Novel cerium oxide nanocomposite and composition for preventing or treating kidney diseases comprising same

Abstract

The present invention relates to a novel cerium oxide nanocomposite and a composition for preventing or treating kidney diseases, comprising same as an active ingredient. The present invention can be used as an excellent therapeutic composition with maximized biostability and in vivo inflammation control efficiency, achieved by modifying the surface of cerium oxide nanoparticles with an optimal content of a pyrrolidone derivative polymer. The cerium oxide nanocomposite of the present invention can be used as an excellent therapeutic composition that significantly improves the survival rate of patients with acute kidney injury, which is an intractable severe disease, by reducing reactive oxygen species and neutrophils in kidney tissue and repairing tubular damage.

Description

Novel cerium oxide nanocomposite and composition for the prevention or treatment of kidney disease containing the same

[0001] The present invention relates to a cerium oxide nanocomposite with improved biocompatibility and a composition for the prevention or treatment of kidney disease, specifically acute kidney injury, comprising the same as an active ingredient.

[0002]

[0003] Due to their minute diameters, nanoparticles exhibit unique optical, electromagnetic, and chemical properties within the body that differ from those of bulk materials; based on these characteristics, they are utilized in various medical fields, including diagnostic imaging, drug delivery systems, and therapeutic agents. In particular, nanoparticles serve not only as carriers to deliver pharmacological components to lesion sites but can also be utilized as pharmacological components themselves, based on their heat-inducing or antioxidant activities. Consequently, active research is being conducted to explore particle compositions, shapes, sizes, and characteristics suitable for diverse medical applications.

[0004] Cerium oxide nanoparticles exhibit thermal stability at high temperatures, and due to their lattice structure, Ce depends on the surrounding oxygen concentration. 4+ / Ce 3+Due to its redox action, it can be utilized in the medical field as an effective therapeutic composition for inflammatory diseases by efficiently removing reactive oxygen species (ROS) that cause oxidative damage to tissues and cells. However, due to the characteristics of nanoparticles with nanoscale diameters, it is a very difficult task to suppress aggregation and maintain uniform particle size, excellent dispersibility, and low surface charge (zeta-potential) during mass production. Accordingly, the development of cerium oxide nanoparticles with new structures capable of maintaining excellent antioxidant effects and optimized particle characteristics even in industrial-scale production to secure therapeutically effective amounts is continuously being pursued.

[0005] Meanwhile, the kidneys are vital organs that maintain the body's homeostasis, regulating body fluid volume, blood ion concentration, and pH, eliminating waste products, and controlling blood pressure and endocrine function. Acute kidney injury (AKI) refers to a clinical syndrome characterized by a rapid decline in renal function due to various complex causes, such as reduced renal blood flow, glomerulonephritis, and the long-term administration of nephrotoxic antibiotics or anticancer drugs. AKI is accompanied by a decrease in glomerular filtration rate (GFR), reduced urine output, azotemia caused by the accumulation of nitrogenous waste products, and electrolyte imbalance. AKI is a common symptom among hospitalized patients that significantly increases morbidity and mortality; even if renal function recovers, depending on the cause and severity of the injury, delayed treatment or recurrence can often lead to chronic kidney disease (CKD) or end-stage renal disease (ESRD). Although inflammatory cytokines and reactive oxygen species have been proposed as pathogenic factors of acute kidney injury, drugs capable of rapidly and effectively eliminating their symptoms have not yet been developed. Currently, renal replacement therapies such as dialysis are applied only when complications arise following conservative treatment, highlighting the need for the development of therapeutic agents capable of effectively controlling inflammation in acute kidney injury tissues.

[0006]

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0008]

[0009] The inventors have made diligent research efforts to develop an excellent nanoparticle-based therapeutic composition capable of efficiently restoring kidney tissue damage and functional decline caused by oxidative stress and inflammatory responses. As a result, the present invention was completed by discovering that when a pyrrolidone polymer of Formula 1 described below is bonded to the surface of cerium oxide nanoparticles in a certain amount to form an outer layer, the dispersibility of the nanoparticles is significantly improved and the surface charge is reduced, thereby maximizing both biocompatibility and the efficiency of removing reactive oxygen species in the kidney.

[0010] Accordingly, the objective of the present invention is to provide a novel cerium oxide nanocomposite and a composition for the prevention or treatment of kidney disease comprising the same as an active ingredient.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a composition for the prevention or treatment of kidney disease comprising a cerium oxide nanocomposite as an active ingredient, comprising the following:

[0015] (a) a cerium oxide nanoparticle core layer; and

[0016] (b) Outer layer comprising a polymer represented by the following chemical formula 1:

[0017] Chemical formula 1

[0018]

[0019] In the above chemical formula, R1 and R2 are each independently hydrogen or oxygen, and represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000.

[0020] According to another aspect of the present invention, the present invention provides a method for preventing or treating kidney disease comprising the step of administering the cerium oxide nanocomposite of the present invention described above to a subject.

[0021] The inventors have made diligent research efforts to develop a nanoparticle-based therapeutic composition capable of efficiently treating various kidney diseases caused by kidney tissue damage due to oxidative stress. As a result, it was discovered that when a pyrrolidone polymer of Formula 1 is bonded to the surface of cerium oxide nanoparticles in a certain amount to form an outer layer, the dispersibility of the nanoparticles is significantly improved and the surface charge converges to 0 mV, thereby maximizing the biocompatibility of the particles as well as the reactive oxygen species removal rate in the kidney and the survival rate of kidney cells, which can be used as an excellent therapeutic composition.

