KR20140133241A - Pharmaceutical Composition for Preventing or Treating Autophagy-associated Diseases, Apoptosis-associated Diseases, Hyperproliferative Disease or Angiogenic Diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating autophagy-associated diseases, apoptosis-associated diseases, hyperproliferative diseases, or angiogenic diseases, the pharmaceutical composition containing: (a) a pharmaceutically effective amount of convallatoxin, a convallatoxin derivative, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention simultaneously and effectively induces autophagy and apoptosis of cells in a dose-dependent manner and an administration time-dependent manner, and thus suppresses hyper-proliferation and angiogenesis of cells in a dose-dependent manner, thereby exhibiting great efficacy in the prevention or treatment of autophagy-associated diseases, apoptosis-associated diseases, hyperproliferative diseases, or angiogenic diseases. Furthermore, the pharmaceutical composition of the present invention increases the phosphorylation of AMP-activated protein kinase (AMPK) and reduces the phosphorylation of mTOR and p70S6K, thereby verifying the induction of autophagy, and thus contributing to the finding of an autophagy induction mechanism of the pharmaceutical composition of the present invention.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating autophage-related diseases, apoptosis-related diseases, hyperproliferative diseases or angiogenesis diseases, and pharmaceutical compositions for preventing or treating autophage-associated diseases, apoptosis-related diseases, hyperproliferative diseases or angiogenic diseases,

The present invention relates to a pharmaceutical composition for preventing or treating autophage-related diseases, apoptosis-related diseases, hypertrophic diseases or angiogenesis diseases. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating autophage-related diseases, apoptosis-related diseases, hypertrophic diseases or angiogenesis diseases comprising convallatoxin or derivatives thereof.

Autophagy

Autophagy is a major intracellular degradation pathway responsible for the removal of long-lived proteins and damaged cellular organelles and maintenance of cellular homeostasis [1]. The process is involved in a variety of diseases such as neurodegenerative diseases including cancer [2], Alzheimer's disease and Parkinson's disease [3], atherosclerosis [4] and age-related diseases [5]. Autophagy can be characterized by dynamic reconstruction of membranes for degradation of cell components. When autophagy is induced, a separate membrane (called a phagophore) surrounds some of the cytoplasmic and intracellular organelles and then carries the ATGs (autophagy related genes) and LC3 (microtubule-associated 1 light chain 3) Together they form a double-membrane-bound structure known as the autophagosome. The intracellular LC-I form is converted to the membrane-bound form LC3-II, which is characteristic of autophagy induction. The outer membrane of autopagos is fused with lysosomes, resulting in the formation of autos lysosomes. Finally, the isolated contents are degraded by lysosomal hydrolase and recycled for cell survival (Fig. 1) [6].

Recently, the role of autophagy during cell death has been discussed in many reports [7, 8]. Programmed cell death (PCD) can be categorized into a number of categories, including type I apoptosis and type II autophage cell death. Autophage is the most common form of PCD and is characterized by membrane blistering, DNA fragmentation, and the formation of apoptotic bodies. Autophage cell death is different from apoptosis [9], or secondary to apoptosis [7]. Based on this background, the induction of apoptosis or apoptosis through autophagy in cancer cells is now a promising target for strategies for cancer therapy.

Auto Phage  Inducing Small molecule

Induction of autophagy can be beneficial for the treatment of various diseases. Identification of autophagy inducing agents is therefore a promising strategy for finding potential therapeutic candidates. Many small molecules that affect autophage activity have been reported [11]. For example, Rapamycin, an oleophilic macrolide antibiotic that deactivates the target (mTOR) of rapamycin, is the most widely used compound to induce autophage [12]. Rapamycin is used to prevent kidney-graft rejection, and temsirolimus, an analog of rapamycin, is in clinical phase 3 for human gliomas [13]. Based on this background, novel small molecules that induce autophage have the potential to contribute to the development of therapeutic uses involving cancer and to present new insights in autophage-related biology.

Natural and cell-based screening

Natural products have become an important raw material for the bioactive of therapeutic drugs including anticancer drugs. For example, vinblastine (VLB) has been isolated in the first day (Catharanthus roseus G. Don, Apocynaceae), which was used for the treatment of diabetes. We studied this for potential hypoglycaemic oral medications, but found that it has activity against lymphocytic leukemia in mice. Subsequently, we found a semi-synthetic analogue of VLB, which was used in conjunction with chemotherapeutic agents for the treatment of cancers such as leukemia, lymphoma, breast cancer and lung cancer [14]. Given this great advantage, the identification and discovery of new natural small molecules as a promising source for a variety of biological studies is emphasized.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive studies for the development of novel pharmaceutical compositions capable of effectively preventing or treating autophage-related diseases. As a result, convallatoxin and its derivatives inhibit proliferation of cells and not only induce autophagy and apoptosis, but also inhibit tubule formation and invasion, angiogenesis), thereby completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating autophagy-related diseases, apoptosis-related diseases, hypertrophic diseases or angiogenesis diseases comprising convalatoxin or its derivatives.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a pharmaceutical effective amount of a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; And (b) a pharmaceutical composition for the prevention or treatment of autophage-related diseases, apoptosis-related diseases, hyperproliferative diseases or angiogenesis diseases comprising a pharmaceutically acceptable carrier.

