KR101083433B1 - Composition for prevention and treatment of atherosclerosis comprising tetrahydroisoquinoline compound - Google Patents

Abstract

The present invention relates to YS49 [1- (α-naphthylmethyl) -6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] and YS51 [1- (β-naphthylmethyl) -6 , 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] relates to a composition for preventing and treating atherosclerosis, which comprises a tetrahydroisoquinoline compound selected as an active ingredient.

According to the present invention, the tetrahydroisoquinoline compound can selectively prevent VCAM-1 expression in arterial endothelial cells induced by LPS by activating PTEN to inhibit Akt phosphorylation, thereby effectively preventing and treating atherosclerosis. .

YS49, YS51, PTEN, Akt, VCAM-1, Atherosclerosis

Description

Composition for prevention and treatment of atherosclerosis comprising tetrahydroisoquinoline compound

The present invention relates to a composition for preventing and treating atherosclerosis comprising at least one tetrahydroisoquinoline compound selected from YS49 and YS51 as an active ingredient.

Arteries deliver blood from the heart to all organs of the body. Artery wall is elastic and smooth inside, so blood flow is effective according to the heart rate. Several components in the blood affect the artery walls, and cholesterol plays a leading role. Cholesterol, triglycerides, etc. are deposited in the walls of the arteries, thickening the walls, narrowing the lumen of the blood vessels through which blood can pass, and loss of elasticity can seriously block the blood vessels completely. Atherosclerosis is a disease that occurs in arteries that can develop into serious heart and brain diseases.

Atherosclerosis is a lesion that results from inflammation caused by the injury of cholesterol and triglycerides in the artery wall. Atherosclerosis is most common in the beginning of the aorta, the coronary arteries, the carotid arteries, and the cerebral arteries to the head, which are relatively high in blood flow and are more likely to be physically impacted and injured by these impacts. Because. Monocytes adhere to the wound arterial endothelial cells, which infiltrate under the endothelium and are converted to macrophage. When there is a lot of fat in the blood, macrophages capture it and turn it into foam cells. Therefore, atherosclerosis patients have many macrophages and foam cells. Leukocytes, such as macrophages, are concentrated at the site of inflammation to remove the inflammation. Macrophages, in the course of this action, leave the wound on the artery wall, and secondary fat plaques tend to form on these wounds. Over time, this can lead to narrowing of the arterial wall, increasing the likelihood of developing diseases such as blood clotting, heart disease, and stroke.

The adhesion of mononuclear cells to the arterial endothelial cells described above requires expression of adhesion molecules such as intracellular adhesion molecule-1 (IMC-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin. Thus, inhibition of expression of adhesive molecules can be an effective atherosclerosis therapy. Recently, however, selective inhibition of VCAM-1 has been reported to be more important in the treatment of atherosclerosis than simultaneously inhibiting adhesion molecules such as VCAM-1 or ICAM-1 in arterial endothelial cells.

Accordingly, the present inventors are exploring a substance capable of selectively inhibiting the expression of VCAM-1, and tetrahydroisoquinoline compounds such as YS49 and YS51 activate PTEN to inhibit phosphorylation of Akt, thereby selectively selecting only expression of VCAM-1. It was confirmed that it can be inhibited and prevented and treated atherosclerosis, and came to complete the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing and treating atherosclerosis, which comprises a tetrahydroisoquinoline compound selected from at least one selected from YS49 and YS51 as an active ingredient.

The active ingredients of the present invention are YS49 [1- (α-naphthylmethyl) -6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] (also called CKD712) and YS51 [1- (β-naphthylmethyl) -6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline]. The compound may be racemate or (S)-or (R) -enantiomer, but since (R) -enantiomer has lower action than (S) -enantiomer, (S) -enan Tiomer is more preferable. The compounds may be included in the composition of the present invention alone or in combination.

In addition, the present invention may include a pharmaceutically acceptable salt of the tetrahydroisoquinoline compound or prodrugs thereof.

