KR102048846B1 - Medicine for treating vascular disease and method for screening the same - Google Patents

Abstract

The present application relates to a pharmaceutical composition for the prevention or treatment of vascular diseases, including EGTA, and to a method for screening for the prevention or treatment of vascular diseases, in particular ethylene glycol-bis (β-aminoethyl ether) -N, N, N ' Prevention of vascular disease, including ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA) and a pharmaceutically acceptable carrier or excipient Or a therapeutic pharmaceutical composition.

Description

MEDICINE FOR TREATING VASCULAR DISEASE AND METHOD FOR SCREENING THE SAME}

The present application relates to a pharmaceutical composition for preventing or treating vascular diseases including EGTA, and to a method for screening for preventing or treating vascular diseases.

A zebrafish is a small sized tropical freshwater fish. Its early development is fast and it is possible to obtain a large amount of embryos. Since the embryos are transparent during the early development, fluorescent markers are used to create internal organs such as blood vessels. The development of tissues can be observed in real time at the cellular level. In addition, they share more than 70% of genes and signaling systems with humans and show great similarity in organ development, which is very useful for the study of human diseases. In particular, the small size of zebrafish embryos and their characteristics as aquatic organisms have been widely used as model animals to search for substances with specific activity by searching the low molecular weight library at the system level.

In particular, zebrafish's vascular system in studies of angiogenesis is very similar structurally, functionally and embryologically compared to other vertebrates including mammals (Isogai et al., 2001). It is actively used for research and mechanism research of disease-causing genes (Wilson et al., Science 2006; Petente et al., Genes and Dev. 2007; Hogan et al., Human Mol. Genetics 2008; Guarani et al., Nature 2011).

DYRK1A (D ual regulated t Y rosinephosphorylation- R egulated K inase 1A) are serine-threonine kinases (Serine-Threonine kinase) as one of, DYRK1A gene site of the Down trisomy (trisomy) commonly found in Down Syndrome It is thought to be a key gene located in the Syndrome Critical Region (DSCR). In the nervous system, DYRK1A is known to regulate the time of differentiation of neurons, axon outgrowth, dendrite formation, synaptic plasticity, and the like. In addition, (1) patients with Down's syndrome with overexpression of DYRK1A reduce the incidence of solid cancer and decrease vascular diseases such as atherosclerosis and diabetic retinopathy. (2) SIRT1, NOTCH1, NFAT, etc. The key signaling proteins related to angiogenesis are regulated by DYRK1A phosphorylation, and (3) DYRK1A shows angiogenesis inhibitory function with another DSCR gene, DSCR1, during VEGF angiogenesis. Plays an important role in the development and maintenance of blood vessels, and it is very likely to function in angiogenesis-related diseases such as solid cancer and stroke, but research on its function is still insufficient.

We search for a Sigma LOPAC library of 1280 FDA-approved bioactive small molecules based on the cerebrovascular phenotypes represented by knockout mutants of the zebrafish dyrk1aa gene, a human DYRK1A homolog. In particular, the aim of this study was to find out the prevention and treatment of cerebrovascular disease and to verify its efficacy.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The first aspect of the present application is ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) and a pharmaceutically acceptable carrier or excipient can be provided a pharmaceutical composition for the prevention or treatment of vascular diseases.

According to a second aspect of the present invention, the method comprises: (a) treating and culturing a test substance in an embryo of zebrafish having a dyrk1aa gene deletion; And (b) it can provide a screening method for the prevention or treatment of vascular diseases comprising the step of selecting a test substance to reduce the phenotype of the vascular disease compared to the untreated control.

According to a third aspect of the present invention, a method of culturing and culturing a harmine in an embryo of zebrafish; (b) treating and culturing the test substance in the zebrafish embryo; And (c) it can provide a screening method for the prevention or treatment of vascular diseases comprising the step of selecting a test substance to reduce the phenotype of the vascular disease compared to the untreated control.

The above-mentioned means for solving the problems are merely exemplary, and should not be construed to limit the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

According to the present invention, a small molecule library using the cerebrovascular defect and cerebrovascular phenotype of zebrafish with DYRK1A suppressed or knocked out can be searched for a drug for preventing or treating vascular diseases such as cerebrovascular disease. In addition, by providing a pharmaceutical composition comprising EGTA discovered during the search process can help in the treatment of diseases including cerebral hemorrhage, cerebrovascular defects, and cerebrovascular injury.