Formula 1

Wherein X ₁ and X ₂ each independently represent oxygen, sulfur or nitrogen; R ₁ , R ₂ And R ₃ each independently represent hydrogen, C ₁ -C ₅ straight or branched chain alkyl, C ₂ -C ₆ alkenyl or C ₃ -C ₈ cycloalkyl; R ₄ represents -COH, -COR ₆ , -COOR ₆ (wherein R ₆ is a C ₁ -C ₅ linear or branched alkyl) or -CONH ₂ ; R ₅ is hydrogen, C ₁ -C ₅ linear or branched alkyl, C ₁ -C ₅ linear or branched alkyl alcohol.

The present inventors have made intensive studies for the development of novel pharmaceutical compositions capable of effectively preventing or treating autophage-related diseases. As a result, convallatoxin and its derivatives inhibit proliferation of cells and not only induce autophagy and apoptosis, but also inhibit tubule formation and invasion, angiogenesis).

As used herein, the term " alkyl " means an unsubstituted or substituted saturated hydrocarbon group and includes, for example, methyl, ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. C _{1 -5} alkyl having a carbon number in the case where means an alkyl group that has an alkyl unit having from 1 to 5 carbon atoms, and, C _{1 -5} alkyl substituted with a substituent is not included.

The term " C ₁ -C ₅ Straight chain or branched chain alkyl "means straight chain or branched chain such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert- quot; means an alkyl group of a branched chain.

The term "alkenyl" represents an unsubstituted straight- or branched-chain having the designated number of carbon atoms or substituted unsaturated hydrocarbon, for example, ethenyl, vinyl, propenyl, allyl, isopropenyl, butenyl, isobutenyl ethenyl, t - unit N - pentenyl, and n - hexenyl. C ₁ -C ₆ The term " alkenyl " means an alkenyl group having an alkenyl unit having 1 to 6 carbon atoms, and when the alkenyl group is substituted with C ₁ -C ₆ alkenyl, the number of carbon atoms of the substituent is not included.

The term " C ₃ -C ₈ Quot; cycloalkyl " means a mono ring or multi rings saturated hydrocarbon consisting of 3 to 8 carbon atoms. But are not limited to, for example, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and the like.

The term "alkenyl" represents an unsubstituted straight- or branched-chain having the designated number of carbon atoms or substituted unsaturated hydrocarbon, for example, ethenyl, vinyl, propenyl, allyl, isopropenyl, butenyl, isobutenyl ethenyl, t - unit N - pentenyl, and n - hexenyl. C ₂ -C ₆ Alkenyl is that does not include the carbon atoms of the substituents, if substituted alkenyl of 2 to 6 carbon atoms alkenyl means an alkenyl group having the unit and, C ₂ -C ₆ of.

According to one embodiment of the present invention, the compound of Formula 1, which is a convalatoxin derivative of the present invention, is represented by Formula 2 below.

(2)

According to one embodiment of the present invention, the compound of Formula 1, which is a convalatoxin derivative of the present invention, is represented by Formula 3 below.

(3)

According to one embodiment of the present invention, the compound of Formula 1, which is a convalatoxin derivative of the present invention, is represented by Formula 4 below.

Formula 4

According to another embodiment of the present invention, in Formula 1-4, X ₁ and X ₂ represent oxygen; R ₁ represents a C ₁ -C ₅ straight or branched chain alkyl; R ₂ And R < ₃ > represent hydrogen; R ₄ represents -COH; R ₅ represents a C ₁ -C ₅ linear or branched alkyl.

According to an embodiment of the present invention, X ₁ and X ₂ in the general formula (1-4) represent oxygen; R ₁ represents C ₁ -C ₃ linear or branched alkyl; R ₂ And R < ₃ > represent hydrogen; R ₄ represents -COH; R ₅ represents a C ₁ -C ₃ linear or branched alkyl.

According to a particular embodiment of the present invention, the compound of formula 1 is convallatoxin of formula 5:

Formula 5

The compounds of the present invention may have one or more chiral centers and / or geometric centers, and thus the present invention encompasses all stereoisomers, i.e., optical isomers, diastereoisomers, and geometric isomers Lt; / RTI >

Convalatoxin or convalatoxin derivatives of the present invention are very effective in preventing or treating autophage-related diseases. Autophage-related diseases refers to diseases that can be treated by inducing autophage, and more specifically, autophage is capable of inhibiting cell growth and differentiation, decreasing mutagenesis, or removing organelles such as mitochondria damaged by reactive oxides All diseases that can be treated. Examples of autophage-related diseases include cancer, atherosclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, oculopharyngeal muscular dystrophy Dentatorubral pallidoluysian atrophy, anterior temporal dementia, progressive supranuclear palsy, x-linked spinobulbarrhea, spondyloarthropathies, dyspepsia, dyspepsia, muscular atrophy, and neuronal intranuclear hyaline inclusion disease.

