JP5467742B2 - A prophylactic and therapeutic agent for left ventricular remodeling comprising ECP as an active ingredient. - Google Patents

Description

  The present invention relates to a prophylactic and / or therapeutic agent for left ventricular remodeling containing ECP as an active ingredient, arrhythmia caused by myocardial necrosis, myocardial necrosis, hypertrophy of the ventricular septal wall, and enlargement of the heart lumen.

Eosinophil cationic protein (ECP) is a protein present in basic granules whose expression increases with the activation of eosinophils. Parasite killing, neurotoxin, lymphocyte proliferation suppression, bactericidal, histamine It has been reported in the past that it has activity relating to release, ribonuclease, shortening of coagulation time, and the like (Non-patent Document 1). Patent Document 1 shows that cardiomyocyte hypertrophy and an increase in heart rate are associated with the addition of ECP to cultured cardiomyocytes. However, sufficient studies have not been made on what kind of heart disease is effective for ECP, and left ventricular remodeling of heart, arrhythmia caused by myocardial necrosis, myocardial necrosis, heart There has been virtually no indication of the effectiveness of ECP in the prevention or treatment of lumen enlargement and the like.
Japanese Patent Application No. 2004-123679 Japanese clinical 1992, 51 (3): 60 (606)

  The present invention provides a new medicine containing ECP as an active ingredient for preventing and / or treating left ventricular remodeling, arrhythmia caused by myocardial necrosis, myocardial necrosis, hypertrophy of the ventricular septum and expansion of the heart lumen. The purpose is to provide.

Inventors of the present application have made extensive studies in view of the above circumstances, and ECP prevents left ventricular remodeling, arrhythmia caused by myocardial necrosis, myocardial necrosis, hypertrophy of the ventricular septal wall, and expansion of the intraventricular lumen and / or Or it was found to be particularly effective for treatment. The present invention has been completed as a result of further research based on such knowledge. That is, the present invention provides the following aspects of the invention.
Item 1. A preventive and / or therapeutic agent for left ventricular remodeling comprising ECP as an active ingredient.
Item 2. A prophylactic and / or therapeutic agent for arrhythmia caused by myocardial necrosis comprising ECP as an active ingredient.
Item 3. A prophylactic and / or therapeutic agent for myocardial necrosis comprising ECP as an active ingredient.
Item 4. A preventive and / or therapeutic agent for hypertrophy of the ventricular septal wall containing ECP as an active ingredient.
Item 5. An agent for preventing and / or treating the expansion of the heart lumen, comprising ECP as an active ingredient.
Item 6. Item 6. The agent according to any one of Items 1 to 5, comprising a therapeutically effective amount of ECP.
Item 7. Item 7. The agent according to any one of Items 1 to 6, wherein the ECP is derived from a human.
Item 8. Item 8. The agent according to any one of Items 1 to 7, which is administered to an affected part of the heart with a catheter.

  The agent containing the ECP of the present invention as an active ingredient is effective in the prevention and / or treatment of left ventricular remodeling, arrhythmia caused by myocardial necrosis, myocardial necrosis, hypertrophy of the ventricular septum, and enlargement of the heart lumen.

  In one embodiment, the present invention is a prophylactic and / or therapeutic agent for left ventricular remodeling comprising ECP as an active ingredient. Left ventricular remodeling refers to the cardiomyocyte being damaged and necrotic as a result of a load on the heart due to coronary artery occlusion, etc., and myocardial cell shedding, thinning, stretching, and subsequent processes in the damaged part This refers to a phenomenon in which morphological changes occur in the left ventricle of the heart, such as enlargement and extension of the healthy heart muscle, enlargement of the lumen, and fibrosis of the stroma. Arrhythmias may occur with left ventricular remodeling.

  In one embodiment, the present invention is a prophylactic and / or therapeutic agent for arrhythmia caused by myocardial necrosis comprising ECP as an active ingredient. An arrhythmia caused by myocardial necrosis is an arrhythmia that occurs as a result of the heart's blood vessels becoming occluded and oxygen and nutrients not reaching the cardiomyocytes that exist ahead of it, resulting in necrosis of the cardiomyocytes.