The salt of the tetrahydroisoquinoline compound means a conventional salt used in pharmaceuticals, and various acids used in the preparation of the salt include hydrochloric acid, bromic acid, sulfuric acid, methanesulfonic acid, propionic acid, succinic acid, glutaric acid, citric acid, Fumaric acid, maleic acid, tartaric acid, glutamic acid, gluconic acid, glucuronic acid, galacturonic acid, ascorbic acid, carbonic acid, phosphoric acid, nitric acid, acetic acid, L-aspartic acid, lactic acid, vanic acid and hydroiodic acid.

As described above, the present invention includes a prodrug of the tetrahydroisoquinoline compound, which is generally a functional derivative of such a prodrug compound, and should be easily changed in order to enter a living body and exhibit drug efficacy. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the existing literature.

The above-described YS49 or YS51 compounds do not affect the expression of ICAM-1, while the expression of VCAM-1 can be suppressed. The reason why the YS49 or YS51 compound selectively reduces the expression of VCAM-1 is because it activates PTEN to inhibit Akt phosphorylation. Thus, YS49 or YS51 compounds can effectively prevent and treat atherosclerosis.

Pharmaceutical compositions containing the tetrahydroisoquinoline compounds of the present invention may be formulated for oral administration such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, sterile injectable solutions, suppositories, and transdermal administrations according to conventional methods. It can be formulated into a formulation for use. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, it is formulated with diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like.

In one embodiment, the tetrahydroisoquinoline compounds of the present invention may be formulated as solid preparations for oral administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. It is formulated by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

In another embodiment, the pharmaceutical composition containing the tetrahydroisoquinoline compound of the present invention may be formulated into a liquid preparation for oral administration. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to conventional inert diluents (e.g., purified water, ethanol, liquid paraffin), various excipients may be used. Wetting agents, sweetening agents, fragrances, preservatives and the like can be included.

In another embodiment, the pharmaceutical compositions containing the tetrahydroisoquinoline compounds of the present invention may be formulated in preparations for parenteral, preferably intraperitoneal administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As a sterile aqueous solution, suitable buffer solutions such as Hanks' solution, Ringer's solution, or physically buffered saline can be used.For non-aqueous solvents, suspensions include propylene glycol, polyethylene glycol, olive oil, Same vegetable oils, injectable esters such as ethyloleate, and the like can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts for controlling osmotic pressure and / or buffers may also be used. Meanwhile, in the case of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like, which are conventional bases thereof, may be used.

Compositions formulated in such a manner can be administered in various amounts, including orally or parenterally (percutaneously, subcutaneously, intravenously, intramuscularly, intraperitoneally) in an effective amount. As used herein, an "effective amount" refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the composition according to the present invention may be appropriately selected depending on the route of administration, subject of administration, age, sex, weight, individual difference and disease state. Preferably, the composition of the present invention may vary the content of the active ingredient according to the degree of disease, preferably 1 μg ~ 100mg / weight kg / day, more preferably 10 μg ~ 10mg / weight kg / day It may be administered in an effective amount of.

According to the present invention, the tetrahydroisoquinoline compound selected from at least one selected from YS49 and YS51 can selectively inhibit only expression of VCAM-1 by activating PTEN to inhibit phosphorylation of Akt, thereby preventing and treating atherosclerosis.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Materials and Methods

1-1. Acquisition of materials

Tissue culture medium 199, fetal bovine serum (FBS), antibiotics (penicillin / streptomycin), glutamine and collagenase were purchased from Gibco-BRL (Rochville, MD). Anti-ICAM-1, anti-VACM-1 and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Crus, CA); Anti-p-PKC, anti-PKC, anti-p-p38, anti-p38, anti-p-Akt and anti-Akt antibodies were provided by Cell Signaling Technology (Beverly, MA). SB203580, SP600125, PD98059, LY294001 and GF109203 were purchased from Calbiochem (San Diego, CA). All other compounds were obtained from Sigma-Aldrich (St Louis, MO).

1-2. Cell culture

HUVEC was obtained from Cascade Biologics (Portland, OR) and supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, 5 U / ml heparin, 100 IU / ml penicillin, 10 μg / ml streptomycin and 50 μg / ml ECGS In cultured medium 199. Cells were seeded in 100 mm dishes and cultured in a humidified 5% CO ₂ incubator. HUVECs were plated in 100 mm dishes at a density of 1 × 10 ⁷ cells. Cells with three to six passages were used. U937 human monocytes were obtained from KCLB (Korea Cell Line Bank, Seoul, Korea) and RPMI supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, 25 mM NaHCO ₃ , 100 IU / ml penicillin and 10 μg / ml streptomycin. Incubated at 1640.