Convalatoxin or convalatoxin derivatives of the present invention are very effective in preventing or treating apoptosis-related diseases. As used herein, the term " apoptosis " is programmed cell death (PCD) that occurs in multicellular organisms (About Apoptosis. Http://www.nih.gov/sigs/aig/Aboutapo.html. Retrieved November 2009 : Apoptosis Interest Group, preferred pronunciation of National Institute of Health and Green, Douglas (2011). Means To An End. New York: Cold Spring Harbor Laboratory Press, ISBN 978-87969-888-1 http: //celldeathbook.wordpress .com /). The biochemical phenomenon of apoptosis changes the shape of the cell and leads to death. These changes include cell membrane bubble formation, cell shrinkage, nuclear breakdown, chromosome condensation, and chromosomal DNA breakage. Unlike cell necrosis, the apoptotic body produced by apoptosis leaks out of the cell, and phagocytic cells are surrounded and removed before it damages the cell. Apoptosis-related diseases means diseases that can be treated by inducing apoptosis, including but not limited to cancer, viral infection, allergic diseases, inflammatory diseases, autoimmune diseases, immune diseases, reperfusion injury and degenerative diseases and the like. According to one embodiment of the present invention, among the apoptosis-related diseases, the virus of the viral infection is a human papillomavirus, a human immunodeficiency virus or a hepatitis virus.

Convalatoxin or convalatoxin derivatives of the present invention are very effective in preventing or treating hyperproliferative diseases. Hyperplasia refers to diseases caused by hyperproliferation of cells, such as cancer, abnormal proliferation, keloid, Cushing's syndrome, hyperalgesia, hyperphagia, intracellular hypertrophy, vitiligo, hypertrophic scarring, Atherosclerosis, recurrent stenosis and stenosis, and the like.

In addition, the convalatoxin or convalatoxin derivative of the present invention effectively inhibits angiogenesis by inhibiting tube formation and invasion, and thus is very effective in preventing or treating angiogenic diseases. The term " angiogenic disease " means a disease caused by blood vessel formation, and includes, for example, cancer, diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascular glaucoma, hypoxia, proliferative retinopathy, psoriasis, hemophilic joints, Atherosclerosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcer, hepatopathy, glomerulonephritis, glomerulonephritis, glomerulonephritis, glomerulonephritis, glomerulonephritis, But are not necessarily limited to, diabetic nephropathy, malignant neuropathies, thrombotic microangiopathy, organ transplant rejection, nephropathies, diabetes, inflammation or neurodegenerative diseases.

According to another embodiment of the present invention, the cancer of the present invention can be used for the treatment of cancer such as pituitary adenoma, glioma, brain tumor, female cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, Cancer of the prostate, cancer of the gallbladder, penis cancer, ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, pediatric cancer, ovarian cancer or cervical cancer It is cervical cancer.

The convalatoxin or convalatoxin derivatives of the present invention induce autophage and apoptosis of cells, inhibit cell proliferation and angiogenesis, exhibit excellent anticancer efficacy, inhibit angiogenesis and invasion, and inhibit cancer metastasis ≪ / RTI >

According to one embodiment of the present invention, the pharmaceutical composition of the present invention simultaneously induces autophagy and apoptosis. According to another embodiment of the present invention, the pharmaceutical composition of the present invention simultaneously induces autophagy and apoptosis, and suppresses cell hyperproliferation. According to another embodiment of the present invention, the pharmaceutical composition of the present invention simultaneously induces autophagy and apoptosis, and inhibits angiogenesis. According to some embodiments of the present invention, the pharmaceutical composition of the present invention simultaneously induces autophagy and apoptosis, and inhibits angiogenesis and cell hyperproliferation.

According to one embodiment of the present invention, the pharmaceutical composition of the present invention increases the phosphorylation of AMP-activated protein kinase (AMPK). Phosphorylation of rapamycin mammalian target (mTOR) is inhibited by increased phosphorylation of AMPK by the pharmaceutical composition of the present invention and phosphorylation of p70S6K downstream of mTOR is also reduced by the pharmaceutical composition of the present invention.

According to one embodiment of the present invention, autophagy induction, induction of apoptosis, hyperproliferative hyperproliferation or angiogenesis inhibition by the pharmaceutical composition of the present invention occurs in a dose-dependent manner.

According to another embodiment of the present invention, the autophagy of the present invention is induced by 0.1-20 nM of the pharmaceutical composition of the present invention. According to another embodiment of the present invention, the autophagy of the present invention comprises the pharmaceutical composition of the present invention in an amount of 0.1-15 nM, 0.1-12 nM, 0.1-10 nM, 0.1-7 nM, 0.1-5 nM, nM. < / RTI > According to another embodiment of the present invention, the autophagy of the present invention comprises the pharmaceutical composition of the present invention in an amount of 0.5-20 nM, 0.5-15 nM, 0.5-12 nM, 0.5-10 nM, 0.5-7 nM, nM or 0.5-3 nM.

According to another embodiment of the present invention, the apoptosis of the present invention is induced by 1-20 nM of the pharmaceutical composition of the present invention. According to another embodiment of the present invention, the apoptosis of the present invention can be achieved by administering the pharmaceutical composition of the present invention 3-20 nM, 3-15 nM, 3-12 nM, 3-10 nM, 3-7 nM or 3-5 nM Lt; / RTI >

According to another embodiment of the present invention, the hyperextension of the present invention is inhibited by 0.1-50 nM of the pharmaceutical composition of the present invention. According to another embodiment of the present invention, the hypertrophic formula of the present invention is a pharmaceutical composition of the present invention comprising 0.1-20 nM, 0.1-15 nM, 0.1-12 nM, 0.1-10 nM, 0.1-7 nM, 0.1-5 nM Or 0.1-3 nM. According to another embodiment of the present invention, the hypervariable formula of the present invention is a pharmaceutical composition of the present invention comprising 0.1-20 nM, 0.5-20 nM, 0.5-15 nM, 0.5-12 nM, 0.5-10 nM, 0.5-7 nM , 0.5-5 nM, or 0.5-3 nM.

