KR102819136B1 - Composition for treating vascular inflammation comprising Shinjulactone A - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular inflammation, comprising syringolactone A as an active ingredient. The composition comprising syringolactone A according to the present invention effectively inhibits atherosclerosis regulated by NF-kB by binding to endothelial cells in a specific manner without affecting immune cells such as macrophages, and thus can be used as a pharmaceutical composition or food composition useful for improving, preventing, or treating tissue-targeting or target-specific inflammation.

Description

{Composition for treating vascular inflammation comprising Shinjulactone A}

The present invention relates to a composition for preventing or treating vascular inflammation comprising succinic acid lactone A; and a food composition for improving or preventing it.

Cardiovascular disease is currently the leading cause of death worldwide. In particular, atherosclerosis is a disease that includes heart attack and stroke and accounts for the majority of deaths due to cardiovascular disease. Atherosclerosis is a chronic inflammatory disease, and immune cell recruitment to the endothelium induces the development of atherosclerotic plaques under hyperlipidemic conditions. Endothelial cells respond to inflammatory stimuli such as blood flow disturbance or multiple inflammatory ligands by activating the key inflammatory mediator NFkB, which transcriptionally upregulates cell adhesion molecules including Vcam-1, Icam-1, E-selectin, and MCP-1 for monocyte recruitment to the endothelium.

IL-1β is an important proinflammatory cytokine produced by macrophages and endothelial cells through the inflammasome pathway, and plays an important role in atherosclerosis. There are experimental results showing that antibody-mediated blockade of IL-1β reduces cardiovascular events related to atherosclerosis in patients. Clinical trials also showed a statistically significant increase in infection rates among patients treated with IL-1β blocking antibodies, suggesting adverse effects of systemic IL-1β blockade on immune function.

Endothelial to mesenchymal transition (EndMT) is a process of endothelial cell dedifferentiation that transforms into a mesenchymal cell type. During this process, endothelial cells lose several endothelial cell-specific marker proteins, such as VE-cadherin and PECAM, and begin to express mesenchymal a-SMA, vimentin, and fibronetin. There is accumulating evidence that EndMT is involved in several vascular diseases, including fibrosis, PAH, and atherosclerosis. Mesenchymal cells induced by EndMT induce the secretion of proinflammatory molecules in atherosclerosis, and affect the regulation of matrix and collagen production and plaque stabilization. In vitro EndMT induction requires inflammatory stimuli, such as TGF-β and impaired blood flow or IL-1β.

Endothelial inflammatory signaling plays a key role in atherogenesis by inducing NFkB-dependent cell adhesion molecule (CAM) expression and monocyte recruitment. Conventional inhibitors, such as Bay 11-782, a well-known NFkB inhibitor, have shown significant cytotoxicity in the long-term treatment of atherosclerosis.

Statins, which are currently widely used as anti-atherosclerosis drugs, have been successful in treating or preventing atherosclerosis by blocking LDL synthesis, but have serious side effects such as muscle pain and increased incidence of diabetes. Some preclinical studies and clinical trials have suggested that anti-inflammatory drugs may be an alternative for treating atherosclerosis, but continued suppression of systemic inflammation may increase the risk of infection.

Therefore, there is an urgent need to develop drug candidates for the prevention or treatment of atherosclerosis that effectively block IL-1β-induced NFkB activation and monocyte recruitment in endothelial cells, while not affecting immune-induced NFkB activation in macrophages.

European Patent Publication EP 3498288 A1 (2019.06.19) US Patent Publication No. US 2017/0000837 A1 (2017.01.05)

The present inventors have made extensive research efforts to develop a drug effective in preventing or treating atherosclerosis without affecting the viability of endothelial cells. As a result, the present invention was completed by elucidating that cinquefoil A efficiently blocks IL-1β-induced NFkB activation and protein cell recruitment in endothelial cells, while having little effect on LPS-induced NFkB activation in macrophages.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating vascular inflammation containing cinnamic acid lactone A as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing vascular inflammation, which contains cinnamic acid lactone A as an effective ingredient.

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

One aspect of the present invention relates to a composition for preventing or treating vascular inflammation, comprising Shinjulactone A as an active ingredient.

In the present invention, cinnamon lactone A is a quassinoid from Ailanthus altissima Swingle, which is used in the treatment of colds and stomach diseases in Asian countries. Unlike the very similar quassinoid Ailanthone (having a carbonyl group at C2 instead of a hydroxyl group), which has been widely studied for its anticancer effects, little is known about the function of cinnamon lactone A except for its weak antitumor activity.

