-Deoxy Phorbol 13-Angelate, 12-Deoxy Phorbol 13-Angelate 20-Acetate, 12-Deoxy Phorbol 13-Isobutyrate , 12-Deoxyphorbol 13-isobutyrate 20-acetate, 12-deoxyphorbol 13-phenylacetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, Phorbol 12-Tigrate 13-decanoate, 12-deoxyphorbol 13-acetate, Phorbol 12-assetate, Phorbol 13-assetate and the like can be mentioned, and PMA can be preferably mentioned. PMA can be obtained by preparing from natural plants such as croton tiglium, chemically synthesizing it, or purchasing a commercial product.

The pharmaceutical for preventing or treating a neurodegenerative disease caused by the aggregation of tau protein of the present invention is not particularly limited as long as it contains the above-mentioned agent for suppressing the aggregation of tau protein of the present invention. The resulting neurodegenerative disease means a disease in which cognitive function is impaired due to aggregation of tau protein in nerve cells. Examples of such neurodegenerative diseases include Alzheimer's disease, frontotemporal dementia, and Down's syndrome, and Alzheimer's disease or frontotemporal dementia can be preferably mentioned. Frontotemporal dementia (FTDL) is mainly classified into FTLD-tau, FTLD-TDP, and FTLD-FUS as clinical types, and FTLD-Ttau includes Pick's disease, FTLD-17, and cortical basal nuclear degeneration. (CBD), progressive supranuclear palsy (PSP), silvery granule dementia (AGD), neurofibrillary senile dementia (SD-NFT), MSTD, WMT-GGI, FTLD-TDP Includes FTD-MND, FTD-TDP, and FTLD-FUS includes axial FTLD-U, BIBD, and NIFID.

The tau protein aggregation inhibitor of the present invention and the pharmaceutical for preventing or treating neurodegenerative diseases caused by the aggregation of tau protein of the present invention are pharmaceutically acceptable excipients usually used for formulation. , Binding agents, lubricants, disintegrants, preservatives, tonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, buffers and the like may be included. The dosage form of the preparation may be a solid preparation such as a powder or a granule.

The method for administering the drug for preventing or treating a neurodegenerative disease of the present invention is not particularly limited as long as the desired preventive or therapeutic effect on the neurodegenerative disease can be obtained, and is intravenous administration, oral administration, intramuscular administration, subcutaneous administration, etc. Percutaneous administration, nasal administration, transpulmonary administration and the like can be mentioned. In addition, the dose of the preventive or therapeutic drug for the neurodegenerative disease of the present invention is not particularly limited, and can be appropriately adjusted according to the physical condition, medical condition, body weight, age, sex, etc. of the subject or the subject animal. The daily dose varies depending on which of the above administration methods is used, but based on the difference in sensitivity, 10 to 500 μg, preferably 20 to 100 μg, and more preferably 40 to 60 μg can be mentioned. Furthermore, the preventive or therapeutic agent for a neurodegenerative disease of the present invention may be used in combination with another preventive or therapeutic agent for a neurodegenerative disease. The number of times of administration and the administration period of the preventive or therapeutic drug for the neurodegenerative disease of the present invention are not particularly limited, and the daily dose can be administered once or divided into several times a day. The origin and living body of the cells and tissues to be administered are not particularly limited, and are preferably mammals, and examples thereof include humans, monkeys, cows, horses, sheep, pigs, dogs, cats, rats, mice, and hamsters. And preferably humans can be exemplified. PMA has already been used for the treatment of leukemia, especially acute myeloid leukemia. For example, in the treatment of leukemia, treatment of leukocytes taken out from a patient with PMA 20 μM and ionomycin and then returned to the patient's body has become established. ing.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

<1. Preparation of mutant tau protein-introduced cells>
In order to analyze the aggregation inhibitory effect of tau protein, first, a model (cell) in which tau protein aggregates was prepared.

(Tau gene cloning)
Genomic DNA was extracted from HeLa cells of a human cervical cancer cell line cultured in DMEM medium (containing 10% fetal bovine serum (FBS) and antibiotics penicillin and streptomycin), and human tau protein was encoded by PCR. Obtained the cDNA to be used. The nucleotide sequence of the forward primer used for PCR is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the obtained cDNA is shown in SEQ ID NO: 3. Using the obtained cDNA as a template, the base was substituted so that arginine, which is the 406th amino acid of tau protein, was replaced with tryptophan, and a cDNA encoding a human mutant tau protein was obtained. The nucleotide sequence of the forward primer used for PCR for substitution is shown in SEQ ID NO: 4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 5. The PCR product was confirmed to contain the correct mutation by examining the nucleotide sequence of the cDNA. The cDNA encoding the human mutant tau protein was incorporated into the pEGFP-N1 vector to prepare a pEGFP-N1-tauR406W vector, and the cDNA of green fluorescent protein (GFP) was linked to the C-terminal side of the cDNA to fluoresce the GFP. The tau protein that can be visualized by is expressed. Further, the above pEGFP-N1-tauR406W vector was incorporated into a pShuttle-CMV vector and then an Ad-Easy vector, expressed in HEK293 cells, and the supernatant and cells were collected to prepare a pEGFP-N1-tauR406W adenovirus vector. The 1240th base in SEQ ID NO: 3 is "c" in the normal form, but when this is changed to "t", the 406th amino acid arginine (R) of tau protein is changed to tryptophan (W) ( R406W: Six types of tau protein are known, and the amino acid number conforms to the longest isoform of 441 amino acids by convention), which causes frontotemporal dementia (FTDL).