According to another embodiment of the present invention, angiogenesis of the present invention is inhibited by 0.1-20 nM of the pharmaceutical composition of the present invention. According to another embodiment of the present invention, the hypervariability of the present invention is inhibited by 0.1-15 nM, 0.1-12 nM, 0.1-10 nM, 0.1-7 nM or 0.1-5 nM of the pharmaceutical composition of the present invention. According to another embodiment of the present invention, the hypertrophic formula of the present invention is a pharmaceutical composition of the present invention comprising 0.5-20 nM, 0.5-15 nM, 0.5-15 nM, 0.5-12 nM, 0.5-10 nM, 0.5-7 nM , 0.5-5 nM, or 1-5 nM.

According to one embodiment of the invention, the induction of autophagy or apoptosis by the pharmaceutical composition of the invention takes place in a time-dependent manner.

Convalatoxin or convalatoxin derivatives of the present invention may be included in the composition of the present invention as a compound of the general formula (1-5) as well as in the form of a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound, for example, ammonium salts, Metal salts, alkaline earth metal salts, transition metal salts, quaternary amine salts or amino acid salts, but are not limited thereto. The term " pharmaceutically effective amount " means an amount necessary to adequately induce autophage, apoptosis, hyperproliferative hyperproliferation, or angiogenesis inhibition to exhibit the pharmacological effect of the therapeutic agent for disease.

Pharmaceutical compositions comprising a convalatoxin or a convalatoxin derivative of the present invention include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and preparations are described in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) a pharmaceutical composition comprising (a) a pharmaceutically effective amount of convalatoxin, a convalatoxin derivative or a pharmaceutically acceptable salt thereof; And (b) a pharmaceutical composition for preventing or treating autophage-related diseases, apoptosis-related diseases, hypertrophic diseases or angiogenesis diseases, which comprises a pharmaceutically acceptable carrier.

(Ii) The pharmaceutical compositions of the present invention simultaneously induce autophagy and apoptosis of cells in a dose-dependent and dose-time-dependent manner, thus inhibiting hyperproliferation and angiogenesis in a dose-dependent manner, Phage-related diseases, apoptosis-related diseases, hypertrophic diseases or angiogenic diseases.

(Ii) The present invention is based on the discovery that the pharmaceutical composition of the present invention induces autophage by increasing phosphorylation of AMP-activated protein kinase (AMPK) and reducing phosphorylation of mTOR and p70S6K, Contributes to clarifying the phage induction mechanism.

