Carbon antioxidant enzyme mimic, Anti-inflammatory therapeutic agent comprising same, and method for preparing same

Abstract

Provided is a carbon antioxidant enzyme mimic, wherein a carbonyl group or an amine group, as a first functional group, and a hydroxyl group, as a second functional group, are bonded on a surface of the carbon antioxidant enzyme mimic. The carbon nanoparticle-based antioxidant enzyme mimic can effectively scavenge reactive oxygen species and reactive nitrogen species without metal ions. In addition, the carbon antioxidant enzyme mimic has an anti-inflammatory effect by reducing inflammatory cytokines and increasing the expression of natural antioxidant enzymes in cells.

Description

Carbon antioxidant enzyme mimetic, anti-inflammatory therapeutic agent containing the same, and method for producing the same

[0001] The present invention relates to a carbon antioxidant enzyme mimetic and a method for producing the same, and more particularly, to a novel carbon antioxidant enzyme mimetic exhibiting catalytic activity for ROS scavenging without metal ions and a method for producing the same.

[0002] Intracellular reactive oxygen species and reactive nitrogen species are mediators of inflammatory responses, and excessive expression of reactive oxygen species and reactive nitrogen species can induce excessive expression of proinflammatory cytokines, which can cause various inflammatory diseases.

[0003] Most proinflammatory cytokine antagonists used as treatments for existing inflammatory diseases have failed due to low therapeutic efficacy and serious side effects, and antioxidant therapies that treat inflammatory diseases by scavenging reactive oxygen species and reactive nitrogen species are being developed. However, existing antioxidants and antioxidant enzymes have the problem that they cannot effectively scavenge intracellular reactive oxygen species and reactive nitrogen species due to their low intracellular activity.

[0004] To address this issue, various nanomaterial-based antioxidant materials are being developed; however, most existing nanomaterials are based on metal oxides, which cause cytotoxicity when treated at high concentrations, limiting their application as antioxidants.

[0005] Therefore, the problem that the present invention seeks to solve is to provide a mimic that can effectively eliminate active oxygen and active nitrogen.

[0006] In order to solve the above problem, the present invention provides a carbon antioxidant enzyme mimic, characterized in that a carbonyl group or an amine group as a first functional group and a hydroxyl group as a second functional group are bonded to the surface of the carbon antioxidant enzyme mimic.

[0007] In one embodiment of the present invention, the first functional group and the second functional group are HOO, which is an oxygen active species. -participate simultaneously in the oxidation reaction.

[0008] In one embodiment of the present invention, the carbon antioxidant enzyme mimic is HOO - can be selectively oxidized or reduced.

[0009] In one embodiment of the present invention, after the oxidation reaction, the first functional group, the carbonyl group, becomes a hydroxyl group, and the second functional group, the hydroxyl group, becomes HOO, an oxygen active species. - Combined with, it reduces it.

[0010] In one embodiment of the present invention, the carbon antioxidant enzyme mimetic is HOO according to the following reaction formula - Remove .

[0011] 2HOO - → O2+H2O2

[0012] In one embodiment of the present invention, the carbon antioxidant enzyme mimic may have crystallinity, and in this case, the crystal grain size of the carbon antioxidant enzyme mimic having crystallinity is 0.2 nm or more.

[0013] The present invention also provides a method for producing a carbon antioxidant enzyme mimic, comprising a step of condensation polymerizing an organic compound by bonding a carbonyl group or an amine group as a first functional group and a hydroxyl group as a second functional group, wherein the surface of a particle obtained after the condensation polymerization step is characterized in that the carbonyl group or an amine group as a first functional group and the hydroxyl group as a second functional group are bonded thereto.

[0014] In one embodiment of the present invention, the condensation polymerization step is a thermal solvent reaction in which heat is applied to the organic compound in a solution, and the thermal solvent reaction is performed by irradiating microwaves, and the crystallinity of the carbon antioxidant enzyme mimic is determined according to the temperature and time of the thermal solvent reaction.

[0015] In one embodiment of the present invention, the organic compound is caffeic acid or norepinephrine.

[0016] According to the present invention, carbon nanoparticle-based antioxidant enzyme mimics can effectively scavenge reactive oxygen species and reactive nitrogen species without the need for metal ions. Furthermore, they exhibit anti-inflammatory effects by reducing inflammatory cytokines and increasing the expression of natural antioxidant enzymes within cells.

[0017] Figure 1 is a schematic diagram for the synthesis of crystalline or amorphous carbon oxidase mimetics (crys-CAM or amo-CAM) via spontaneous solvothermal reaction of caffeic acid.

