KR20150113272A - Pharmaceutical compositions for the prevention and treatment of the nephrotoxicity induced by anticancer agent - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating nephrotoxicity caused by an anticancer agent and, more specifically, a silent information regulator 2 (SIRT2) inhibitor according to the present invention can be helpfully used in a medicine for preventing or treating cancer and a health functional food for preventing or treating cancer, due to having an effect of increasing anticancer efficacy while reducing nephrotoxicity, which is a side effect of cisplatin, through inhibiting apoptosis and expression of inflammation-related factors when administered together with cisplatin.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of nephrotoxicity induced by anticancer agents,

The present invention relates to a pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by an anticancer agent. More particularly, the SIRT2 gene inhibitor according to the present invention reduces renal toxicity, which is a side effect of cisplatin when administered together with cisplatin, It is an object of the present invention to provide medicines for the prevention and treatment of cancer diseases and health functional foods for prevention and improvement of cancer diseases.

Cancer is a disease that causes about 7 million deaths annually worldwide. In Korea, according to the Statistical Yearbook of the Year 2000 (2000 death data analysis) of Korea National Statistical Office, 23.5%, which is the number one cause of death in Korea. Currently, several methods such as surgery, radiation therapy, and gene therapy are used to treat cancer, but chemotherapy is one of the most commonly used treatments.

Cancer chemotherapy is a systemic treatment. Most of them are administered by injection or oral anticancer drug. Therefore, micrometastasis is a treatment that spreads throughout the body rather than local effects. Therefore, systemic side effects are more frequent and more severe than surgery or radiation therapy. Using chemotherapy or most anticancer drugs can not distinguish normal cells from cancer cells by using the sensitivity difference between normal cells and cancer cells to exhibit dose-limiting toxicity There is a problem.

Due to the above-mentioned problems, researches on target anticancer drugs are active. In the prior art for the above anticancer agent, at least one selected from the group consisting of integrin [beta] 3 neutralizing antibody, integrin [beta] 3 siRNA, Src inhibitor and Src siRNA is A pharmaceutical composition for inhibiting anticancer drug resistance, an anticancer adjuvant and the like, which are contained as active ingredients.

However, the target anticancer drug has a problem that it can not kill cancer cells, and instead, drugs that inhibit the growth of cancer cells and inhibit the growth of cancer cells are necessary.

On the other hand, cis-diammine-dichloroplatinum (II) is widely used as a chemotherapeutic agent for the treatment of ovarian cancer, bladder cancer, lung cancer, head and neck cancer, testicular cancer and the like (Rosenberg B., Cancer, pp. 2303-2315, 1985). It is known that cisplatin produces an active oxygen species to attack cancer cells, induce inter-intrastrand cross-linking of DNA and DNA adduct formation in cancer cells, and exhibit anticancer effects. However, there is evidence that side effects such as loss of hearing, neurotoxicity, and renal toxicity are present above the limited amount of drug during the course of treatment (Mollman et al., 1998; Screnci and McKeage, 1999) Toxicity is also known to be frequently observed (Cerosimo RJ, Ann. Pharm., 27: pp438-441, 1993; Cavalli F. et al., Cancer Treat. Rep., 62: pp2125-2126, al., J. Clin. Oncol., 5: pp318-319, 1987). Such adverse effects by cisplatin include increased lipid peroxidation due to active oxygen species produced by cisplatin (Matsushima H. et al., J. Lab. Clin. Med., 131: pp518-526, 1998; Koc A. et al Inhibition of Antioxidant Enzyme Activity in Tissues (Sadzuka Y. et al., Biochem. Pharmacol., 43: pp 1873-1875, Mol. Cell Biochem., 278 1992), depletion of glutathione (Zhang JG and Lindup WE, Biochem. Pharmcol. 45: pp2215-2222, 1993) and collapse of intracellular Ca homeostasis (Zhang JG and Lindup WE, Toxicology in Vitro, : pp205209, 1996).

Recently, it has been observed that cisplatin-induced renal toxicity is effectively inhibited when cisplatin and glutathione ester are coadministered (Babu E. et al., MoI. Cell Biochem., 144: pp7-11, 1995) There is much interest in reducing cisplatin-induced toxicity by ingesting antioxidants through diet (Appenroth D. et al., Arch. Toxicol., 71: pp677-683, 1997; Bogin E. et al., Eur J. Clin. Chem. Clin. Biochem., 32: pp 843-851, 1994; Rao M. et al., J. Biochem., 125: pp383-390, 1999).