In the present invention, the new lactone A may include a compound represented by the following chemical formula I, but is not limited thereto.

[Chemical Formula I]

In the present invention, the new lactone A has a molecular weight of 378.4 and a molecular formula of C ₂₀ H ₂₆ O ₇ .

In the present invention, vascular inflammation may be caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia, but is not limited thereto.

The term “pharmaceutically effective amount” as used herein means an amount sufficient to achieve the efficacy or activity of synthracite A.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition according to the present invention can be administered to mammals, including humans, by various routes. The administration route can be any route commonly used, and for example, it can be administered by oral, cutaneous, intravenous, intramuscular, subcutaneous, etc. routes, and for example, it can be administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

The composition of the present invention may additionally contain, in addition to the mixed extract, pharmaceutically suitable and physiologically acceptable auxiliary agents such as carriers, excipients, and diluents.

Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations can be prepared by mixing the extract with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Oral preparations include suspensions, solutions, emulsions, syrups, and ointments, and in addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and transdermal preparations. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

Suppository formulations that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

In specific embodiments of applying the composition of the present invention to humans, the composition of the present invention may be administered alone, but may generally be administered mixed with a pharmaceutical carrier selected in consideration of the mode of administration and standard pharmaceutical practice.

For example, the compositions of the present invention may be administered orally, intrabuccally or sublingually in the form of tablets containing starch or lactose, or in the form of capsules alone or containing excipients, or in the form of elixirs or suspensions containing flavoring or coloring chemicals. Such liquid preparations may be formulated with pharmaceutically acceptable excipients such as suspending agents (e.g., methylcellulose, semi-synthetic glycerides such as witepsol, or glyceride mixtures such as mixtures of apricot kernel oil and PEG-6 esters or mixtures of PEG-8 and caprylic/capric glycerides).

The dosage of the composition containing the extract of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist.

In the present invention, the pharmaceutical composition may be administered in a range of 1 to 10 mg/day, 1 to 8 mg/day, for example, 5 mg/day of cinnamic acid A per kg of body weight.

Another aspect of the present invention relates to a food composition for improving or preventing vascular inflammation, comprising Shinjulactone A as an active ingredient.

In the present invention, the new lactone A may include a compound represented by the following chemical formula I, but is not limited thereto.

[Chemical Formula I]

In the present invention, the new lactone A has a molecular weight of 378.4 and a molecular formula of C ₂₀ H ₂₆ O ₇ .

In the present invention, vascular inflammation may be caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia, but is not limited thereto.

When the food composition of the present invention is used as a food additive, the food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. In general, when producing food or beverage, the food composition of the present invention may be added in an amount of 15 wt% or less, for example, 10 wt% or less, relative to the raw material.

The above beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The ratio of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The ratio of these additives may also be appropriately selected by those skilled in the art.

In the above food composition, any content overlapping with the pharmaceutical composition is omitted in consideration of the complexity of this specification.

The present invention relates to a composition for preventing or treating vascular inflammation, comprising syringolactone A as an active ingredient. The composition comprising syringolactone A according to the present invention effectively inhibits atherosclerosis regulated by NF-kB by binding to endothelial cells in a specific manner without affecting immune cells such as macrophages, and thus can be used as a pharmaceutical composition or food composition useful for preventing or treating tissue-targeting or target-specific inflammation.

Figure 1 is a structural formula showing the chemical structure of Shinjulactone A according to one embodiment of the present invention.
Figure 2 is a diagram showing the results of immunoblotting of p-p65 and actin according to one embodiment of the present invention.
Figure 3 is a graph showing the quantitative results of immunoblotting of p-p65 and Actin according to one embodiment of the present invention.
Figure 4 is a diagram showing monocyte adhesion to endothelial cells according to one embodiment of the present invention.
Figure 5 is a graph showing the results of counting monocyte adhesion to endothelial cells according to one embodiment of the present invention.
Figure 6 is a diagram showing the results of immunoblotting of p-p65 and actin after treatment with succinic acid lactone A according to one embodiment of the present invention.
Figure 7 is a graph showing the quantitative results of immunoblotting of p-p65 and actin after treatment with succinimidyl lactone A according to one embodiment of the present invention.
Figure 8 is a graph showing the results of measuring cell viability after treatment with Shinjulactone A according to one embodiment of the present invention.
Figure 9 is a diagram showing the results of evaluating the levels of endothelial markers and mesenchymal markers by treating with IL-1β and TGF-β1 after treating with succinyl lactone A according to one embodiment of the present invention.
Figure 10 is a graph showing the quantitative results of immunoblotting after treatment with succinic acid A and IL-1β and TGF-β1 according to one embodiment of the present invention.
Figure 11 is a diagram showing cell-to-cell adhesion of BAEC cells on day 2 and day 5 after treatment with IL-1β and TGF-β1 following treatment with syringamine A according to one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Immunoblotting