  In one embodiment, the present invention is a prophylactic and / or therapeutic agent for myocardial necrosis comprising ECP as an active ingredient. Myocardial necrosis refers to necrosis of cardiomyocytes due to coronary artery occlusion or various causes.

  In one embodiment, the present invention is a preventive and / or therapeutic agent for ventricular septal hypertrophy comprising ECP as an active ingredient. The enlargement of the ventricular septal wall means that as a result of left ventricular remodeling, an excessive load is applied to the myocardium on the healthy side, resulting in the enlargement of the myocardial cells. Give.

  In one embodiment, the present invention is an agent for preventing and / or treating the expansion of the heart lumen, which comprises ECP as an active ingredient. Enlargement of the heart lumen is the final form of left ventricular remodeling. The enlarged myocardium cannot cover the heart's pump function, resulting in a heavy load on the heart, resulting in an increase in the volume of the heart. The cavity is enlarged (increased left ventricular end-diastolic volume). When the heart lumen expands, the heart's pumping function is significantly impaired, leading to a state of heart failure that not only lowers the patient's quality of life but also forced to be hospitalized for heart failure, resulting in significant medical and economic losses. The burden on the patient and family also increases.

  One pathological change characteristic of left ventricular remodeling is fibrosis. Since fibrosis occurs in the space after cardiac muscle cells killed by myocardial infarction are treated with phagocytic cells, the larger the fibrosis area, the greater the damage to the myocardium, and the subsequent left ventricular remodeling may proceed. is expected.

  In the present invention, the ECP is effective as long as it has the effect of treating or preventing arrhythmia caused by left ventricular remodeling, myocardial necrosis, enlargement of the ventricular septal wall, enlargement of the intraventricular cavity, and myocardial necrosis. There is no particular limitation. ECP can be isolated from cells of various mammals including humans (white blood cells and hematopoietic stem cells) according to a known method. Further, since the amino acid sequence of ECP and the gene sequence encoding the same have been clarified (for example, human: GenBank / X15161, chimpanzee: GenBank / AF294028, gorilla: GenBank / U24097), a known peptide synthesis method was used. Synthesis and production in host cells using genetic recombination techniques are also possible. Furthermore, as long as the effect as a preventive or therapeutic agent of the present invention is exhibited, the ECP of the present invention may be a modified product obtained by performing substitution, addition, or deletion of one or more amino acids on a known amino acid sequence. .

  For example, when producing recombinant ECP by in vitro transcription and translation, DNA encoding ECP is inserted into a vector having an RNA polymerase promoter to produce an expression vector, and this vector contains RNA polymerase corresponding to the promoter. ECP can also be produced by adding to an in vitro translation system such as rabbit reticulocyte lysate or wheat germ extract. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II.

  When recombinant ECP is expressed in microorganisms such as Escherichia coli, an expression vector obtained by recombining the above DNA fragment with an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism Is then introduced into E. coli and the fusion peptide is isolated from the culture obtained. Examples of the expression vector include pUC system, pBluescript II, pET expression system, pGEX expression system and the like.

  When recombinant ECP is expressed in eukaryotic cells, a DNA fragment encoding ECP is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, and the like. And then introduced into eukaryotic cells, the fusion peptide can be expressed in transformed eukaryotic cells. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2, and the like. As eukaryotic cells, mammalian cultured cells such as monkey kidney cells COS7 and Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus egg cells, etc. are generally used, but can express the target protein. Any eukaryotic cell can be used.

  In order to introduce the expression vector into the host cell, a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used.

  Isolation and purification of ECP from the culture can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, sonication, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Illustrative examples include an appropriate combination of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.

  ECP is a secreted protein. For example, in the case of human ECP, the 23 amino acid sequence on the N-terminal side is a secretion signal. Therefore, when expressing recombinant ECP in prokaryotic cells or eukaryotic cells, considering the recovery efficiency of recombinant ECP from the culture, the C-terminal side of the secretory signal sequence (for example, 28th Arg and later) It is preferable to express the active region.

  The ECP of the present invention can be administered orally or parenterally, but oral administration or administration by injection is preferred. In one embodiment of the present invention, ECP is preferably administered systemically by infusion or the like.

