KR101664106B1 - A pharmaceutical composition comprising polymyxin B as a active ingredient for prevention or treatment of cardiovascular diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, which comprises a polymyxin B compound as an active ingredient, and more particularly to a pharmaceutical composition for preventing and treating cardiovascular diseases comprising a polymyxin B compound as an active ingredient ≪ / RTI >

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing and treating cardiovascular diseases including a polymyxin B compound,

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, which comprises a polymyxin B compound as an active ingredient, and more particularly to a pharmaceutical composition for preventing and treating cardiovascular diseases comprising a polymyxin B compound as an active ingredient ≪ / RTI >

Cardiovascular disease is the leading cause of death in Korea as well as the world. It is expected that the number of cardiovascular disease patients will continue to increase due to the increase in adult diseases and the aging population. However, the treatment for heart failure, myocardial infarction, Almost never.

Recently, seven subtypes of G-protein-coupled receptor kinases (GRKs) have been found and phosphorylated the intracellular C-terminal regions of beta adrenergic receptors and adenosine receptors to induce desensitization of receptors ). ≪ / RTI > GRK5 (G-protien-coupled receptor kinase 5) is expressed specifically in cardiac muscle and is recognized as a major regulator of cardiac signal transduction and function. GRK5 activity itself is directly enhanced by stimuli that cause cell hypertrophy (e. G., Epinephrine, angiotensin II, endothelin) (Huang et al., Front Biosci, 2011, 16, 3047-60). Unlike other GRKs, GRK5 has a nuclear localization sequence (NLS) within the catalytic domain of GRK5. GRK5, activated by external stimuli, migrates to the nucleus and class II HDAC5 (Histone deacetylase 5) and induce transcription of heart failure-associated genes (Marini et al., Proc Natl Acad Sci USA, 2008, 105, 12457-62).

In addition, GRK5 mRNA and protein overexpression was observed in 31 patients with heart failure following cardiac transplantation, and cardiovascular events such as cell hypertrophy and stress fiber induction were observed. (Aguero et al, J Card Fail, 2011, 18, 53-61) have been demonstrated to correlate with various pathologic markers of disease.

Therefore, the need for the development of a GRK5 inhibitor is that GRK5 overexpressed transgenic mice exhibit excessive myocardial hypertrophy and cardiac dysfunction, resulting in heart failure and death. In the case of mice expressing GRK5 deficient in nucleus-targeting sequences, GRK5 (Marini et al., Proc Natl Acad Sci USA, 2008, 105, 12457-62) in overtly transgenic mice (Tg GRK5 mice). In addition, the GRK5-deficient mouse (GRK5-KO) showed a slight hypothermia and hypoactivity as a whole and was found to be resistant to myocardial hypertrophy and cardiac dysfunction (Gold et al, Circ Res , 2012, 111, 1048-53). Currently, no clinically available GRK5 inhibitors have been reported yet, and recent trends have led to the development of inhibitors with higher selectivity and no GRK5 inhibition without cross-activity with other AGC kinase families such as PKA, PKC, and receptor tyrosine kinase Research is underway.

On the other hand, polymyxin B is a peptide antibiotic substance first discovered in the 1950s and is produced from Bacillus polymyxa and is a selective antibiotic for Gram-negative infections. The pharmacological mechanism of polymyxin B binds to the negatively charged site of the outer wall of the cell, causing a change in the structure and increasing the permeability of the cell wall. In the present study, we investigated the effects of antioxidants on cell proliferation and cell proliferation. Recently, it has attracted attention as an excellent therapeutic effect for multidrug-resistant gram-negative bacteria. Recently, it has been applied to polymyxin B-immobilized fiber colums for the prevention of septic shock after cardiac surgery or for improving liver function after ischemic injury (Murakami et al., Circ J, 2009, 73, 658-661; Sato et al., 2009, 15, 4571-4575), but the effects of polymyxin B on cardiovascular disease, including direct inhibition of GRK5 and concomitant myocardial fibrosis, have not yet been reported.

Accordingly, the present inventors have completed the present invention by discovering that polymyxin B can be used as an effective ingredient of a therapeutic agent for heart failure diseases by inhibiting GRK5 activity in cardiac cells.

KR 2009-7004841

(Marini et al, Proc Natl Acad Sci U SA, 2008, 105, 12457-62). (Aguero et al, J Card Fail, 2011, 18, 53-61)

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cardiovascular diseases comprising a polyamicin B compound as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases comprising a polyamicin B compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

[In the above formula (1)

R is (C ₁ -C ₂₀ ) alkyl and the alkyl may be further substituted with (C ₁ -C ₁₀ ) alkyl.

The present invention provides a composition, wherein said compound selectively inhibits the activity of GRK5.

The present invention provides a composition wherein the compound inhibits cell hypertrophy.

또는

인 것을 특징으로 하는 조성물을 제공한다. The present invention provides a compound of formula

or

≪ / RTI >

The present invention provides a composition characterized in that said compound is contained at a concentration of 0.003 to 100 μM based on the total weight of the composition.

The present invention provides a composition wherein the cardiovascular disease is selected from the group consisting of cardiovascular diseases caused by cardiac hypertrophy, heart failure, ischemic heart disease, angina pectoris, arteriosclerosis, myocardial infarction, myocardial fibrosis and the like.

The present invention has an excellent effect as an inhibitor of GRK5, which is correlated with various pathologic markers of cardiovascular diseases such as polymyxin B, which is a selective antibiotic against Gram-negative bacteria, and induces cell hypertrophy and actin stress fiber And found a possibility as a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing the polyamicin B compound as an active ingredient. The compounds inhibit cardiomyocyte hypertrophy, which is a direct pathology of heart failure, by inhibiting GRK5 activity and inhibit the formation of actin stress fibers in cardiac cells, thereby preventing or treating cardiovascular diseases such as myocardial infarction, heart failure and myocardial fibrosis It can be used as a highly effective therapeutic agent.

Figure 1 shows the effect of GRK inhibitory activity on polymyxin B at various concentrations.
Figure 2 shows the result of analysis of the inhibition pattern of polymyxin B due to inhibition of GRK5.
FIG. 3 shows the results of inhibiting the formation of actin stress fibers by concentration of polymyxin B. FIG.
Fig. 4 shows the results of experiments confirming the effect of the polyamicin B on the cell hypertrophy.

Hereinafter, the present invention will be described in more detail.

Polymyxin is a lipopeptide-based remedy that has excellent therapeutic effects against infectious diseases of multi-drug resistant bacteria (super bacteria). It has been found more than 60 years ago, but despite its excellent sterilizing power, it is limited due to toxicity (kidney toxicity and neurotoxicity) As antibiotics resistant to various antibiotics have emerged as a big issue in the world, it is a new antibiotic.

These polymyxins are components of gram-negative bacillus produced by Bacillus polymyxa , peptide antibiotics A, B, C, D and E, which are effective against P. aeruginosa.

The polyamicin B mainly composed of B ₁ (C ₅₆ H ₉₈ N ₁₆ O _13, molecular weight of 1203.49) and B ₂ (C ₅₅ H ₉₆ N ₁₆ O _13, molecular weight of 1189.47) was practically used and antibiotic It is characterized by a polypeptide structure, narrow antimicrobial spectrum, almost exclusively acting on Gram-negative bacteria, and acts on sterilizing power like penicillin. Oral and intramuscular injections are rapidly absorbed into the body while excretion is relatively late. Clinically, almost all of the other antibiotics are useful for infections caused by ineffective P. aeruginosa. Side effects include kidney failure, perception, and neuropathy.

The present invention can be used for preventing or treating cardiovascular diseases such as myocardial fibrosis mitigation or heart failure disease treatment by inhibiting GRK5 activity in cardiac cells by using the aforementioned antibiotic polymyxin B The present invention has been completed to provide a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof.

The present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases comprising a polyamicin B compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

[In the above formula (1)

R is (C ₁ -C ₂₀ ) alkyl and the alkyl may be further substituted with (C ₁ -C ₁₀ ) alkyl.

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cardiovascular diseases comprising the polymyxin B compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

또는 More preferably, in Formula 1, R is

or

인 것이 가장 바람직하다.

