KR102671325B1 - RABA nanoparticles for preventing and treating reperfusion injury and pharmaceutical compositions comprising the same - Google Patents

Abstract

The present invention provides a composition for preventing and treating IR damage containing an atRA-based hybrid prodrug (RABA) and nanoparticles thereof as active ingredients for the prevention and treatment of ischemia/reperfusion (IR) damage having the structure shown in Formula 1 below. to provide:
[Formula 1]

Description

RABA and pharmacological compositions comprising the same for preventing and treating ischemia/reperfusion injury {RABA nanoparticles for preventing and treating reperfusion injury and pharmaceutical compositions comprising the same}

The present invention relates to RABA as a self-assembling prodrug for the prevention and treatment of ischemia/reperfusion (IR) damage, and provides an effective means for preventing and treating IR damage.

In various surgical procedures such as organ transplantation and transplantation of tissues containing blood vessels, it is necessary to ligate the blood vessels for a long time to prevent blood loss, and then remove the ligation at the end of the surgery to re-supply blood. In this process, organs experience unavoidable ischemia/reperfusion (IR) injury. Reactive oxygen species (ROS) are rapidly generated, leading to oxidative damage to organs, resulting in oxidative damage to organs. It is known that damage to organs occurs due to inflammatory reactions.

In particular, liver IR damage is characterized by the generation of ROS, activation of inflammatory pathways, and infiltration and activation of lymphocytes and/or monocytes (Y. Zhai, H. Petrowsky, JC Hong, RW Busuttil, JW Kupiec-Weglinski, Ischaemia -reperfusion injury, in liver transplantation-from bench to bedside, Nature Reviews Gastroenterology & Hepatology 10 (2013) 79-89). In the initial stage, damaged cells produce ROS, which then activates resident macrophages (Kupffer cells) to produce more ROS and cytokines. It is known that NADPH oxidase in liver cells generates a large amount of ROS, including H ₂ O ₂ , hydroxyl radicals, and superoxide anions, soon after reperfusion of ischemic tissue. ROS are believed to be the main cause of the development of liver IR damage because excess ROS causes oxidative stress, which leads to apoptotic cell death and worsening liver damage (D. Lee, S. Park, S. Bae, D. Jeong, M. Park, C. Kang, W. Yoo, MA Samad, Q. Ke, G. Khang, PM Kang, Hydrogen peroxide-activatable antioxidant prodrug as a targeted therapeutic agent for ischemia-reperfusion injury , Scientific Reports 5 (2015) 16592 et al.).

During IR injury, a series of pro-inflammatory cytokines are produced, such as tumor necrosis factor-alpha (TNF-a), interleukine-6 (IL-6), and IL-1b. , they activate white blood cells and also stimulate liver cells to secrete pro-inflammatory cytokines, causing serious liver damage. ROS also stimulate the secretion of high mobility group box-1 (HMGB1), a nuclear protein involved in transcription (DL Tang, YZ Shi, R. Kang, T. Li, WM Xiao, HC Wang, XZ Xiao, Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1, Journal of Leukocyte Biology 81 (2007) 741-747). HMGB1 is severely up-regulated as early as 1 hour after liver IR injury, serving as an early mediator of inflammation-dependent IR in the liver, heart, and kidney (H. Wang, ZF Xi, L. Deng, YX Pan, K. He , Q. Xia, Macrophage Polarization and Liver Ischemia-Reperfusion Injury, International Journal of Medical Sciences 18 (2021) 1104-1113 et al. Numerous evidences suggest that inhibition of HMGB1 alleviates liver injury and regulates downregulation of proinflammatory cytokines (H. Wang, ZF Xi, L. Deng, YX Pan, K. He, Q. Xia, Macrophage Polarization and Liver Ischemia-Reperfusion Injury, International Journal of Medical Sciences 18 (2021) 1104-1113 et al. Therefore, targeting HMGB1 with an antioxidant that depletes ROS is an effective strategy for inflammation-related diseases such as liver IR damage (GY Zhao, C. Fu, L. Wang, L. Zhu, YT Yan, Y. Xiang, F. Zheng, FL Gong, S. Chen, G. Chen, Down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 translocation and release and protects against liver ischemia-reperfusion injury, Scientific Reports 7 (2017) 46272 et al.