1A and 1B show the results of observing increased cerebral hemorrhage (FIG. 1A) and decreased cerebral angiogenesis (FIG. 1B) when zebrafish embryos were treated with Harmin, an inhibitor of DYRK1A, according to one embodiment of the present disclosure.
Figures 2a to 2c shows a candidate search strategy (FIG. 2A), a list of effective small molecules (FIG. 2B) and the frequency and cytotoxicity test results of cerebral hemorrhage embryos (FIG. 2C) using the Sigma LOPAC library according to an embodiment of the present application will be.
3A and 3B show the results of induction of cerebral hemorrhage experiments under normal conditions (FIG. 3A) and high temperature conditions (FIG. 3B) of dyrk1aa-deficient mutant zebrafish embryos according to an example of the present disclosure.
4A to 4D show the incidence of posterior cerebellar cerebrovascular CtAs (FIGS. 4A and 4C) and the decrease in cerebral hemorrhage phenotype after treatment with EGTA in dyrk1aa-deficient mutant zebrafish embryos according to one embodiment of the present disclosure (FIGS. 4B and 4D). ).

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present disclosure. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present application.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless specifically stated otherwise.

As used throughout this specification, the terms "about", "substantially" and the like are used at, or in the sense of, numerical values when a manufacturing and material tolerance inherent in the stated meanings is indicated, Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term "step to" or "step of" does not mean "step for."

Throughout this specification, the term "combination of these" included in the expression of the makushi form means one or more mixtures or combinations selected from the group consisting of constituents described in the expression of the makushi form, wherein the constituents It means to include one or more selected from the group consisting of.

Throughout this specification, the description of "A and / or B" means "A, B, or A and B."

Hereinafter, a pharmaceutical composition for preventing or treating vascular diseases of the present application and a screening method for preventing or treating vascular diseases will be described in detail with reference to embodiments, examples, and drawings. However, the present application is not limited to these embodiments, examples and drawings.

The first aspect of the present application is ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) and a pharmaceutically acceptable carrier or excipient can be provided a pharmaceutical composition for the prevention or treatment of vascular diseases.

According to the exemplary embodiment of the present application, the vascular disease may be cerebrovascular disease, but may not be limited thereto.

According to one embodiment of the present application, the cerebrovascular disease may be selected from cerebral hemorrhage, cerebrovascular defect, and cerebrovascular injury, but may not be limited thereto.

According to one embodiment of the present application, EGTA may be included in the pharmaceutical composition at a concentration of about 5 to about 20 nM, but may not be limited thereto. For example, the EGTA may be about 1 to about 100 nM, about 10 to about 100 nM, about 1 to about 10 nM, about 1 to about 50 nM, about 5 to about 20 nM, or about 8 to about 15 nM. When included in a concentration may exhibit a more desirable effect, but may not be limited thereto.

EGTA is known as a representative calcium chelator (calcium chelator). Calcium, an essential ion component in vivo, is an ion that is involved in almost all life phenomena including muscle contraction and neurotransmission. Lack of such calcium ions is known to cause a variety of diseases, such as heart arrhythmias, ataxia. Therefore, the treatment of excessive high concentrations of EGTA, the lack of calcium components in the body, may affect the homeostasis in vivo, causing side effects, too low EGTA concentration may not have a sufficient effect.

According to a second aspect of the present invention, the method comprises: (a) treating and culturing a test substance in an embryo of zebrafish having a dyrk1aa gene deletion; And (b) it can provide a screening method for the prevention or treatment of vascular diseases comprising the step of selecting a test substance to reduce the phenotype of the vascular disease compared to the untreated control.

According to one embodiment of the present application, the zebrafish from which the dyrk1aa gene is deleted is a homozygous (homozygote) mutant zebrafish having a dyrk1aa − / − genotype, and (1) a tail that binds to the base sequence of the dyrk1aa gene in the zebrafish embryo. Heterozygote having a dyrk1aa +/- genotype by inserting a forward and reverse DNA pair comprising a tale effector and a nucleotide sequence encoding the nucleotide sequence Fok I nuclease, respectively, into the embryo ) Obtaining zebrafish; And (2) may be prepared by a method comprising the step of crossing the heterozygot zebrafish of step (1), but may not be limited thereto.