1-1. Preparing reagents

In this study, the antibodies used for immunoblotting were phospho-NFkB p65 (S ⁵³⁶ , Cell signaling technology), beta-actin (Cell signaling technology), anti-rabbit IgG, and anti-mouse IgG (R&D systems). LPS (1ug/ml used), IL-1β (10ng/ml or 20ng/ml used), and TGF-b1 (10ng used) were purchased from Sigma. Shinjulactone A used in this experiment was purified as a single component from the bark of the Korean Institute of Oriental Medicine.

1-2. Cell culture

Primary bovine aortic endothelial cells (BAEC) were isolated and maintained in Dulbecco's Modified Eagle Medium (DMEM, GenDEPOT) supplemented with 10% FBS, penicillin, and streptomycin. All cell types were maintained at 37 °C in a 5% CO ₂ incubator. THP-1 cells were maintained in RPMI-1640 supplemented with 10% FBS, penicillin, streptomycin, and 0.05 mM β-mercaptoethanol. Macrophage Raw 264.7 cells were cultured in DMEM containing 10% FBS and penicillin and streptomycin.

1-3. Transition from endothelium to mesenchyme

One day after re-application, cells were pretreated with succinyl lactone A (10 uM) and DMSO as a control for 1 h before incubation with TGF-β1 (10 ng/ml) and IL-1b (10 ng/ml). Medium was changed every 2 days to supply fresh ligands. After 2 and 5 days, cells were harvested for examination of morphology and immunoblotting using EndMT marker antibodies.

1-4. Immunoblotting

Equal amounts of protein samples were loaded into the wells of a 10% polyacrylamide SDS-page gel and denatured with SDS-running buffer. The proteins were transferred to NC membranes (Nitrocellulose Filter, GE Healthcare Life science) and blocked with 5% skim milk. The membranes were incubated with p-p65 and actin antibodies overnight at 4 °C. After washing three times for 10 min with TBST, anti-rabbit IgG and anti-mouse IgG secondary antibodies were incubated for 1 h at room temperature. After washing three times for 10 min each with TBST, images were acquired using a chemiluminescent substrate (Thermofisher). The results were quantified using Image J and Prism5 software.

1-5. Monocyte adhesion

One day after cell seeding, endothelial cells (ECs) were added with cinnamaldehyde lactone A (1 μM, 10 μM, Figure 1 ) or DMSO as a control. After 1 h, they were treated with IL-1β (20 ng/ml). After 6 h, THP-1 cells were centrifuged and resuspended in 5% BSA (Bovine Serum Albumin, GeorgiaCHem) in HBSS (Hanks's Balanced Salt Solution, Gibco). After washing the cells once with PBS, the resuspended THP-1 cells were cultured on the endothelial cell monolayer for 30 min in a 5% CO ₂ , 37 °C incubator. After 30 min, unbound THP-1 cells were washed four times with 0.5% BSA HBSS. After fixing the cells with a 4% solution of paraformaldehyde in PBS (USB) for 10 min, bound monocytes were counted under a microscope. Results were quantified using Prism 5 software.

1-6. Cell viability

Bovine aortic endothelial cells were treated with cisplatinum A (1–10 μM) or BAY 11-782 (1–10 μM) and maintained at 37 °C in a 5% CO ₂ incubator for 5 days. Live cells were counted every 24 h after trypan blue staining (Thermofisher) using a Countess II FL automated cell counter, and the results were quantified using Prism 5 software.

Example 2. Evaluation of inhibition of vascular inflammation by Shinjulactone A

To investigate the effect of cisplatin A on vascular inflammation, NF-kB activation was examined. NF-kB activation was monitored by immunoblotting for pS ⁵³⁶ -p65, which was gradually decreased with increasing doses of cisplatin A compared to the DMSO control, and the results are shown in Table 1 and Figures 2 and 3.

Shinjulactone A Bay um 0 0.2 1.0 5.0 10.0 1.0 IL-1B (20ng) - + - + - + - + - + - + Mean 1 3.50291 0.83343 1.82712 0.7456 1.68598 0.61719 1.04549 0.4273 0.51869 0.41948 0.88859 SE 0 1.96012 0.17113 0.79784 0.18058 0.6001 0.17132 0.11664 0.06226 0.06493 0.13752 0.19378

As shown in Figures 2 and 3, the NF-kB inhibitor Bay 11-782 almost completely blocked NF-kB activation at a concentration of 1 uM, and 5 uM cinthoxyluminal A induced the same level of NFkB inhibition.