Fig. 1 shows a schematic diagram of an auto-phage [10]. (a, b) indicates that cytoplasmic proteins and cell organelles are isolated by pagophores; (c) represents the formation of a double membrane vesicle, an autoparge species; (d) represents an autoserosome fused with an autopagosomalisosol; (e) shows decomposition of the cargo-containing oxylosome.
Figure 2 shows the scheme of screening of autophagy inducers.
Fig. 3 shows screening of active small molecules derived from a natural material library which inhibits the proliferation of HeLa cells at high concentrations. 67 Natural Crude Extracts inhibiting the growth of HeLa cells were selected from 3000 natural product libraries.
Fig. 4 shows an active small molecule screening derived from a natural product library inhibiting proliferation at low concentrations. Five natural Crude extracts inhibiting the growth of HeLa cells at low concentrations were selected from 67 natural product libraries with anti-proliferative activity at high concentrations.
Figure 5 shows the active small molecule screening from natural products that induces LC3 conversion. One of the natural crude extracts leading to LC3 conversion, NP13-07, was selected from five natural products with anti-proliferative activity at 1 μg / mL. NP13-07 is Antiaris toxicaria (Pers.) Lesch . (Moraceae). Rapamycin (Rapa) was used as a positive control.
Figure 6 shows the chemical structure of convallatoxin. It is the chemical structure of convalatoxin , an ingredient in A. toxicaria . The compound is a steroid aglycon having a lactone ring and a glycoside, which is? -L-rhamnoside.
Figure 7 shows anti-proliferative activity of A. toxicaria crud extract and convalatoxin in HeLa cells. HeLa cells were treated with A. toxicaria crude extract and convalatoxin for 72 hours and cell growth was measured using MTT colorimetric assay. Figure 7a shows the effect of A. toxicaria on HeLa cell proliferation And Fig. 7B shows the effect of convalatoxin on HeLa cell proliferation.
Figure 8 shows the effect of convalatoxin on the viability of HeLa cells. Was tested using trypan blue assay. CVT represents convalatoxin.
Figure 9 shows the detection of convalatoxin-induced autophage vacuole by MDC staining. HeLa cells were treated with convalatoxin for 24 hours and stained with MDC. The effect of convalatoxin on the accumulation of autophage vacuole was monitored using a fluorescence microscope and the fluorescence intensity was measured by Image J software (Ver. 1.41). CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 10 shows the detection of convalatoxin-induced autophage vacuole by LysoTracker red staining. HeLa cells were treated with convalatoxin for 24 hours and nuclei and autophage vacuoles were stained with Hoechst 33342 and LysoTracker Red, respectively. FIG. 10A shows the effect of convalatoxin on the accumulation of LysoTracker red using a fluorescence microscope and FIG. 10B shows the measurement of LysoTracker red fluorescence intensity by High Content Screening analysis (CellomicsArrayScanVTI). CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 11 shows the conversion of LC3-I to LC3-II in convalatoxin-treated HeLa cells. Figure 11a shows HeLa cells treated with various concentrations of convalatoxin and 10 [mu] M rapamycin for 24 hours and Figure 11b shows a significant increase in LC3-II in a time-dependent manner in 10 nM convalatoxin. The level of tubulin was used as an internal control. CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 12 shows the effect of convalatoxin on phosphorylation of AMP-activated protein kinase (AMPK). The level of expression of phosphorylated AMPK in HeLa cells by Western blotting is shown. CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 13 shows the effect of convalatoxin on phosphorylation of mTOR and p70S6K. Detection of phosphorylation levels of mTOR and p70S6K by western blot. CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 14 shows the increase of the sub-G0 / G1 cell population by convalatoxin treatment. Convalatoxin treatment causes an increase in the percentage of subG0 / G1 cells in a dose-dependent manner. HeLa cells were treated with various convalatoxin doses for 48 hours and cells treated by FACs analysis were recovered. CVT represents convalatoxin, and Rapa represents rapamycin.
Figure 15 shows DNA fragmentation detection in convalatoxin-treated-HeLa cells. HeLa cells were treated with 20 nM convalatoxin and 10 μM camptothecin for 24 hours. Camptothecin was used as a positive control. CVT represents convalatoxin, Campt represents camptothecin.
Figure 16 shows detection of PARP and caspase-3 cleavage in convalatoxin-treated HeLa cells. PARP and caspase-3 truncations characteristic of apoptosis were observed in a dose-dependent manner (Figure 16a) and in a dose time-dependent manner (Figure 16b). Tubulin was used as a positive control. CVT represents convalatoxin.
Figure 17 shows the effect of apoptosis inhibitors on convalatoxin-treated-HeLa cells. HeLa cells were pretreated with DMSO or the caspase family inhibitor Z-VAD-FMK (50 [mu] M) for 1 hour before treatment with convalatoxin or camptothecin. After 24 hours, the percentage of dead cells by trypan blue staining was evaluated. CVT represents convalatoxin, Campt represents camptothecin.
Figure 18 shows the anti-proliferative activity of convalatoxin against HUVECs. HUVECs were treated with convalatoxin for 72 hours and cell growth was measured using MTT colorimetric assay. CVT represents convalatoxin.
Figure 19 shows the anti-angiogenic activity of convalatoxin in vitro . 19A is an inhibitory activity of convalatoxin against endothelial cell invasion. Figure 19b shows the effect of convalatoxin on the tube forming ability of HUVECs. The arrow shows the broken tube formed by VEGF-induced HUVECs after convalatoxin treatment. The basal level invasion (Figure 19a) and capillary formation (Figure 19b) of the remaining HUVECs in serum-free medium were normalized to 100%. CVT represents convalatoxin.
Figure 20 shows the in vivo anti-angiogenic activity of convalatoxin. EtOH control, RA (1 μg / ml) and (0.5 ng / ml) were applied to CAM and the membranes were observed. Arrows indicate inhibition of neovascularization by convalatoxin treatment. The inhibition rate was calculated as the percentage of inhibited eggs relative to the total number of eggs tested. CVT represents convalatoxin, and RA represents retinoic acid.
Figure 21 is a schematic representation of the convalatoxin signaling pathway.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Materials and Experiments

 Experimental material

Convallatoxin was purchased from Sigma-Aldrich (USA). DMEM (Dulbecco's modified Eagle medium) and fetal bovine serum (FBS) were purchased from Invitrogen (USA). EBM-2 (Endothelial basal media-2) was purchased from Cambrex Bio Science (USA). Hoechst 33342 and Lysotracker red were purchased from Invitrogen (USA). Monodansylcadaverine (MDC) was purchased from BioChemika (Switzerland). The caspase family inhibitor Z-VAD-FMK was purchased from Promega (USA). Transwell plates, recombinant human vascular endothelial cell growth factor (VEGF) and Matrigel were purchased from Corning (USA), KOMA Biotech (Seoul) and BD Biosciences (USA) respectively . Anti-phosphotyrosine-3 and anti-tubulin antibodies are useful in the treatment and / or prophylaxis of cancer, including, but not limited to, anti-LC3, anti-mTOR, anti-phosphorylated mTOR, anti-p70S6K, anti-phosphorylated p70S6K, anti-AMPK, They were purchased from MBL (Japan), Cell Signaling (USA) and Millipore (USA) respectively.

Experimental Method

Cell culture

HeLa (human cervical cancer) cells were cultured and maintained in DMEM containing 10% FBS and 1% antibiotic. Human umbilical vascular endothelial cells (HUVECs) were cultured and maintained in EBM-2 medium supplemented with 10% FBS for 7-11 passages. Two cell lines were cultured in a solution of 5% CO ₂ Lt; / RTI >

Cell proliferation and survival analysis

Cells were subcultured in 96-well plates and incubated overnight, then treated with various concentrations of convalatoxin for 72 hours. Cell proliferation was induced by adding 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT: 3- (4,5-dimehylthiazol- tetrazolium bromide) colorimetric analysis. Cell viability assays were assessed using trypan blue staining.