SIRT2 (Sirtuin 2), on the other hand, is one of the sirtuin protein family and performs important cell survival functions under certain conditions. However, the biological function of the SIRT2 protein in relation to inflammation and oxidative stress is unclear.

SIRT2 (silent information regulator 2), known as sirtuin, is a NAD + -dependent deacetylase modulator in biological processes such as life span, aging, cancer development, neurodegeneration and metabolic disease [Michan, S .; Sinclair, D. Biochem. J. 404: 1-13; 2007, Finkel, T. et al. Nature 460: 587-591; 2009, Donmez, Z .; Guarente, L. Aging Cell 9: 285-290; 20101-3]. The SIRT2 gene family is well conserved from bacteria to eukaryotes. Seven types of SIRT have been identified in humans (Frye, RA Biochem. Biophys. Res. Comm. 272: 793-798; 2000]. Most of the seven human SIRTs have focused on SIRT1. Overexpression of SIRT1 increases cell survival under DNA damage and oxidative stress [Oberdoerffer, P. et al. Cell 135: 907-918; 2008.]. In addition, the neuroprotective role of SIRT1 is well established in Alzheimer's disease and amyotrophic lateral sclerosis [Chen, J. et al. J. Biol. Chem. 280: 40364-40374; 2005, Kim, D. et al. EMBO J. 26: 3169-3179; 2007]. However, the biological functions and mechanisms of SIRT2 in inflammation and oxidative stress are not well known.

On the other hand, many chemotherapeutic agents including cisplatin are known to attack active cancer cells by generating active oxygen species, and the produced reactive oxygen species are also known to act on normal cells and cause damage. Therefore, antioxidant substances are likely to reduce toxicity induced by chemotherapeutic agents. In addition, it is considered that hepatotoxicity and renal toxicity caused by external harmful substances that produce active oxygen species or radicals can be effectively suppressed, but there is a problem in that research is lacking.

Further, as described above, there is a problem in that research on the biological function and mechanism of SIRT2 in inflammation and oxidative stress is insufficient, and there is a problem in that the renal toxic disease caused by cisplatin, which is a typical anticancer drug, There is a problem in that a method for preventing and treating a nephrotoxic disease caused by an anticancer agent is very insufficient.

It is an object of the present invention to solve the above-mentioned problems, and it is an object of the present invention to provide a method for preventing and treating nephrotoxic diseases caused by an anticancer agent which inhibits renal toxicity by an anticancer drug while improving anti- And to provide a therapeutic pharmaceutical composition.

A second problem to be solved by the present invention is to provide an anticancer adjuvant having an inhibitory activity against renal toxicity induced by an anticancer agent.

A third problem to be solved by the present invention is to provide a health functional food for preventing and improving a renal toxic disease caused by an anticancer agent.

In order to achieve the first object, the present invention provides a pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by an anticancer agent comprising an SIRT2 inhibitor as an active ingredient.

According to a preferred embodiment of the present invention, the anticancer agent may be cisplatin.

According to another preferred embodiment of the present invention, the SIRT2 inhibitor may be an antisense oligonucleotide specific to the SIRT2 gene, an siRNA, an aptamer or an antibody.

According to another preferred embodiment of the present invention, the SIRT2 inhibitor may be AGK2 or AK-1.

According to another preferred embodiment of the present invention, the pharmaceutical composition may inhibit renal damage by inhibiting the expression of molecules ICAM-1 and VCAM-1 associated with apoptosis and inflammatory response.

In order to achieve the second object, the present invention provides an anti-cancer adjuvant comprising, as an active ingredient, a SIRT2 inhibitor having an inhibitory activity against renal toxicity induced by an anti-cancer agent.

In order to achieve the third object, the present invention provides a health functional food for preventing or ameliorating a renal toxic disease caused by an anticancer agent comprising an SIRT2 inhibitor as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by an anticancer agent, wherein the SIRT2 inhibitor according to the present invention inhibits apoptosis and inhibits inflammation-related factors ICAM-1 and VCAM- 1 expression, it has an excellent effect of inhibiting the renal damage caused by the cisplatin, which is an anticancer drug, and reducing the renal toxicity, and enhancing the anticancer efficacy, and when it is combined with the anticancer drug, it increases the anticancer efficacy of the anticancer drug, The present invention relates to a pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by the above-mentioned diseases.

