CN105169414A - Application of AMO-143 in preparation of medicines for treating long QT syndrome - Google Patents

Abstract

The invention discloses an application of AMO-143 in preparation of a pharmaceutical composition for preventing and treating a long QT syndrome and promoting hERG expression, and the pharmaceutical composition for treating the long QT syndrome. The pharmaceutical composition is characterized by containing the AMO-143.

Description

The application of AMO-143 in preparation treatment long QT syndrome medicine

Technical field

The present invention relates to the application of AMO-143 in preparation treatment long QT syndrome medicine, more particularly, the present invention relates to AMO-143 and preparing the application in the medicine promoting hERG to express in treatment long QT syndrome.

Background technology

Long QT syndrome (LQTS) is the one group of clinical syndrome caused because coding Myocardial ion channel protein gene mutation causes myocardial cell membrane ion channel dysfunction, clinically with QT interval prolongation and ventricular tachycardia, torsade de pointes (Torsadedepoints, TdP) and paroxysmal syncope, SCD for feature.What LQTS caused because of gene mutation is referred to as congenital LQTS (congenitalLongQTSyndrome, cLQTS), and the day after tomorrow obtain produce be referred to as acquired LQTS (acquiredLongQTSyndrome, aLQTS).13 genes are found that there is up to now relevant with cLQTS, wherein hERG (huamnether-a-Go-Gorelatedgene, hERG) the congenital long QT of 2 type that sudden change causes levies (LQT2) and accounts for always suddenly change 45%, is the second common mutations gene.In clinical position, find that in institute, SCD a big chunk reason, all caused by aLQTS, has higher mortality rate because itself and the morbidity of Torsade de points are closely related.The generation of current known aLQT is relevant to many factors, and these factors comprise medicine, bradycardia/speed, coronary heart disease, myocardial hypertrophy, heart failure, electrolyte exception and genetic predisposition, and wherein the aLQTS of drug-induced is common cause.

Research shows that the quick active Delayed Rectifier Potassium Current (rapidlyactivatingcomponentofdelayedrectifierpotassiumcur rent, IKr) of hERG gene code plays a crucial role in normal action potential process of repolarization.HERG gene expression amount reduces or passage dyssynthesis, gate characteristic changing all can make I _krfunction is lowered, and the outer phase current minimizing of repolarization causes lengthening of action potential, finally causes LQT2 and aLQTS.It simultaneously or multi-medicament produces the molecular target of heart condition reaction, is closely related the most with QT interval prolongation and TdP.

The Therapeutic Method of current LQTS mainly takes Drug therapy, left cardiac sympathetic nerve remove art and implant cardioverter-defibrillator, but these methods all exist limitation, and can not effect a radical cure.Therefore find effective treatment means, there is important clinical meaning.And take gene therapy to be one of method of radical cure in hereditary.

Microrna (miRNA) is the endogenous non-coding tiny RNA that a class is about 21-25nt, by combining with mRNA complementation, form RNA and induce silencing complex (RNA-inducedsilencingcomplex, RISC), the expression of target gene is regulated at post-transcriptional level, translation or the promotion mRNA of Profilin matter decompose, thus play reverse regulating action to the expression of target gene.In recent years, a series of research confirms, miRNA can targeting channel protein, and causing ion channel dysequilibrium, bring out arrhythmia, is potential Arrhythmia target spot.At present the research that ion channel expresses directly or indirectly being affected on miR-1 and miR-133 more, causing ARR generation mainly through suppressing the target gene of direct coding ion channel.Use the function that can suppress corresponding miRNA with the oligonucleotide sequence AMOs of corresponding miRNA complementation.Research finds that ARR generation is relevant with miRNA-1 up-regulated under several pathological condition, and studies discovery AMO-1 reversible miRNA-1 at present to the toxic action of heart, proves that it can be used as the potential treatment means of ischemic arrhythmia.

Summary of the invention

HERG gene is positioned on No. 7 chromosomes, and the alpha subunit of IKr passage of encoding in heart plays an important role in action potential 3 phase process of repolarization.Research has proved that hERG changing function can cause the generation of LQT2.In current technology, there is no medicine by promoting that hERG expresses and LQTS.The present inventor utilize-RegRNA website, bioinformatics website ( http:// regrna.mbc.nctu.edu.tw), input the target gene hERG determined, dope than score value the miRNA that several are relevant to hERG according to corresponding, selected miR-143, and make antisense nucleotide AMO-143 according to its nucleotide sequence.When carrying out intervention experiment, finding that AMO-143 can in the expression promoting hERG, treatment arrhythmia, thus completing the present invention.

The change of the gene expression profile relevant to arrhythmia can affect heart pathology and physiology, the function of directly or indirectly impact.A lot of molecule can change hERG channel protein function, but these regulatory mechanisms lack specificity mostly, and it can change gate or the dynamic characteristic of other ion channel simultaneously.MiRNAs can mediated gene transcribe after negative regulation, miRNA can targeting channel protein to have research to confirm, causing ion channel dysequilibrium, bring out arrhythmia, is potential Arrhythmia target spot.In research before, have report miR-212 can lower Kir2.1 and express, obviously change the density of IKr, but whether miRNAs works and it be unclear that in the pathogenic process of LQT2 and aLQTS.