[0018] Figure 2 is a TEM image of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0019] Figure 3 is an FFT pattern of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0020] Figure 4 is an AFM image of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0021] FIG. 5 is a UV-Vis spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention.

[0022] Figure 6 is a Raman spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention.

[0023] Figure 7 is an XPS spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, where k on the left represents amorphous and i on the right represents crystalline.

[0024] Figure 8 shows the results of analyzing the scavenging activity of crys-CAM, amo-CAM, and Trolox of Example 1 of the present invention for ABTS radicals and the mechanism of carbon antioxidants.

[0025] Figure 9 shows the concentration required to scavenge ABTS radicals by 50% (SC50) and the scavenging rate constants of crys-CAM and amo-CAM of Example 1 of the present invention for extracellular peroxide.

[0026] Figure 10 shows the results of the concentration-dependent scavenging activity analysis of crys-CAM and amo-CAM of Example 1 for extracellular superoxide.

[0027] Figure 11 is a Lineweaver-Burk plot for superoxide scavenging by the SOD-like crys-CAM of Example 1.

[0028] Figure 12 shows the results of analyzing the amount of molecular oxygen converted from superoxide by the SOD-like crys-CAM of Example 1.

[0029] Figure 13 shows the results of analyzing the scavenging activity of crys-CAM and amo-CAM of Example 1 for extracellular nitric oxide, and Figure 14 shows the results of analyzing the scavenging activity of crys-CAM and amo-CAM for hydroxyl radicals.

[0030] Figure 15 is a schematic diagram of the reaction of treating crys-CAM of Example 1 with NaBH4 and 1,3-propane sultone (PS), and Figure 16 is the result of analyzing the SOD-like activity of pure crys-CAM and crys-CAM of Example 1 treated with NaBH4 and 1,3-propane sultone (PS).

[0031] Figure 17 is a drawing explaining the mechanism of SOD-like activity of crys-CAM of Example 1.

[0032] Figure 18 shows the results comparing the scavenging ability of intracellular superoxide, nitric oxide, hydroxyl radical, and hydrogen peroxide levels in cells treated with amo-CAM (a) and crys-CAM (b).

[0033] Referring to Figure 19, it can be seen that Example 2 having an amine group also has antioxidant properties identical / similar to Example 1.

[0034] Figure 20 shows the results of measuring the secretion levels of TNF-a (acute inflammation), IL-6 (chronic inflammation), IL-12p40 (chronic inflammation), and IL-10 (anti-inflammation) treated with amo-CAM (a) and crys-CAM (b).

[0035] Figure 21 shows the results of Western blot analysis of the Nrf2 signaling pathway in LPS-stimulated BMDMs treated with amo-CAM or crys-CAM.

[0036] Figure 22 shows the results of intracellular activity of SOD, i) CAT, and j) GPx, which are innate antioxidant enzymes related to Nrf2, in LPS-stimulated BMDM.

[0037] Figures 23 to 26 show the results of Western blot analysis of the effects of mo-CAM and crys-CAM on mitochondrial ROS-generating proteins in LPS-stimulated BMDM under various concentration conditions, and the results of analysis of the activities of complex I, complex III, and NADPH oxidase.

[0038] Figure 27 is an optical image and a fluorescent image of RAW264.7 cells treated with PBS, amo-CAM, or cry-CAM, which illustrate the endocytosis pathway of the mimic according to the present invention and the enhancement of anti-inflammatory immunity by carbon oxidase.

[0039] Figure 28 is a graph quantifying the fluorescence intensity in the fluorescence image of Figure 27.

[0040] Figure 29 shows the results showing the effect of an endocytosis inhibitor on the cellular uptake of crys-CAM in Raw264.7 cells.

[0041] Figure 30 is a snapshot of the top and side views of cell membranes adsorbing crys-CAM (red beads) and Figure 20 is amo-CAM (blue beads) over time.

[0042] Figure 31 shows the height change of the membrane bilayer adsorbing crys-CAM (1 and 16 ea.) or amo-CAM (1 and 16 ea.).

[0043] Figure 33 shows the number of crys-CAM or amo-CAM entities within the membrane bilayer without clustering according to liver progression, and Figure 34 is a diagram illustrating a mechanism for enhancing anti-inflammatory and innate anti-inflammatory immunity induced by SOD-like crys-CAM.

[0044] Figure 35 is a graph showing the overall experimental schedule for rheumatoid arthritis (RA) mouse modeling and intra-articular injection of crys-CAM (or amo-CAM) into the RA mouse model.

[0045] Figure 36 is an optical image of the joints of mice treated with PBS, amo-CAM or crys-CAM on day 37, Figure 37 is the paw thickness over time, and Figure 38 is the clinical CIA score calculated for the joints of RA mice treated with PBS, amo-CAM or crys-CAM on day 38.

[0046] Referring to Figures 36 to 38, it can be seen that the crystalline mimic continues to have excellent therapeutic effects as the treatment progresses.

[0047] Figure 39 is an H&E stained image of the joints of RA mice treated with PBS, amo-CAM, or crys-CAM on day 4, and Figure 40 is a histological score of the corresponding H&E stained images.

[0048] Figures 41 and 42 show the secretion levels of TNF-a, IL-6, IL-12p40 and IL-10 in the synthetic fluid of RA mice treated with PBS, amo-CAM or crys-CAM on day 14, respectively, and the relative expression levels of mRNA of Nrf2, HO-1 and iNOS.

[0049] Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0050] Before describing the present invention in detail, it should be noted that the terms or words used in this specification should not be interpreted as being unconditionally limited to their usual or dictionary meanings, and the inventor of the present invention may appropriately define and use the concepts of various terms in order to describe his or her invention in the best possible manner.

[0051] Furthermore, it should be noted that these terms and words should be interpreted with meanings and concepts that are consistent with the technical idea of ​​the present invention.

[0052] That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention.

[0053] It should be noted that these terms are defined taking into account the various possibilities of the present invention.

[0054] Additionally, in this specification, a singular expression may include a plural expression unless the context clearly indicates a different meaning.

[0055] Also, it should be noted that even if similarly expressed in plural, it can contain singular meaning.

[0056] Throughout this specification, whenever a component is described as "including" another component, it may mean that the component may further include any other component, rather than excluding any other component, unless specifically stated otherwise.

[0057] Furthermore, if a component is described as being "internal to, connected to, or installed within" another component, it is understood that the component may be directly connected to, or installed in contact with, the other component.

[0058] In order to solve the above-described problem, the present invention provides an antioxidant material using carbon as a basic element as a carbon nanoparticle-based antioxidant enzyme mimic capable of effectively scavenging intracellular active oxygen and active nitrogen by controlling the crystallinity of carbon nanoparticles.

[0059] In the present invention, the carbon antioxidant enzyme mimic refers to a structure having antioxidant enzyme properties such as SOD, which is a material having carbon as a basic skeleton.

[0060] The carbon antioxidant enzyme mimic according to the present invention has an excellent antioxidant effect through the surface functional group of the structure obtained by condensation polymerization of an organic compound without using metal ions.

[0061] To this end, the surface of the mimic according to the present invention is bonded with a first functional group, a carbonyl group or an amine group, and a second functional group, a hydroxyl group, which is a polymer obtained by polymerizing caffeic acid (hydroxyl group, Example 1) or norepinephrine (Example 2).

[0062] The present invention will be described in more detail below through preferred embodiments and experimental examples. However, the scope of the present invention is not limited by the following embodiments and experimental examples.

[0063] Example 1

[0064] Synthesis of carbon antioxidase mimics (CAM)

[0065] 81 mg of caffeic acid, an organic small molecule having a functional group capable of condensation reaction, was dissolved in 15 mL of deionized water, and 200 μL of 1 M NaOH was added. The resulting mixture was stirred while maintaining the temperature at 150°C for 10 minutes under microwave irradiation to obtain an amorphous carbon-based mimic (amo-CAM).

[0066] For the synthesis of crystalline carbon-based antioxidant mimic (crys-CAM), the same reaction was carried out at 210°C for 20 min. After the reaction, the solution was dialyzed against 5 L of water using a cellulose membrane for 3 h, and this process was repeated 5 times to obtain crystalline carbon-based antioxidant mimic and amorphous carbon-based mimic, respectively. Thereafter, the crys-CAM or amo-CAM solution was filtered through a PVDF filter with 100 nm pores to obtain a filtrate containing the desired product.

[0067] Figure 1 is a schematic diagram for the synthesis of crystalline or amorphous carbon oxidase mimetics (crys-CAM or amo-CAM) via spontaneous solvothermal reaction of caffeic acid.

[0068] Referring to Figure 1, it can be seen that the crystallinity of the carbon antioxidant enzyme mimic, which is a structure obtained from caffeic acid, changes depending on the microwave irradiation time and temperature.

[0069] That is, it can be seen that the crystallinity of the carbon structure obtained from caffeic acid varies depending on the irradiation time and temperature of the microwave. In addition, the SOD mimetic, which is an antioxidant enzyme according to one embodiment of the present invention, has a carbonyl group, a hydroxyl group, and a carboxyl group on the surface. In particular, the carboxyl group and the hydroxyl group formed on the surface of the crystalline mimetic are oxygen active species ( - 00H) and oxidizes it to generate O2. Afterwards, the two hydroxyl groups form oxygen active species (- 00H) and recombines to produce H2O2.

[0070] The above reactions can be summarized as follows.

[0071] 2HOO - → O2+H2O2

[0072] Therefore, the mimic according to the present invention is the HOO - It can be selectively oxidized or reduced, and this is due to its structural characteristic of having both a carbonyl group and a hydroxyl group.

[0073]

[0074] Example 2

[0075] A carbon structure was synthesized in the same manner as in Example 1, except that norepinephrine of the following chemical formula was used. It can be seen that an amine group was used instead of a carboxyl group.

[0076]

[0077] Experimental Example 1

[0078] Measurement of ROS / RNS scavenging ability

[0079] To measure the superoxide scavenging activity of the examples or comparative examples, superoxide anions were generated by the xanthine (Xan) / xanthine oxidase (XOD) reaction. To this end, a mixture was prepared with 50 μL of Xan (1 mM), 125 μL of crys-CAM (0 to 200 μM), and 15 μL of WST-8, and 50 μL of XOD (0.1 U / mL) was added. The mixture was shaken at 25°C for 2 hours, and then the absorption at 460 nm was measured using a microplate reader.

[0080] To evaluate the activity of CAM against OH radicals, 6 mM H2O2 and 0.1 mg / mL TMB, 20 μL of FeSO4 (400 μM), and 200 μL of crys-CAM (0–200 μM) were mixed and stirred at 25°C for 1 h. The reaction was stopped by adding 15 μL of 2 M H2SO4 to the mixture, and the solution absorbance at 450 nm was measured using a microplate reader.

[0081] For the NO scavenging activity of the examples or comparative examples, NO was generated with sodium nitroprusside (SNP). First, 125 μL of SNP (20 mM) and 125 μL of crys-CAM (0 to 200 μM) were mixed together in a PBS solution (0.1 M, pH 7.4), and stirred at 25°C for 2 hours. Then, 7.5 μL of nitrate reductase and 7.5 μL of enzyme cofactor were added to the crys-CAM solution, and stirred at 25°C for 1 hour. 50 μL of Griess reagent R1 and 50 μL of Griess reagent R2 were added to the crys-CAM solution, and the solution was stirred at 25°C for 30 minutes, and the absorbance of the solution was measured at 540 nm using a microplate reader.

[0082]

[0083] Intracellular measurement of intracellular ROS and RNS

[0084] Intracellular ROS levels were assessed by intracellular analysis.

[0085] For this purpose, cells were cultured in serum-free medium and loaded with redox-sensitive dyes, which are as follows:

[0086] 10 μM 2',7'-dichlorofluorescein diacetate (DCFH-DA for H2O2; Calbiochem), OH580 probe (OH radical detection assay kit, ab219931; Abbacam), 2 μM dihydroethidium (DHE for O2; Calbiochem), or 10 μM 4,5-diaminofluorescein diacetate (DAF-2DA for NO; Calbiochem)

[0087] After rapid and thorough washing with pulse spin, cells were immediately analyzed using a FACSCalibur (BD Biosciences, San Jose, CA). Data analysis and plotting were performed using CellQuest software (BD Biosciences).

[0088]

[0089] Measurement of natural enzyme activity within cells

[0090] Total protein amounts and activities of four antioxidant enzymes, SOD, GPx, CAT, and glutathione reductase (GSR), were assessed in all seminal plasma samples according to previously published methods.

[0091] SOD, GPx, and GSR activities were measured using Randox Kits (Randox Laboratories Ltd, Crumlin, UK) adapted for the Olympus AU400 system. Specifically, GPx activity (Ransel kit) was determined by monitoring NADPH oxidation in the presence of cumene hydroperoxide. GSR activity (Glut Red kit) was measured by monitoring NADPH oxidation, and SOD activity (Ransod kit) was assessed using the XOD method. CAT activity was determined indirectly by observing H2O2 consumption based on a published method.

[0092]

[0093] Experimental results

[0094] In this experiment, it was confirmed that the carbon-based antioxidant enzyme mimetic according to one embodiment of the present invention effectively scavenged ABTS radicals both intracellularly and extracellularly, exhibiting a high antioxidant effect. Notably, these results are significantly higher than those of Trolox, a representative organic antioxidant molecule, and this will be described in more detail using the drawings below.

[0095] Figure 2 is a TEM image of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0096] Referring to FIG. 2, it can be seen that the carbon-based antioxidant enzyme mimic according to the present invention has a size of 5.2 nm.

[0097] Figure 3 is an FFT pattern of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0098] When Fig. 3 is examined, it can be confirmed that an additional pattern corresponding to 0.23 nm appears in the crystalline carbon-based antioxidant enzyme mimic.

[0099] Figure 4 is an AFM image of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, with the upper part being amorphous and the lower part being crystalline.

[0100] Referring to FIG. 4, it can be seen that the mock-up according to the present invention has a thickness of 1.9 nm.

[0101] FIG. 5 is a UV-Vis spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention.

[0102] Referring to Figure 5, it can be confirmed that the crystalline carbon-based antioxidant enzyme mimic has a higher absorbance at 450 nm, indicating higher crystallinity.

[0103] Figure 6 is a Raman spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention.

[0104] Referring to Figure 6, it can be confirmed that the crystalline carbon-based antioxidant enzyme mimic has a higher crystallinity as indicated by a lower ratio of the D / G peak.

[0105] Figure 7 is an XPS spectrum of a crystalline or amorphous carbon-based antioxidant enzyme mimic manufactured according to the present invention, where k on the left represents amorphous and i on the right represents crystalline.

[0106] Referring to FIG. 7, it can be seen that the antioxidant enzyme mimic according to the present invention has a carboxyl group, a carbonyl group, and a hydroxyl group regardless of crystallinity.

[0107] Figure 8 shows the results of analyzing the scavenging activity of crys-CAM, amo-CAM, and Trolox of Example 1 of the present invention for ABTS radicals and the mechanism of carbon antioxidants.

[0108] Figure 9 shows the concentration required to scavenge ABTS radicals by 50% (SC50) and the scavenging rate constants of crys-CAM and amo-CAM of Example 1 of the present invention for extracellular peroxide.

[0109] Referring to the results of FIGS. 8 and 9, it can be seen that both the crystalline and amorphous antioxidant enzyme mimetics according to the present invention exhibit a stronger antioxidant effect than Trolox, a well-known organic antioxidant, and the speed of the antioxidant action is also very fast.

[0110] Figure 10 shows the results of the concentration-dependent scavenging activity analysis of crys-CAM and amo-CAM of Example 1 for extracellular superoxide.

[0111] Figure 11 is a Lineweaver-Burk plot for superoxide scavenging by the SOD-like crys-CAM of Example 1.

[0112] Figure 12 shows the results of analyzing the amount of molecular oxygen converted from superoxide by the SOD-like crys-CAM of Example 1.

[0113] Figure 13 shows the results of analyzing the scavenging activity of crys-CAM and amo-CAM of Example 1 for extracellular nitric oxide, and Figure 14 shows the results of analyzing the scavenging activity of crys-CAM and amo-CAM for hydroxyl radicals.

[0114] Figure 15 is a schematic diagram of the reaction of treating crys-CAM of Example 1 with NaBH4 and 1,3-propane sultone (PS), and Figure 16 is the result of analyzing the SOD-like activity of pure crys-CAM and crys-CAM of Example 1 treated with NaBH4 and 1,3-propane sultone (PS).

[0115] Figure 17 is a drawing explaining the mechanism of SOD-like activity of crys-CAM of Example 1.

[0116] Referring to Figure 17, the SOD mimetic, which is an antioxidant enzyme according to one embodiment of the present invention, has a carbonyl group, a hydroxyl group, and a carboxyl group on the surface. In particular, the carboxyl group and the hydroxyl group formed on the surface of the crystalline mimetic are oxygen active species ( - 00H) and oxidizes it to generate O2. Afterwards, the two hydroxyl groups form oxygen active species ( - 00H) and recombines to produce H2O2.

[0117] The above reactions can be summarized as follows.

[0118] 2HOO - → O2+H2O2

[0119] Figure 18 shows the results comparing the scavenging ability of intracellular superoxide, nitric oxide, hydroxyl radical, and hydrogen peroxide levels in cells treated with amo-CAM (a) and crys-CAM (b).

[0120] Referring to Figure 18, it can be seen that the crystalline analogue of the present invention among the analogues having an antioxidant effect is effectively introduced into cells and has an antioxidant effect.

[0121] Figure 19 is a drawing showing the antioxidant properties of a mimic having an amine group synthesized according to Example 2.

[0122] Referring to Figure 19, it can be seen that Example 2 having an amine group also has antioxidant properties identical / similar to Example 1.

[0123]

[0124] Experimental Example 2

[0125] Molecular Dynamics Simulation of Cell Permeability of Carbon-Based Antioxidant Enzyme-Like Mimetics (CAMs)

[0126] All simulations and analyses were performed using the GROMACS-2018.6 simulation package, utilizing the MARTINI-2.2 coarse-grained (CG) force field, where each CG bead has a few (three or four) heavy atoms clustered together.

[0127] For the carbon-based antioxidant enzyme-like mimetic (CAM) according to the present invention, the hydrocarbon, hydroxyl group (-OH) and anionic carbonyl group (C=O) of the aromatic ring were modeled as bead types of hydrophobic “SC2”, polar “SP1” and negatively charged “SQa”, respectively.

[0128] The crystalline Crys-CAM is composed of 51 SC2, 10 SP1 and 4 SQa beads, whereas the amorphous amo-CAM is composed of 29 SC2, 20 SP1 and 2 SQa beads, similar to the CAM structure characterized experimentally.

[0129] To mimic the membrane of human macrophages, the phospholipid bilayer model was composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; 28 mol%), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPC; 10 mol%), 1-palmitoyl-2-oleoyl-sn-glycero-3-lac-glycero-L (POPG; 8 mol%), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI; 5.5 mol%), 1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS; 3.5 mol%), sphingomyelin (SM(d18:1 / 18:0), 10.5 mol%), and cholesterol (CHOL; 34.5 mol%), which was reported as the experimentally observed lipid composition. Considering that anionic lipids exist in the cytoplasmic (inner) leaflet of human cell membranes, anionic POPG, POPS, and POPI lipids were located in the inner leaflet, whereas COL, POPC, and SM lipids were more abundant in the outer leaflet.

[0130] A single CAM molecule or 16 CAM molecules were initially placed on the outer leaflet surface of an equilibrated bilayer, which was solvated by ~8100 CG water beads (representing 32,400 actual water molecules) in a periodic box measuring 14 × 14 × 9 nm3. Sufficient counter ions (Na+) were added to achieve electroneutrality, and then 100 Na+Cl- ions were further added to produce an anion concentration of 0.14 M NaCl, which is close to the experimental conditions. A cutoff of 1.1 nm was used for the Lennard-Jones potential, which has a smooth zero transition between 0 and 1.1 nm. For the Coulomb potential, a cutoff of 1.1 nm with a relative dielectric constant of 15 was used. A temperature of 310 K and a pressure of 1 bar were maintained in an NPxyPzT ensemble with semi-isotropic pressure coupling using a velocity-rescaled thermostat and a Parrinello-Rahman barostat. The LINCS algorithm was used to constrain the coupling length. Simulations were performed for 5 μ with a time step of 20 fs on a computing facility supported by the National Supercomputing Center (KSC-2023-CRE-019) with supercomputing resources including technical support. The last 2 μ trajectories were averaged for analysis.

[0131]

[0132] Preparation of a mouse model of collagen-induced arthritis

[0133] Type II collagen (Chondrex Inc., WA, USA) was dissolved at a concentration of 2 mg / mL and emulsified with complete Freund's adjuvant (CFA, Chondrex Inc.) containing heat-inactivated Mycobacterium tuberculosis. Mice were immunized by intradermal injection of 100 μL of the collagen / CFA emulsion. Twenty-one days after the first immunization, the mice received a second intradermal injection of 100 μL of the type II collagen emulsion and incomplete Freund's adjuvant (IFA, Chondrex Inc.). Subsequently, 1 week after the second immunization, 50 μL of a partial LPS solution (1 mg / mL, pH 7.4 in PBS) was injected intraperitoneally to enhance inflammatory signals as described previously. Simultaneously, CIA mice received intra-articular injections of crys-CAM (16 mg / kg), amo-CAM (16 mg / kg), or PBS.

[0134]

[0135] Arthritis Score

[0136] One week after the second immunization, trained investigators performed blinded assessments of arthritis scores. The severity of arthritis was assessed daily, and each paw was scored based on the degree of erythema and swelling, with each paw receiving a score ranging from 0 (baseline) to 4. For histological analysis, mice were euthanized, and hind paws were fixed in 10% buffered formalin, decalcified with 10% (w / v) EDTA, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for histological examination.

[0137]

[0138] Experimental Example 2

[0139] In this experimental example, the therapeutic effect of the carbon-based antioxidant enzyme analogue mimetic described above according to one embodiment of the present invention on rheumatoid arthritis was tested. As a result, unlike the amorphous carbon-based antioxidant enzyme analogue mimetic (amo-CAM), the crystalline carbon-based antioxidant enzyme analogue mimetic (crys-CAM) has excellent endocytosis and removes active oxygen and active nitrogen after entering cells, thereby having a therapeutic effect on rheumatoid arthritis. This will be described in more detail using the drawings below.

[0140] Figure 20 shows the results of measuring the secretion levels of TNF-a (acute inflammation), IL-6 (chronic inflammation), IL-12p40 (chronic inflammation), and IL-10 (anti-inflammation) treated with amo-CAM (a) and crys-CAM (b).

[0141] Referring to Figure 20, it can be seen that the level of inflammatory cytokines is greatly reduced when crys-CAM, a crystal, is treated.

[0142] Figure 21 shows the results of Western blot analysis of the Nrf2 signaling pathway in LPS-stimulated BMDMs treated with amo-CAM or crys-CAM.

[0143] Referring to Figure 21, it can be seen that only the crystalline carbon-based antioxidant enzyme mimic expresses the Nrf2 mechanism within the cell, which is also consistent with the results of Figure 9.

[0144] Figure 22 shows the results of intracellular activity of SOD, i) CAT, and j) GPx, which are innate antioxidant enzymes related to Nrf2, in LPS-stimulated BMDM.

[0145] Referring to Figure 22, it can be seen that only the crystalline carbon-based antioxidant enzyme mimic according to the present invention increases the expression amount of the antioxidant enzyme through Nrf2 mechanism expression.

[0146] Figures 23 to 26 show the results of Western blot analysis of the effects of mo-CAM and crys-CAM on mitochondrial ROS-generating proteins in LPS-stimulated BMDM under various concentration conditions, and the results of analysis of the activities of complex I, complex III, and NADPH oxidase.

[0147] Referring to FIGS. 23 to 26, it can be seen that the carbon-based antioxidant enzyme mimic according to the present invention does not affect the mechanism for generating active oxygen.

[0148] Figure 27 is an optical image and a fluorescent image of RAW264.7 cells treated with PBS, amo-CAM, or cry-CAM, which illustrate the endocytosis pathway of the mimic according to the present invention and the enhancement of anti-inflammatory immunity by carbon oxidase.

[0149] Figure 28 is a graph quantifying the fluorescence intensity in the fluorescence image of Figure 27.

[0150] Referring to Figures 27 and 28, it can be seen that only the crystalline carbon-based antioxidant enzyme mimic according to the present invention is incorporated into cells.

[0151] Figure 29 shows the results showing the effect of an endocytosis inhibitor on the cellular uptake of crys-CAM in Raw264.7 cells.

[0152] Referring to FIG. 29, it can be seen that the crystalline carbon-based antioxidant enzyme mimic according to the present invention is incorporated into cells through macropinocytosis.

[0153] Figure 30 is a snapshot of the top and side views of cell membranes adsorbing crys-CAM (red beads) and Figure 20 is amo-CAM (blue beads) over time.

[0154] Figure 31 shows the height change of the membrane bilayer adsorbing crys-CAM (1 and 16 ea.) or amo-CAM (1 and 16 ea.).

[0155] Referring to FIGS. 30 to 31, it can be seen that the crystalline carbon-based antioxidant enzyme mimic according to the present invention forms clusters on the cell surface and strongly interacts with the cell membrane to be incorporated into the cell.

[0156] Figure 33 shows the number of crys-CAM or amo-CAM entities within the membrane bilayer without clustering according to liver progression, and Figure 34 is a diagram illustrating a mechanism for enhancing anti-inflammatory and innate anti-inflammatory immunity induced by SOD-like crys-CAM.

[0157] Figure 35 is a graph showing the overall experimental schedule for rheumatoid arthritis (RA) mouse modeling and intra-articular injection of crys-CAM (or amo-CAM) into the RA mouse model.

[0158] Figure 36 is an optical image of the joints of mice treated with PBS, amo-CAM or crys-CAM on day 37, Figure 37 is the paw thickness over time, and Figure 38 is the clinical CIA score calculated for the joints of RA mice treated with PBS, amo-CAM or crys-CAM on day 38.

[0159] Referring to Figures 36 to 38, it can be seen that the crystalline mimic continues to have excellent therapeutic effects as the treatment progresses.

[0160] Figure 39 is an H&E stained image of the joints of RA mice treated with PBS, amo-CAM, or crys-CAM on day 4, and Figure 40 is a histological score of the corresponding H&E stained images.

[0161] Figures 41 and 42 show the secretion levels of TNF-a, IL-6, IL-12p40 and IL-10 in the synthetic fluid of RA mice treated with PBS, amo-CAM or crys-CAM on day 14, respectively, and the relative expression levels of mRNA of Nrf2, HO-1 and iNOS.

[0162]

[0163] The present invention relates to a carbon antioxidant enzyme mimetic, an anti-inflammatory therapeutic agent comprising the same, and a method for producing the same, and is recognized to have industrial applicability.

Claims

1. As a carbon antioxidant enzyme mimic, A carbon antioxidant enzyme mimic characterized in that a carbonyl group or an amine group as a first functional group and a hydroxyl group as a second functional group are bonded to the surface of the carbon antioxidant enzyme mimic.

2. In paragraph 1, The first and second functional groups are oxygen-active species, HOO - A carbon antioxidant enzyme mimic characterized by simultaneously participating in the oxidation reaction.

3. In paragraph 2, The above carbon antioxidant enzyme mimic is, HOO above - A carbon antioxidant enzyme mimetic characterized in that it can selectively oxidize or reduce.

4. In paragraph 2, After the above oxidation reaction, the first functional group, the carbonyl group, becomes a hydroxyl group, and the second functional group, the hydroxyl group, becomes an oxygen active species, HOO. - A carbon antioxidant enzyme mimetic characterized by combining with and reducing it.

5. In paragraph 4, The above carbon antioxidant enzyme mimic is HOO according to the following reaction formula - A carbon antioxidant enzyme mimetic characterized by removing . <h2 style=";text-align:left;direction:ltr">2HOO<h2 style=";text-align:left;direction:ltr"> - <h2 style=";text-align:left;direction:ltr"> → O2+H2O2 6. In paragraph 1, The carbon antioxidant enzyme mimic can have crystallinity, and the carbon antioxidant enzyme mimic having crystallinity is characterized in that the crystal grain size is 0.2 nm or more.

7. A method for producing a carbon antioxidant enzyme mimic, A method for producing a carbon antioxidant enzyme mimic, comprising a step of condensation polymerizing an organic compound by combining a carbonyl group or an amine group as a first functional group and a hydroxyl group as a second functional group, wherein the surface of a particle obtained after the condensation polymerization step is characterized in that the carbonyl group or an amine group as a first functional group and the hydroxyl group as a second functional group are combined.

8. In paragraph 7, A method for producing a carbon antioxidant enzyme mimic, characterized in that the above condensation polymerization step is a thermal solvent reaction in which heat is applied to the organic compound in a solution.

9. In paragraph 8, A method for producing a carbon-based antioxidant enzyme analogue, characterized in that the above thermal solvent reaction is carried out by irradiating microwaves, and the crystallinity of the carbon antioxidant enzyme analogue is determined according to the temperature and time of the thermal solvent reaction.

10. In paragraph 7, A method for producing a carbon-based antioxidant enzyme-like mimic, characterized in that the organic compound is caffeic acid or norepinephrine.

11. An anti-inflammatory therapeutic agent comprising a carbon antioxidant enzyme mimic according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient, An anti-inflammatory therapeutic agent characterized in that the carbon-based antioxidant enzyme analogue mimic has crystallinity.

12. In paragraph 11, An anti-inflammatory treatment agent characterized in that the intracellular effect of the carbon-based antioxidant enzyme analogue mimic occurs due to the above crystallinity.

13. In paragraph 12, An anti-inflammatory agent characterized in that the above anti-inflammatory agent is a treatment for rheumatoid arthritis.

14. A method for manufacturing an anti-inflammatory treatment agent, A method for producing an anti-inflammatory agent, comprising a step of condensation polymerizing an organic compound having a first functional group that is a carboxyl group or an amine group and a second functional group that is a hydroxyl group, wherein the particles obtained after the condensation polymerization step are characterized in that they have crystallinity.

15. In paragraph 14, A method for producing an anti-inflammatory agent, characterized in that the above condensation polymerization step is a thermal solvent reaction in which heat is applied to the organic compound in a solution.

16. In paragraph 15, A method for producing an anti-inflammatory therapeutic agent, characterized in that the above thermal solvent reaction is carried out by irradiating microwaves, and the crystallinity of the carbon-based antioxidant enzyme analogue is determined according to the temperature and time of the thermal solvent reaction.

17. In paragraph 14, A method for producing an anti-inflammatory treatment agent, characterized in that the organic compound is caffeic acid.

18. In paragraph 17, A method for manufacturing an anti-inflammatory treatment agent, characterized in that a carbonyl group and a hydroxyl group are bonded to the surface of the particles obtained after condensation polymerization of the above caffeic acid.

19. In any one of paragraphs 14 to 18, A method for manufacturing an anti-inflammatory treatment agent, characterized in that the above anti-inflammatory treatment agent is a treatment agent for rheumatoid arthritis.

Images