MITOCHONDRIA-TARGETING COMPOUND AND COMPOSITION COMPRISING SAME FOR TREATING OR PREVENTING AGING-RELATED DISEASES

Abstract

Provided are a mitochondria-targeting compound and a composition including the same for the treatment or prevention of an aging-related disease. A compound according to an aspect or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition including the same are effectively used to prevent or treat aging-related diseases by specifically inducing apoptosis of senescent cells.

Claims

1. A compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof: ##STR00005## wherein, in Formula 1, R.sub.1 is —OH or —SH, R.sub.2 is a cationic moiety, and R.sub.3 is a mitochondria-targeting moiety, and in Formula 2, R.sub.4 is —OH or —SH, and R.sub.5 is a mitochondria-targeting peptide.

2. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the cationic moiety is an oxonium ion, quaternary ammonium, or quaternary phosphonium.

3. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the mitochondria-targeting moiety is triphenylphosphonium (TPP), dequalinium, guanidinium, triethylammonium, pyridinium, 3-phenylsulfonyl furoxan, F16, 2,3-dimethylbenzothiazolium iodide, rhodamine 19, or rhodamine 123.

4. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein R.sub.2 is connected to amide or R.sub.3 directly or through a linker.

5. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the mitochondria-targeting peptide is Leu-Leu-Arg-Ala-Ala-Leu-Arg-Lys-Ala-Ala-Leu (LLRAALRKAAL), Met-Leu-Arg-Ala-Ala-Leu-Ser-Thr-Ala-Arg-Arg-Gly-Pro-Arg-Leu-Ser-Arg-Leu-Leu (MLRAALSTARRGPRLSRLL), Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys (MLSLRQSIRFFK), Leu-Ser-Arg-Thr-Arg-Ala-Ala-Ala-Pro-Asn-Ser-Arg-Ile-Phe-Thr-Arg (LSRTRAAAPNSRIFTR), Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN), Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN), Lys-Leu-Ala-Lys-Leu-Ala-Lys (KLAKLAK), Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKRGD), or Lys-Leu-Ala-Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKLAKRGD).

6. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the compound represented by Formula 1 is represented by Formula 3: ##STR00006## wherein, in Formula 3, m and n are each from 1 to 20.

7. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the compound represented by Formula 2 is one selected from compounds represented by Formulae 4 to 7: ##STR00007##

8. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein R.sub.1 of Formula 1 or R.sub.4 of Formula 2 is oxidized to form a bond with a plurality of other compounds.

9. The compound or pharmaceutically acceptable salt thereof of claim 8, wherein the oxidation is caused by reactive oxygen species (ROS).

10. A pharmaceutical composition for preventing or treating aging-related diseases, the pharmaceutical composition comprising, as an active ingredient, the compound or pharmaceutically acceptable salt thereof of claim 1.

11. The pharmaceutical composition of claim 10, wherein the aging-related disease is at least one selected from senile cardiovascular disease and disorder, senile lung disease, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, osteoarthritis, senile eye disease, and skin aging.

12. The pharmaceutical composition of claim 11, wherein the senile eye disease is senile macular degeneration.

13. The pharmaceutical composition of claim 10, wherein the compound of the pharmaceutical composition induces apoptosis in mitochondria in senescent cells.

14. A method of preventing or treating aging-related diseases, comprising administering, to a subject in need thereof, the compound or pharmaceutically acceptable salt thereof of claim 1.

15. A use of the compound or pharmaceutically acceptable salt thereof of claim 1 in the preparation of a composition for preventing or treating aging-related diseases.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0055] FIG. 1 shows a diagram schematically illustrating the apoptosis mechanism of the present disclosure.

[0056] FIG. 2 shows a chemical structure of SP-101 according to an embodiment of the present disclosure.

[0057] FIG. 3 shows a diagram schematically showing that the thiol group of the present disclosure is oxidized to form a disulfide bond.

[0058] FIG. 4 shows scanning electron microscope (SEM) and transmission electron microscopy (TEM) images in which SP-101 is oxidized by ROS to form macromolecules.

[0059] FIG. 5 shows a graph showing the ROS production of HeLa, IMR90, and senescent cells.

[0060] FIG. 6 shows a graph showing the cytotoxicity of SP-101.

[0061] FIG. 7 shows photographs of senescent cells observed under a microscope showing that senescent cells are reduced by SP-101.

[0062] FIG. 8 shows a photograph of the result of confirming the effect of SP-101 inhibiting the generation of choroidal neovascularization.

[0063] FIG. 9 shows a graph of the result of confirming the effect of SP-101 inhibiting the generation of choroidal neovascularization.