1-3. Cell viability

Cell viability was measured by MTT assay. Exponential cells were seeded at 1 × 10 ⁴ cells / sphere on 24 platelets. After several treatments, 20 μl of 5 mg / ml MTT solution was added to each sphere (0.1 mg / sphere) and left to incubate for 4 hours. The supernatant was aspirated and the formazan crystals of each sphere were dissolved for 30 minutes at 37 ° C. with 200 μl of dimethyl sulfoxide, and the optimum density was read with a Microplate Reader (Bio-Rad, Hercules, CA) at 570 nm.

1-4. Western blot analysis

Cells were harvested and lysed with buffer containing 0.5% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5 and protease inhibitors. Protein concentration of each sample was measured using a BCA protein assay kit (Pierce, Rockford, IL). To detect VCAM-1 and ICAM-1, 20 μg of total protein was electrophoresed on a 10% polyacrylamide gel and phospho-PTEN, phospho-Akt, phospho-PKC or phospho-p38 was detected. To do this, 30 μg of total protein was electrophoresed on 12% polyacrylamide gels. The gel was then transferred to a polyvinylidene difluoride (PVDF) membrane at 15 V for 60-75 minutes by semidry electrophoretic transfer. The PVDF membrane was blocked with 5% bovine serum albumin (BSA) at 4 ° C. overnight. The cells were left standing with a primary antibody diluted 1: 500 in Tris buffered saline / Tween20 (TBS-T) containing 5% BSA and then incubated with secondary antibody for 1 hour at room temperature. Anti-rabbit IgG was used as secondary antibody (diluted 1: 5000 in TBST containing 1% BSA). Signals were detected by ECL (Amersham, Piscataway NJ).

1-5. RT-PCR for ICAM-1 and VCAM-1 Detection

Total RNA from HUVEC was extracted using TRIzol reagent (Invitrogen, Carlsbad CA) by a single-step guanidine thiocyanate-phenol-chloroform extraction procedure. A negative control without reverse transcriptase was used to verify that amplification did not continue from residual genomic DNA. PCR amplification was performed using specific oligonucleotide primers for cDNA corresponding to 100 ng of starting mRNA. PCR products were electrophoresed on agarose gels stained with 2% ethidium bromide.

1-6. Transfection

Transient transfection with VCAM-1-luciferase and ICAM-1-luciferase was performed using Lipofectin (Gibco-BRL. Rockville, MD). Specifically, it is as follows. 5 × 10 ⁵ cells were plated on 60 mm plates the day before transfection and grown to about 70% confluency. Cells were transfected with empty vectors (pGL3 and / or pcDNA3) or 1 μg of VCAM-1-luciferase or ICAM-1-luciferase + 0.5 μg of pRL-TK-luciferase. Transfection was performed for 4 hours. The transfected cells were washed with 4 ml of 1 × PBS (phosphate buffered saline, pH 7.4) and then stimulated at 1 μg / ml. The cells were cultured in serum-free medium 199 until recovery of the cells. Luciferase activity of each sample was normalized using pRL-TK Luciferase activity.

1-7. Luciferase Essay

After several treatments, cells were washed twice with cold PBS, lysed with lysis buffer contained in the Dual Luciferase Kit (Promega, Madison, Wis.) And manufactured using a TD-20 / 20 luminometer (Turner Designs, Sunnyvale, CA). Luciferase activity was measured according to the protocol. All transfections were performed in 3 replicates. Data are presented as the ratio between firefly (Firefly) and Renilla luciferase activity.

1-8. siRNA technology

Small interfering RNA (siRNA) for human VCAM-1, human PTEN and scramble was provided from Santa cruz Biotechnology (Santa Cruz, Calif.) And performed according to the manufacturer's protocol.