Brief Description of the Drawings Figure 1 is a graph of BUN measured to confirm the function of kidney by cisplatin in SIRT2 gene-deficient mice.
FIG. 2 is a graph showing creatinine measured in order to confirm the function of kidney by cisplatin in SIRT2 gene-deficient mice
FIG. 3 is a photograph showing the histological damage of kidney due to administration of cisplatin through PAS staining.
4 is a graph showing the survival rate of cisplatin administration.
FIG. 5 shows the results of Western blotting, which confirmed the effect of SIRT2 gene expression on apoptosis by caspase-3 expression. The WT shown in Fig. 5 means a control group in which SIRT2 exists, and KO means a control group lacking SIRT2. The first lane treated the vehicle to mice in which SIRT2 was present, the second lane treated vehicle in mice lacking SIRT2, the third to sixth lanes treated cisplatin in mice with SIRT2, and the SIRT2 mice in the seventh to tenth lanes Lacked mice were treated with cisplatin. The expression of caspase-3, which was not expressed in the vehicle-treated control group, was found to be increased when cisplatin was administered to mice in which SIRT2 was present, and significantly decreased in mice lacking SIRT2.
FIG. 6 shows the result of Western blotting in which apoptosis-modulating effect by SIRT2 gene expression was confirmed by p53 expression pattern. It was found that acetylation of p53, which was increased when cisplatin was treated with SIRT2, decreased in mice lacking SIRT2.
FIG. 7 shows the results of western blotting in which inflammatory molecule-mediated effects of SIRT2 gene expression were confirmed by the expression patterns of ICAM-1 and VCAM-1. The first and second lanes did not express ICAM-1 and VCAM-1 when treated with vehicle, with or without SIRT2. In the third to fifth lanes, cisplatin treatment of mice with SIRT2 showed increased expression of ICAM-1 and VCAM-1. However, it was confirmed that ICAM-1 and VCAM-1 decreased in the sixth to eighth lanes, which were mice lacking SIRT2, when treated with cisplatin, as compared to mice with SIRT2.
8 is a photograph of a cell in which SIRT2 inhibitor is confirmed to inhibit cytotoxicity by cisplatin.
FIG. 9 is a graph quantifying the results of FIG.
10 is a graph showing the cell proliferation effect of SIRT2 inhibitor by XTT through cell proliferation test.

Hereinafter, the present invention will be described in more detail.

As described above, studies on the biological functions and mechanism of SIRT2 in inflammation and oxidative stress are insufficient, and there is a problem in that it is related to the intracellular interaction and signaling and regulation mechanism of SIRT2 with the renal toxic disease caused by the typical anticancer drug cisplatin There is a problem in that a method for preventing and treating a renal toxic disease caused by an anticancer drug is very insufficient.

Thus, according to a preferred embodiment of the present invention, a pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by an anticancer agent containing SIRT2 inhibitor as an active ingredient is provided, and the above-mentioned problems are solved.

Thus, there is an excellent effect of inhibiting renal damage caused by the conventional anticancer drug cisplatin, reducing the renal toxicity, and enhancing the anticancer efficacy. When the anticancer drug is used in combination with the anticancer drug, it is confirmed that the anticancer efficacy of the anticancer drug is enhanced. And can be usefully used as a pharmaceutical composition for the prevention and treatment of renal toxic diseases.

The present invention includes a SIRT2 inhibitor as an active ingredient.

"SIRT2" of the present invention is one of the sirtuin protein family and performs important cell survival functions under certain conditions. However, the biological function of the SIRT2 protein in relation to inflammation and oxidative stress is unclear.

SIRT2 (silent information regulator 2), known as sirtuin, is a NAD + -dependent deacetylase modulator in biological processes such as life span, aging, cancer development, neurodegeneration and metabolic disease [Michan, S .; Sinclair, D. Biochem. J. 404: 1-13; 2007, Finkel, T. et al. Nature 460: 587-591; 2009, Donmez, Z .; Guarente, L. Aging Cell 9: 285-290; 20101-3]. The SIRT2 gene family is well conserved from bacteria to eukaryotes. Seven types of SIRT have been identified in humans (Frye, RA Biochem. Biophys. Res. Comm. 272: 793-798; 2000]. Most of the seven human SIRTs have focused on SIRT1. Overexpression of SIRT1 increases cell survival under DNA damage and oxidative stress [Oberdoerffer, P. et al. Cell 135: 907-918; 2008.]. In addition, the neuroprotective role of SIRT1 is well established in Alzheimer's disease and amyotrophic lateral sclerosis [Chen, J. et al. J. Biol. Chem. 280: 40364-40374; 2005, Kim, D. et al. EMBO J. 26: 3169-3179; 2007]. However, the biological functions and mechanisms of SIRT2 in inflammation and oxidative stress are not well known.