[0064] FIG. 10 shows a chemical structure of SH-KLAKLAKRGD according to an embodiment of the present disclosure.

[0065] FIG. 11 shows a graph and TEM image showing the molecular size of SH-KLAKLAKRGD to form a macromolecule.

[0066] FIG. 12 is a graph showing the cytotoxicity of SH-KLAKLAKRGD:

[0067] FIG. 12A is a graph showing the cytotoxicity of SH-KLAKLAKRGD in senescent cells, and FIG. 12B is a graph showing the cytotoxicity of SH-KLAKLAKRGD in normal cells.

MODE OF DISCLOSURE

[0068] Hereinafter, the present disclosure will be described in more detail through Examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited to these examples.

Example 1. Synthesis and Analysis of SP-101, a Mitochondria-Targeting Compound

[0069] SP-101, a mitochondria-targeting compound having a targeting ability specific to mitochondria, was prepared.

[0070] Specifically, 0.8 g of methyl 3,5-dihydroxybenzoate, 0.2 g of dimethylcarbamothioic chloride, and 0.2 g of DABCO were dissolved in 100 mL of DMF, and stirred at room temperature for 24 hours. The mixture was purified by HPLC to obtain white powder. 0.5 g of the resulting methyl 3,5-bis((dimethylcarbamothioyl)oxy)benzoate was dissolved in 10 mL of diphenyl ether and stirred under reflux for 24 hours. The resultant mixture was purified by HPLC to obtain yellow powder. 0.2 g of the obtained methyl 3,5-bis((dimethylcarbamoyl)thio)benzoate and 0.1 g of KOH were dissolved in 10 ml of methanol and 10 ml of water, and stirred under reflux for 24 hours. The mixture was purified by HPLC. 0.2 g of the obtained 3,5-dimercaptobenzoic acid and 2 g of Ph.sub.3CCl were dissolved in 10 ml of methanol and stirred under reflux for 24 hours. The mixture was purified by HPLC. 0.1 g of the obtained 3,5-bis(tritylthio)benzoic acid), 0.1 g of (3-((3-aminopropyl)dimethylammonio)propyl)triphenylphosphonium), and 0.1 g of EDC were dissolved in 10 ml of methanol and stirred at room temperature for 24 hours. The mixture was purified by HPLC. 0.1 g of the obtained (3-((3-(3,5-bis(tritylthio)benzamido)propyl)(metheyliumyl)(methyl)ammonio)propyl)triphenylphosphonium, 0.05 g of TIPS, and 0.05 g of TFA were dissolved in 10 ml of DCM and stirred at room temperature for 24 hours. The resultant mixture was purified by HPLC.

[0071] As a result, (3-((3-(3,5-dimercaptobenzamido)propyl)dimethylammonium)propyl)triphenylphosphonium was obtained, which is hereinafter referred to as ‘SP-101’ (FIG. 2).

[0072] FIG. 2 shows a chemical structure of SP-101 according to an embodiment of the present disclosure.

Example 2. Synthesis and Analysis of SH-KLAKLAKRGD, a Mitochondria-Targeting Compound

[0073] SH-KLAKLAKRGD, a mitochondria-targeting compound having a targeting ability specific to mitochondria, was prepared by solid phase peptide synthesis.

[0074] Specifically, 200 mg MBHA amide resin was used, and the first amino acid loading capacity was 0.106 mmol.

[0075] First, 200 mg MBHA amide resin was put into a syringe with a filter inserted and swelled in DMF for 30 minutes. Then, the fluorenylmethoxycarbonyl protecting group (Fmoc) was removed therefrom for 50 minutes by using 3 ml of 20% piperidine in DMF (Fmoc-deprotection). The result was washed with DMF and DCM, three times for each. A D amino acid solution (5 eq amino acid, 5 eq 0.5M HBTU, and 10 eq DIPEA) was added, followed by D amino acid coupling for 1 hour and 50 minutes. The result was washed with DMF and DCM, three times for each. In the same way, in this stated order, a G amino acid, a R amino acid, a R amino acid without deprotection, a K amino acid, an A amino acid, a L amino acid, a K amino acid, an A amino acid, a L amino acid, and a K amino acid were subjected to coupling. At this time, in the case of Fmoc-Arg(pbf)-OH, the amino acid coupling process was performed once again without the deprotection process (#2, #3). After the synthesis of NH2-KLAKLAKRGD was completed, 2 eq disulfide 4, 2 eq HBUT, and 4 eq DIPEA were dissolved in DMF and reacted for 24 hours or more. The result was washed with DMF and DCM, three times for each. Reagent R (4.5 ml of TFA, 0.25 ml of thioanisole, 0.15 ml of 1,2-ethanedithiol, and 0.1 ml of anisole) was used as a peptide cleavage cocktail, and the cleavage process was performed for 2 hours and 30 minutes. The cleavage solution was precipitated in 40 ml of cold ether. After recovering the precipitate by centrifugation, the precipitate was washed once more with 45 ml of cold ether. The obtained crude was dried, and then, a methanol solution having a concentration of 20 mg/ml was prepared. The crude was separated using HPLC, and a C18-reverse phase column was used.