1-9. Monocyte adhesion assay

48 hours prior to assay, HUVECs were inoculated into the two-neck chamber. The medium was changed before stimulation with LPS. U937 monocytes (3 × 10 ⁷ ) were incubated for 30 minutes at 37 ° C. in RPMI 1640 medium containing 2% FBS and 10 mg / ml fluorescent dye BCECF / AM (Boehringer, Mannheim, Germany). Fluorescently labeled cells were precipitated and resuspended (7.5 × 10 ⁵ / ml) in Medium 199 (M199H) with 10 mM HEPES buffer. HUVECs were washed three times with M199H and then incubated at 37 ° C. prior to addition of loading cells. After 30 minutes, the cell suspension was recovered and the HUVECs were washed with M199H. Fluorescence images were screened using a high resolution video camera (DXC-960MD; Sony) attached to a BH-2 Olympus microscope (Melville, NY). After selecting an image with a width of 0.2 mm in the primary screened area, the immunoreactivity of this image was measured using SigmaGel 1.0 (Jandel Scientific, Germany). The analysis was repeated three times for the same area and the results are shown as mean values.

1-10. Stimulation with LPS in vivo

Twenty four 7-8 week old male Sprague-Dawley rats (220-250 g body weight, Sam Tako Inc., Osan, Korea) were randomly divided into four groups: (1) saline (ip, n = 6), (2) LPS (10mg / kg ip, n = 6), (3) LPS Plus (S) -YS49 (5mg / kg ip, n = 6), (4) LPS Plus (S) -YS49 (10mg / kg ip, n = 6) (20 mg / kg. ip n = 4). (S) -YS49 was administered 1 hour before LPS injection. After 6 hours, rats were anesthetized with ketamine (30 mg / kg) and xylazine (6 mg / kg), and the thoracic artery was recovered and subjected to immunohistochemistry.

1-11. Immunohistochemistry

Immunohistochemistry was performed as known. Specifically, it is as follows. Samples were fixed in 4% paraformaldehyde and cryoprotected in 20% sucrose, then tissues were embedded in OCT compound (Sakura Finetsch Co., Tokyo, Japan) and snap-frozen in iso-pentene It was then prechilled in liquid nitrogen. A series of frozen sections (10 μm) were enclosed in gelatin-coated slides and boiled for 3 minutes on 10 mM sodium straight. Sections were blocked for 2 hours at room temperature in 0.1 M TBS containing 1% BSA, then incubated with antibody overnight at 4 ° C. and washed three times. Immunofluorescence histochemistry was performed using secondary antibody (1: 300) conjugated with FITC and observed by confocal microscopy (Olympus, Tokyo, Japan).

Example 2: Results

2-1. (S) -YS49 and (S) -YS51 compounds inhibit VCAM-1 but not ICAM-1 induced by LPS in HUVEC

As a result of confirming using the MTT assay, (S) -YS49 and (S) -YS51 showed a cell death rate of about 56% only at high concentration (A in FIG. 1A). LPS promoted expression of VCAM-1 and ICAM-1 in a time dependent manner (FIG. 1A B). Pretreatment with (S) -YS49 or (S) -YS51 compounds dose-dependently reduced the expression of VCAM-1 but not ICAM-1 in HUVECs stimulated with LPS (FIGS. 1B-D). In the case of (S) -YS49, similar results were confirmed in VCAM-1 and ICAM-1 luciferase activity (B of FIG. 1B). Cells were not cytotoxic when left incubated with (S) -YS49 or (S) -YS51 compound (100 μM) for 8-12 hours, so that in all examples thereafter (S) -YS49 and (S)- YS51 compound was treated for 8 hours.

2-2. Akt, p38 and PKC signaling pathways are involved in the different regulation of VCAM-1 and ICAM-1 in HUVECs stimulated by LPS.

To characterize how the (S) -YS49 compound regulates the expression of adhesion molecules differently, SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), PD98059 (ERK1 / 2 inhibitor), LY294001 (PI3K / Akt inhibitor) and GF109203X Pharmacological inhibitors such as (PKC inhibitor) were used. Among them, PI3K / Akt, p38 and PKC inhibitors specifically inhibited VCAM-1 but not ICAM-1 in cells treated with LPS (FIG. 2A). LPS activated Akt, p38 and PKC from 15-30 minutes (FIG. 2B). As shown in FIG. 2C, (S) -YS49 significantly inhibited Akt phosphorylation in HUVEC activated with LPS, but did not affect the amount of phospho-p38 or phospho-pan-PKC.

