CN104491879B - A kind of GEM 132 medical composition and its uses of miRNA 2861 - Google Patents

Abstract

The present invention relates to a kind of GEM 132 medical composition and its uses of miRNA 2861.MiRNA 2861 antisense base sequences are 5 ' CCGCCCGCCGCCAGGCCCC 3 '；It is 10 that pharmaceutical composition, which includes infection titer,¹⁶It is cholesterol, nano particle or liposome that PFU, which is overexpressed the antisense nucleoside acid vectors of miRNA 2861, virus or auxiliary material, the carrier,；Viral vector is the one or more in adenovirus vector, slow virus carrier, retroviral vector；The auxiliary material is the one or more in mannitol, phosphate buffer, physiological saline.By oral or injectable in prevention and treatment myocardial hypertrophy, myocardial fibrosis, coronary heart disease and heart failure.Drug regimen composition formula of the present invention is reasonable, and pharmacology is reliable, and manufacture craft is simple, and therapeutic effect is obvious, has a wide range of application, and use environment is friendly.

Description

A miRNA-2861 antisense nucleotide pharmaceutical composition and its application

Technical field:

The invention relates to a new drug of endogenous non-coding small RNAs and its application, in particular to a pharmaceutical composition of miRNA-2861 antisense nucleotide for treating heart disease and its application, and belongs to the technical field of biopharmaceuticals.

Background technique:

Cardiovascular diseases mainly include hypertension, coronary heart disease and congestive heart failure, etc. Cardiovascular disease is the number one killer of human health. Seventeen million people die of cardiovascular disease every year worldwide, and the mortality rate is close to The sum of all cancer mortality rates. With the improvement of people's living standards and the change of diet structure, the mortality rate of cardiovascular disease is on the rise, surpassing cancer as the number one cause of death. Prevention and treatment of cardiovascular disease is still a major task of medicine and biology. Cardiac hypertrophy is a general term for the thickening of myocardial tissue at the tissue level and the increase in the volume of cardiomyocytes at the cellular level. The disease is caused by the co-stimulation of some physiological and pathological factors, and it is a comprehensive manifestation of many cardiovascular diseases; myocardial hypertrophy is a kind of cardiomyopathy, which is a response of myocardial cells to hemodynamic increase. Various conditions can cause increased hemodynamics in the body, such as high blood pressure, heart valve disease. Long-term overload of hemodynamic stimulation can cause cardiomyocyte remodeling process characterized by the increase of cardiomyocyte volume, that is, cardiac hypertrophy. Now it is generally believed that there are two kinds of cardiac hypertrophy, one is normal physiological hypertrophy, such as the cardiac hypertrophy that occurs with the development of companions after birth, and myocardial hypertrophy caused by physical exercise; the other is pathological cardiac hypertrophy, other Early features are thickening of the ventricular wall and interventricular septum, and enhanced myocardial contractility, so it is considered as compensatory hypertrophy. If the condition has not been relieved, ventricular wall-interstitial fibrosis, myocardial cell systolic dysfunction, and energy loss will occur in the later stage of myocardial hypertrophy. Abnormalities in metabolism, gene expression, and electrophysiological features ultimately lead to heart failure. In view of the fact that the occurrence of myocardial hypertrophy is a progressive and irreversible process, and will eventually lead to heart failure, modern medicine has regarded it as a milestone myocardial morphological change in the occurrence of clinical lesions in the heart, and it is considered to be a risk of heart disease and sudden cardiac death. factor.

With the recent upsurge in miRNA research and application, endogenous non-coding small RNAs have been revealed as a central factor in the regulation of gene expression, participating in many important physiological processes. For miRNA, the characteristics of the regulation method determine that its target genes will not only One is the one-to-many method, which enriches the research content of miRNA function; miRNA plays an important role in maintaining the normal physiological function of the heart, and the abnormal expression of miRNA in the heart is related to the occurrence of many heart diseases. Therefore, using miRNA as a target for the treatment of heart diseases and developing related drugs has potential clinical application value. ; Although more and more miRNAs have been discovered as biomarkers and therapeutic targets of human diseases, the key miRNAs in heart diseases of cardiac hypertrophy and myocardial fibrosis have not been identified, and their functions are still important for scientific research in this field. People challenge.

Invention content:

The purpose of the present invention is to overcome the deficiencies in the prior art, seek to provide a miRNA-2861 antisense nucleotide for the preparation of a pharmaceutical composition for the diagnosis, prevention or treatment of heart disease and its use, to determine or discover the regulation of cardiomyocyte hypertrophy miRNAs expressed in the heart, further determine their role in heart diseases, and apply them to the diagnosis and prevention of these heart diseases.

In order to achieve the purpose of the above invention, the antisense nucleotide sequence of miRNA-2861 designed in the present invention is 5'-CCGCCCGCCGCCAGGCCCC-3'; the pharmaceutical composition is made into a pharmaceutical composition with an infection titer of 10 ¹⁶ PFU overexpressing miRNA- 2861 antisense nucleotide carrier, virus or adjuvant, the carrier is cholesterol, nanoparticles or liposomes; the viral vector is one or more of adenoviral vectors, lentiviral vectors, and retroviral vectors; the The auxiliary material is one or more of mannitol, phosphate buffer saline, and physiological saline;

The pharmaceutical dosage forms are one or more of oral preparations, injection preparations, tablets, and dry powder dosage forms, preferably small water injections for injection or freeze-dried powder for injection.