[0076] As a result, as shown in FIG. 10, SH-KLAKLAKRGD was obtained.

[0077] FIG. 10 shows a chemical structure of SH-KLAKLAKRGD according to an embodiment of the present disclosure.

Experimental Example 1. Confirmation of Macromolecular Formation of SP-101 by ROS

[0078] It was confirmed that SP-101 was oxidized by ROS to form macromolecules.

[0079] Specifically, 1 mM SP-101 and 10 mM SP-101 were each dissolved in water and stirred for 24 hours. A drop of an aqueous solution of SP-101 was placed on a formvar/carbon-coated copper grid and evaporated under atmospheric conditions. The sample was stained with a 2 wt % uranyl acetate solution, evaporated for 1 minute, and then the excess solution was removed therefrom by using a filter paper. The specimens were observed with a JEM-1400 transmission electron microscopy (TEM) and a scanning electron microscopy (SEM), each operating at 120 kV.

[0080] As a result, it was confirmed that SP-101 was oxidized to form a macromolecule (FIGS. 3 and 4).

[0081] FIG. 3 shows a diagram schematically showing that the thiol group of the present disclosure is oxidized to form a disulfide bond.

[0082] FIG. 4 shows scanning electron microscope (SEM) and transmission electron microscopy (TEM) images in which SP-101 is oxidized by ROS to form macromolecules.

Experimental Example 2. Confirmation of ROS Over-Expression in Senescent Cells

[0083] It was confirmed whether ROS was over-expressed in senescent cells compared to other cells.

[0084] Specifically, HeLa cells, IMR90 cells, and aged chondrocyte cells were seeded at a density of 4×10.sup.4 cells/100 μL per well on a Lab Tek II slide chamber in DMEM (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin. Then, the cells were incubated overnight under conditions of 5% CO.sub.2 and 37° C. Subsequently, by using the Cellular ROS assay kit, the cell plate was treated with 100 μL/well of a red working solution (ROS). The fluorescence increase was monitored at Ex/Em=520/605 nm (590 nm was blocked) using the bottom reading mode.

[0085] As a result, it was confirmed that ROS was over-expressed in senescent cells compared to HeLa and IMR90 cells (FIG. 5).

[0086] FIG. 5 shows a graph showing the ROS production of HeLa, IMR90, and senescent cells.

Experimental Example 3. Confirmation of Cytotoxicity of SP-101

[0087] Cytotoxicity was analyzed to observe the killing effect of SP-101 on senescent cells.

[0088] Specifically, the cell viability of IMR90 and senescent cells against SP-101 was evaluated by identifying the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to insoluble formazan. Cells were seeded at a density of 5×10.sup.3 cells per well in a 96-well plate and cultured overnight, followed by treatment with different concentrations of SP-101 in DMEM medium containing 10% FBS and cultured for 24 hours. Then, the cells were cultured with MTT, and then the crystallized formazan was quantified by measuring the absorbance thereof at 595 nm by using an ELISA plate reader. Results were expressed as percent viability=[(A550 (treated cells)−background value)/(A550 (untreated cells)−background value)]×100.

[0089] As a result, it was confirmed that in the concentration range of 5 μM to 30 μM of SP-101, more senescent cells were killed than normal cells, IMR90 (FIG. 6).

[0090] These results indicate that SP-101 kills senescent cells more specifically and effectively than normal cells.

[0091] FIG. 6 shows a graph showing the cytotoxicity of SP-101.

Experimental Example 4. Confirmation of Reduction in Senescent Cells Due to SP-101

[0092] It was confirmed under a microscope that senescent cells were reduced by SP-101.

[0093] Specifically, SA-b-galactosidase staining was used. Cells that were not treated with anything were used as a control, and those treated with 30 μM of SP-101 were used as an experimental group. SA-gal staining was performed using a kit (Biovision, K320-250) according to the manufacturer's instructions. Senescent cells were identified as blue-stained cells under an optical microscope.