Is most preferable.

The inventors of the present invention conducted studies to develop a preventive or therapeutic agent for cardiovascular diseases such as myocardial fibrosis or heart failure. As a result, overexpression of GRK5 mRNA and protein was observed in heart failure patients after heart transplantation, fiber induction, etc., and that the antibiotic polymyxin B directly inhibits GRK5, thereby effectively preventing or preventing cardiovascular diseases including heart failure or myocardial fibrosis, And confirmed the possibility as a therapeutic agent.

The polymyxin B compound directly inhibits GKR5 activity and inhibits excessive production of actin stress fibers in the cardiac cells due to the activity of GRK5, whereby the polymyxin B compound acts as a GRK5 inhibitor , And can be effectively used for the prevention or treatment of cardiovascular diseases.

In addition, since the excessive activity of GRK5 is recognized as one of the main factors of cardiac hypertrophy, which is a direct pathological condition of heart failure, which is one of the cardiovascular diseases, the ability of the polyamicin B compound according to the present invention to inhibit cell hypertrophy, B compound as an active ingredient as an agent for the prevention or treatment of cardiovascular diseases.

The present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases, which comprises a polymyxin B compound as an active ingredient. In one embodiment of the present invention, 10 mu M, more preferably 0.1 to 3 mu M, but it is not limited thereto.

When the effective concentration of the polymyxin B compound is significantly inhibited actin stress fibrous formation, the therapeutic efficacy of cardiovascular diseases such as heart failure, myocardial fibrosis or myocardial infarction may be more excellent.

The pharmaceutical composition for preventing or treating cardiovascular diseases comprising the polyamicin B compound of the present invention as an active ingredient may further comprise a pharmaceutically acceptable additive in addition to the effective ingredient.

The above additives may be formulated together with carriers, excipients or diluents, and may be formulated into tablets, hard or soft capsules, granules, chewing tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups and preparations for oral administration Aerosols, sterile injectable solutions or sterile powders, and the like. Formulations suitable for this purpose may be administered parenterally, e. G. Subcutaneously, intravenously, intramuscularly or intraperitoneally. Parenteral administration preparations such as injectable solutions or suspensions, injectable preparations such as ready-to-use injectable dry powders which can be used as injectable distilled water for injections, or topical preparations such as ointments are preferred. At this time, a diluent such as a filler, an extender, a gum arabic, a binder such as corn starch, microcrystalline cellulose or gelatin, a wetting agent, a disintegrating agent such as alginic acid, corn starch or potato starch, a surfactant, Excipients such as oz may be used together.

When the pharmaceutical composition for prevention or treatment of cardiovascular diseases comprising the polymyxin B according to the present invention as an active ingredient is to be used as a therapeutic agent, the effective dose may be variously related to the age, sex, health condition, It can be determined by taking factors into consideration, and it can be administered once or several times a day.

In providing a pharmaceutical composition for preventing or treating cardiovascular diseases comprising the polyamicin B compound of the present invention as an active ingredient, the cardiovascular diseases specifically include heart hypertrophy, heart failure, ischemic heart disease, angina pectoris, arteriosclerosis, Myocardial infarction, myocardial infarction, cardiovascular disease, myocardial fibrosis, and the like. However, the present invention is not limited thereto.

Hereinafter, the present invention will be described more specifically by way of examples. It is intended to assist the understanding of the present invention and is not intended to limit the scope of the present invention in any way.

Example 1: Inhibitory effect of G-protein coupled receptor kinase 5 (G-protein coulped receptor kinase 5 (GRK5)

An experiment for confirming the inhibitory activity of polymyxin B against GRK5 (G-protein coupled receptor kinase 5) was carried out using polyamicin B satisfying the following formula (2). The experiment was carried out using a differential scanning calorimetry (LANCE kinase system, Perkin-Elmer).

(2)

(50 mM HEPES pH 7.5, 10 mM MgCl ₂ , 1 mM EGTA, 2 mM DTT, 0.01% Tween 20, and Perkin-Elmer) buffered with buffer solution to induce phosphorylation reaction and fluorescence (Perkin-Elmer), 260 μg / ml of GRK5 (# PV3824, Invitrogen), and 10 mM ATP (Invitrogen) were added to a solution of 5 μM Ulight-histone H3 (Thr3) peptide (AR T KQTARK S TG, (Sigma-aldrich).