According to the exemplary embodiment of the present application, the dyrk1aa gene of step (1) may include a nucleotide sequence represented by SEQ ID NO: 1, but may not be limited thereto. The gene may be a homologous gene of the human DYRK1A gene, and the human DYRK1A gene may be located in a Down Syndrome Critical Region (DSCR), a common trisomy site known as a direct cause of Down syndrome. The zebrafish embryo is preferably a first cell phase, but may not be limited thereto.

The DNA pair of step (1) preferably acts on the serine / threonine kinase domain of the fifth exon of the dyrk1aa gene, and in the right and left directions of the 5 ′ terminal portion of the domain. It may be preferred to be a working DNA pair, but may not be limited thereto.

For example, the preparation of the mutant zebrafish inserts dyrk1aa TALEN RNA into wild-type zebrafish embryos of the first cell phase, and agrose gel (agarose) for cleavage by T7E1 assay or StuI restriction enzyme about 24 hours after development. gel) to confirm the activity of dyrk1aaTALEN RNA may be included. Specifically, in the case of mutant zebrafish injected with dyrk1aa TALEN RNA, the activity of dyrk1aa TALEN RNA can be confirmed by confirming that PCR DNA is cleaved by T7E1.

The method of treating and culturing the test substance in the zebrafish embryo is not particularly limited, and various methods known in the art may be used. Among them, the culture is preferably incubated for 1 hour to 3 hours at a temperature in the range of 30 ℃ to 40 ℃, more preferably incubated for 1.5 hours to 2.5 hours at a temperature within the range of 33 ℃ to 38 ℃, Most preferably, it is incubated for about 2 hours at a temperature of about 35 ℃.

In the mutant zebrafish, phenotypes such as hemorrhage and angiogenesis defects, which are hardly observed in the wild type, can be confirmed in the middle brain, the whole brain, the rear brain, or the eye. In addition, the development of cerebrovascular vessels (particularly central arteries) can be tracked through confocal imaging using fluorescently labeled blood vessel-specific fluorescence transformants (Tg (kdrl: egfp)).

According to a third aspect of the present invention, there is provided a method for preparing a zebrafish, comprising: (a) treating and culturing a harmine in an embryo of zebrafish; (b) treating and culturing the test substance in the zebrafish embryo; And (c) it can provide a screening method for the prevention or treatment of vascular diseases comprising the step of selecting a test substance to reduce the phenotype of the vascular disease compared to the untreated control.

According to one embodiment of the present application, the harmin may be treated at a concentration of about 5 to about 100 μM, but may not be limited thereto. For example, the harmin may have a concentration of about 1 to about 200 μM, about 1 to about 50 μM, about 10 to about 50 μM, about 25 μM to about 50 μM, about 10 to about 200 It may be treated with a concentration of μM, a concentration of about 10 to about 100 μM, or a concentration of about 10 to about 25 μM, but may not be limited thereto.

According to one embodiment of the present application, the test substance may be treated at a concentration of 0.1 μM to 500 μM, but may not be limited thereto. For example, the test substance may have a concentration of about 1 to about 100 μM, a concentration of about 10 μM to about 100 μM, a concentration of about 20 μM to about 100 μM, a concentration of about 0.1 μM to about 100 μM, about 0.1 μM To a concentration of from about 50 μM, to a concentration of about 20 μM to about 40 μM, or to a concentration of about 30 μM, but may not be limited thereto.

According to one embodiment of the present application, the vascular disease may be cerebrovascular disease, but may not be limited thereto.

According to the exemplary embodiment of the present application, the cerebrovascular disease may be selected from the cerebrovascular disease is cerebral hemorrhage, cerebrovascular defect, and cerebrovascular injury, but may not be limited thereto.