[0022] In this specification, the term “core layer” refers to the innermost layer in a multilayer composite having only one surface in contact with another layer.

[0023] In this specification, the term “multilayer composite” refers to a composite composed of multiple layers made of different components, and includes, without limitation, a laminated multilayer structure, a core-shell multilayer structure, and a combination thereof. Specifically, the multilayer composite of the present invention is a core-shell multilayer structure in which a nanoparticle is present in the center and a polymer of Formula 1 surrounds it on the outside.

[0024] In this specification, the term “outer layer” refers to a layer that surrounds the core layer of a core-shell structure and is further from the center than the core layer. The outer layer does not necessarily have to be a layer that is in direct contact with the core layer, and there may be additional layers that are closer to the core layer than the outer layer (e.g., an inner layer, etc.), and the outer layer also does not necessarily have to be the outermost layer, and there may be additional outermost layers located further from the core than the outer layer.

[0025] According to the present invention, the boundary between the core layer and the outer layer may or may not be clearly distinguished. If the boundary between the core layer and the outer layer is not clear, each component may be mixed in the area of ​​the boundary between the two layers or in the entire section.

[0026] In this specification, the term “polymer” refers to a synthetic or natural polymer compound in which monomers of the same or different types are sequentially combined. Accordingly, polymers include homopolymers (polymers in which one type of monomer is polymerized) and copolymers prepared by the polymerization of at least two different monomers, and copolymers include copolymers (polymers prepared from two different monomers) and polymers prepared from more than two different monomers. Specifically, the polymer of Formula 1 used in the present invention is a homopolymer.

[0027] In this specification, the term “alkyl” means a straight-chain or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl means an alkyl group having alkyl units having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the number of carbon atoms of the substituent is not included.

[0028] In this specification, the term “biocompatibility” refers to a property that does not cause short- or long-term side effects when administered into the body and comes into contact with organ cells, tissues, or body fluids. Specifically, it includes tissue compatibility and blood compatibility, which do not cause tissue necrosis or blood coagulation upon contact with biological tissues or blood, as well as biodegradability, which disappears after a certain period following administration, and excretability, which is the property of being excreted from the body without accumulating after administration. Accordingly, the term “biocompatible dispersion stabilizer” refers to a component that possesses the aforementioned biocompatibility while improving the dispersibility of particles.

[0029] In this specification, the term “biodegradable” means the property of naturally degrading when exposed to a physiological solution of pH 6-8, and specifically means the property of being able to degrade over time by body fluids, degrading enzymes, or microorganisms in the body.

[0030] According to a specific embodiment of the present invention, the cerium oxide nanoparticles used in the present invention may be selected from the group consisting of cerium oxide (III) (Ce2O3) nanoparticles, cerium oxide (IV) (CeO2) nanoparticles, and mixtures thereof.

[0031]

[0032] According to a specific embodiment of the present invention, in Chemical Formula 1, R1 is hydrogen, R2 is oxygen, and l is 1. According to the octet rule, when R1 is hydrogen When it is a single bond and R2 is oxygen It is obvious that is a double bond. The compound with chemical formula 1, where R1 is hydrogen, R2 is oxygen, and l is 1, is polyvinylpyrrolidone (PVP).

[0033]

[0034] According to a specific embodiment of the present invention, the polymer represented by Formula 1 and the cerium oxide nanoparticles have a content ratio of 7:1 to 11:1.

[0035] According to the present invention, the inventors observed that when the polymer of Formula 1 and cerium oxide nanoparticles in the nanocomposite of the present invention have a content ratio of 7:1 to 11:1 (7 - 11 mg PVP / mg Ce), they exhibited optimal biocompatibility, dispersibility, and maximized inflammation control effects, thereby displaying the best particle characteristics. This confirmed that, compared to conventional cerium oxide nanocomposites in which dispersion stabilizers such as PGA (polyglutamic acid) must be additionally modified in the outermost layer, biocompatibility and therapeutic effects are significantly improved, while the efficiency and economic feasibility of mass production through structural simplification can be dramatically improved.

[0036] More specifically, the polymer represented by the above chemical formula 1 and the cerium oxide nanoparticles have a content ratio of 7.5:1 to 10.9:1, more specifically, a content ratio of 8:1 to 9.5:1, even more specifically, a content ratio of 8.5:1 to 9:1, and most specifically, a content ratio of about 8.7:1.

[0037] According to a specific embodiment of the present invention, the nanocomposite of the present invention additionally comprises a polyfunctional ligand represented by the following chemical formula 2:

[0038] Chemical formula 2

[0039]

[0040] In the above chemical formula, n is an integer from 3 to 7.

[0041] In this specification, the term “multi-functional ligand” refers to a molecule having two or more active functional groups that acts as a linker between two or more molecules by binding to said molecules. The multi-functional ligand of Formula 2 used in the present invention has a carboxyl group capable of binding to cerium oxide nanoparticles and an amine group capable of binding to the compound of Formula 1 of the outer layer (e.g., PVP), thereby enabling the nanocomposite of the present invention to be formed more efficiently and stably. Accordingly, the multi-functional ligand of Formula 2 may exist within the outer layer or between the core layer and the outer layer; in this case, the carboxyl group of the multi-functional ligand may be aligned directionally so that it faces the core, and the amine group faces the compound of Formula 1 (Fig. 1a).

[0042] Specifically, in the above chemical formula 2, n is 5. The compound of chemical formula 2 where n is 5 is 6-aminohexanoic acid (6-AHA).

[0043]

[0044] According to a specific embodiment of the present invention, the nanocomposite of the present invention has an average particle size of 5 nm to 80 nm. More specifically, it has an average particle size of 10 nm to 50 nm, even more specifically, an average particle size of 15 nm to 30 nm, and most specifically, an average particle size of about 20 nm.

[0045] According to a specific embodiment of the present invention, the nanocomposite of the present invention has a surface charge (Zeta potential, mV) of -1.0 to 1.0 mV, and more specifically, has a surface charge (Zeta potential, mV) of -0.5 to 0.5 mV. According to the present invention, the nanocomposite of the present invention exhibits a significantly low surface charge converging to 0 mV, thereby minimizing interaction with the in vivo environment of water-soluble phosphorus when injected into the human body, and thus can exhibit excellent biocompositeness.

[0046]

[0047] In this specification, the term “renal disease” (or “kidney disease”) refers to a pathological condition in which damage or dysfunction occurs in kidney tissue, resulting in the kidney's inability to normally perform its inherent biological functions, such as excretion, metabolic regulation, endocrine function, and the maintenance of homeostasis within the body. The decline in function due to kidney damage leads to swelling of the kidney and related peripheral organs, renal atrophy, changes in fluid volume, electrolyte imbalance, metabolic acidosis, impaired gas exchange, impaired anti-infective function, and the accumulation of uremic toxins. Examples of such renal diseases include, but are not limited to, acute kidney injury, chronic renal disease, nephritis, glomerulonephritis, pyelonephritis, renal failure, kidney stones, and kidney cancer.

[0048] According to a specific embodiment of the present invention, a kidney disease that can be prevented or treated with the composition of the present invention is an inflammatory kidney disease.

[0049] In this specification, the term “inflammatory renal disease” refers to a pathological condition in which tissue damage and / or functional decline occur due to unwanted immune or excessive inflammatory responses in kidney tissue or kidney cells; it is also referred to as nephritis to distinguish it from nephrosis, a non-inflammatory renal disease. If nephritis persists, it leads to complications such as chronic renal failure, hypertension, damage to other surrounding organs, and metabolic abnormalities. Nephritis is primarily characterized by glomerulonephritis caused by autoimmune reactions and pyelonephritis caused by bacterial infections; immunosuppressants, anti-inflammatory agents, and vasoactive agents are mainly used to treat the former, while quinolone antibiotics are primarily used to treat the latter. However, long-term administration of anti-inflammatory agents or immunosuppressants can cause side effects such as ulcers, edema, increased susceptibility to infection, worsening hypertension, and hepatotoxicity; therefore, there is a need to develop superior treatments for nephritis that are safer and exhibit significant therapeutic effects even with short-term administration at low doses.

[0050] According to a specific embodiment of the present invention, the kidney disease that can be prevented or treated by the composition of the present invention is acute kidney injury (AKI) or chronic kidney disease (CKD).

[0051] In this specification, the term “Acute Kidney Injury” refers to a kidney disease accompanied by a rapid decline in kidney function, and encompasses all rapid declines in kidney function caused by complex and diverse causes, such as acute tubular necrosis, interstitial nephritis, glomerulonephritis, vasculitis, ischemia, and secondary damage caused by nephrotoxic agents. Acute kidney injury includes, but is not limited to, ischemic-reperfusion-associated acute kidney injury, sepsis-associated acute kidney injury, and nephrotoxic acute kidney injury. Acute kidney injury is a severe disease that significantly increases the risk of death even in cases of mild, reversible damage. In cases of persistent acute kidney injury, it causes fluid, electrolyte, and acid-base imbalances as well as loss of hormonal regulation, and leads to multiple organ failure, including dysfunction of the central nervous system, immune function, and blood coagulation function, thereby greatly worsening the patient's prognosis.

[0052] In this specification, the term “Chronic Kidney Disease (CKD)” refers to a disease in which the kidney’s ability to filter metabolic waste products from the blood gradually decreases over several months to several years, and as time passes, the blood becomes more acidic, anemia develops, and nerve damage and bone tissue degeneration progress, increasing the risk of atherosclerosis.

[0053] According to a specific embodiment of the present invention, a kidney disease that can be prevented or treated by the composition of the present invention is acute kidney injury.

[0054] According to a more specific embodiment of the present invention, the acute kidney injury is sepsis-associated acute kidney injury (Sepsis-Associated AKI, SA-AKI).

[0055] In this specification, the term “sepsis-associated acute renal injury” refers to a state of acute renal decline or renal injury caused directly or indirectly by sepsis or septic shock, and refers to renal dysfunction accompanied by changes in clinical indicators such as decreased glomerular filtration rate, elevated blood creatinine, and decreased urine output, caused by one or more of the following: excessive inflammatory response induced by systemic infection, abnormal immune response, microvascular dysfunction, hemodynamic changes, metabolic abnormalities, and oxidative stress. Such sepsis-associated acute renal injury may involve ischemic injury, inflammation-mediated injury, immune cell infiltration, endothelial dysfunction, tubular dysfunction, etc., either alone or in combination; however, it is not limited to a specific pathogenesis or clinical severity and encompasses all forms of acute renal dysfunction that occur in temporal and causal association with sepsis.

[0056]

[0057] According to a more specific embodiment of the present invention, the acute kidney injury is ischemic-reperfusion associated acute kidney injury (Ischemic-Reperfusion Associated AKI).

[0058] In this specification, the term “ischemia-reperfusion-associated acute kidney injury” refers to an acute decline in renal function or kidney injury that occurs in association with an ischemia state in which blood supply to kidney tissue is temporarily reduced or blocked, and a subsequent reperfusion process in which blood flow is restored. Ischemia-reperfusion-associated acute kidney injury is characterized by a lack of oxygen supply during the ischemia period, increased production of reactive oxygen species during the reperfusion process, induction of an inflammatory response, activation of immune cells, microvascular dysfunction, endothelial cell damage, tubular epithelial cell damage, or apoptosis, and as a result, is accompanied by changes in clinical indicators associated with kidney dysfunction, such as a decrease in glomerular filtration rate, an increase in blood creatinine, a decrease in urine output, or abnormalities in electrolyte homeostasis. In the present invention, ischemia-reperfusion-associated acute renal injury is not limited to specific pathogenesis or clinical severity, but encompasses all forms of acute renal dysfunction resulting directly or indirectly from renal ischemia and reperfusion situations induced by various causes such as surgery, organ transplantation, shock, trauma, hypotension, and cardiovascular disease.

[0059] More specifically, the ischemia-reperfusion-associated acute renal injury is any one selected from the group consisting of cardiac surgery-associated acute renal injury (Cardiac Surgery-Associated AKI), kidney transplantation-associated acute renal injury (Kidney Transplantation-Associated AKI), and contrast-induced acute renal injury (Contrast-induced AKI).

[0060]

[0061] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0062] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. The composition of the present invention serves to inhibit, eliminate, or alleviate the progression of symptoms caused by kidney disease, specifically acute kidney injury, by reducing reactive oxygen species in the kidney, improving the tubules, and reducing the number of neutrophils infiltrating the kidney. Accordingly, the composition of the present invention may serve as a therapeutic composition for kidney disease in itself, or it may be applied as an adjuvant for the treatment of kidney disease when administered together with other pharmacological components. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic aid” or “therapeutic adjuvant.”

[0063] In this specification, the term “administration” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject’s body, and has the same meaning as “implantation” or “injection.”

[0064] In the present invention, the term “therapeutic effective amount” refers to the content of a composition contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the composition of the present invention is to be administered, and includes the meaning of “preventive effective amount.”

[0065] In this specification, the term “object” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0066] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0067] The pharmaceutical composition of the present invention can be administered through various routes of administration, specifically by parenteral administration, more specifically by parenteral administration, and even more specifically by intravenous, arterial, subcutaneous, abdominal, intradermal, intramuscular, intraventricular, spinal, inhalation, nasal, joint cavity, or local administration.

[0068] Suitable dosages of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity. Preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001 to 1000 mg / kg for adults.

[0069] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0070]

[0071] The features and advantages of the present invention are summarized as follows:

[0072] (a) The present invention provides a cerium oxide nanocomposite and a composition for the prevention or treatment of kidney disease comprising the same as an active ingredient.

[0073] (b) The present invention can be used as an excellent therapeutic composition in which both biocompatibility and efficiency of controlling inflammation in the body are maximized by modifying the surface of cerium oxide nanoparticles with a pyrrolidone derivative polymer at an optimal content.

[0074] (c) The cerium oxide nanocomposite of the present invention can be used as an excellent therapeutic composition that significantly improves the survival rate of patients with acute kidney injury, a severe intractable disease, by reducing reactive oxygen species and neutrophils in kidney tissue and repairing tubular damage.

[0075]

[0076] Figure 1 is a diagram showing a schematic diagram (Figure 1a) and transmission electron microscope observation results (Figure 1b) of the cerium oxide nanocomposite of the present invention, respectively.

[0077] Figure 2 shows the results of analyzing the particle size after synthesis and the changes in dispersion stability and surface charge in a biomimetic environment according to the content of PVP (polyvinylpyrrolidone), a polymer modified on the outer surface of the cerium oxide nanocomposite of the present invention (Figure 2a), and the results of converting the content of PVP in the dried cerium oxide nanocomposite of the present invention into unit cerium (Figure 2b), respectively.

[0078] Figure 3 is a figure showing the results of measuring the particle size over time using a dynamic light scattering device after exposing the cerium oxide nanocomposite of the present invention to a biomimetic environment.

[0079] Figure 4 shows the results of comparing the surface charges of a conventionally developed cerium oxide nanocomposite (CX213) and the cerium oxide nanocomposite of the present invention.

[0080] Figure 5 shows the results of observing the reaction residue (Figure 5a) and particle size change (Figure 5b) according to the concentration of the sodium chloride aqueous solution used in the purification process of the cerium oxide nanocomposite of the present invention, respectively.

[0081] Figure 6 shows the results of a comparative analysis of the therapeutic effects of a conventionally developed cerium oxide nanocomposite (CX213) and the cerium oxide nanocomposite (CX301) of the present invention on severe cerebral infarction by observing the survival rate after intravenous injection in a rat model of severe cerebral infarction (MIS).

[0082] Figure 7 shows the results of a comparative analysis of the therapeutic effects of a conventionally developed cerium oxide nanocomposite (CX213) and the cerium oxide nanocomposite of the present invention (CX301) on severe cerebral infarction through infarction volume.

[0083] Figure 8 shows the results of measuring changes in reactive oxygen species (Figure 8a) and changes in cell viability (Figure 8b) in acute kidney injury-induced HK2 cells treated with the cerium oxide nanocomposite of the present invention, respectively.

[0084] Figure 9 shows the results of measuring changes in reactive oxygen species (Figure 9a) and changes in cell viability (Figure 9b), respectively, in HK2 cells that were induced with acute kidney injury by H2O2 after the nanocomposite was pretreated to confirm the preventive effect of the cerium oxide nanocomposite of the present invention on kidney disease.

[0085] Figure 10 is a figure showing the results of quantitatively measuring the degree of tubular improvement in mice with induced intra-abdominal infection after confirming it with H&E staining (Figure 10a) and then measuring it (Figure 10b).

[0086] Figure 11 shows the results of confirming changes in the number of neutrophils in a sepsis-associated acute kidney injury mouse model induced by intra-abdominal infection using immunohistochemical staining (Figure 11a) and then quantitatively measuring them (Figure 11b).

[0087] Figure 12 shows the PAS (eriodic Acid-Schiff) staining (Figure 12a) and the quantitative evaluation results of tubular damage scores (Figure 12b), respectively, after renal ischemia-reperfusion-associated acute kidney injury mouse model with induced renal ischemia.

[0088] Figure 13 shows the results of evaluating the degree of cell death in kidney tissue in an ischemia-reperfusion-associated acute kidney injury mouse model, including the DAPI staining results (Figure 13a) and the results of measuring the ratio of TUNEL-positive cells to total cells (Figure 13b), respectively.

[0089] Figure 14 shows the results of quantitatively measuring changes in neutrophil counts (Figure 14b) after confirming them by immunohistochemical staining (Figure 14a) in an ischemia-reperfusion-associated acute kidney injury mouse model.

[0090] Figure 15 shows the results of measuring changes in the concentrations of blood urea nitrogen (BUN) (Figure 15a) and creatinine (Figure 15b), which are key markers of renal function decline, in an ischemia-reperfusion-associated acute kidney injury mouse model.

[0091]

[0092] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0093]

[0094] Examples

[0095] Example 1: Synthesis of Cerium Oxide Nanocomposites

[0096] A first solution was prepared by dissolving 6-aminohexanoic acid (37.75 g, Sigma-Aldrich, St. Louis, MO) in deionized water (1.7 L). While stirring the first solution, ethyl alcohol (1.4 L) was added, and 132.83 g of polyvinylpyrrolidone (PVP, Ashland) was weighed and added, after which the mixture was heated to 70°C in air to prepare a second solution. Meanwhile, a third solution was prepared by dissolving cerium(III) nitrate hexahydrate (Ce(NO3)3·6H2O, 31.05 g, Alfa Aeser, Ward Hill, MA) in ethyl alcohol (2.9 L) at room temperature (approx. 20°C). Subsequently, the third solution was added to the second solution to prepare a fourth solution. The temperature of the fourth solution was maintained at 70°C for 2 hours, and then lowered to 45°C. Through this process, cerium oxide nanoparticles with 6-aminohexanoic acid and polyvinylpyrrolidone bonded to their surfaces were obtained (Figs. 1a and 1b). Subsequently, the nanoparticles were washed 5 times with a mixture of acetone and an aqueous sodium chloride solution to remove unreacted materials.

[0097]

[0098] Example 2: Exploration of the optimal content of polyvinylpyrrolidone used as the outer layer polymer

[0099] To determine the optimal content of polyvinylpyrrolidone dispersion stabilizer forming the outer layer of the cerium oxide nanoparticles of the present invention, the particle formation tendency according to PVP content and the stability of dispersion in a biomimetic environment were analyzed. During the synthesis process of the cerium oxide nanocomposite of Example 1, the particle size of the synthesized product and the dispersion stability (Z-Average, nm) and surface charge (Zeta-potential, mV) in a biomimetic environment containing protein and sodium bicarbonate were measured by dynamic light scattering analysis while adjusting the PVP content ratio from 15 to 30 mg PVP / total batch mL. As a result, it was found that optimized particle characteristics were observed when PVP was applied at a content ratio of 20–25 mg PVP / total batch mL (Fig. 2a).

[0100] Additionally, to more clearly measure the derived optimal PVP content range based on the final product, the nanocomposite, the inventors freeze-dried the entire synthesized nanocomposite and converted the PVP content in the dried nanocomposite into units of cerium. As a result, the best particle characteristics were observed when the PVP and cerium oxide nanoparticles in the final nanocomposite had a content ratio of 7.5 to 10.9 (7.5 - 10.9 mg PVP / mg Ce), and it was confirmed that the optimal content ratio was 8.7 (Fig. 2b). This compositional ratio explored in the present invention can be considered the result of finding the most suitable conditions when considering the influence of nanocomposite size on dispersion, function, and biological stability.

[0101]

[0102] Experimental Example 1: Comparison of biostability and dispersibility between conventionally developed particles and the particles of the present invention in a biomimetic environment

[0103] To compare the biostability (dispersibility) in a biomimetic environment of a ceria nanocomposite (CX213) previously developed by the inventors, in which polyglutamic acid (PGA) is modified in the outermost layer as an additional dispersion stabilizer, and the cerium oxide nanoparticles (CX301) of the present invention, in which the introduction of such an outermost polymer layer is omitted, changes in particle size were analyzed over time in 0.05 M PBS (phosphate buffer saline). Samples were collected at 0, 20, 40, and 60 minutes, respectively, and the particle size was analyzed using dynamic light scattering equipment. As a result, it was found that as the reaction time progressed, the CX213 nanocomposite exhibited particle aggregation reaching several hundred nm, whereas the CX301 of the present invention maintained a constant particle size over time (Fig. 3).

[0104]

[0105] Experimental Example 2: Surface charge analysis between conventionally developed particles and the particles of the present invention

[0106] The inventors intended to evaluate the differences in biocompatibility and dispersibility depending on the presence or absence of PVP optimization by measuring the surface charge of CX213, which was previously developed by the inventors, and CX301 of the present invention. After dispersing the respective synthesized cerium oxide nanocomposites in deionized water and analyzing the surface characteristics of the ceria nanocomposites using dynamic light scattering equipment, the surface charge was -17.5 mV for CX213 and 0 mV for CX301 of the present invention, showing a clear difference between the two particles (Fig. 4). Through this, it was found that the present invention minimizes interactions in the in vivo environment compared to CX213, which contains an outermost PGA polymer and has a strong negative charge, as the surface charge converges to 0 mV.

[0107]

[0108] Experimental Example 3: Evaluation of the effect of adding sodium chloride solution during purification on reaction residue concentration and particle size

[0109] In order to measure the reaction residue and particle size of the final product obtained by adding sodium chloride solutions of different concentrations during the purification process, the concentration of the aqueous sodium chloride solution was adjusted to 0, 0.25, 0.5, 1.0, 2.5, and 5 M and added to the reaction solution during the preparation process of the cerium oxide nanocomposite of Example 1. Consequently, the residual content of the reaction residue, particularly nitrate, was measured using a nitrate indicator pad (Jonson). In addition, the particle size was confirmed using dynamic light scattering equipment. As a result, as shown in Figure 5a, it was found that using a 0.25 M aqueous sodium chloride solution during the purification process was most effective in removing the reaction residue, particularly nitrate. This indicates that the manufacturing process of the present invention is efficient for both reducing residue in pharmaceuticals and removing toxicity caused by nitrate. In addition, the particle size was also optimized (20 nm) when using a 0.25 M sodium chloride aqueous solution, confirming that the addition of sodium chloride has a significant effect on the formation stability of the particles (Fig. 5b).

[0110]

[0111] Experimental Example 4: Evaluation of the therapeutic effect of non-infectious inflammatory diseases through survival rate

[0112] To compare and evaluate the therapeutic effects of CX213, a conventionally developed cerium oxide nanocomposite, and CX301 of the present invention prepared in Example 1 above on non-infectious inflammatory diseases, Sparague-Dawley (SD) rats (Coatech Co., Ltd.) were anesthetized with isoflurane, and severe cerebral infarction (MIS), a representative non-infectious inflammatory disease, was induced by ligating the left middle cerebral artery with 4-0 proline sutures. One hour after the induction of severe cerebral infarction, the two types of cerium oxide nanocomposites were intravenously injected at a dose of 0.5 mg Ce / kg over 5 minutes, and an equal volume of physiological saline was injected as a control. The mortality of SD rats was periodically checked from the time of MIS induction until 7 days later, and their survival rates were measured. The experiment was conducted on 18 SD rats in Example 1 and 15 SD rats in the control group, respectively, and the average value was expressed as the survival rate. As shown in Figure 6, the CX213 nanocomposite showed a 2.25-fold increase in survival rate compared to the control group (control group: 28.57%, CX213 administration group: 64.28%), whereas the group injected with CX301 of the present invention showed a survival rate increase of up to 6 times (control group: 13.33%, CX301 administration group: 80.00%).

[0113] Through this, it was observed that CX301, a novel cerium oxide nanocomposite of the present invention, has a therapeutic effect 2.6 times greater than that of CX213 previously developed by the inventors, thereby confirming that it can be usefully utilized as an efficient therapeutic composition for non-infectious inflammatory diseases.

[0114]

[0115] Experimental Example 5: Evaluation of the therapeutic effect of non-infectious inflammatory diseases through infarction volume

[0116] Brain tissue was collected from the SD rats of Experimental Example 5 to obtain coronal sections, and Nissl staining was performed to check the degree of infarction. After staining the tissue, the volume was calculated based on the area of ​​each section. As a result, it was confirmed that the CX213 administration group showed an 18.58% reduction in infarction volume compared to the control group, while the CX301 administration group of the present invention showed a 51.22% reduction. Accordingly, the cerium oxide nanocomposite of the present invention demonstrated a therapeutic effect 2.76 times greater than that of conventionally developed nanocomposites in terms of infarction volume (Fig. 7). Through this, it was confirmed from various angles that the CX301 nanocomposite of the present invention not only significantly increases the survival rate of the disease model but also greatly reduces the infarction volume of actual brain tissue, thereby exhibiting a significantly superior therapeutic effect.

[0117]

[0118] Experimental Example 6: Therapeutic effect on additional non-infectious inflammatory diseases - Acute kidney injury

[0119] Acute Kidney Injury (AKI) is a disease characterized by a rapid decline in kidney function and involves kidney tissue damage and failure caused by sepsis, kidney transplantation, etc. To confirm whether the cerium oxide nanocomposite of the present invention exhibits therapeutic and preventive effects against AKI, the inventors measured reactive oxygen species (ROS) and cell viability at the cellular level. First, acute kidney injury was induced in HK2 cells, which are proximal renal tubule cells that suffer the most direct and severe damage due to acute kidney injury, by treating them with 500 μM of hydrogen peroxide (H2O2). One hour after treatment, the cerium oxide nanocomposite of the present invention was treated at a concentration of 0.01 to 0.5 μM. Two hours later, ROS in HK2 cells was measured using a DCF-DA assay. The DCF-DA assay is a biochemical analysis method that measures the degree of fluorescence exhibited by 2',7'-dichlorofluorescein diacetate (DCF-DA) reacting with intracellular ROS, allowing for the accurate evaluation of intracellular antioxidant effects. As a result of the measurement, the amount of intracellular ROS in cells treated with H2O2 increased by approximately 257% compared to the control group not treated with H2O2. In contrast, when the cerium oxide nanocomposite of the present invention was post-treated, a 55% ROS removal rate was observed at 0.01 μM and an approximately 82.5% ROS removal rate was observed at 0.5 μM compared to cells treated only with H2O2 (Fig. 8a). Furthermore, to measure changes in the viability of HK2 cells, HK2 cells were treated with 500 μM of H2O2, and then the cerium oxide nanocomposite of the present invention was treated at a concentration of 0.01–0.1 μM one hour later, followed by a CCK-8 assay six hours later.The CCK-8 assay is a biochemical analysis method utilizing WST-8, a tetrazolium derivative compound that emits fluorescence upon reduction by NADH and NADPH enzymes within living cells, and can accurately evaluate the viability of target cells through the fluorescence generated. As a result of the measurement, cells treated with H2O2 showed a viability of approximately 45.79% compared to the control group not treated with H2O2; however, when treated with the cerium oxide nanocomposite of the present invention, a viability 3.7% higher than that of H2O2-treated cells was observed at a minimum of 0.01 μM, and a viability improvement 204% higher at a maximum of 0.5 μM was observed, resulting in a cell viability of 93% compared to the control group not treated with H2O2 (Fig. 8b).

[0120] To confirm the prophylactic protective effect of the cerium oxide nanocomposite of the present invention, HK2 cells were first treated with the cerium oxide nanocomposite of the present invention at a concentration of 0.01 to 0.1 μM. After 1 hour of treatment, 2 hours after treatment with 500 μM of H2O2, ROS levels in HK2 cells were measured using the DCF-DA assay to confirm the intracellular antioxidant effect. As a result of the experiment, the amount of intracellular ROS increased by approximately 191% in cells treated with H2O2 compared to the control group not treated with H2O2, whereas when the cerium oxide nanocomposite of the present invention was pre-treated, an ROS removal rate of approximately 65.2% was observed at a maximum concentration of 0.1 μM compared to cells treated only with H2O2 (Fig. 9a).

[0121] For the viability experiment, the cerium oxide nanocomposite of the present invention was treated at a concentration of 0.01 to 0.1 μM. After 1 hour of treatment, a CCK-8 assay was performed to evaluate cell viability after 3 hours of treatment with 200 μM of H2O2. As a result, cells treated with H2O2 showed a viability of approximately 83% compared to the control group not treated with H2O2, whereas treatment with the cerium oxide nanocomposite of the present invention showed a viability improvement of approximately 31.2% compared to cells treated with H2O2 at a maximum concentration of 0.1 μM, resulting in a cell viability of 109% compared to the control group not treated with H2O2 (Fig. 9b). Through these results, it was found that the cerium oxide nanocomposite of the present invention can be used as an efficient therapeutic and preventive composition for acute kidney injury.

[0122]

[0123] Experimental Example 7: Therapeutic effect on acute kidney injury in vivo_Sepsis-associated acute kidney injury

[0124] The inventors intended to further verify whether the cerium oxide nanocomposite of the present invention exhibits a significant therapeutic effect against AKI even in vivo. A Sepsis-Associated Acute Kidney Injury (SAKI) model was constructed by anesthetizing mice with 2.5% isoflurane, ligating and perforating the cecum to induce intra-abdominal infection. One hour after disease induction, a single intravenous dose of 0.5 mg / kg of the cerium oxide nanocomposite of the present invention was administered, and the kidneys of surviving individuals were excised after 24 hours. To confirm the extent of renal tubular damage, the excised kidneys were stained using hematoxylin, which stains the cell nucleus, and eosin, which stains the cytoplasm. As a result, approximately 40% improvement in the renal tubules was confirmed in the group treated with the cerium oxide nanocomposite of the present invention compared to the control group treated with physiological saline (control group: 2.55, experimental group: 1.53) (Figs. 10a and 10b). In addition, immunohistochemistry (IHC) staining was performed using an MPO antibody, a neutrophil marker, to confirm changes in neutrophil cells infiltrating within the kidney. As a result, it was observed that there was a difference of approximately 6.16 times in the number of infiltrated neutrophil cells between the control group treated with physiological saline and the group treated with the cerium oxide nanocomposite of the present invention (control group: 245.7 cells / mm2, experimental group: 39.9 cells / mm2) (Figs. 11a and 11b). Through these results, it was comprehensively confirmed that the cerium oxide nanocomposite of the present invention can be used as an efficient therapeutic composition for acute kidney injury.

[0125]

[0126] Experimental Example 8: Therapeutic Effect of Acute Renal Injury in Human Biomedical Data_Ischemia-Reperfusion-Associated Acute Renal Injury

[0127] To comprehensively verify whether the cerium oxide nanocomposite of the present invention has a therapeutic effect on AKI caused by various factors, mice were anesthetized with 2.5% isoflurane by inhalation, the abdomen was incised to expose bilateral renal vessels, and renal ischemia was induced by ligating the renal artery for 20 minutes using a microvascular clamp. Subsequently, an ischemic-reperfusion-associated acute kidney injury (IRI) model was established by removing the clamp to restore blood flow. Immediately after inducing ischemic-reperfusion injury, the cerium oxide nanocomposite of the present invention was administered by a single intravenous injection at a dose of 0.5 mg / kg based on cerium concentration. Physiological saline was administered as a control under the same conditions. After 24 hours had elapsed following reperfusion, the experimental animals were sacrificed and the kidneys were extracted. Periodic Acid-Schiff (PAS) staining was performed on the excised kidney tissue to evaluate the degree of renal tubular damage caused by acute kidney injury. Quantitative analysis of the tubular damage score revealed that the tubular damage index improved by approximately 37.4% in the group treated with the cerium oxide nanocomposite of the present invention compared to the control group (control group: 2.02, experimental group: 1.26) (Figs. 12a and 12b).