5 μg / ml (final concentration: 0.5 μg / ml) of 260 μg / ml GRK5 (# PV3824, Invitrogen), 5 μM substrate Ulight-H3 (Thr3) peptide (AR T KQTARK S TG, Perkin- 400 nM (final reaction concentration: 80 nM) and 40 μM (final reaction concentration: 20 μM). As a negative control, 2 μl of 5% DMSO, 2 μl of substrate and 1 μl of phosphorylation buffer were used. For positive control, 2 μl of 5% DMSO, 2 μl of substrate solution, 1 mg of GRK5 solution Lt; / RTI > As experimental groups, 2 μl of the polyamicin B of the above formula (2), 2 μl of the substrate solution and 1 μl of the GRK 5 solution were used. Before the phosphorylation reaction, pretreatment was carried out between the compound and the enzyme for about 10 minutes, and then phosphorylation reaction was induced by adding 5 A of ATP. Each test substance, enzyme, substrate, and ATP were 10%, 20%, and 50% of the total volume during the reaction, so they were prepared at a high concentration of 10, 5, and 2 times before the addition. After that, it was shaken for 3 minutes and induced phosphorylation reaction at room temperature for 1 hour. Then, 5 인 of phosphorylation stop solution (40 mM EDTA) and 5 형 of fluorescent detection solution (8 nM europium-labeled anti-phospho H3-antibody) for inducing a differential fluorescence reaction were successively added, The reaction was induced. The anti-phospho H3 antibody labeled with a fluorescent substance Europium (Eu) is diluted in the fluorescence detection solution, and the binding reaction between the phosphorylated site of the Thr residue of the substrate and the anti- Sex fluorescent reaction was induced. Time-resolved fluorescence resonance energy transfer (TR-FRET) values were measured using a multilabel counter (Envision, Perkin-Elmer) at an ambient temperature of 1 hour nm, 665 nm, excitation wavelength: 320 nm), to the expression (1) and a differential property and calculating the fluorescent reaction rate and inhibition rate, and as a result the concentration of polymyxin B by inhibiting the GRK5 50% in the in vitro according to the 2 IC ₅₀ (Fig. 1).

Equation 1

Equation 2

As shown in FIG. 1, as a result of evaluating the effect of GRK5 as a GRK5 inhibitor on various concentrations of polymyxin B (0.003 μM to 100 μM), GRK5 activity was inhibited in a concentration-dependent manner and the IC ₅₀ value was analyzed to be 1.1 μM ).

Since the overexpression of GRK5 is frequently observed in patients with cardiovascular diseases and correlates with various pathological markers of cardiovascular diseases and the degree of GRK5 expression can be used as a measure of symptoms of the disease, Of the present invention can be effectively used for the treatment of cardiovascular diseases of polymyxin B through direct inhibition of GRK5 expression.

Example 2: Analysis of the inhibitory effect of polymyxin B on GRK5 inhibitory effect

In order to analyze the inhibitory mode of the polymyxin B according to Example 1 of the present invention according to GRK5 inhibition (Final reaction concentration: 0.5 μg / ml), 400 nM substrate (final reaction concentration: 80 nM), various concentrations of ATP (final reaction concentration: 2.5 to 40 μM) and polyamicin B (final reaction concentration: 0 μg / ml). 1.5, and 3 μM) was induced in the same manner as in Example 1, and the differential fluorescence reaction rate at 0, 20, 40, and 60 minutes was measured in order to measure the phosphorylation reaction rate. The initial reaction rate (the amount of phosphorylated substrate per minute, nM / min) and the amount of phosphorylated substrate (ATP) per minute were calculated using the regression line between the differential fluorescence response rate and the phosphorylated substrate amount, Were applied to double reciprocal plots on the basis of the amount (μM) of polymyxin B. The inhibition pattern of polymyxin B by GRK5 inhibition was analyzed.