According to one embodiment of the present application, the test substance may be selected from the group consisting of compounds, microbial cultures, microbial extracts, nucleic acids, antibodies, proteins, peptides, aptamers and natural extracts, but may not be limited thereto. For example, the compound may include a low molecular weight material, and in addition, a library known in the art to which the present invention belongs may be used without limitation.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[ Example ]

One. Harmin ( harmine Inhibition of cerebrovascular development by treatment

After 24 hours of development, zebrafish embryos were treated with inhibitors of DYRK1A at concentrations of 10, 25 and 50 μM, respectively, and incubated in a 28.5 ° C. thermostat for 28 hours, and then embryos 52 hours after development were observed under a microscope. As a result, it was confirmed that the symptoms of hemorrhage (hemorrhage) appeared in up to 65.7% of the embryos depending on the concentration of harmin (upper part of Figure 1a).

The same experiments were carried out by fluorescence-specific fluorescent transformant Tg (kdrl: egfp) embryos at different concentrations, and confocal microscopy showed that cerebral vascular development was inhibited according to the concentration of the harmin . It was confirmed through. In particular, the degree of cerebrovascular injury and development was analyzed using the number of central arteries (CtAs) distributed in the hindbrain among the cerebrovascular vessels. In wild type, the germination degree of central arteries appearing regularly in about 6-7 was reduced (15-31%) by 1-2 and by 25 and 50 μM Harmin treatment (Fig. 1A bottom and 1b). This means that the development of cerebrovascular system is not normal. Therefore, it was confirmed that the developmental disorder of cerebrovascular disease was induced by cerebral hemorrhage by treatment of harmin.

2. Candidate Drug Discovery

Among the two phenotypes of cerebral hemorrhage and cerebrovascular injury, which are manifested by the inhibition of DYRK1A by harmin, we use a relatively observable phenotype of cerebral hemorrhage to find a candidate molecule that can alleviate the symptoms and to analyze the mechanism involved. Drug drug screening was performed.

When five zebrafish embryos were treated at 30 μM per well of a 48-well plate, optical microscopy confirmed that 2-3 out of 5 embryos exhibited cerebral hemorrhage. Under these conditions, a small molecule of 10 μM compound library (Sigma, 1280 LOPAC) was treated with Harmin, respectively, to search for candidate drugs that could accelerate or alleviate the symptoms of cerebral hemorrhage caused by Harmin. . The increase in the number of 2-3 embryos with cerebral hemorrhage increased to 4-5 when 30 μM of Harmin was treated, and the symptoms of cerebral hemorrhage were accelerated. The search proceeded as shown to be (FIG. 2A).

 As a result, 171 drug candidates that could increase or decrease the symptoms of cerebral hemorrhage were identified from a total of 1280 compound libraries. Of the 171 candidates, 128 were found to accelerate cerebral hemorrhage, and 43 drugs were found to improve the symptoms of cerebral hemorrhage (FIG. 2B). In particular, EGTA was identified as the most potent candidate drug that can effectively alleviate the symptoms of cerebral hemorrhage while reducing the frequency of cerebral hemorrhage embryos to zero among the low molecular weight substances found to alleviate the symptoms of cerebral hemorrhage in embryos (FIG. 2C). Figure 2c shows the data of some of the drugs that improved the symptoms of cerebral hemorrhage, EGTA not only reduced the frequency of cerebral hemorrhage embryos to 0, but no cytotoxicity was observed. On the other hand, other drugs were not suitable because the frequency of cerebral hemorrhage embryos was not sufficiently reduced or cytotoxicity was observed.

3. dyrk1aa  Deleted mutation Zebrafish  Induction of cerebral hemorrhage

The brain hemorrhage and cerebrovascular development of zebrafish embryos caused by the DYRK1A-inhibiting effect by harmin was confirmed in the mutant individuals with dyrk1aa deletion. Was it possible to alleviate the symptoms of. For this, we examined the incidence of cerebral hemorrhage in mutant zebrafish embryos that lacked the dyrk1aa, a zebrafish homolog of human dyrk1a. As a result, it was confirmed that cerebral hemorrhage symptom appeared in 20% or more of all individuals in the mutant compared to spontaneous hemorrhage within 2 to 5% in wild type (FIG. 3A). At this time, zebrafish embryos cultured at 28.5 ° C. for up to 48 hours at 35 ° C. were observed to increase the heart rate temporarily, resulting in a sharp increase in cerebral hemorrhage phenotype in dyrk1aa mutants compared to the control group (FIG. 3b) These cerebral hemorrhage-inducing conditions were used to observe the cerebral hemorrhage phenotype more clearly. In general, cerebral hemorrhage occurs at 22.4% in dyrk1aa mutants 52 hours after development at 28.5 ° C, whereas cerebral hemorrhage increases to 37-39% in embryos subjected to high temperature conditions of 35 ° C for 2 hours after 48 hours of development. Was confirmed (FIG. 3B).