  The ECP of the present invention can be used alone, but it is desirable to appropriately formulate and use it according to symptoms and dosage forms, and pharmacologically acceptable carriers and additives are added according to known pharmaceutical techniques. Can do. Examples of the dosage form include ordinary oral preparations and parenteral preparations such as liquids (injections, nasal drops, syrups, dry syrups, etc.), tablets, troches, capsules (hard, soft, microcapsules). Etc.), powders, fine granules, granules, ointments, suppositories, etc., drug delivery systems (for example, sustained release agents, etc.) and the like can also be used. Especially, it is desirable that it is an oral administration agent or an injection, and it is desirable to use as an injection especially.

  Various known carriers can be appropriately selected as the carrier used in formulating the various dosage forms. For example, in the case of oral dosage form, sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoate, strawberry, Flavors such as flavor and peppermint can be used.

  In the case of an injection, it can be formulated using a carrier comprising a salt solution, a glucose solution, a mixture of salt water and a glucose solution, various buffers, or the like. Alternatively, it may be formulated in a powder state and mixed with the liquid carrier at the time of use to prepare an injection solution. When used as an injection, the injection site is not particularly limited, and may be performed on an artery, vein, muscle (skeletal muscle, smooth muscle, heart muscle, etc.), skin, subcutaneous, or the like. Local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusate injection and the like can be employed as necessary.

  For example, it is desirable to inject the agent of the present invention directly into the vicinity of an ischemic heart using a catheter. As catheters that can be used in the present invention, catheters for cardioangiography (CAG), therapeutic catheters (perfusion catheters), microcatheters and thrombus aspiration catheters, etc., can be used to administer ECP through the lumen. Any type of balloon catheter may be used, and a balloon catheter that gradually penetrates from the inside of the balloon can be used. Another method is to specify an infarcted myocardium from the heart lumen and use a system (NOGA system) that can be inserted directly into the same site from the lumen side. In addition, if there is a drug delivery system that specifically allows the drug to reach the infarcted myocardium, it can be administered to the target site by administering it from the periphery.

  Furthermore, the preventive or therapeutic agent of the present invention can be used in combination with an antiplatelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, a hyperlipidemia therapeutic agent and the like.

  Although it varies depending on the therapeutically effective amount of ECP in the agent of the present invention, the age and weight of the patient, symptoms, administration route, etc., and is not particularly limited as long as the desired effect is exhibited, the blood ECP concentration is 10 nmol to 0 It is desirable to administer an amount of about 1 mmol, preferably about 5 nmol to 0.5 mmol.

  Regarding the timing of administration of the preventive or therapeutic agent of the present invention, it is desirable to administer as soon as possible after myocardial infarction, but it can be administered at any time until the completion of left ventricular remodeling. .

EXAMPLES Hereinafter, although this invention is further demonstrated based on an Example, this invention is not limited to these.
Example 1
Effect of ECP on the heart of mice with occluded coronary arteries

  In a rat experimental myocardial infarction model, immediately after the infarction was created, 150 μl of ECP at 10 μg / ml was directly administered into the myocardium at about 15 infarcted sites and infarct margins. As a control, 10 μg / ml of RNase A was directly administered intramyocardially to a rat myocardial infarction model as in the case of ECP administration. The ECP used was produced in E. coli using the human ECP gene. 7 days after administration, the heart function was measured by echocardiography 7 days later, the effect on left ventricular remodeling, etc. was examined, the heart was removed, formalin fixed, sectioned, histological We examined and compared the fibrosis area. The result of HE staining and Azan staining of a heart section of a model not treated with ECP is shown in FIG.