To date, there are no drugs that can effectively prevent or treat IR damage. Therefore, in order to effectively prevent IR damage, it is necessary to suppress the inflammatory response while removing high concentrations of reactive oxygen species.

atRA (all -trans retinoic acid) is a type of active metabolite of vitamin A that plays an important role in cell growth and differentiation and is used to treat acute promyelocytic leukemia. Additionally, it has been reported to exert antioxidant effects, and interest in its application is increasing.

However, atRA causes toxicity to normal cells when administered at high doses. In addition, the development of effective drug delivery technology is necessary due to low selectivity for the target site and low solubility. Furthermore, atRA is the most abundant ROS in IR injury and has no ability to scavenge H ₂ O ₂ , which acts as a second signaling molecule in the pathological process of IR injury in the liver. Therefore, there is a need to develop strategies to accurately deliver atRA to the target site and maximize its therapeutic effect.

Hybrid prodrugs are chemical compounds containing two or more structural fragments with different biological functions and complex activities, and are proposed to be effective drugs by creating two or more distinct drug molecular structures and providing complex modes of activity.

In the last 20 years, numerous nanocarriers have been developed as drug carriers. However, drug carrier-mediated drug delivery has disadvantages such as low drug loading capacity, early drug release, and complexity of scale-up. Additionally, most drug carriers act as excipients or do not play any therapeutic role, and may induce toxicity and immunogenicity during degradation and metabolism.

Self-assembling prodrug-based drug self-delivery is attracting attention as a new paradigm in the field of controlled drug delivery for highly efficient anticancer treatment. In drug self-delivery, the drug has a nano size and achieves intracellular delivery on its own without the need for additional excipients.

Self-assembly is the formation of nanostructures by self-assembly of small molecules through various nano-covalent interactions such as hydrophobic interactions, van der Waals interactions, and pi-pi stacking.

A prodrug is defined as a bioreversible inactive compound that is converted to an active drug upon administration and exhibits its therapeutic activity without toxic side effects.

Self-assembling prodrug-based drug self-delivery is a combination of prodrug and molecular self-assembly and has been realized by accurately controlling the molecular structure of the prodrug. A key design feature of self-assembled prodrugs is amphiphilic balance. They have both hydrophobic and hydrophilic regions and can spontaneously assemble in an aquatic environment. As a result, prodrug self-assembly can protect the drug from rapid exhaustion and inhibit the early release of the drug. Additionally, self-assembling prodrugs play a unique role in high drug loading efficiency of 100% and simple manufacturing process.

An example of a self-assembling prodrug for drug self-delivery is a prodrug amphiphile that binds hydrophilic irinotecan to hydrophobic chlorambucil to form a nanostructure through self-assembly, which is a prodrug amphiphile that creates a nanostructure without any carrier. Self-delivery was realized. (Huang, P. et. al., Combination of Small Molecule Prodrug and Nanodrug Delivery: Amphiphilic Drug-Drug Conjugate for Cancer Therapy. Journal of the American Chemical Society 2014, 136, 11748-11756.)

The inventors of the present application made efforts to develop an effective material for suppressing IR damage using atRA and arrived at the present invention.

The present invention is a means for maximally improving the therapeutic effect of atRA, atRA-based hybrid prodrug RABA, which is a hybrid prodrug of atRA and hydroxybenzyl alcohol (HBA), and its The purpose is to provide nanoparticles.

The present invention provides an all- trans retinoic acid (atRA)-based hybrid prodrug (RABA) for preventing and treating ischemia/reperfusion (IR) damage, having the structure shown in Formula 1 below:

[Formula 1]

The RABA can form nanoparticles through self-assembly.

The RABA nanoparticles are formed by nano-precipitating RABA in a solvent.

The nanoparticles may be composed of 100% RABA by weight without the presence of other excipients or auxiliaries.

The RABA produces atRA and hydroxybenzyl alcohol (HBA) in the presence of H ₂ O ₂ .

The RABA has the ability to remove reactive oxygen species (ROS).

The RABA preferentially accumulates in the liver.

The RABA preferentially accumulates in IR-damaged liver.

The RABA inhibits the production of pro-inflammatory substances.

The pro-inflammatory substances are TNF-α, IL-1β, IL-6 or HMGB1.

The RABA has suitable biostability within the range of use for therapeutic effect.

The present invention provides a composition for preventing and treating ischemia/reperfusion (IR) damage containing atRA-based hybrid prodrug (RABA) nanoparticles as an active ingredient.

According to the present invention, efficient and excellent effects can be achieved by providing an all- trans retinoic acid (atRA)-based hybrid prodrug (RABA) for the prevention and treatment of ischemia/reperfusion (IR) damage. In other words, the RABA can be manufactured into nanoparticles made of pure RABA through a self-assembly method without using additional substances such as excipients or auxiliaries, so the drug loading efficiency is very excellent. It is reported that the RABA nanoparticles preferentially accumulate in the liver and can achieve a preventive or therapeutic effect on IR damage through removal of reactive oxygen species and inhibition of production of pro-inflammatory substances.

Therefore, according to the present invention, an effective pharmacological composition can be provided by using RABA as an active ingredient for preventing and treating IR damage.

Figure 1 is a schematic diagram of the production, decomposition process, and ischemia/reperfusion damage inhibition of the RABA nanoparticles of the present invention.
Figure 2a shows the manufacturing process of RABA prepared in Examples and ¹ H NMR data of the prepared RABA.
Figure 2b shows the decomposition process of RABA induced by H ₂ O ₂ and ¹ H NMR data of HBA produced therefrom.
Figure 3 shows the mass spectrometry measurement results of RABA.
Figure 4 is particle size analyzer data of RABA nanoparticles.
Figure 5a is an SEM photo of RABA nanoparticles.
Figure 5b is an SEM image of atRA deposits.
Figure 5c is an SEM image of RABA nanoparticles after incubation with H ₂ O ₂ .
Figure 6 shows the measured HBA release rate from RABA nanoparticles in the presence and absence of H ₂ O ₂ .
Figure 7 shows the H ₂ O ₂ removal ability of RABA nanoparticles compared to atRA and HBA.
Figure 8 is a fluorescence image of RAW 264.7 cells treated with RABA nanoparticles labeled with Nile red.
Figure 9a shows the cytotoxicity evaluation results of RABA nanoparticles on RAW 264.7 cells.
Figure 9b shows the results of evaluating the cytoprotective effect of RABA nanoparticles in RAW 264.7 cells stimulated with H ₂ O ₂ .
Figure 10 is data showing the generation of intracellular ROS and the antioxidant effect of RABA nanoparticles in cells stimulated with H ₂ O ₂ .
Figures 11a to 11c show the results of measuring the amount of inflammatory cytokines (TNF-α, IL-1β, IL-6) in H ₂ O ₂ stimulated RAW 264.7 cells.
Figure 12a is a confocal laser scanning microscope image for observing the movement of HMGB1 in H ₂ O ₂ stimulated RAW 264.7 cells, and Figure 12b shows this quantitatively.
Figure 13 shows the results of (a) ALT analysis (b) AST analysis (c) histological analysis through photographs and H&E staining of the extracted liver in a mouse liver IR injury model.
Figure 14 shows (a) histological analysis results of liver stained with TUNEL, (b) quantified TUNEL intensity, and (c) histological analysis of liver tissue stained with caspase-3 antibody and PARP-1 antibody in the liver IR injury model. Results and (d) quantification of the levels of caspase-3 and PARP-1.
Figure 15 shows the inhibitory effect of RABA nanoparticles on the expression of (a) TNF-a, (b) IL-1b, and (c) IL-6 in liver IR damage.
Figure 16 quantifies HMGB1 levels in liver tissue in IR injury.
Figure 17 quantifies F4/80 levels in liver tissue in IR injury.
Figure 18 quantifies the level of DHE intensity in liver tissue in IR injury.
Figure 19 is an in vitro fluorescence image of major organs 24 hours after intravenous administration of IR780-labeled RABA nanoparticles.
Figure 20 shows changes in ALT/AST levels in serum and histological data of major organs due to administration of RABA nanoparticles.

The present invention provides an atRA-based hybrid prodrug (RABA) for the prevention and treatment of ischemia/reperfusion (IR) injury, which has the structure shown in Formula 1 below:

[Formula 1]

The RABA is a self-assembling H ₂ O ₂ -activatable prodrug and, as an example, can be synthesized from a one-step reaction of atRA and 4-(bromomethyl)phenylboronic acid.

Boronate/boronic acid (boron functional group) undergoes rapid H ₂ O ₂ -induced oxidation, so by combining it with atRA, atRA has the ability to remove H ₂ O ₂ .

Since the boron functional group in RABA is weakly polar and hydrophilic, RABA becomes amphipathic when combined with the boron functional group. Amphipathic RABA self-assembles in aqueous solution to form nanoparticles. Moreover, RABA has structural flexibility. Therefore, RABA is an antisolvent, typically self-assembling in water to form stable colloidal nanoparticles. Therefore, the present invention provides nanoparticles formed by self-assembly of RABA. The nanoparticles have a narrow size distribution and are formed into a stable spherical nanostructure.

As shown in Chemical Formula 1, RABA contains an ester link that is easy to decompose. This produces retinoic acid (atRA) and hydroxybenzyl alcohol (HBA) in the presence of H ₂ O ₂ . Therefore, RABA can not only effectively remove H ₂ O ₂ generated at high concentrations in IR damage, but also generates retinoic acid (atRA) and hydroxybenzyl alcohol (HBA) after decomposition to exert antioxidant and anti-inflammatory effects, thereby protecting the long-term health. Damage can be prevented.

RABA forms nanoparticles through self-assembly in an aqueous solution due to its amphiphilic nature, so the preparation method for the RABA nanoparticles of the present invention is very simple. Additionally, there is no need to add additional ingredients such as excipients to form nanoparticles from RABA. Therefore, the nanoparticles produced at this time may be composed of 100% by weight of RABA. This means that the drug content can be manufactured at 100% by weight without the need to add other ingredients such as excipients in the particles. Therefore, the nanoparticles of the present invention can maximize pharmacological effects. In other words, maximum effect can be achieved with minimal use. As an example, the RABA nanoparticles of the present invention exert therapeutic effects when administered in an amount of 5.0 mg/kg in animal experiments, which is a much smaller amount than the conventional atRA administered in an amount of 15 mg/kg. is the use of The reason that the RABA nanoparticles of the present invention exert sufficient therapeutic effect at a lower dose compared to conventional drugs is that the RABA nanoparticles are 'H ₂ O ₂ -activatable prodrug self-assembly' and have a high drug loading (~ 100%), possible from the simultaneous release of targeted pharmaceutically active drugs and their coordinated therapeutic action. Furthermore, the RABA nanoparticles of the present invention are administered intravenously before reperfusion, which is clinically applicable in realizing a preventive effect in patients with liver IR damage.

Figure 1 briefly shows the process of manufacturing and decomposing the nanoparticles of the present invention. First, nanoparticles are manufactured through self-assembly of RABA, and RABA decomposes in the presence of H ₂ O ₂ and water and acts on ischemia/reperfusion damaged liver to suppress damage.

RABA nanoparticles effectively protect the liver by inhibiting the expression of pro-inflammatory TNF-a, IL-1b, IL-6, and HMGB1 in IR-damaged liver and inhibiting the infiltration of macrophages. Therefore, liver cell damage and death are significantly inhibited by RABA nanoparticles. RABA has a higher H ₂ O ₂ removal capacity compared to atRA or HBA. This makes RABA nanoparticles have a much higher hepatoprotective effect against IR damage compared to atRA or HBA. RABA nanoparticles reach the IR damaged site and undergo H ₂ O ₂ -induced degradation, simultaneously releasing atRA and HBA to the same site, resulting in a cooperative therapeutic effect.

The hallmark of liver IR injury is an increase in macrophages due to monocyte invasion and differentiation of macrophages induced by monocytes. Since the liver has a large number of macrophages, preferential accumulation of RABA nanoparticles in the liver is helpful for their potential therapeutic action. In the present invention, we confirm and present that RABA nanoparticles preferentially accumulate in the liver compared to other organs.

Therefore, the present invention provides a composition for inhibiting IR damage containing the RABA nanoparticles as an active ingredient. The composition may contain additional drugs, adjuvants, or excipients for pharmacological effects in addition to the nanoparticles of the present invention.

The present invention will be described in more detail through examples below. However, the present invention should not be considered limited thereto.

Example

1. Preparation and observation of RABA

atRA (3.33 mmol, 1.00 g) and 4-(bromomethyl)phenylboronic acid (3.33 mmol, 0.71 g) were mixed with cesium carbonate (4.99 mmol, 1.60 g) at room temperature. ) was dissolved in 10 mL THF. After reaction at 75°C for 24 hours, the reaction mixture was filtered and purified by silica gel chromatography (ethyl acetate/hexane=1:1). RABA was obtained as a light yellow powder, and the chemical structure observed using ¹ H NMR is shown in Figure 2a. Additionally, as a result of measurement using a mass spectrometer (Figure 3), the obtained molecular weight of RABA was 435.20 m/z[M+H] ⁺ .

RABA, which has an excellent leaving group at the benzylic position, undergoes rapid self-immolation caused by H ₂ O ₂ as shown in FIG. 2b while boronic acid is converted to quinone metate by 1,6-elimination. quinone methide (QM)). Next, QM reacts with water to produce HBA, which has potential antioxidant and anti-apoptotic activities. The ¹ H NMR data of HBA generated from the decomposition of RABA induced by H ₂ O ₂ is shown in Figure 2b. The appearance of a new peak corresponding to the proton of HBA shows that RABA is triggered by H ₂ O ₂ to generate atRA and HBA.

2. Preparation of RABA nanoparticles and reactivity to H ₂ O ₂

RABA (1.00x10 ² mg) was dissolved in 1.00 mL of methanol and the solution was precipitated in 20.0 mL of water with constant stirring. The remaining methanol was removed and freeze-dried to obtain RABA nanoparticles in the form of yellow powder. The shape and size distribution of the obtained nanoparticles were analyzed using SEM (H-7650, Hitachi, Japan) and particle size analyzer (90 Plus, Brookhaven Instrument Corp. Holtsville, NY, USA). Figure 4 is a result obtained from a particle size analyzer, and Figure 5a is an SEM photo of RABA nanoparticles. It was confirmed that the nanoparticles were produced in a spherical shape with a mean hydrodynamic diameter of ~230 nm.

Meanwhile, for comparison, an SEM image of atRA precipitated using the same method as the RABA nanoparticle production process is shown in Figure 5b. It can be seen that this exists as a large sediment. Additionally, an SEM image of RABA nanoparticles after incubation with H ₂ O ₂ is shown in Figure 5c. It was no longer outdated. In other words, it was proven that RABA nanoparticles decompose in response to H ₂ O ₂ .

Meanwhile, in order to evaluate the reactivity of RABA nanoparticles to H ₂ O _{2 ,} the release rate of HBA was measured during the decomposition process induced by H ₂ O ₂ (FIG. 6). Most of the HBA was released from RABA nanoparticles within 12 hours in the presence of H ₂ O ₂ , and ~20% of HBA was released within 72 hours in the absence of H ₂ O ₂ .

Next, the H ₂ O ₂ removal ability of RABA nanoparticles was measured by changing the concentration (FIG. 7). As a result, it was confirmed that most of H ₂ O ₂ was removed in the presence of 100 mg/mL RABA nanoparticles. On the other hand, H ₂ O ₂ was hardly removed in atRA (65 mg/mL) and HBA (27 mg/mL).

3. Therapeutic effect of RABA nanoparticles in H ₂ O ₂ stimulated RAW 264.7 cells

To obtain fluorescent RABA nanoparticles, Nile red (2.00 mg) along with RABA nanoparticles (1.00x10 ² mg) were dissolved in 1 mL methanol. RAW 264.7 cells were treated with the Nile red labeled RABA nanoparticles to confirm cellular uptake, and H ₂ O ₂ -mediated degradation of RABA nanoparticles was observed. Fluorescence images were obtained with a confocal laser scanning microscope (LSM 880, Carl Zeiss, Oberkochen, Germany) (FIG. 8). After 1 hour of nanoparticle treatment, a weak fluorescence image was obtained, and after 24 hours, the fluorescence image became significantly stronger. Additionally, the fluorescence intensity further improved with the addition of H ₂ O ₂ , which is due to the rapid release of Nile red following the decomposition of RABA by H ₂ O ₂ .

The cytotoxicity of RABA nanoparticles on RAW 264.7 cells (3 10 ⁵ cells/well) was evaluated (Figure 9a). The results confirm that atRA, HBA, and RABA nanoparticles do not exhibit the same cytotoxicity.

Next, to evaluate the cytoprotective effect of RABA nanoparticles, RAW 264.7 cells (3 10 ⁵ cells/well) were each incubated with atRA, HBA, and RABA nanoparticles for 24 hours under 200 mM H ₂ O ₂ stimulation. . Cell activity was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Figure 9b). It was confirmed that the cell activity in the presence of RABA nanoparticles was significantly increased compared to the cell activity in the presence of atRA, HBA, and their combination.

The antioxidant effect of RABA nanoparticles was evaluated by staining cells with DCFH-DA (dichlorofluorescein-diaceatate, Sigma Aldrich, St. Louis, MO, USA). RAW 264.7 cells (2 10 ⁵ cells/well) cultured in a 24 well plate were stimulated with 200 mM H ₂ O ₂ and atRA, HBA and RABA nanoparticles were immediately administered thereto. After 6 hours of incubation, cells were stained with 5 mM DCFH-DA. DCF fluorescence intensity was obtained using a fluorescence image and Image J program (Figure 10). In the figure, the green probe is caused by ROS. It can be seen that cells stimulated with H ₂ O ₂ produce a large amount of ROS, and this disappears by administration of RABA. On the other hand, when atRA, HBA, and their combination were administered, the green probe remained. From this, the antioxidant effect of RABA nanoparticles was confirmed.

To evaluate the anti-inflammatory effect, culture medium of RAW 264.7 cells treated with atRA, HBA and RABA nanoparticles 10 minutes before H ₂ O ₂ stimulation was used. The amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) was measured using an ELISA kit (FIGS. 11A to 11C). H ₂ O ₂ stimulation significantly increased the amount of inflammatory cytokines TNF-α, IL-1β, and IL-6, and while atRA, HBA, and their combination only slightly alleviated this, there was a significant increase in RABA nanoparticle-treated cells. A decrease was confirmed.

To observe the movement of HMGB1 in H ₂ O ₂ stimulated RAW 264.7 cells, after treatment with atRA, HBA and RABA nanoparticles, RAW 264.7 cells were stimulated with 200 mM H ₂ O ₂ . Cells were washed with PBS and fixed with formalin. Fixed cells were incubated with rabbit anti-HMGB1 polyclonal antibody (Cusabio, Houston, TX, USA) and goat anti-rabbit IgG cross-absorbed secondary antibody labeled with Alexa Fluor 488. Images were obtained with a confocal laser scanning microscope (FIG. 12a). In cells without H ₂ O ₂ stimulation, HMGB1 was observed in the nucleus. Stimulation with H ₂ O ₂ rapidly increased the level of HMGB1 in the cytoplasm after 6 hours, and treatment with HBA and atRA effectively reduced it, and it was confirmed that RABA nanoparticles suppressed the level of HMGB1 in the cytoplasm in proportion to the concentration. It has been done. Figure 12b shows this quantitatively. From this, the minimum amount of HMGB1 secreted in the culture medium was measured 12 hours after treatment with 50 mg/mL RABA nanoparticles. This shows that RABA nanoparticles effectively and significantly inhibit H ₂ O ₂ -mediated movement and release of HNGB1.

4. Therapeutic effects of RABA nanoparticles in the body

ICR mice (Orient Bio (Seongnam, Korea)) were used as control group, ischemia/reperfusion (IR), IR + atRA (3 mg/kg), IR + HBA (2 mg/kg), IR + atRA (3 mg) + HBA. (2 mg/kg), IR + RABA (5 mg/kg). After anesthesia, a midline laparotomy was made, and the exposed portal vein was ligated with a vascular clip for 30 minutes to cause liver ischemia. atRA (3 mg/kg), HBA (2 mg/kg), and RABA nanoparticles (5 mg/kg) were administered intravenously into the tail vein. After 30 minutes, the vascular clip was removed and perfusion was performed. Liver tissue and serum were collected 24 hours after reperfusion to evaluate the therapeutic effect.

Liver function was confirmed by measuring serum levels of liver enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) (Asan Pharma, Seoul, Korea) (Figures 13(a) and (b)). ALT and AST levels were significantly reduced in the group administered RABA nanoparticles. On the other hand, in the atRA and HBA administration groups, the effect was so small that it could be ignored.

In addition, to confirm the protective effect of RABA nanoparticles against liver IR damage, the liver was cut and stained with hemotoxylin and eosin (H&E) for histological observation (Figure 13 (c) ). Necrosis (marked with yellow dots) was found over a wide area in the liver where IR damage occurred, and it was confirmed that the necrosis area was significantly reduced in the RABA nanoparticle-administered group compared to the atRA and HBA-administered groups.

To confirm the hepatoprotective effect from IR-induced apoptosis, the left lobe of the liver was collected and fixed in 10% formalin. Fixed tissues were embedded in OCT blocks and cut into 6 mm thick sections. Tissue sections were stained with TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling, Promega, WI, USA) (Figures 14(a) and (b)). In the IR injury group, TUNEL-positive cells were observed compared to sham (normal group, negative control), while significantly fewer TUNEL-positive cells were observed in the group treated with RABA nanoparticles compared to the atRA and HBA treated groups.

To confirm the anti-apoptotic effect of RABA nanoparticles, tissues were blocked with 5% bobbin serum albumin (BSA) and immunostaining was performed to measure the levels of Caspase-3 and PARP-1, important mediators of cell death. (Figures 14(c) and (d)). In the IR injury group, the levels of Caspase-3 and PARP-1 in the liver were significantly increased, and RABA nanoparticles significantly reduced the levels of Caspase-3 and PARP-1, which are associated with apoptosis. On the other hand, a negligible effect was observed in the atRA and HBA treatment groups.

To evaluate the anti-inflammatory effect, the amount of inflammatory cytokines (TNF-α, IL-1β, IL-6) was measured using an ELISA kit (FIGS. 15(a) to (c)). IR damage significantly increased the amount of inflammatory cytokines TNF-α, IL-1β, and IL-6, and while atRA, HBA, and their combination somewhat alleviated this, a significant decrease was observed in cells treated with RABA nanoparticles. .

The movement of HMGB1 was observed in IR damaged liver tissue through immunostaining and quantified (FIG. 16). Liver tissue showed slight HMGB1 staining in sham-operated mice, whereas a significantly increased number of HMGB1-positive cells were observed in the IR group. Additionally, while HBA and atRA showed a slight effect, RABA nanoparticles were confirmed to significantly inhibit IR-induced up-regulation of HMBG1.

Next, to evaluate the effect of RABA nanoparticles on macrophage infiltration, histological observation was made using the macrophage-specific marker F4/80 (Santa Cruz Biotechnology, Dallas, TX, USA) (FIG. 17). A high level of macrophage infiltration was observed in the IR-damaged group, which was significantly reduced by pretreatment with RABA nanoparticles. On the other hand, the effect was negligible in atRA and HBA. From this, it can be seen that RABA nanoparticles significantly reduce macrophage infiltration induced by liver IR damage.

In addition, dihydroethidium (DHE) staining was performed to evaluate the antioxidant effect of RABA nanoparticles by inhibiting ROS generation (FIG. 18). A significant increase in hepatic DHE staining was observed in the IR injury group compared to the Sham group. This means that IR damage causes excessive production of ROS. On the other hand, RABA nanoparticles significantly inhibited ROS generation, which was a much better result than atRA and HBA.

IR780-labeled RABA nanoparticles were administered intravenously to healthy mice and liver IR-damaged mice, and the biodistribution of RABA nanoparticles in the liver was observed from fluorescence images of the cut major organs 24 hours later (FIG. 19). In both groups, a higher distribution of RABA nanoparticles was confirmed in the liver than in other organs. This confirms that RABA nanoparticles naturally target the liver and that the liver has the largest distribution of macrophages in contact with the blood, which plays a primary role in removing foreign substances. Furthermore, the distribution of RABA nanoparticles was much higher in the IR-damaged liver than in the liver of healthy mice, showing that more macrophages were activated and entered the liver during the IR damage process.

Meanwhile, the accumulation of RABA nanoparticles was high in the kidneys of the IR-damaged group, which means that IR-damaged mice in the liver suffer kidney damage due to adjacent tubular necrosis and inflammation, and that various pro-inflammatory mediators secreted during the IR damage process are transported to distant sites. It causes inflammation in organs and causes dysfunction of various organs (H. Naito, T. Nojima, N. Fujisaki, K. Tsukahara, H. Yamamoto, T. Yamada, T. Aokage, T. Yumoto, T. Osako, A. Nakao, Therapeutic strategies for ischemia reperfusion injury in emergency medicine, Acute Medicine & Surgery 7 (2020) e5010 et al.) is related to. Therefore, the significantly increased accumulation of RABA nanoparticles in the kidneys of the IR group could be explained by hepatic IR injury-mediated renal injury accompanied by increased macrophage infiltration.

Finally, to evaluate the biocompatibility of RABA nanoparticles, 5 mg/kg of RABA nanoparticles were administered intravenously to mice for 7 days, and then blood and liver were collected. The acute in vivo toxicity of RABA nanoparticles was evaluated by comparing changes in serum ALT/AST levels with those of healthy mice (Figures 20(a) and (b)). RABA nanoparticles did not cause significant changes in ALT/AST levels. Additionally, RABA nanoparticles did not cause pathological disease in the histological data of major organs (Figure 20(c)). From these results, it was confirmed that RABA nanoparticles have appropriate biostability within the range of use for therapeutic effects.

Claims (11)

A composition for preventing or treating ischemia/reperfusion (IR) damage containing as an active ingredient an all- trans retinoic acid (atRA)-based hybrid prodrug (RABA) having the structure shown in Formula 1 below:
[Formula 1]

In paragraph 1,
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the RABA forms nanoparticles by self-assembly by nano-precipitating in a solvent.
In paragraph 1,
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the RABA forms nanoparticles composed of 100% by weight of RABA without the presence of other excipients or auxiliaries.
In paragraph 1,
The RABA is a composition for preventing or treating ischemia/reperfusion (IR) damage, characterized in that it generates atRA and hydroxybenzyl alcohol (HBA) in the presence of H ₂ O ₂ .
In paragraph 1,
The RABA is a composition for preventing or treating ischemia/reperfusion (IR) damage, characterized in that it has the ability to remove reactive oxygen species (ROS).
In paragraph 1,
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the RABA preferentially accumulates in the liver.
In paragraph 1,
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the RABA preferentially accumulates in IR damaged liver.
In paragraph 1,
The RABA is a composition for preventing or treating ischemia/reperfusion (IR) damage, characterized in that it inhibits the production of pro-inflammatory substances.
In paragraph 8:
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the pro-inflammatory substance is TNF-α, IL-1β, IL-6, or HMGB1.
In paragraph 1,
A composition for preventing or treating ischemia/reperfusion (IR) damage, wherein the RABA has appropriate biostability within the range of use for therapeutic effects.
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