(Cell culture)
4 mL of the above DMEM medium and 1 × 10 ⁶ mouse neuroblast Neuro-2a cells were placed in a culture dish having a diameter of 6 cm and cultured overnight.

(Differentiation of Neuro-2a)
The culture medium of Neuro-2a in which the cells were placed and cultured for 24 hours was replaced with a medium containing 2% FBS and 20 μM retinoic acid, and cultured for 72 hours.

<2. Aggregation of mutant tau protein>
The pEGFP-N1-tauR406W adenovirus vector was added to the differentiated Neuro-2a medium and infected for 6 hours. A fluorescence micrograph of the obtained mutant tau protein-introduced cells is shown in FIG.

As shown in FIG. 1, it was revealed that the mutant tau protein forms aggregates in cultured neuroblasts.

<Agglutination suppression by active HSF1>
Activated HSF1 (Ad-HSF1 RDT: Nakai et al., Expressed in the adenovirus prepared by the inventors 6 hours after culturing the above-mentioned mutant tau protein-introduced cells in the differentiated Neuro-2a culture medium. EMBO J 19: 1545-1554 (2000)) was added to a concentration of 0, ^{2.0, 8.0, 20.0 × 10 6 pfu / mL, respectively, and cultured for 24 hours.} Further, the cultured cells were collected by centrifugation and pelletized, and then crushed with a buffer solution containing NP40 (manufactured by Nakarai Co., Ltd.) to prepare a mutant tau protein solution, and then the anti-HSF1 prepared by the inventors. Soluble tau protein and insolubilization using antibody HSF1j (Fujimoto et al., Molecular Cell 48: 182-194 (2012)), anti-GFP antibody (manufactured by Nakarai), and anti-betaactin antibody (manufactured by Sigma-aldrich). The amount of tau protein precipitated in the cells was detected. In addition, these cells were harvested and at the same time Western blots were performed on each cell to compare the amount of soluble tau protein and insolubilized and intracellularly precipitated tau protein. The results are shown in FIG.

FIG. 2A is the result of examining the proportion of cells having aggregates after culturing, and FIG. 2B is the result of Western blot analysis. According to the result of FIG. 2 (a), the aggregation of the human mutant tau protein in the cell is suppressed as the concentration of active HSF1 increases, and the result of FIG. 2 (b) increases the concentration of active HSF1. As a result, it became clear that the insoluble human mutant tau protein decreased.

<Agglutination suppression by PMA>
From the results shown in FIG. 2 above, it was clarified that the active HSF1 is involved in the aggregation of human mutant tau protein. Therefore, the present inventor suspects that there is a substance that suppresses the aggregation of tau protein among the enormous factors involved in the upstream or downstream pathway of HSF1, and based on the inventor's many years of experience and intuition, the compound was prepared. After narrowing down, we considered that PMA might be involved in the aggregation of human tau protein, and investigated the aggregation inhibitory effect of PMA.

(Addition of PMA)
Neuro-2a differentiated at the same time as the addition of pEGFP-N1-tauR406W adenovirus vector or 6 hours after the vector addition so that PMA (manufactured by Sigma-Aldrich) dissolved in DMSO (dimethyl sulfoxide) has a final concentration of 20 μM. It was added to the culture medium. As a control without PMA, DMSO was added so that the volume was the same.

(Judgment of the effect of PMA)
The cells expressing the mutant tau protein were counted using fluorescence as an index, and then the number of cells forming aggregates in the cells was counted. In addition, the percentage of cells that produced aggregates was compared between cells without PMA and cells with PMA added. The results of adding PMA to the Neuro-2a culture medium at the same time as the addition of the pEGFP-N1-tauR406W adenovirus vector are shown in FIG. 3, when PMA was added to the medium 6 hours after the addition of the pEGFP-N1-tauR406W adenovirus vector. The results are shown in FIG.

As shown in FIG. 3, when PMA was added to the medium at the same time as the addition of the pEGFP-N1-tauR406W adenovirus vector, the PMA was added to the medium 6 hours after the addition of the pEGFP-N1-tauR406W adenovirus vector as shown in FIG. When added, the proportion of cells that produced aggregates was also reduced. Therefore, it was clarified that PMA can suppress the aggregation of tau protein and decompose the aggregation of tau protein. In addition, PMA was usually used when stimulating a specific protein in combination with ionomycin, but from the results shown in FIGS. It became clear that there is.

When tau protein aggregates, it forms a structure called neurofibrillary tangle, which causes neuronal cell death, which is a neurodegenerative disease and dementia represented by Alzheimer's disease and frontotemporal dementia. Many studies have shown that it causes. From the results shown in FIGS. 3 and 4, PMA can be used not only to suppress the aggregation of tau protein but also to decompose it. Therefore, by using the PMA of the present invention, it is possible to prevent or treat, preferably prevent and treat, neurodegenerative diseases such as Alzheimer's disease.

The present invention can be used in the field of treatment or prevention of neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia.

Claims (2)

A drug for preventing or treating frontotemporal dementia caused by aggregation of tau protein containing phorbol ester as an active ingredient. The pharmaceutical agent for preventing or treating frontotemporal dementia according to claim 1, wherein the phorbol ester is phorbol 12-millistart 13-acetate.

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