[0128] Next, to evaluate the degree of apoptosis in kidney tissue, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) analysis was performed, and the ratio of TUNEL-positive cells to total cells was analyzed through DAPI staining. As a result of quantitative analysis of the ratio of TUNEL-positive cells, it was confirmed that the cerium oxide nanocomposite-treated group of the present invention showed a 53% reduction in the level of apoptosis compared to the control group, demonstrating a significant renal cell protective effect even in acute kidney injury induced by ischemia-reperfusion (control group: 18.97%, experimental group: 8.97%) (Figs. 13a and 13b).

[0129] Additionally, IHC for MPO, a neutrophil marker, was performed to evaluate the degree of infiltration of inflammatory immune cells within kidney tissue. Quantitative analysis of the number of MPO-positive cells per unit area revealed that the cerium oxide nanocomposite-treated group of the present invention showed a 38.5% reduction in neutrophil infiltration compared to the control group, confirming that the inflammatory response in ischemia-reperfusion-induced acute kidney injury is effectively suppressed (Control group: 123.6 cells / mm²). 2 , experimental group: 76.0 pieces / mm 2 )(Figs. 14a and 14b).

[0130] Furthermore, the inventors intended to verify whether the cerium oxide nanocomposite of the present invention could significantly alter blood biomarkers associated with renal function decline in ischemia-reperfusion-associated acute renal injury. To this end, an ischemia-reperfusion-associated acute renal injury mouse model was established, and immediately after induction, the cerium oxide nanocomposite of the present invention was administered as a single intravenous dose at a dose of approximately 0.5 mg / kg based on cerium concentration. As a control, physiological saline was administered under the same conditions. Seven days after drug administration, blood was collected to evaluate renal function, and the concentrations of blood urea nitrogen (BUN) and creatinine, which are blood biomarkers closely associated with acute renal injury, were analyzed. As a result, the group administered the cerium oxide nanocomposite of the present invention showed a 99% reduction in the increase of BUN and creatinine compared to the control group, exhibiting levels similar to those before injury, thereby confirming that renal function damage caused by ischemia-reperfusion was effectively alleviated (BUN - control group: 2.18 mg / dl, experimental group: 0.04 mg / dl / creatinine - control group: 183.3 mg / dl, experimental group: 32.1 mg / dl) (Figs. 15a and 15b). Through these results, it was confirmed that the cerium oxide nanocomposite of the present invention effectively inhibits acute renal injury at a functional level by improving blood renal function biomarkers as well as providing renal tissue protection effects in an ischemia-reperfusion-related acute renal injury model.

[0131]

[0132] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for the prevention or treatment of kidney disease comprising a cerium oxide nanocomposite as an active ingredient, comprising the following: (a) a cerium oxide nanoparticle core layer; and (b) Outer layer comprising a polymer represented by the following chemical formula 1: Chemical formula 1 In the above chemical formula, R1 and R2 are each independently hydrogen or oxygen, and represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000.

2. A composition according to claim 1, wherein the cerium oxide nanoparticles are selected from the group consisting of cerium oxide (III) (Ce2O3) nanoparticles, cerium oxide (IV) (CeO2) nanoparticles, and mixtures thereof.

3. A composition according to claim 1, characterized in that in the above chemical formula 1, R1 is hydrogen, R2 is oxygen, and l is 1.

4. A composition according to claim 3, characterized in that the polymer represented by Chemical Formula 1 and the cerium oxide nanoparticles have a content ratio of 7:1 to 11:

1.

5. A composition according to claim 1, characterized in that the nanocomposite additionally comprises a polyfunctional group ligand represented by the following chemical formula 2: Chemical formula 2 In the above chemical formula, n is an integer from 3 to 7.

6. A composition according to claim 5, characterized in that n in Chemical Formula 2 is 5.

7. A composition according to claim 1, characterized in that the nanocomposite has an average particle size of 5 nm to 80 nm.

8. A composition according to claim 1, characterized in that the nanocomposite has a surface charge (Zeta potential, mV) of -1.0 to 1.0 mV.

9. A composition according to claim 1, characterized in that the kidney disease is acute kidney injury (AKI) or chronic kidney disease (CKD).

10. A composition according to claim 9, characterized in that the acute kidney injury is sepsis-associated acute kidney injury (Sepsis-Associated AKI).

11. A composition according to claim 9, characterized in that the acute kidney injury is ischemic-reperfusion-associated acute kidney injury (Ischemic-Reperfusion Associated AKI).

12. A composition according to claim 11, wherein the ischemia-reperfusion-associated acute renal injury is selected from the group consisting of cardiac surgery-associated acute renal injury (Cardiac Surgery-Associated AKI), kidney transplantation-associated acute renal injury (Kidney Transplantation-Associated AKI), and contrast-induced acute renal injury (Contrast-induced AKI).

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