As shown in FIG. 2, the inhibition pattern of the polymyxin B inhibited by GRK5 inhibition was analyzed by ATP-non competitive inhibitor (ATP), indicating that polymyxin B is not an ATP binding site (Fig. 2). ≪ tb >< TABLE >

Example 3: Test for inhibiting actin stress fiber formation

Actin stress fibrosis is a direct cell morphology of cardiac cells and vasoconstriction that occurs after myosin L chain phosphorylation. Recently, beta-arrestin (RhoA-ROCK) (Rho phosphorylase ) Signal transduction activity, the mechanism of excessive actin stress fibrosis formation in cardiac cells has been reported. Therefore, in order to verify GRK5 inhibition of polymyxin B, actin stress fiber formation inhibition test in cardiac cells was performed as follows.

The cardiac cell H9c2 cells cultured in a chamber slide (16 well) were pretreated with the test substance of the polymyxin B according to Example 1 for 1 hour and stimulated with angiotensin II (300 nM) Lt; RTI ID = 0.0 > F-actin < / RTI > After washing with PBS (phosphate buffered saline), the cells were fixed with 4% paraformaldehyde (Sigma-aldrich) for 30 minutes. The immobilized cells were resuspended in 0.5% triton X-100 solution for 5 minutes and then treated with a blocking solution containing 1% BSA (bovine serum albumin) for 30 minutes at room temperature. F-actin stress fibers and nuclei were stained using Alexa fluor 586 phalloidin and Hoechst staining reagents and cell images were collected by optical microscopy.

As shown in FIG. 3, actin stress fiber formation strongly induced by angiotensin II (300 nM) was inhibited in a concentration-dependent manner by pretreatment of polyamicin B for 2 hours. In particular, polyamicin B compound Statistically significant inhibition of actin stress fiber formation was observed at concentrations above 3 μM. These results demonstrate the efficacy of the polymyxin B compound to effectively inhibit the formation of actin stress fibers in cardiac cells observed in patients with cardiovascular diseases, thus confirming the possibility of its usefulness as a therapeutic agent for cardiovascular diseases of polyamicin B.

Example 4: Celluar hypertrophy inhibition assay

One of the major factors of cardiac hypertrophy, which is a direct pathology of heart failure, is the overactive activity of GRK5. Therefore, an experiment for verifying the inhibition of GRK5 through inhibition of cell hypertrophy of polymyxin B was carried out as follows.

Cardiac cell H9c2 cells were pretreated with the test substance of polyamicin B according to Example 1 for 1 hour and continuously administered angiotensin II (300 nM), a cardiac cell hypertrophy factor, for 4 days. After washing with PBS (phosphate buffered saline), cells were fixed with 1% glutaraldehyde for 30 minutes. Fixed cells were stained with 0.1% Crystal vial (Sigma-aldrich) for 10 min. Cell images were collected using a Nikon microscope equipped with a digital camera and Image-Pro PLUS software (MediaCybernetics, Silver Spring) And the number and size of cells were analyzed. It was confirmed that angiotensin II inducing cardiac cell hypertrophy induces about 44% of cardiac hypertrophy by continuously administering angiotensin II for 4 days. The degree of cardiac hypertrophy by the angiotensin II- (%), And the effect of inhibiting cardiac hypertrophy at each concentration is shown in FIG. 4 as a graph.

As shown in FIG. 4, it was confirmed that polymyxin B inhibited angiotensin II-induced cardiac hypertrophy in a concentration-dependent manner. In particular, when the concentration of polymyxin B was more than 1 μM, a statistically significant inhibitory effect Respectively.

Claims (6)

A polymyxin B compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, wherein the polymyxin B compound selectively inhibits the activity of GRK5 for the prophylaxis and treatment of cardiac hypertrophy or myocardial fibrosis A pharmaceutical composition.
[Chemical Formula 1]

[In the above formula (1)
R is (C ₁ -C ₂₀ ) alkyl and the alkyl may be further substituted with (C ₁ -C ₁₀ ) alkyl.
delete delete The method according to claim 1,
Wherein R is

or

≪ / RTI >
5. The method of claim 4,
Wherein said compound is contained at a concentration of from 0.003 to 100 μM based on the total weight of the composition.
delete

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