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Medicinal compositions

A technology for compositions and drugs, applied in drug combinations, pharmaceutical formulations, animal/human peptides, etc., and can solve problems such as unclear detailed mechanisms

Inactive Publication Date: 2005-03-09
TAISHO PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although PS2V shows the possibility of being one of the important factors of neuronal death in sAD, the detailed mechanism of PS2V generation and disease crisis is still not very clear.

Method used

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Examples

Experimental program
Comparison scheme
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experiment example 1

[0160] Experimental example 1 Cell culture, hypoxic stimulation and transient transfection

[0161] According to the literature of Sato et al. [Sato N et al., J. Biol. Chem., 276, 2108-2114 (2001)], cell culture and hypoxic stimulation were performed as follows.

[0162] Human neuroblastoma SK-N-SH cells were cultured in αMEM (GIBCO BRL) containing 10% fetal bovine serum in 5% CO 2 , Culture at 37°C. The above cells are at 176.6cm 2 When the dishes were confluent, the cultures were replaced with serum-containing α-MEM for serum-free α-MEM. The medium-changed cultures were incubated for an additional 4 hours.

[0163] HEK-293T cells and HeLa cells were cultured in Dulbecco's minimal medium (GIBCO BRL) containing 10% fetal bovine serum in 5% CO 2 , Culture at 37°C. The above cells are respectively at 176.6cm 2 At confluence in the dish, the culture was replaced with serum-free Dulbecco's minimal medium in the culture. The medium-changed cultures were incubated for an addi...

experiment example 2

[0167] Experimental example 2 Preparation of total RNA and RT-PCR

[0168] According to the literature of Sato et al. [Sato N et al., J.Neurochem., 72, 2498-2505, (1999)], prepare neuroblastoma SK-N-SH cells and HEK-293T cells under various stresses respectively as follows Cell and HeLa cells total RNA, and RT-PCR.

[0169] Using RNeasy total RNA kit (Qiagen), according to the manufacturer's instructions, total RNA was extracted and purified from SK-N-SH cells and HEK-293T cells in normoxia, hypoxia exposure or HMG-I overexpression, respectively. .

[0170]Next, a reverse transcription reaction was performed at 42° C. for 1 hour using the obtained total RNA and mouse Moloney leukemia virus reverse transcriptase (Promega). Nested PCR was then performed using the obtained reaction product as a template. The brief process of PCR is: 95°C for 30 seconds-60°C for 30 seconds-72°C for 1 minute (the final cycle is 2 minutes) as a cycle, a total of 30 cycles. Primer ps251 [5'-attca...

experiment example 3

[0172] Experimental Example 3 Preparation of nuclear extract

[0173] According to the modified method of Shreiber et al. [ Yoneda, Y. et al., Neuroscience, 90, 519-533 (1999 )], the nuclear extract was prepared as follows.

[0174] The buffers and other solutions used were filtered and sterilized through Steritop (Milipore) with a pore size of 220 nm before each use.

[0175] Unless otherwise specified, the homogenate of each cell structure substance was mixed with 10mM KCl, 1mM EDTA, 1mM EGTA, 5mM dithiothreitol (DTT) and 1mM (p-amidinophenyl) methanesulfonyl fluoride (PMSF). 50 volumes [315 μl / plate] of 10 mM HEPES-NaOH (pH 7.9) at 2°C.

[0176] Next, 10% ethylphenyl polyethylene glycol (Nonidet P-40) was added to the obtained homogenate so that the final concentration was 0.6%. The resulting mixture was centrifuged at 15000 rpm for 5 minutes. Next, the obtained precipitate was suspended in 10 volumes [0.1 ml] of 20 mM Tris-HCl (pH 7.5) containing 400 mM KCl, 1 mM EDTA, ...

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Abstract

To provide a means useful for treating or preventing a disease such as a brain disorder or a neurodegenerative disease even more efficiently and even more sustainedly. The present invention relates to an inhibitor capable of inhibiting a binding between HMG-I protein and exon 5 of presenilin-2 mRNA, an agent for suppressing neuronal death, capable of suppressing neuronal death, a pharmaceutical composition which is useful for treatment or prevention of a disease caused by the generation of a splice variant that lacks exon 5 of presenilin-2 mRNA, a method for treating or preventing the disease and a use of the inhibitor.

Description

technical field [0001] The invention relates to an inhibitor capable of hindering the combination of HMG-I protein and presenilin-2 mRNA exon 5, a neuron death inhibitor capable of inhibiting neuronal cell death, and splicing variation caused by presenilin-2 mRNA exon 5 defect An effective pharmaceutical composition for the treatment or prevention of a disease caused by the production of the body, a method for the treatment or prevention of the disease, and the use of the inhibitor. Background technique [0002] In Alzheimer's disease (AD), one of the neurodegenerative diseases, the disease of more than 90% of AD patients is classified as sporadic Alzheimer's disease (sAD). An abnormal protein (PS2V) encoded by a variant of the presenilin-2 gene is known to exist in apoptotic pyramidal cells of the cerebral cortex and hippocampal CA1 region of sAD patients, respectively [Sato, N. et al., J. Biol. Chem., 276, 2108-2114 (2001)]. [0003] PS2V was shown to be induced by hypox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K31/7105A61K31/711A61K38/00A61K45/00A61K48/00A61P9/10A61P25/00A61P25/16A61P25/28A61P43/00C07K14/47
CPCC07K14/4711C07K14/4702A61K48/00A61K38/00A61P21/04A61P25/00A61P25/16A61P25/28A61P43/00A61P9/10
Inventor 远山正弥片山泰一真部孝幸今泉和则池田阳子
Owner TAISHO PHARMACEUTICAL CO LTD
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