  In the comparative examination of the fibrosis area, the fibrosis was 23.2 ± 6.03% in the non-administered group, and the fibrosis area was small as 16.1 ± 3.65% in the ECP administration group (comparison of 5 cases each). This suggests that fibrosis is suppressed by ECP administration. A healthy heart is shown in FIG. In the echocardiography, the pump function of the heart administered with ECP was the stroke rate (EF) = 63.8%, and the heart administered with RNase A was EF = 51.5%. Therefore, it was found that the EF was markedly improved by 10% or more compared with the control. As for the left ventricular cavity, LVDd = 8.77 mm in the heart administered with ECP and LVDd = 9.70 mm in the heart administered with RNase A. Therefore, the improvement in the size of the left heart lumen was similarly observed, and the effect of suppressing left ventricular remodeling was observed. Furthermore, from the results of FIG. 1, histologically, myocardial necrosis is significantly reduced in the heart administered with ECP, and it is considered that the infarcted myocardium has survived or regenerated extensively.

Example 2
Immediately after the creation of an infarction using a rat experimental myocardial infarction model, as in Example 1, a physiological saline was directly injected into the infarcted region, an ECP (10 μg / ml) was directly injected, and An Alzette osmotic pump (2ML-1) was implanted in the back and ECP (1.25μg / ml) was administered systemically, and echocardiography (short axis and long axis methods) was performed 7 days after closing the chest. The cardiac function was measured and the effect on left ventricular remodeling etc. was examined. The results are shown in FIGS. As shown in FIGS. 3 and 4, there was no difference in the body weight and heart rate (HR) of the rats between the groups depending on the presence or absence of ECP, but EF (ejection rate) and FS (fractional) indicating the heart pump function. Regarding shortening), the value was significantly higher in both the case of direct administration of ECP and the case of administration of pump compared to the control. In other words, it maintained good heart function. The heart-to-weight ratio was also significantly better than the control, and as a result of suppression of left ventricular remodeling, it was considered that cardiac hypertrophy was suppressed (the heart-body weight ratio was low). In addition, since the above effects were obtained even by systemic administration using an osmotic pump, it is considered that ECP can be applied to the heart even if systemic administration is performed, and major side effects due to systemic administration have so far been reached. It has not been confirmed.

Example 3
Experiment on differentiation induction of mouse embryonal carcinoma cell line P19 1) Cell culture and recovery 5 × 10 ⁵ P19CL6 cells were seeded on a cell culture dish with a diameter of 60 mm, and 5 mL differentiation induction medium (α-MEM medium / 10% FBS / 50 μg / mL streptomycin / 50 units / mL penicillin / 0.9% DMSO) and two types of media obtained by adding ECP to the medium at a concentration of 1 μg / mL. Cells were collected at regular time intervals and total RNA was extracted.

2) Total RNA extraction and evaluation Total RNA extraction was performed using QIA shuffledder (QIAGEN) and RNeasy Mini Kit (QIAGEN). First, 700 μL of RLT solution (cell lysate contained in the kit) containing β-ME was added to cells on the dish to lyse the cells. The lysate was transferred to a QIA shredder column and centrifuged (10000 rpm, 2 minutes, room temperature) to disrupt the cells. The crushed liquid was added to the RNeasy Mini Kit column and centrifuged (10000 rpm, 15 seconds, room temperature). 700 μL of RW liquid (washing liquid) contained in the kit was added to the column, and centrifugation (10000 rpm, 15 seconds, room temperature) was performed again. Next, 700 μL of the RPE solution (washing solution) of the same kit was added to the column, and centrifugation (10000 rpm, 15 seconds, room temperature) was performed. Finally, 50 μL of RNase free water was added to the column, and centrifugation (10000 rpm, 15 seconds, room temperature) was performed. The concentration of the recovered RNA solution was measured with a spectrophotometer. Moreover, RNA was analyzed by EXPERION (BioRad).

3) Synthesis of cDNA 5 μg of the collected total RNA and 100 mM oligo-dT _18-21 , 10 mM dNTPs (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) were mixed well, heated at 65 ° C. for 5 minutes, and rapidly cooled on ice. Then, 5 × buffer of Superscript II kit (Invitrogen) and 0.1M dithiothreitol (DTT) were mixed in the reaction solution, heated at 42 ° C. for 2 minutes, and further added with reverse transcriptase Superscript II, Reaction was performed for 50 minutes to synthesize cDNA. After completion of the reaction, the reverse transcriptase was inactivated by heating at 70 ° C. for 15 minutes.
4) Polymerase Chain Reaction (PCR)
Using the obtained cDNA as a template, PCR was performed with primers specific to each gene. PCR reaction conditions are as follows.