Example 3. New lactone A-mediated monocyte attachment

Adhesion of monocytes to endothelial cells leads to migration of endothelial cells and further inflammation. Monocyte THP-1 cells were added to endothelial cells treated with cinquefoil A and incubated for 30 minutes. Unbound monocytes were then washed away, and the attached monocytes were counted and quantified, and the results are shown in Figures 4 and 5.

As shown in Figures 4 and 5, monocyte adhesion to bovine aortic endothelial cells induced by IL-1β was significantly reduced in cells treated with cisplatin A, supporting the anti-inflammatory activity of cisplatin A.

Example 4. Blockade of LPS-induced NF-kB activation in macrophages

LPS is well known to induce the inflammasome pathway by NF-kB activation in macrophages. We tested whether the lactone A-dependent NF-kB inhibition was specific to endothelial cells, and the results are shown in Figs. 6 and 7.

As shown in Figures 6 and 7, we were able to confirm that syringamine A, as a Bay compound, does not suppress inflammation in endothelial cells by directly modulating the NF-kB activation machinery, but rather modulates cell type-specific or ligand-specific signals.

Example 5. Cytotoxicity evaluation of Shinjulactone A

The toxicity of cultured bovine aortic endothelial cells treated with 1 to 10 uM of cinquefoil lactone A was analyzed compared to the control and BAY 11-782 (1 to 10 uM) treated cells. The cells were observed for 5 days after treatment. The viable cells were counted every 24 hours using an automated cell counter after trypan blue staining, and the results are shown in Table 2 and Fig. 8.

days DMSO Shinjulactone A BAY 1 uM SE 1 uM SE 10 uM SE 1 uM SE 10 uM SE 1 85.24752 1.43275 88.25719 3.68963 91.96803 1.16663 84.80286 1.3052 58.47164 7.10787 2 85.35791 2.12067 87.34491 2.9429 87.75641 1.98796 85.91787 2.47553 42.48043 17.96584 3 89.77519 0.33776 89.19176 0.74297 86.9964 3.70314 84.25221 2.59972 48.21648 15.40378 4 79.91293 3.86046 81.92299 3.93849 86.53237 2.53093 85.28011 0.75721 59.52763 13.0193 5 85.36943 1.43605 86.00349 3.4075 82.57991 2.7129 84.72315 3.83799 43.77423 12.10106

As can be seen in Table 2 and Fig. 8, the viability of cells treated with succinyl lactone A (1 to 10 uM) was 90% higher than that of untreated cells, indicating that succinyl lactone A (1 to 10 uM) is not toxic to cells.

Example 6. Evaluation of the reduction of endothelial-mesenchymal transition by Shinjulactone A

The effect of cisplatin A on endothelial-mesenchymal transition was investigated. Specifically, endothelial cells treated with 0 uM and 10 uM cisplatin A, respectively, were treated with IL-1β and TGF-β1, and the levels of endothelial and mesenchymal markers were evaluated, and the results are shown in Figures 9 to 11.

As shown in Figs. 9 to 11, the down-regulation of VE-cadherein and the up-regulation of a-SMA were partially blocked after 5 days of treatment with cisplatinum A. In the absence of IL-1β and TGF-β1, the endothelial cells had a cobblestone-like phenotype and were closely attached to each other. In addition, when IL-1β and TGF-β1 were simultaneously treated for 2 days, the endothelial cells showed a spindle-shaped morphology and loss of cell-cell adhesion, but there was no significant change in cell morphology when treated with cisplatinum A compared to the untreated control and the co-treated group. In addition, in the sample after 5 days, cisplatinum A still maintained the cell-to-cell adhesion of vascular endothelial cells.

Claims (6)

A pharmaceutical composition for preventing or treating vascular inflammation, comprising shinjulactone A as an active ingredient. A pharmaceutical composition for preventing or treating vascular inflammation, wherein in claim 1, the new lactone A comprises a compound represented by the following chemical formula I.
[Chemical Formula I]
A pharmaceutical composition for preventing or treating vascular inflammation, wherein in claim 1, the vascular inflammation is caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia. A food composition for improving or preventing vascular inflammation, comprising shinjulactone A as an active ingredient. A food composition for improving or preventing vascular inflammation, wherein in claim 4, the new lactone A comprises a compound represented by the following chemical formula I.
[Chemical Formula I]
A food composition for improving or preventing vascular inflammation, wherein in claim 4, the vascular inflammation is caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia.