KR20180105474A - Hybrid anticancer prodrug for creating cinnamaldehyde or cinnamic acid, and method for preparing the same - Google Patents

Abstract

The present invention relates to a hybrid anticancer prodrug for producing cinnamaldehyde or cinnamic acid; a preparation method for the same; a pharmaceutical composition comprising the prodrug as an effective component for preventing or treating cancer; a functional health food comprising the prodrug as an effective component for preventing or alleviating cancer; a drug delivery vector comprising the prodrug; and an imaging contrast agent comprising the prodrug. Unlike cinnamaldehyde which has low anticancer effects due to a short half-life in blood, nanoparticles of cinnamaldehyde-conjugated maltodextrin according to the present invention disintegrate under mildly acidic conditions around cancer cells to induce the formation of reactive oxygen, thereby releasing cinnamaldehyde exhibiting anticancer effects, and thus increase anticancer effects. In addition, the nanoparticles of cinnamaldehyde-conjugated maltodextrin, as disintegrated by hydrogen peroxide in cancer tissues, form carbon dioxide bubbles, such that the nanoparticles achieve resonance with ultrasound frequencies, thereby enhancing ultrasound signals and enhancing an ultrasound image. Further, polymer nanoparticles of cinnamaldehyde-conjugated maltodextrin, as a drug delivery vector, gather regular anticancer agents to create a synergistic effect inanticancer effects through inducing apoptosis.

Description

(Hybrid anticancer prodrug for creating cinnamaldehyde or cinnamic acid, and method for preparing the same)

The present invention relates to a hybrid anticancer prodrug that produces cinnamic aldehyde or cinnamic acid, a method for producing the same, a pharmaceutical composition for preventing or treating cancer containing the prodrug as an active ingredient, a composition comprising the prodrug as an active ingredient A drug delivery vector containing the prodrug, and an ultrasound imaging contrast agent including the prodrug.

Cancer accounts for 13% of all deaths and is one of the leading causes of death worldwide. The main therapeutic business of cancer is chemotherapy using various chemical agents such as dogsorubicin, cis palatin, paclitaxel and camptothecin. However, they have low solubility and are not specific for tumor cells, resulting in adverse effects, limiting clinical use. Various drug delivery strategies have been developed to improve drug delivery and biodistribution of existing drugs using nanotechnology and biodegradable polymers. Over the past few decades, nanometer-sized multifunctional particles have emerged as one of the promising systems for controlling and targeting targeted anticancer drugs. However, the development of smart nanoparticles that can specifically target tumor sites and release drugs in a controlled manner to enhance the therapeutic efficacy of drugs remains a major challenge.

Cinnamaldehyde is the main active ingredient of cinnamomum cassia, a plant of the genus Lauraceae, which has been used to treat dyspepsia, gastritis, blood circulation disorders and inflammation in both the east and the west. Cinnamon bark It is a major component. Shin Nam Aldehyde contains α, β-carbonyl, known as micheal receptor pharmacophore, and produces reactive oxygen species (ROS) to reduce the mitochondrial membrane potential , Which induces apoptosis through the release of cytochrome C from the cell to the cytosol, has been demonstrated to have anticancer ability through a mechanism dependent on caspase. Despite the excellent anticancer ability of cinnamaldehyde and its derivatives, however, phagocytosis is rapidly phagocytosed in vivo by hepatic macrophages and has a short half-life of less than 1.5 hours (minutes, about 5 minutes) There is a disadvantage that there is no ability to be able to. Therefore, in order to apply Shin-Nam aldehyde to chemotherapy in clinical practice, it is required to develop physicochemical modification or new drug delivery system to enhance anticancer effect.

Numerous soluble and biodegradable polymers have been extensively studied in pharmaceutical and medical premises. Polysaccharides such as chitosan, hyaluronic acid, dextrin and maltodextrin have great advantages as platforms for drug delivery that include well known biocompatibility, non-toxicity, non-immunogenicity and broad availability. Maltodextrin is a polysaccharide composed mainly of glycoside-linked D-glucose and has been widely used as a food additive. In particular, maltodextrin contains a large amount of OH groups in the main chain, which facilitates chemical modification.

The inventors of the present invention have found that Shin Nam aldehyde capable of amplifying ultrasound signals by resonance with ultrasound frequency by generating CO ₂ upon decomposition by hydrogen peroxide and having high anticancer activity while accelerating degradation to weak acid which is a specific environment of cancer Polymeric nanoparticles of conjugated maltodextrin have been developed.

The present invention provides a hybrid anticancer prodrug that produces cinnamic aldehyde or cinnamic acid, a method for producing the same, a pharmaceutical composition for preventing or treating cancer containing the prodrug as an active ingredient, a pharmaceutical composition comprising the prodrug as an active ingredient A drug delivery vector containing the prodrug, and an ultrasound imaging contrast agent containing the prodrug. The present invention also provides an ultrasound imaging contrast agent comprising the prodrug.

The present invention provides a hybrid carcinoma precursor which produces cinnamaldehyde or cinnamic acid, which is a hybrid anticancer prodrug inducing apoptosis of cancer cells and a method for producing the same.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the above-mentioned hybrid anticancer prodrug as an active ingredient.

In addition, the present invention provides a food composition for preventing or ameliorating cancer comprising the hybrid anticancer prodrug as an active ingredient.

The present invention also provides a drug delivery system comprising the hybrid anticancer prodrug as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the hybrid anticancer prodrug and the anticancer agent as an active ingredient.

The present invention also provides an imaging contrast agent comprising the hybrid anticancer prodrug as an active ingredient.

Shin Nam Aldehyde has low anticancer effect due to a short half-life in blood. However, the present invention invented nanoparticles of cinnamaldehyde-conjugated maltodextrin, and decomposed under weak acidic conditions inside and outside cancer cells to induce production of active oxygen, Can release the cinnamaldehyde, which can exert its anti-cancer effect. In addition, it decomposes by the hydrogen peroxide of the cancer tissue, and CO ₂ gas bubble is formed, and the nanoparticles resonate with the ultrasonic frequency and increase the ultrasound signal to increase the ultrasound image. Furthermore, conjugated maltodextrin polymer nanoparticles, which are conjugated with cinnamaldehyde, have a synergistic effect on the anticancer effect through the induction of apoptosis by collecting general anticancer drugs as drug carriers.

Figure 1 shows the synthesis and characterization of CMD, wherein (a) is the synthesis of the cinnamic aldehyde derivative 2, (b) is the 1 H NMR spectral result of compound 22 recorded in CDCl 3, and (C) (D) is the 1 H NMR spectrum of CMD in DMSO-d6 and (e) the 1 H NMR spectrum of CMD after acid-induced hydrolysis.
(A) shows the hydrodynamic diameter of CMD nanoparticles determined by DLS, (b) is a representative scanning electron micrograph of CMD nanoparticles, and (C) (D) is the result of the hydrodynamic diameter of the CPT-CMD nanoparticles determined by DLS, and (e) is the result of the hydrodynamic diameter of the CPT-CMD nanoparticles determined by DLS. (F) shows the result of a change in the hydrodynamic diameter of CPT-CMD nanoparticles suspended in PBS in the presence or absence of FBS (10%).
Figure 3 shows the pH-dependent release of CA (cinnamaldehyde) to CMD nanoparticles.
Figure 4 shows ROS production in cancer cells treated with CPT-CMD nanoparticles. (A) is a representative micrograph of cells that produce ROS after CMD or CPT-CMD nanoparticles, (b) .
FIG. 5 shows the cytotoxicity of CMD and CPT-CMD nanoparticles on various cell lines. FIG. 5 (a) shows cytotoxicity of CMD nanoparticles treated with SW620DP with different amounts of CA (cinnamaldehyde) (C) shows HEK293 cells, (d) shows DU145 cells, and (e) shows cytotoxicity of CMD nanoparticles to SW620 cells.
6 shows flow cytometric analysis of apoptosis of SW620 cells treated with CMD nanoparticles and CPT-CMD nanoparticles.
Figure 7 shows the Western blot results of the apoptosis-related proteins of SW620 cells treated with CMD nanoparticles and CPT-CMD nanoparticles.
FIG. 8 shows the therapeutic anticancer activity of CPT-CMD nanoparticles. (A) shows a total image of tumor-bearing mice 4 weeks after treatment. CA (cinnamaldehyde) and CPT were administered at 5 mg / kg and 3 mg / kg (B) is the result of quantifying the change of tumor during treatment.
FIG. 9 is a representative image of a tumor tissue, and FIG. 9 (a) is a representative image of H & E stained tumor tissue, and FIG. 9 (b) is a representative image of TUNEL stained tumor tissue.
Figure 10 is a representative image of H & E staining of organs in tumor bearing mice.
11 is an ultrasound image of a tumor using CMD nanoparticles.

In general, cinnamaldehyde is known to induce apoptosis through the production of reactive oxygen species (ROS), but it has weak cytotoxicity to normal cells. However, its use has been limited by the short half-life in the blood of cinnamaldehyde and by its lower activity compared to conventional anti-cancer drugs. Therefore, in order to overcome these disadvantages, a new hybrid anticancer precursor was prepared by binding a hydroxyl group of maltodextrin to cinnamaldehyde through an acetal linkage.

The present invention provides a hybrid anticancer prodrug, which produces cinnamaldehyde or cinnamic acid, and which produces the following chemical formula 1 and / or chemical formula 2:

[Chemical Formula 1]

(2)

(3)

In Formula 3, n may be 5 to 100, and most preferably 15 to 20.

The formula 1 may be cinnamaldehyde, and the formula 2 may be cinnamic acid.

The above Formulas (1) and (2) can be produced by hydrogen peroxide (H ₂ O ₂ ) and an acidic pH. In particular, they can be produced specifically in cancer cells,

The acidic pH cleaves the acetal bond of the present invention to release cinnamal aldehyde, and the released cinnamaldehyde can generate ROS to promote apoptosis.

In addition,

 (a) reacting cinnamic aldehyde with an acidic solution to prepare a compound having an acetal bond represented by the following formula 4;

[Chemical Formula 4]

(b) reacting the compound prepared in step (a) with carbonyldiimidazole to prepare a cinnamaldehyde releasing compound; And

(c) reacting maltodextrin with the cinnamyl aldehyde releasing compound prepared in step (b).

A representative example of the method for producing the hybrid anticancer prodrug of the present invention can be represented by the following Reaction Scheme 1.

[Reaction Scheme 1]

There is provided a composition for preventing or treating cancer comprising as an active ingredient a hybrid anticancer prodrug represented by the following formula (3).

(3)

The composition comprises a pharmaceutical composition and a food composition.

Hybrid anticancer prodrugs of the present invention are produced by sequential release of cinnamic acid and cinnamic aldehyde by H ₂ O ₂ and acidic pH, thereby accumulating large amounts of reactive oxygen species (ROS), which accelerate apoptosis, Lt; / RTI >

Said cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer, colorectal cancer, myeloid leukemia, thyroid cancer, MDS, bladder carcinoma, epidermal carcinoma, melanoma, breast cancer, prostate cancer, head and neck cancer, uterine cancer, A cancer selected from the group consisting of gastric cancer, laryngeal cancer, esophageal cancer, bladder cancer, oral cancer, cancer of the mesenchymal origin, sarcoma, teratocarcinoma, neuroblastoma, renal carcinoma, liver cancer, non- Hodgkin's lymphoma, multiple myeloma, Or more. Most preferably, it can be colon cancer.

The composition of the present invention may contain one or more known active ingredients having an effect of preventing or treating cancer together with the hybrid anticancer prodrug.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the hybrid anticancer prodrug of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, and may be administered once or several times a day.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of cancer.

The food composition of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

There is provided a drug delivery system comprising as an active ingredient a hybrid anticancer prodrug drug represented by the following formula (3).

(3)

The drug may be one or more drugs selected from the group consisting of paclitaxel, docetaxel, doxorubicin, cisplatin, carboplatin, 5-FU, etoposide and camptothecin.

There is provided an ultrasound imaging contrast agent comprising a hybrid anticancer prodrug represented by the following formula (3) as an active ingredient.

(3)

Dosages and formulations used for administration of the contrast agents and / or pharmaceutical compositions of the present invention can be readily determined by those skilled in the art of diagnosis or treatment of diseases. It is understood that the dosage of the contrast agent will depend on the age, sex, health and weight of the recipient, the type of treatment involved, the number of treatments, if any, and the nature of the desired effect. For any mode of administration, the dosage regimen required to achieve the beneficial effects described herein as well as the actual amount of contrast agent delivered will also be dependent, in part, on the bioavailability of the contrast agent, the disorder being treated or diagnosed, the desired therapeutic or diagnostic dose And other factors that will be apparent to those skilled in the art. In the present invention, the dosage administered to an animal, particularly a human, should be sufficient to exhibit the desired therapeutic or diagnostic response over an appropriate period of time in the animal.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Methyl ((5-methyl-2- styryl -1,3- dioxan -5- yl ) methyl) carbonate

1-1. Shin Nam Aldehyde  The derivative (5-methyl-2- styryl -1,3- dioxan -5- yl ) Preparation of methanol (1)

Tris (hydroxymethyl) ethane (4.08 g) was dissolved in 70 ml of dry benzene. To the reaction solution, cinnamaldehyde (2.86 mL) and p-toluenesulfonic acid (40 mg) were added and reacted at 85 ° C for 6 hours. After that, the reaction solution was cooled at room temperature, and 1 mL of triethyleneamine was added to terminate the reaction. The benzene in the reaction mixture was evaporated using a rotary evaporator and purified by column chromatography (hexane / ethyl acetate = 4/1) to obtain the title compound (1).

1-2. Shin Nam Aldehyde  The emission compound (5-methyl-2- styryl -1,3- dioxan -5-yl) methyl 1H-imidazole-1-carboxylate (2)

1,1'-Carbonyldiimidazole (4.1 g) and the cinnamaldehyde derivative (2) (3.0 g) prepared in the above 1-1 were dissolved in 50 mL of dry dichloromethane And reacted at room temperature for 30 minutes. The reaction mixture was evaporated under reduced pressure to remove dichloromethane, and the residue was purified by column chromatography using ethyl acetate as an eluting solvent to obtain the title compound (2) (FIG. 1A). Fig. 1b shows the result of confirming compound 2 by HNMR.

1.3 Maltodextrin  conjugated Methyl ((5-methyl-2- styryl -1,3- dioxan -5-yl) methyl) carbonate (3)

Maltodextrin (0.3 g) and various amounts of Compound 2 (1.1 g, 1.7 g or 2.2 g) were dissolved in DMSO containing DMAP as a catalyst. The bonding reaction was maintained at room temperature for 2 days. To the reaction mixture was added deionized water (25 mL) and then centrifuged at 10000 x g 4 ° C for 15 minutes. The supernatant was separated and the pellet was precipitated in cold hexane. The final product CMD was obtained by vacuum drying. A large amount of cinnamaldehyde is linked to the backbone of maltodextrin as a terminal group through an acetal linkage. Chemical organisms were identified by 1 H NMR (FIGS. 1C and 1D) and molecular weights were confirmed using gel permeation chromatography. The presence of aromatic protons and acetal protons represents a successful junction of cinnamaldehyde and maltodextrin. At acidic conditions (pH 6.5), the intensity of the acetal proton signal gradually decreased over time and a new peak corresponding to the aldehyde proton of cinnamaldehyde appeared at about 9.6 ppm (FIG. 1e). Carboxyl protons (9.1 ppm) of cinnamic acid also showed acid-induced oxidation of aldehydes. This observation suggests that methyl (5-methyl-2-styryl-1,3-dioxan-5-yl) methyl carbonate undergoes acid- triggered cleavage of acetal bonds to release cinnamaldehyde .

1.4 Methyl ((5-methyl-2- styryl -1,3- dioxan -5- yl ) methyl) carbonate Nanoparticles Preparation and Characterization

100 mg of CMD dissolved in 1 mL of DCM was added to 10 mL of 5 (w / v)% PVA solution. The mixture was sonicated for 1 minute with an ultrasonic mill and homogenized for 1.5 minutes with a homogenizer. The primary emulsion was emulsified in 15 mL of 0.5% (w / v)% PVA solution for 1.5 minutes using a homogenizer. The DCM was removed with a rotary evaporator and the nanoparticle suspension was meadowed at 12000 x g 4 ° C for 4 min and washed three times with deionized water. The nanoparticles were obtained by freeze-thawing. For the preparation of CPT-CMD nanoparticles, 10 mg of CPT was added to 1 mL of DCM containing 100 mg of CMD. The process of nanoparticle formulation was the same as the blank CMD nanoparticle formulation. The morphology and size of nanoparticles were observed using a scanning electron microscope (SEM, JSM-6400, JEOL, Japan) with an accelerating voltage of 20 KV and a particle size analyzer (90 Plus, Brookhaven Instrument Corp., Holtsville, NY) 2b). The CMD nanoparticles showed a single hydrodynamic diameter of ~ 230 nm and a smooth and smooth surface distribution. The stability of CMD nanoparticles was evaluated by observing the distribution of size and size of CMD during incubation in PBS with or without fetal bovine serum (FBS). After 96 hours of incubation in PBS, CMD nanoparticles hydrated and swelled into the hydrophobic interior due to the diffusion of water, resulting in a significant increase in size. However, the CMD nanoparticles had a nearly constant hydrodynamic size for 72 hours under aqueous conditions (Fig. 2c) and showed excellent stability. CPT-CMD nanoparticles were also prepared using the same single emulsion method. CPT was loaded at 8 wt% concentration with an encapsulation efficiency of ~ 80%. When CPT was encapsulated as CMD nanoparticles, the size of the nanoparticles increased without morphological changes (Fig. 2d and 2e). The hydrodynamic diameter of the CPT-CMD nanoparticles increased in 96 hours, but the size and size distribution were not finely influenced by FBS (FIG. 2f). This shows excellent stability in the physiological environment. To investigate the pH-dependent degradation of CMD nanoparticles, CMD nanoparticles were suspended in buffers at pH 7.4 and 5.5. The pH-dependent release of cinnamaldehyde from CMD nanoparticles was observed by UV-vis spectroscopy. The CMD nanoparticles showed pronounced release of cinnamaldehyde at pH 5.5 (Fig. 3), and 80% of the CMD nanoparticles were released within 24 hours. This implies that CMD nanoparticles release acid-induced decomposition and cinnamaldehyde.

2. Experimental Example

2.1 CMD  By nanoparticles ROS  produce

The effect of CMD nanoparticles inducing the production of ROS in cancer cells was first measured by the release of cinnamaldehyde. SW620 cells were inoculated into confocal dishes (SPL Life Sciences, Korea) at a density of 3 x 105 / dish. After 24 hours of incubation, the cells were treated with varying amounts of CMD (50, 100 or 100 μg / mL) or CPT-CMD nanoparticles for 48 hours .The cells were washed twice with fresh phosphate buffer (PBS) , 2 μM of DCFH-DA ((2 ', 7'-dichlorofluorescein-diacetate) dissolved in DMSO was added and rinsed twice with PBS after 20 minutes of incubation The images were analyzed using a confocal microscope CMT nanoparticles induce the production of ROS in a concentration-dependent manner, as shown in Figure 4. CPT also induced the production of large amounts of ROS at a concentration of 20 μg / mL, CPT-CMD nanoparticles (200 μg / ml) showed significantly higher ROS production than empty CMD nanoparticles and CPT, indicating that CMD and CPT Lt; RTI ID = 0.0 > ROS < / RTI >

2.2 Cell culture

Human prostate cancer cell line (DU145), human colon cancer cell line (SW620), mouse macrophage RAW264.7 and human embryonic kidney cell line (HEK 293) were cultured in 10% fetal bovine serum medium at 37 DEG C, 5% CO2.

2.3 Cytotoxicity analysis

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was used to assess the cytotoxicity of CMD nanoparticles. Human prostate cancer cell line (DU145), human colon cancer cell line (SW620), mouse macrophage RAW264.7 and human embryonic kidney cell line (HEK 293) were cultured in 24-well plates for 24 hours. Cells with 90% confluency were treated with various concentrations of CMD or CPT-CMD nanoparticles for 48 h. Cells were then added with 100 μL of MTT solution per well and incubated for 4 hours. The resulting formazan crystal was dissolved in 200 占 퐇 of DMSO. After incubation for 10 minutes, the absorbance at 570 nm was measured using a microplate reader (Biotek Instruments, Winooski, VT) to determine cell viability by comparing untreated cells. We first examined the effect of cinnamaldehyde content on the toxicity of CMD nanoparticles to cancer cells (SW620 cells). As shown in Fig. 5A, higher toxicity was observed as the synnal aldehyde was compounded more. Thus, CMD with the highest (90%) degree of junction was selected for further study. The present inventors next evaluated the cytotoxicity of CMD nanoparticles against various cell lines. Empty CMD nanoparticles were almost non-toxic to normal cells, mouse macrophages RAW264.7 (Figure 5b) and HEK 293 cells (Figure 5c). In contrast, CMD nanoparticles showed dose-dependent toxicity to cancer cells, DU145 cell line and SW620 cell line (Figures 5d and 5e). CMD nanoparticles at a concentration of 200 μg / mL have been reduced by more than 50% of cell viability. CPT-CMD nanoparticles showed significantly higher anticancer activity than CPT, cinnamaldehyde, and vacant CMD nanoparticles as a result of the anticancer effects of oxidative stress producing CMD nanoparticles with CPT payload.

2.4 Flow cell  analysis

To further investigate the apoptosis induced by CPT-CMD nanoparticles, Annexin V-FITC was used as a cell death probe and propidium iodide was used as a survival probe to analyze cancer cells after 24 hours of treatment using flow cytometry. Cells with 90% confluency were treated with varying amounts of CMD or CPT-CMD nanoparticles for 24 hours. The cells were washed twice with fresh medium and collected at a concentration of 1 × 10 5 in 1 × binding buffer. 10 μl each of Annexin V-FITC and propidium iodide was added to the cells. They were incubated for 15 minutes and treated with 400 μL of 1 X binding buffer solution. The stained cells were evaluated using a flow cytometer (FACS calibrator, Becton Dickinson, San Jose, Calif.). A total of 1 x 104 cells per sample were counted in the assay. As previously reported, CPT and cinnamaldehyde induced moderate apoptosis at a given concentration. Empty CMD nanoparticles induced concentration-dependent apoptosis (Figure 6), evidenced by an increasing population in the upper right quadrant. As expected, CPT-CMD nanoparticles caused significantly higher levels of apoptosis than empty CMD nanoparticles and CPT, indicating the combined anticancer activity of CMD and CPT payloads.

2.5 Western Blasting

SW620 cells were cultured in a 6-well plate with a cell density of 1.5 x 106 / well for 24 hours. After incubation for 48 h with varying amounts of CMD nanoparticles, the cells were washed twice with fresh PBS. Proteins were extracted from the cells using lysis buffer according to the manufacturer's protocol. The protein (25 ㎍) was separated by electrophoresis and transferred to a PVDF membrane. Capase-3, PARP and β-actin (Santa Cruz Biotechnology, Dallas, TX) were used as the primary antibodies and HRP binding goat anti-mouse (Millipore, Billerica, MA) was used as the secondary antibody. Target protein bands were expressed using Super Signal Ultra chemiluminescence reagent (Pierce, Rockford, Ill.) And imaged on film in the dark. The apoptosis induced by CPT-CMD nanoparticles was further confirmed by measuring levels of apoptosis-related caspase-3 and poly (ADP ribose) polymerase-1 (PARP-1). Figure 7 shows the effect of CPT-CMD nanoparticles on the activation of caspase-3 (caspase-3) and PARP-1 in SW620 cells. CPT induced activation of caspase-3 and PARP-1 as evidenced by reduced levels of pro-caspase-3 and PARP-1, but the cleaved caspase-3 and cleaved PARP -1. ≪ / RTI > CMD nanoparticles also induced caspase-3 and PARP-1 cleavage in a concentration dependent manner. However, the CPT-CMD nanoparticles demonstrate that caspase-3 and PARP-CMD nanoparticles exert excellent apoptosis inducing potential as a synergistic action of oxidative stress leading to CMD nanoparticles having CPT payloads, 1 was induced more. The results are also in good agreement with flow cytometry and MTT analysis results (Figures 5 and 6).