[0094] As a result, it was confirmed that senescent cells were reduced in the experimental group treated with SP-101 (FIG. 7).

[0095] Taking the above results together, it can be seen that phenyldithiol of SP-101 was oxidized by ROS over-expressed in senescent cells to form macromolecules, thereby disrupting the mitochondrial membrane and specifically killing senescent cells.

[0096] FIG. 7 shows photographs of senescent cells observed under a microscope showing that senescent cells were reduced by SP-101.

Experimental Example 5. Confirmation of the Inhibitory Effect of SP-101 on Choroidal Neovascularization

[0097] The CNV model was used to confirm whether SP-101 has an effect of inhibiting the production of choroidal neovascularization.

[0098] Specifically, the mice were anesthetized by intraperitoneal injection of ketamine hydrochloride and xylazine hydrochloride. By using a diode laser with a wavelength of 810 nm, eight photocoagulation spots, each having the size of 75 μm, the intensity of 250 mW, and the time of 50 ms, were made in concentric circles among the main retinal vessels around the optic nerve. The rupture of the Bruck's membrane was confirmed by identifying that cavitation bubbles had been formed in the choroid. Immediately after laser irradiation, the experimental groups treated with 10 μM, 50 μM, and 100 μM of SP-101, and phosphate buffered saline and Eylea, which were used as control groups, were injected into the vitreous cavity. Two weeks after laser irradiation, fluorescein angiography was performed thereon using a confocal scanning laser fundus camera. After fluorescein-dextran was rapidly injected intravenously, images thereof were taken at the beginning (2 min) and late (10 min).

[0099] As a result, compared with the volume of choroidal neovascularization, it was identified that the volume of the groups treated with 10 μM and 100 μM of SP-101 was decreased by about twice compared to the Eylea control group (FIGS. 8 and 9). These results mean that SP-101 eliminates senescent cells and factors secreted by senescent cells are thus removed, thereby inhibiting the occurrence of new blood vessels, and SP-101 is effective about twice as effective as Eylea, so choroidal neovascularization was effectively inhibited.

[0100] FIG. 8 shows a photograph of the result of confirming the effect of SP-101 inhibiting the generation of choroidal neovascularization.

[0101] FIG. 9 shows a graph of the result of confirming the effect of SP-101 inhibiting the generation of choroidal neovascularization.

Experimental Example 6. Confirmation of Self-Assembly by Formation of SH-KLAKLAKRGD

[0102] SH-KLAKLAKRGD (1 mM and 10 mM) was dissolved in water and stirred for 24 hours. A drop of SH-KLAKLAKRGD aqueous solution was placed on a formvar/carbon coated copper grid and allowed to evaporate at ambient conditions. The sample was stained with 2 wt % uranyl acetate solution and evaporated for 1 minute, and the excess solution was removed with filter paper. The specimen was observed using a JEM-1400 TEM operating at 120 kV.

[0103] As a result, as shown in FIG. 11, the size of the polymerized SH-KLAKLAKRGD was 350±100 nm, and the TEM image showed a macromolecule in which peptides were polymerized.

[0104] FIG. 11 shows a graph and TEM image showing the molecular size of SH-KLAKLAKRGD to form a macromolecule.

Experimental Example 7. Confirmation of Cytotoxicity of SH-KLAKLAKRGD

[0105] Cytotoxicity was analyzed to observe the killing effect of SH-KLAKLAKRGD on senescent cells.

[0106] Specifically, the cell viability of IMR90 and senescent cells against SH-KLAKLAKRGD was evaluated by identifying the reduction of MTT to insoluble formazan. Cells were seeded at a density of 5×10.sup.3 cells per well in a 96-well plate and cultured overnight, followed by treatment with different concentrations of SH-KLAKLAKRGD in DMEM medium containing 10% FBS and cultured for 24 hours. Then, the cells were cultured with MTT, and then the crystallized formazan was quantified by measuring the absorbance thereof at 595 nm by using an ELISA plate reader. Results were expressed as percent viability=[(A550 (treated cells)−background value)/(A550 (untreated cells)−background value)]×100.

[0107] As a result, it was confirmed that the cell viability in senescent cells was lower than that in IMR90, which is a normal cell (FIG. 12).

[0108] These results indicate that SH-KLAKLAKRGD kills senescent cells more specifically and effectively than normal cells.

[0109] FIG. 12 is a graph showing the cytotoxicity of SH-KLAKLAKRGD:

[0110] FIG. 12A is a graph showing the cytotoxicity of SH-KLAKLAKRGD in senescent cells, and FIG. 12B is a graph showing the cytotoxicity of SH-KLAKLAKRGD in normal cells.