4. Zebrafish  On the Cerebrovascular Disease Phenotype in Embryos EGTA  effect

To examine the effects of the identified EGTA, 20 mutant zebrafish embryos that were dyrk1aa deleted 24 hours after each well in each 6-well plate were added, and EGTA was treated at 1, 10 and 100 nM concentrations for 48 hours. Incubated in an incubator at 28.5 ° C until. In order to more clearly observe the symptoms of cerebral hemorrhage and the relief of symptoms by EGTA, EGTA-treated zebrafish embryos were incubated for 2 hours at 35 ° C and further stabilized for 1 hour at room temperature, followed by 4% form. O-Dianisidine staining, which is a staining method capable of detecting hemoglobin activity by fixing with aldehyde and IX PBST solution at room temperature for 4 hours, was performed. After 0.6 mg / ml O-Dianisidine staining solution was treated for about 10 minutes and washed three times with 1X PBST (1XPBS containing 0.1% Tween 20), cerebral hemorrhage was visually confirmed on a microscope. At this time, it was observed that 37% of the symptoms of cerebral hemorrhage seen in the control group were alleviated to about 23% under EGTA 10 nM treatment conditions (FIGS. 4B and 4D).

Furthermore, dyrk1aa mutant fluorescent transformant Tg (kdrl: egfp) zebrafish embryos 52 hours after development were placed in fixed solution (4% formaldehyde, 4% sucrose, 0.15 mM CaCl ₂ , 1XPBS) and fixed at room temperature for 4 hours, The incidence of cerebrovascular CtAs in the posterior brain was confirmed by confocal microscopy (FIG. 4C). For detailed analysis, the cerebrovascular of wild-type and dyrk1aa-deleted mutant fluorescent transformants was quantified by comparing the length of CtAs and the degree of branching points of CtAs (FIG. 4A). In mutant zebrafish embryos, the length and branching of CtAs were reduced by 76% and 57.4%, respectively, compared to wild type, and the length and branching of CtAs were also recovered by EGTA treatment. Could. In particular, the CtAs length was increased by about 10.6% and the branch by about 18.6% under the EGTA concentration of 10 nM, and the development of cerebrovascular development was restored to about 86%. Overall, EGTA, which has been shown to restore the phenotype of cerebral hemorrhage by inhibition of DYRK1A by harmin through low-molecular substance discovery, could be applied to cerebral hemorrhage and cerebrovascular injury of zebrafish embryos genetically deleted from dyrk1aa. Confirmed.

From the fact that EGTA concentrations resulted in more favorable results at 10 nM conditions than at 100 nM conditions, considering the properties of EGTA, a calcium chelating agent, it influenced in vivo homeostasis at excessively high concentrations, causing side effects or 10 nM. It can be interpreted that the range included is the concentration range having the optimum therapeutic effect.

The above description of the present application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

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Claims (12)

Ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid, EGTA ) And a pharmaceutically acceptable carrier or excipient,
A pharmaceutical composition for preventing or treating vascular disease selected from cerebral hemorrhage, cerebrovascular defect, and cerebrovascular injury.
delete The method of claim 1,
The EGTA is contained in a concentration of 8 to 15 nM, pharmaceutical composition.
The method of claim 1,
Pharmaceutical composition, wherein the EGTA is included in a concentration of 10 nM.
delete delete delete delete (a) treating and incubating the germine of zebrafish at a concentration of 30 to 60 μM;
(b) treating the zebrafish embryo with the test material and incubating for 1 hour to 3 hours at a temperature in the range of 30 ° C to 40 ° C; And
(c) selecting a test substance that reduces the phenotype of vascular disease as compared to the untreated control,
A method for screening a prophylactic or therapeutic agent for vascular disease selected from cerebral hemorrhage, cerebrovascular defect, and cerebrovascular injury.
The method of claim 9,
The method of screening is to treat the harmin at a concentration of 50 μM.
delete delete

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