Visualization and analysis of cellular autophage vacuole

To detect autophage vacuoles, cells were treated with 50 μM of MDC, a fluorescent dye known as a marker specific for autophagy organelles, for 30 minutes [15]. After incubation, the cells were washed three times with PBS (phosphate buffered saline) and analyzed immediately by fluorescence microscopy. Lysotracker Red [16], a fluorescent dye for labeling functional lysosomes, was applied to the cells at 50 nM for 30 min (1: 1000) with Hoechst 33342 and then washed and visualized by fluorescence microscopy or by ArrayScan VTI HCS Reader (Cellomics, USA). To identify individual cells by Hoechst-labeled nuclei, the target activation protocol of HCS Reader was used and then the fluorescence intensity of each cell was automatically analyzed.

DNA content analysis

Cells treated with convalatoxin were harvested and fixed in 70% ethanol for 30 min on ice. After centrifugation (2000 rpm), the cell pellet was washed once with PBS and incubated at 37 ° C with 50 μg / mL propidium iodide (PI) and 100 μg / mL RNase A. After 1 hour, the DNA content of 10,000 items was measured using FAC Scan (Becton Dickinson, USA). Data were analyzed by Cell Quest software (Becton Dickinson, USA).

DNA Fragmentation Analysis

To detect DNA fragmentation, cells were harvested and washed with PBS. This was then dissolved in 0.3 M Tris-HCl at pH 7.5 containing 0.01 M EDTA, 0.1 M NaCl and 0.2 M sucrose and incubated with 10% SDS (sodium dodecyl sulfate) at 65 ° C for 30 minutes.

5 M potassium acetate was added to the ice, and the supernatant was recovered after centrifugation. Next, the supernatant was incubated with 20 μg / mL RNase A for 1 hour, phenol / chloroform extraction and ethanol precipitation were performed for DNA extraction. After electrophoresis on 2% agarose gel at 50 V for 1 hour, it was visualized by staining with ethidium bromide.

Caspase  Inhibitor analysis

Cells were plated in 12-well plates and incubated overnight at 37 ° C. Z-VAD-FMK (caspase family inhibitor) or DMSO was pretreated for 1 hour before treatment with convalatoxin or camptothecin for 24 hours. Cell viability was determined by trypan blue staining.

In-vitro capillary tube formation analysis

Matrigel was coated on a 48-well culture plate and polymerized at 37 [deg.] C for 2 hours. HUVECs (6 x 10 ⁴ cells / well) were dispensed onto the matrigel surface and treated with VEGF (30 ng / mL). Then, the compound was added and incubated at 37 ° C for 3-18 hours. Morphological changes and tube formation of the cells were observed with a microscope (IX71, Olympus, Japan) and photographed (DP70, Olympus, Japan).

In bito Chemical invasion  analysis

Involvement of HUVECs in vitro was tested using a transwell chamber system with an 8.0 μm for-size polycarbonate filter [17]. The bottom of the filter was coated with 10 μL gelatin (1 mg / mL) and the top was coated with 10 μL Matrigel (3 mg / mL). Convalatoxin was added to the lower chamber containing VEGF (30 ng / mL) and HUVECs were placed on top of the filter. Thereafter, the chamber was incubated at 37 ° C. After 18 hours, the cells were fixed with 70% methanol and stained with hematoxylin and eosin. The invasion of the cells was determined by counting the total number of invading cells at the bottom of the filter using an Olympus IX70 microscope at 100x magnification.

CAM ( Chick Chorioallantoic membrane ) analysis

CAM analysis was performed as disclosed in the prior art [18]. The fertilized eggs were kept in a wet incubator at 37 ° C for 3 days. After CAM and egg yolk fell off the membrane of the egg, about 4-5 mL of egg albumin was removed using a hypodermic needle. On day 5, a 2.5 cm diameter window was made with razor and tweezers and compound-mounted Thermanox coverslips (NUNC, USA) were placed on the CAM surface. After 2 days, 2-3 mL Intralipose (Greencross, Korea) was injected into the lower part of the CAM and observed under a microscope. Retinoic acid (RA) was used as a positive control.

Western Blot  analysis

Cell lysates were separated by 8-12.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and stained with polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using standard electroblotting procedures. . The blots were then blocked and immunoblotted with primary antibody overnight at 4 ° C. Immune markers were detected by an enhanced chemiluminescence kit (GE Healthcare, UK) according to the manufacturer's instructions.

Statistical analysis

The results were expressed as mean ± standard deviation (SE). Student's t-test was used to determine the statistical significance between the control and test groups. P <0.05 was considered statistically significant.

Experiment result

Natural Library Screening

To detect novel autophagy inducing agents that inhibit cancer cell proliferation, 3,000 species of crude extracts of natural plant libraries were screened using a cell-based screening system (Figure 2). The anti-proliferative activity (FIGS. 3 and 4) and the LC3 conversion (FIG. 5) at high dose and low dose of these were investigated in HeLa cells. First, 67 proliferation of Crude extracts of natural products inhibiting the growth of HeLa cells were found using the proliferation assay. Second, crude extracts of five natural products inhibiting the growth of HeLa cells were selected by analysis of cell proliferation at low concentrations. As a result, by LC conversion detection, one of the five natural product crude extracts, Antiaris Toxicaria (Pers.) Lesch. (Moraceae) was selected and convalatoxin , one of the cardiac glycosides, strongly exhibiting the activity from the natural substance crude extract was found (Fig. 6). The plants grow throughout the tropical regions of Southeast Asia [20] and contain a complex mixture of radial glycosides [21]. Coronary glycosides are well known as Na ⁺ / K ⁺ -ATPase inhibitors, some of which are used to treat congestive heart failure and atrial arrhythmias [22]. The present invention discloses the anticancer activity that induces both the properties of convalatoxin and its autophagy and apoptosis.