(Primers specific to each gene)
GATA4;
forward 5′-ACTCTGAGGGCGAGATGGG-3 ′ (SEQ ID NO: 1)
reverse 5'-CTCGGGCATTACGACGCCACAG-3 '(SEQ ID NO: 2)
Wnt3a;
forward 5′-AAGCAGGCTCTGGGGCAGCTA-3 ′ (SEQ ID NO: 3)
reverse 5'-GACGGTGGTGCAGCTTCCA-3 '(SEQ ID NO: 4)
Wnt5a;
forward 5′-TTTTTCTCCTTCGCCCCAGTGTGT-3 ′ (SEQ ID NO: 5)
reverse 5'-GGCTCATGGCGTTCACCAC-3 '(SEQ ID NO: 6)
Wnt8a;
forward 5′-TCCCAAGGCCTATCTGACCTAC-3 ′ (SEQ ID NO: 7)
reverse 5'-CCGGCCCTGTTGTTGTGA-3 '(SEQ ID NO: 8)
α-Actin ;
forward 5′-CCCGTTACGCCCTCTGGCCGTA-3 ′ (SEQ ID NO: 9)
reverse 5'-ACTCCTGCTTGCTGATCCACA-3 '(SEQ ID NO: 10)
Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH);
forward 5'-CCCTTCATTGACTCCAACTAC-3 '(SEQ ID NO: 11)
reverse 5'-CCACCCTTCTTGATGCATCAT-3 '(SEQ ID NO: 12)
PCR reaction composition:
cDNA solution 1.0 μL
10 x PCR buffer 2.0 μL
10 mM dNTPs 0.5 μL
Primer forward (10 μM) 1.0 μL
Primer reverse (10 μM) 1.0 μL
Taq polymerase (New England Biolabs) 0.2 μL
Ultrapure water (UPW) 14.3 μL
PCR reaction conditions: after 5 min treatment at 95 ° C., after repeating the reaction 30 times in this order under the conditions of 95 ° C. for 1 min, 55 ° C. for 1 min, 75 ° C. for 1 min, perform the following analysis The reaction was maintained at 4 ° C until

  After the PCR reaction, the PCR product was confirmed by agarose gel electrophoresis. The results are shown in FIGS. The confirmed genes are GATA4, Wnt3a, Wnt5a, and Wnt8a, which are known to have significant changes in expression during the development of the myocardium. Expression of GATA4, Wnt3a, Wnt5a, and Wnt8a genes was induced early in the presence of ECP. Among these, the expression of Wnt3a, Wnt5a, and Wnt8a attenuated after elapse of time, but it was observed that the attenuation occurred early in the presence of ECP. These phenomena correlate with the activity of ECP that promotes the induction of myocardial differentiation.

  Furthermore, it usually takes 12-14 days in the presence of 0.9% DMSO for the cells to beat, but shortened to 7-8 days in the presence of 0.9% dimethyl sulfoxide (DMSO) and ECP. . In addition, with DMSO alone, pulsatile cells are discontinuously present in a lump and each lump pulsates independently, but when ECP is present, the pulsating cells pulsate in a sheet form, and the cells in the sheet Were observed to propagate pulsations in a coordinated manner. This suggests the prevention or treatment effect of arrhythmia by ECP.

FIG. 1 shows the results of HE staining and Azan staining of a heart section administered with EPC and a heart section not administered with EPC. FIG. 2 shows a section of a healthy mouse heart stained with Azan. FIG. 3 shows the effect of administration of ECP. FIG. 4 shows the effect of administration of ECP. FIG. 5 shows the effect of EPC addition on gene expression. FIG. 6 shows the effect of EPC addition on gene expression.

Claims (4)

An agent for preventing and / or treating left ventricular remodeling after myocardial infarction, comprising ECP as an active ingredient. The agent of claim 1 , comprising a therapeutically effective amount of ECP. The agent according to claim 1 or 2 , wherein the ECP is derived from a human. The agent according to any one of claims 1 to 3 , which is administered to an affected part of the heart with a catheter.

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