The miRNA-2861 antisense nucleotide pharmaceutical composition of the present invention is used for preventing and treating myocardial hypertrophy, myocardial fibrosis, coronary heart disease and heart failure through oral administration or injection.

The expression of the pharmaceutical composition involved in the present invention is significantly up-regulated in hypertrophic cardiomyocytes and cardiac hypertrophy, and the expression of miRNA-2861 is enhanced by constructing miRNA-2861 overexpression adenovirus, and it is found that miRNA-2861 overexpression can induce cardiomyocyte hypertrophy at the cellular level Compared with the control group, the expression of hypertrophy genes increased significantly; in hypertrophic cardiomyocytes, compared with the control group, there was a significant increase. The detection of hypertrophy indicators found that the indicators of myocardial hypertrophy, such as cell surface area, protein Compared with the control group, the ratio of /DNA and the recombination of sarcomeres also have a significant increase, which can induce the occurrence of cardiac hypertrophy, as a biomarker for early diagnosis and early prevention of cardiac diseases such as cardiac hypertrophy and myocardial fibrosis; Hypertrophy and myocardial fibrosis have protective effects, found at the cellular level by transfecting miRNA-2861 antisense nucleotides, which can inhibit hypertrophy-stimulating factor-induced cardiomyocyte hypertrophy, including hypertrophy indicators such as cell surface area, protein/DNA ratio The recombination of sarcomeres and sarcomeres was also significantly reduced compared with the stimulation group; at the animal level, injection of antisense nucleotides of exogenous miRNA-2861 can also inhibit the hypertrophic effects of hypertrophic models, including cardiac phenotype, heart body weight ratio , myocardial cell cross-sectional area (WGA staining) and myocardial fibrosis (Masson staining) in the marginal zone were significantly reduced, and cardiac function was also significantly improved.

Compared with the prior art, the pharmaceutical composition of the present invention has reasonable formula, reliable pharmacology, simple manufacturing process, obvious therapeutic effect, wide application range and friendly environment.

Description of drawings:

Figure 1 shows the changes in the expression level of miRNA-2861 during cardiomyocyte hypertrophy induced by cardiac hypertrophy stimulating factors PE (phenylephrine) and Ang Ⅱ (angiotensin Ⅱ);

Figure 2 shows the inhibition of PE-induced hypertrophy at the cellular level by treating primary cells transfected with miRNA-2861 antisense nucleotides with PE;

Figure 3 is a schematic diagram of the experimental results of the sarcomere reorganization of cardiomyocytes after PE treatment of primary cells transfected with miRNA-2861 antisense nucleotides;

Figure 4 is a schematic diagram of the detection results of the mouse intravenous injection of miRNA-2861 antisense nucleotide (antagomir) can inhibit the hypertrophy model induced by PE;

Fig. 5 is a schematic diagram of the detection results of the mouse cardiac hypertrophy model induced by intravenous injection of miRNA-2861 antisense nucleotide (antagomir) into mice can inhibit aortic constriction (TAC).

detailed description:

The present invention will be further elaborated below through the embodiments and in conjunction with the accompanying drawings.

Embodiment 1,

The miRNA-2861 antisense nucleotide overexpression adenovirus vector and the negative control adenovirus were constructed. In this example, the miRNA-2861 antisense nucleotide sequence was artificially synthesized and subcloned into the pSilencer Adeno 1.0-CMV system of Ambion Company. Construction of miRNA-2861 antisense nucleotide overexpression adenovirus. Its miRNA-2861 antisense nucleotide sequence is shown in the following SEQ ID NO: 1: 5'-CCGCCCGCCGCCAGGCCCC-3'

According to the company's instructions, the Pac I linearized vector and the linearized adenovirus backbone (AdenovirTs LacZ Backbone) were co-transfected into HEK-293 cells, the adenovirus was packaged, the virus was amplified and collected, and the virus titer was determined; the empty vector Co-transfect HEK-293 cells with the adenovirus backbone according to the above steps, and package the negative control adenovirus.

Dilute the above obtained recombinant adenovirus phosphate buffer overexpressing miRNA-2861 antisense nucleotides to an infectious titer of 1×10 ¹⁶ PFU/ml, and aseptically distribute 1 ml into ampoules.