Antiaris toxicaria ( Pers .) Lesch . ( Moraceae ) of Crud  The extract and Convalatoxin  Dose-dependent HeLa  It inhibits cell proliferation.

A. toxicaria on HeLa cells And the crude extract of convalatoxin were investigated. Cells were treated with various concentrations of crude extract and convalatoxin for 3 days and cell growth was measured using MTT colorimetric assay. Crude extracts of A. toxicaria showed dose-dependent anti-proliferative activity (Figure 7a). It was confirmed whether the antiproliferative activity was due to convalatoxin, which is a component of the extract. As shown in Figure 7b, it was shown to strongly inhibit HeLa cell proliferation (IC ₅₀ = 10 nM). Next, cell viability was measured at 24 hours, 48 hours, and 72 hours using trypan blue assay. Convalatoxin decreased cell viability in a dose and time-dependent manner (Figure 8). To confirm cell growth inhibitory activity and viability by convalatoxin treatment, subsequent studies were performed using a dose range of 1-20 nM.

Convalatoxin HeLa  Cell Auto Phage  Strongly induces activity.

In order to detect the autophage activity of convalatoxin, autophage vacuole was investigated using MDC and LysoTracker Red which are fluorescent dyes known as specific markers for autophage organelles. After treatment with convalatoxin for 24 hours, MDC fluorescence intensity was observed in HeLa cells (Figure 9). The accumulation of convalatoxin-induced LysoTracker Red increased (Figure 10). The conversion of the cytoplasmic LC3-I to the autopagos-related LC3-II, well known as an autopagosomal marker, was assessed by treatment with convalatoxin. Conversion of LC3 was significantly enhanced in convalatoxins over a wide concentration range for 24 hours (FIG. 11A). In addition, HeLa cells treated with 10 nM convalatoxin showed an increase in LC3-II in an administration time-dependent manner (Fig. 11B).

Next, the effect of convalatoxin on the AMP-activated protein kinase (AMPK) pathway regulating autophage was investigated (Fig. 12) [23]. The phosphorylation rate of AMPK was increased in a dose-dependent manner. Thereafter, the phosphorylation of the mammalian target (mTOR) of rapamycin inhibited by AMPK was inhibited and the phosphorylation of p70S6K, the downstream pathway of mTOR, was also reduced by convalatoxin treatment (Fig. 13) [24]. Taken together, these results indicate that convalatoxin is a potent inducer of autophagy through the AMPK / mTOR pathway.

Convalatoxin HeLa  In a cell Auto Phage  As well as Apoptosis  .

To determine whether convalatoxin-induced apoptosis was associated with autophagy as well as apoptosis, DNA content analysis was performed in HeLa cells (Fig. 14). Convalatoxins increased the subG0 / G1 population in a dose-dependent manner. Second, DNA sequenced was also detected in convalatoxin-treated cells, such as camptothecin-treated cells (Figure 15). Third, caspase-3 and PARP cleavage, characteristic of apoptosis, were examined by western blot (Fig. 16). Caspase-3 and PARP were cleaved in convalatoxin-treated cells. After pretreatment with caspase-3 and the apoptosis inhibitor Z-VAD-FMK or DMSO, trypan blue analysis was performed to determine the viability of the convalatoxin-treated cells. The number of cells killed by Z-VAD-FMK decreased in convalatoxin and camptothecin-treated cultures (FIG. 17). Finally, concluding, these results demonstrate that convalatoxins induce not only autophagy but also apoptosis in HeLa cells.

Convalatoxin  Strongly inhibits angiogenesis in in vitro and in vivo.

Angiogenesis is the process of forming new blood vessels from existing blood vessels and plays an important role in a variety of diseases including solid tumor growth and metastasis [25]. Thus, inhibition of angiogenesis has been highlighted as a promising strategy for cancer therapy [26].

The effect of convalatoxin on the proliferation of human umbilical vascular endothelial cells (HUVECs) was investigated. Cells were treated with various concentrations of convalatoxin for 3 days and cell growth was measured using MTT colorimetric assay. Convalatoxin showed anti-proliferative activity in a dose-dependent manner (Figure 18). The IC ₅₀ of convalatoxin for HUVECs was 4 nM. Next, the angiogenic activity of convalatoxin was observed and tube formation analysis was performed using VEGF (vascular endothelial growth factor), one of the angiogenic factors. Serum HUVECs were stimulated by VEGF in the presence or absence of convalatoxin. In Fig. 19A, convalatoxin inhibited the invasion of VEGF-induced HUVECs in a dose-dependent manner. Tubule formation by VEGF was also inhibited in convalatoxin-treated HUVECs (Figure 19b). These results show that inhibit VEGF- induced angiogenesis in which the toxin paint cone bits.