Our research shows the negative regulate of hERG gene by miR-143.Bioinformatics Prediction hERG and miR-143 has binding site in coding region, and two luciferase report gene display miR-143 can be combined with this site of hERG.In this experiment, process LAN miR-143 reduces mRNA and the protein expression level of hERG.Protein degradation may be that it is determined by the combination degree of miRNA and genes of interest again because mRNA degrades or suppresses caused by its translation.The miRNA of a lot of animal can not be combined completely with its object mRNA, usually suppresses the synthesis of destination protein, but its mRNA that but do not degrade.And miR-143 suppresses consistent with protein degradation levels to hERGmRNA, may overlap with its match height, with the hERGmRNA that degrades for main relevant.Use the specific inhibitor AMO-143 of miR-143 can correct the inhibitory action of miR-143 to hERGmRNA and protein expression.The experimental result of laser co-focusing is tested with qRT-PCR and Westernblot and is conformed to.To sum up this experimental verification miR-143 can targeting hERG, negative regulation its express, its AMOs can suppress this regulation and control, recovers the expression of hERG.Accordingly, we intend with miR-143 is potential therapy target, by disturbing its level, and the expression of regulation and control target, thus treat corresponding arrhythmia.Research before shows that siRNA can correct E637K electric current and recover its dynamic characteristic.Therefore, this new AMOs (AMO-143) can as the new specific treatment regimens of heritability or acquired long QT syndrome.We need in animal or human's myocardial cell, verify the regulating and controlling effect of these miRNA to hERG.

Detailed description of the invention

Materials and methods

1.1 experimental apparatus and reagent

1.1.1 major experimental instrument

Table 1 experimental apparatus title and manufacturer

1.1.2 experimental drug and reagent

Table 2 experimental drug title and manufacturer

1.2 experimental technique

Real-time fluorescence quantitative PCR

From the U2OS cell of transfection, total serum IgE is extracted with Trizol reagent.By RNA subsequently with aseptic Rnase-free process, the level of hERG gene mRNA uses the assessment of Taqman probe to detect with qRT-PCR.Primer and probe are from ABI company cat.#4331182 (numbering: Hs04234270-g1).Reverse transcription condition be set as initial step 95 DEG C 10 minutes, subsequently 95 DEG C of 40 circulations in 15 seconds, and at 60 DEG C maintain 1 minute.Real-time PCR reactions independently repeats three times.Result 2-Δ Δ CT relative quantitation method analyzes 30.

Westernblot

Extract the step immunoblotting assay of hERG albumen by previously described.In brief, first in transfection pcDNA-hERG to U2OS cell.The expressed further transfection of hERG cell miRNA or miRNA separately adds corresponding AMOs.Transfection is harvesting after 48 hours.The albumen of equivalent is poured in the 7.5%SDS polyacrylamide gel prepared, then transfers on nitrocellulose filter.Membrane closure with 5% defatted milk powder, then by antibody overnight incubation at 4 DEG C of the anti-hERG of rabbit polyclonal.In TBST, film is washed three times, then at room temperature hatch 1 hour with goat anti-rabbit igg.Trace process with SuperSignalWestPico chemical luminous substrate (PierceBiotechnology company, the U.S.), and uses the imaging system of GE company.

Experiment shows, we adopt Bioinformatics Prediction, utilize luciferase reporter gene method, qRT-PCR, Westernblot, laser co-focusing to demonstrate miR-143 can express by negative regulate hERG, corresponding AMOs can reticent its to the inhibitory action of hERG.Our experiment be understand further hERG albumen regulatory mechanism and from gene angle long QT syndrome treated and provides new visual angle.But, these results of study need animal model, preclinical and clinical in verify further.

Claims (8)

1.AMO-143 promotes the application in the pharmaceutical composition that hERG expresses at preparation control long QT syndrome.

2. apply as claimed in claim 1, wherein said AMO-143 is the Antisensedigonucleotsequence sequence of miR-143.

3. apply as claimed in claim 1 or 2, wherein said long QT syndrome is selected from the congenital long QT of 2 type and levies, one or more in acquired long QT syndrome.

4. apply as claimed in claim 1 or 2, wherein said long QT syndrome is ARR one, can cause sudden cardiac death.

5. treat a pharmaceutical composition for long QT syndrome, it is characterized in that containing AMO-143.

6. pharmaceutical composition as claimed in claim 5, wherein said AMO-143 is the Antisensedigonucleotsequence sequence of miR-143.

7. the pharmaceutical composition as described in claim 5 or 6, wherein said described long QT syndrome is selected from the congenital long QT of 2 type and levies, one or more in acquired long QT syndrome.

8. the pharmaceutical composition as described in claim 5 or 6, wherein said described long QT syndrome is ARR one, can cause sudden cardiac death.