Embodiment 2,

Changes in the expression level of miRNA-2861 under the stimulation of PE and Ang Ⅱ. For the hypertrophy model of cardiomyocytes in this example, the established method was used to cultivate the primary cardiomyocytes of rat suckling mice (rat suckling mice were purchased from Beijing Medical University, Da The mouse strain is Wistar, and the preparation of primary cardiomyocytes of rat suckling rats can be found in the following literature: W.-Q.Tan, et al, Foxo3aInhibits Cardiomyocyte Hypertrophy through Transactivating Catalase J BiolChem.2008October 31; 283(44):29730-29739) , the cardiomyocytes were treated with PE and Ang Ⅱ, and the total RNA of the cells was extracted at different times of culture, and the expression level of miRNA-2861 was detected by real-time fluorescent quantitative PCR technology, as shown in Figure 1A and 1B, where Figure 1A is The expression level of miRNA-2861 over time in the primary rat neonatal cardiomyocytes treated with PE; The expression level of miRNA-2861 in untreated rat primary cardiomyocytes was used as the benchmark, and the expression level of miRNA-2861 in rat primary cardiomyocytes during PE or Ang Ⅱ treatment. The expression level of miRNA-2861 was significantly increased within 6 hours of PE and Ang Ⅱ treatment.

Embodiment 3,

miRNA-2861 antisense nucleotides can inhibit hypertrophy induced by PE at the cellular level. In this example, primary cardiomyocytes were transfected with miRNA-2861 antisense nucleotides (anta-2861), which can effectively inhibit miRNA -2861 expression, at the same time the cells were treated with PE, and at the same time treated with the negative control (anta-NC) of miRNA-2861 antisense nucleotide, after 24 hours, the cardiac hypertrophy index was detected on the cardiomyocytes , such as cardiomyocyte surface area ( FIG. 2A ) and protein/DNA ( FIG. 2B ), the experimental results prove that miRNA-2861 antisense nucleotides can effectively inhibit cardiomyocyte hypertrophy induced by PE.

Embodiment 4,

miRNA-2861 antisense nucleotides can inhibit the recombination of cardiomyocyte sarcomeres induced by PE at the cellular level. This example transfects primary cardiomyocytes with miRNA-2861 antisense nucleotides (anta-2861) and negative After the control (anta-NC), the cardiomyocytes were treated with PE, and after 24 hours of treatment, the sarcomere reorganization detection was performed on the cardiomyocytes, as shown in Figure 3, the experimental results proved that miRNA-2861 antisense nucleotides can Effectively inhibit PE-induced sarcomere reorganization of cardiomyocytes.

Embodiment 5,

Intravenous injection of miRNA-2861 antisense nucleotides (antagomir) in mice can inhibit the hypertrophy model induced by PE. In this example, after the mice were treated with the PE buried pump for two weeks, the hearts of the mice had obvious hypertrophy phenotype. At the same time, the addition of miRNA-2861 antisense nucleotide (anta-2861) can significantly weaken the hypertrophic effect induced by PE, including the cross-sectional area of cardiomyocytes (WGA staining) (as shown in Figure 4A) and heart weight ratio (as shown in Figure 4B ); Simultaneous injection of miRNA-2861 antisense nucleotide negative control (anta-NC) and PE in mice could not weaken the hypertrophic effect of PE, and the experimental results confirmed that miRNA-2861 antisense nucleotide could inhibit The occurrence of cardiac hypertrophy induced by PE.

Embodiment 6,

Intravenous injection of miRNA-2861 antisense nucleotides (antagomir) in mice can inhibit the mouse heart hypertrophy model induced by aortic constriction (TAC). ) method can induce hypertrophy phenotype after three weeks; adding miRNA-2861 antisense nucleotide (anta-2861) can significantly weaken the hypertrophic effect induced by TAC; adding miRNA-2861 antisense nucleotide The negative control (anta-NC) group has no significant change compared with the TAC group, that is, it cannot weaken the hypertrophic effect induced by TAC (Figure 5), including the heart weight ratio (Figure 5A) and the cross-sectional area of cardiomyocytes (WGA (Wheat germ agglutinin) staining) ( FIG. 5B ); the experimental results confirmed that miRNA-2861 antisense nucleotides can inhibit TAC-induced cardiac hypertrophy at the overall level.

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sequence listing

SEQ ID NO:1

5'-CCGCCCGCCGCCAGGCCCC-3'

Claims (4)

1. The use of a miRNA-2861 antisense nucleotide pharmaceutical composition in the preparation of medicines for treating heart disease, characterized in that the antisense nucleotide sequence of miRNA-2861 is 5'-CCGCCCGCCGCCAGGCCCC-3', The pharmaceutical composition includes an antisense nucleotide carrier or virus overexpressing miRNA-2861 with an infection titer of 10 ¹⁶ PFU, and the carrier is cholesterol, nanoparticles or liposomes; the virus is an adenovirus vector, a lentivirus One or more of vectors and retroviral vectors. 2. the purposes of a kind of miRNA-2861 antisense nucleotide pharmaceutical composition according to claim 1 in the preparation of the medicine for the treatment of heart disease, it is characterized in that said heart disease is myocardial hypertrophy, myocardial fibrosis, coronary heart disease or heart failure. 3. The use of a miRNA-2861 antisense nucleotide pharmaceutical composition according to claim 1 in the preparation of medicines for treating heart diseases, characterized in that the dosage form of the pharmaceutical composition is an oral preparation or an injection preparation. 4. The use of a miRNA-2861 antisense nucleotide pharmaceutical composition according to claim 1 in the preparation of medicines for treating heart diseases, characterized in that the dosage form of the pharmaceutical composition is tablet or dry powder.