2-3. PTEN is involved in the different regulation of (S) -YS49 compounds on LPS-induced expression of VCAM-1 and ICAM-1 in HUVECs

PTEN is a natural negative regulator of PI3K / Akt signaling and its activity depends on its phosphorylation status. Thus, it was confirmed whether the (S) -YS49 compound can modulate PTEN activity in cells treated with LPS. As shown in FIG. 3A, the (S) -YS49 compound significantly inhibited PTEN phosphorylation, thereby upregulating PTEN activity by dephosphorylation. Next, we investigated whether upregulation of PTEN is important for the inhibitory effect of p-Akt and VCAM-1 by (S) -YS49 compounds. Transfection with siPTEN (small interfering PTEN RNA) reduced the expression of PTEN as shown in FIG. 3B. The (S) -YS49 compound significantly inhibited the phosphorylation of Akt induced by LPS (p-Akt) and the expression of VCAM-1 in HUVEC. However, it did not inhibit the expression of p-Akt and VCAM-1 in the presence of siPTEN (FIGS. 3C and 3D). This means that the (S) -YS49 compound downregulates expression of VCAM-1 through tight regulation of the PTEN / PI3k / Akt signaling cascade.

2-4. Effect of (S) -YS49 or (S) -YS51 Compound on Monocyte Adhesion

From the above results, it was confirmed that (S) -YS49 or (S) -YS51 compound can inhibit the expression of the adhesion molecule in the activated HUVEC. Therefore, it was further investigated whether the (S) -YS49 or (S) -YS51 compound can regulate the adhesion of monocytes to endothelial cells. As shown in FIGS. 4A and 4B, the (S) -YS49 or (S) -YS51 compounds significantly inhibited adhesion of U937 cells with HUVECs stimulated by LPS.

2-5. Effect of (S) -YS49 Compound on Expression of VCAM-1 and ICAM-1 in LPS Administered Rat Aorta

To confirm the results of the in vitro experiment described above, animal experiments were performed. Sprague-Dawley rats were stimulated with LPS for 6 hours after treatment with (S) -YS49 compound. As shown in FIG. 5, the (S) -YS49 compound (10 or 20 mg / kg) significantly inhibited the expression of VCAM-1 induced by LPS in aortic endothelial cells, but did not affect the expression of ICAM-1. Did.

(A) of FIG. 1A shows the effect of (S) -YS49 and (S) -YS51 on HUVEC cell viability, and (B) shows that LPS expresses expression of VCAM-1 and ICAM-1 in a time dependent manner. It is shown to promote.

(A)-(E) of FIG. 1B show the effect of (S) -YS49 and (S) -YS51 on the expression of VCAM-1, ICAM-1 induced by LPS in HUVEC.

2 shows that the PI3k / Akt pathway is involved in the inhibitory effect of VCAM-1 mediated by (S) -YS49 in HUVECs induced by LPS.

3 shows that PTEN is involved in the inhibitory effect of (S) -YS49 on HUVECs induced by LPS.

Figure 4a shows the inhibitory effect of (S) -YS49 on the adhesion of U937 monocytes to HUVEC.

Figure 4b shows the inhibitory effect of (S) -YS51 on the adhesion of U937 monocytes to HUVEC.

5 shows that ICAM-1 (A) and VCAM-1 (B) are differently regulated by (S) -YS49 in blood vessels of rats treated with LPS.

Claims (4)

(s) -YS49 [(s) -1- (α-naphthylmethyl) -6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] and (s) -YS51 [(s ) -1- (β-naphthylmethyl) -6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] at least one selected from the group of tetrahydroisoquinoline (tetrahydroisoquinoline) as an active ingredient Atherosclerosis prevention and treatment composition comprising. The method of claim 1, A composition characterized in that it is formulated as a preparation for intraperitoneal administration. The method of claim 1, The tetrahydroisoquinoline compound tetrahydroisoquinol water inhibits the expression of VCAM-1 composition. The method of claim 1, The tetrahydroisoquinoline compound is characterized in that for treating atherosclerosis due to adhesion of monocytes to vascular endothelial cells.

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