In addition, anti - angiogenic activity of convalatoxin was investigated in in vivo using chick chorioallantoic membrane (CAM) assay. In general, advanced CAMs exhibit capillaries of a broad network. Convalatoxin-treated CAMs, on the other hand, showed inhibition of capillary blood vessel formation without symptoms of thrombosis or hemorrhage (Fig. 20). Retinoic acid (RA) was used as a positive control. These data demonstrate that convalatoxins strongly inhibit angiogenesis in both in vitro and in vivo without the effect of cytotoxicity.

conclusion

The present invention relates to anti- toxicaria (Pers.) Lesch. Convalatoxin [19], a natural small molecule contained in the extract, induced apoptosis through autophagy and apoptosis in HeLa cells. Convalatoxin inhibited the proliferation of HeLa cells in a dose-dependent manner. Convalatoxin-induced autophagy was also investigated by MDC staining, Lysotracker red staining and LC3 conversion. Phosphorylation of AMPK in convalatoxin-treated HeLa cells was increased in a dose-dependent manner and mTOR / p70S6K was inhibited suggesting that convalatoxin induced autophage through the AMPK / mTOR signaling pathway. In addition, convalatoxin increased sub-G0 / G1 cell populations and induced DNA fragmentation. PARP and caspase-3 cleavage were detected in convalatoxin-treated cells. In addition, viability of convalatoxin-treated cells increased in the Z-VAD-FMK-pretreatment culture. These results suggest that convalatoxin induces apoptosis through both autophagy and apoptosis. Furthermore, convalatoxins inhibit the growth of HUVECs in a dose-dependent manner. Convalatoxin decreased VEGF-induced chemoattractant and tubular formation in HUVECs, and CAM analysis showed anti-angiogenic activity of convalatoxin. These data indicate that convalatoxins strongly inhibit angiogenesis in vitro and in vivo (Figure 21). These results show that convalatoxin can be used as a potent therapeutic anti-angiogenic agent.

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 While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (13)

(a) a pharmaceutical effective amount of a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; And (b) a pharmaceutical composition for preventing or treating autophage-related diseases, apoptosis-related diseases, hyperproliferative diseases or angiogenesis diseases comprising a pharmaceutically acceptable carrier,
Formula 1

Wherein X ₁ and X ₂ each independently represent oxygen, sulfur or nitrogen; R ₁ , R ₂ And R ₃ each independently represent hydrogen, C ₁ -C ₅ straight or branched chain alkyl, C ₂ -C ₆ alkenyl or C ₃ -C ₈ cycloalkyl; R ₄ represents -COH, -COR ₆ , -COOR ₆ (wherein R ₆ is a C ₁ -C ₅ linear or branched alkyl) or -CONH ₂ ; R ₅ is hydrogen, C ₁ -C ₅ linear or branched alkyl, C ₁ -C ₅ linear or branched alkyl alcohol.
2. The pharmaceutical composition according to claim 1, wherein the compound of Formula 1 is represented by Formula 2:
(2)

2. The pharmaceutical composition according to claim 1, wherein the compound of Formula 1 is represented by Formula 3:
(3)

The pharmaceutical composition according to claim 1, wherein the compound of formula (1) is represented by the following formula (4):
Formula 4

5. The compound according to any one of claims 1 to 4, wherein X ₁ and X ₂ represent oxygen; R ₁ represents a C ₁ -C ₅ straight or branched chain alkyl; R ₂ And R &lt; ₃ &gt; represent hydrogen; R ₄ represents -COH; R ₅ is A pharmaceutical composition, characterized in that represents an alkyl of straight or branched-chain C ₁ -C _5.
The composition of claim 1, wherein the compound of formula (1) is a convallatoxin of formula (5)
Formula 5

The method of claim 1, wherein the auto-phage related disorder is selected from the group consisting of cancer, atherosclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, Dentatorubral pallidoluysian atrophy, anterior temporal dementia, progressive supranuclear palsy, vertebral apical muscular dystrophy, osteoarthritis of the spinal cord, an x-linked spinobulbar muscular atrophy, and a neuronal intranuclear hyaline inclusion disease.
The pharmaceutical composition according to claim 1, wherein the apoptosis-related disease is selected from the group consisting of cancer, viral infection, allergic disease, inflammatory disease, autoimmune disease, immune disease, reperfusion injury and degenerative disease.
The method of claim 1, wherein the hyperproliferative disease is selected from the group consisting of cancer, hyperproliferation, keloid, Cushing's syndrome, hyperalgesia, hyperalgesia, hyperalgesia, vitiligo, hypertrophic scarring, squamous cell hyperplasia, blackening, arteriosclerosis, atherosclerosis, Recurrent stenosis and stenosis. &Lt; Desc / Clms Page number 15 &gt;
The method according to claim 1, wherein the angiogenic disease is selected from the group consisting of cancer, diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascular glaucoma, hypochromia, proliferative retinopathy, psoriasis, hemophilic joints, capillary vessels in atherosclerotic plaque Atherosclerosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcer, hepatopathy, glomerulonephritis, diabetic nephropathy, malignant neoplasia, malignant neoplasia, Neuropathic pain, thrombotic microangiopathy, organ transplant rejection, nephropathology, diabetes, inflammation and neurodegenerative diseases.
11. The method of any one of claims 7 to 10, wherein the cancer is selected from the group consisting of pituitary adenoma, glioma, brain tumor, female cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, Cancer of the prostate, cancer of the endometrium, gallbladder cancer, penile cancer, ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, pediatric cancer, ovarian cancer and cervical cancer &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.
2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition simultaneously induces autophagy and apoptosis.
2. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition increases phosphorylation of AMP-activated protein kinase (AMPK).

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