2.6 Mouse tumor xenograft model

SW620 cells were injected into the back of nude BALB / c mice (4 weeks old, Orient Bio, Seoul, Korea). (5 mg / kg), CPT (3 mg / kg), CMD nanoparticles (10 or 20 mg / kg) and CPT-CMD Daily for 3 days. The body weight and tumor volume of the mice were measured every 3 days. All animal tests were carried out according to the guidelines of the Institutional Animal Ethics Committee with the approval of the Chickenbuck Animal Protection Committee (CBU2014-00024). When the tumor reached 50 mm ³ , the mice were treated with cinnamaldehyde, CPT, empty CMD nanoparticles, or CPT-CMD nanoparticles. Untreated mice developed tumors faster than 30 days. Sinannal aldehyde (5 mg / kg) slightly inhibited tumor growth, but was not significant (Figures 8a and 8b). Empty CMD nanoparticles showed a concentration-dependent inhibitory effect on tumor growth. CMD nanoparticles showed a marginal inhibitory effect on tumor growth at a dose of 10 mg / kg. However, administration of 20 mg / kg of CMD nanoparticles significantly inhibited tumor growth but was less effective than the clinically relevant dose of CPT (3 mg / kg). However, CPT-CMD nanoparticles significantly inhibited tumor growth at doses of 10 mg / kg and showed significantly higher therapeutic effects than CPT and empty CMD nanoparticles (20 mg / kg). In vivo high anticancer activity of CPT-CMD nanoparticles against empty CMD nanoparticles is in good agreement with the in vitro cancer activity. The beneficial anti-cancer effect of all agents was achieved without a change in body weight over 30 days (Figure 8c). The anticancer activity of CPT-CMD nanoparticles was determined by hematoxylin and eosin (H & E) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (Figure 9). The antitumor activity of the CPT-CMD nanoparticles was confirmed by a large number of dead cells that had no nucleus and destroyed the cell membrane. CPT-CMD nanoparticles caused more damage to the tumor than other treatment groups. CPT-CMD nanoparticles also caused significant apoptotic cell death, as evidenced by the large number of TUNEL-positive cells. This observation indicates that CPT-CMD nanoparticles induce cancer cell death through apoptosis. To evaluate the biocompatibility of CPT-CMD nanoparticles, major organs of tumor-bearing mice were excised. Figure 10 shows an image of the H & E stain tissue of the organs. There was no histological evidence for toxicity of CMD nanoparticles and CPT-CMD nanoparticles compared to the control group. The results of this initial toxicity study show that CMD nanoparticles are either non-toxic or minimal toxic.

2.7 CMD  Ultrasound imaging of tumor using nanoparticles

CMD nanoparticles (2 mg / mL) were placed in an agarose phantom, and ultrasound images were plotted over time. Without hydrogen peroxide, there was no change in the ultrasound image signal, but the addition of hydrogen peroxide (1 mM) increased the ultrasound image signal from 1 minute and continued for about 30 minutes (FIG. 11 a). Ultrasonographic imaging of the tumor using real CMD nanoparticles. CMD nanoparticles (100 ug) were directly injected into tumor-bearing mice, and ultrasound images were obtained over time (Fig. 11 (b)).

Claims (10)

A hybrid anticancer prodrug drug represented by the following formula (1) or (2):
[Chemical Formula 1]

(2)

(3)

In the above formula (3), n is 5 to 100. The hybrid anticancer prodrug drug according to claim 1, wherein the compound of formula (1) is cinnamic aldehyde and the compound of formula (2) is cinnamic acid. The hybrid anticancer prodrug drug according to claim 1, wherein the formula (1) or ( ₂ ) is produced by hydrogen peroxide (H ₂ O ₂ ) and an acidic pH. (a) reacting cinnamic aldehyde with an acidic solution to prepare a compound having an acetal bond represented by the following formula 4;
[Chemical Formula 4]

(b) reacting the compound prepared in step (a) with carbonyldiimidazole to prepare a cinnamaldehyde releasing compound; And
(c) reacting the maltodextrin with the cinnamyl aldehyde releasing compound prepared in step (b). A pharmaceutical composition for the prevention or treatment of cancer comprising a hybrid anticancer prodrug represented by the following formula (3) as an active ingredient:
(3)

In the above formula (3), n is 5 to 100. The method of claim 5, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer, colorectal cancer, myeloid leukemia, thyroid cancer, MDS, bladder carcinoma, epidermal carcinoma, melanoma, breast cancer, prostate cancer, head and neck cancer, Which is composed of uterine cancer, ovarian cancer, brain cancer, stomach cancer, laryngeal cancer, esophageal cancer, bladder cancer, oral cancer, cancer of the mesenchymal origin, sarcoma, teratocarcinoma, neuroblastoma, renal carcinoma, liver cancer, non-Hodgkin's lymphoma, multiple myeloma, Or a pharmaceutically acceptable salt, solvate or prodrug thereof. A health food composition for preventing or ameliorating a cancer, comprising a hybrid anticancer prodrug represented by the following formula (3) as an active ingredient:
(3)

In the above formula (3), n is 5 to 100. A drug delivery system comprising a hybrid anticancer prodrug represented by the following formula (3) as an active ingredient:
(3)

In the above formula (3), n is 5 to 100. 9. The drug delivery system according to claim 8, wherein the drug is at least one drug selected from the group consisting of paclitaxel, docetaxel, doxorubicin, cispletin, carboplatin, 5-FU, etoposide and camptothecin, ≪ / RTI > An ultrasound imaging contrast agent comprising a hybrid anticancer prodrug represented by the following formula (3) as an active ingredient:
(3)

In the above formula (3), n is 5 to 100.

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