In addition, the anticancer agent of the present invention may be cisplatin.

Cis-diammine-dichloroplatinum (II) is widely used in clinical practice as a chemotherapeutic agent for the treatment of ovarian cancer, bladder cancer, lung cancer, head and neck cancer, testicular cancer and the like (Rosenberg B., Cancer, 55: pp2303- 2315, 1985). It is known that cisplatin produces an active oxygen species to attack cancer cells, induce inter-intrastrand cross-linking of DNA and DNA adduct formation in cancer cells, and exhibit anticancer effects. However, there is evidence that side effects such as loss of hearing, neurotoxicity, and renal toxicity are present above the limited amount of drug during the course of treatment (Mollman et al., 1998; Screnci and McKeage, 1999) Toxicity is also known to be frequently observed.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of renal toxic diseases induced by anticancer drugs by identifying the intracellular interaction of SIRT2 induced by anticancer drugs and the molecular mechanism involved in signal transduction and regulatory mechanisms to provide.

Specifically, as shown in Example 2, in order to examine the effect of SIRT2 gene expression on renal toxicity by cisplatin, cisplatin was administered to mice lacking SIRT2 gene to examine renal function and damage degree. As a result, , It was confirmed that BUN and creatinine were increased in kidney damage. In addition, it was confirmed that the BUN and creatinine increased by the cisplatin in the SIRT2 gene-deficient mouse were remarkably reduced (see FIGS. 1 and 2). In addition, as shown in FIG. 3, in the kidney of cisplatin-treated WT (SIRT2 + / +) mice treated with cisplatin, the epithelial cells (SIRT2 - / -) mice showed a significant reduction in kidney damage, although tissue damage such as skin flaking, brush border loss, and tubular cast formation were observed. In addition, the survival rate was also increased (see FIG. 4).

This suggests that inhibition of renal damage by cisplatin when the SIRT2 gene is absent is suppressed.

In the present invention, said treatment means, unless otherwise stated, means reversing, alleviating, inhibiting the progress of, or preventing the disease or condition to which said term applies, or one or more symptoms of said disease or condition And the term " treatment " used in the present invention refers to the treatment when the treatment is defined as described above.

Therefore, the SIRT2 inhibitor according to the present invention inhibits apoptosis through the regulation of p53 acetylation pathway and reduces the expression of ICAM-1 and VCAM-1, which are inflammatory reaction-related factors increased by cisplatin, The present invention relates to a pharmaceutical composition for prevention and treatment of nephrotoxic diseases induced by an anticancer drug by confirming that it has an excellent effect of inhibiting damage and decreasing renal toxicity while enhancing the anticancer efficacy and when it is used in combination with an anticancer drug, And can be usefully used as a composition.

Specifically, as shown in Example 3, in order to examine the effect of cisplatin-induced apoptosis on caspase-3 when the SIRT2 gene is lacked, since loss of kidney by cisplatin is generally affected by apoptosis, And acetylation of p53 were confirmed by Western blotting. As a result, cleaved caspase-3 increased in the group treated with cisplatin in the WT mouse, and it was confirmed that the cleaved caspase-3 was significantly decreased in the SIRT2 gene-deficient mouse, and the expression of acetyl p53 was also decreased 6).

This suggests that apoptosis is reduced in SIRT2 gene - deficient mouse kidney tissue, which is regulated by p53 acetylation pathway.

Furthermore, as shown in Example 4, it was confirmed that the expression of ICAM-1 and VCAM-1 genes, which are molecules involved in the inflammation response by cisplatin, was regulated by SIRT2 gene. As a result, ICAM- 1 and VCAM-1 gene expression was decreased when cisplatin was administered to mice lacking SIRT2 gene (see FIG. 7).

The pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by the anticancer agent according to the present invention may comprise a pharmaceutically effective amount of SIRT2 inhibitor alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents .

The pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of a renal toxic disease caused by an anticancer drug.

The pharmaceutically effective amount of a SIRT2 inhibitor according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of the renal toxic disease symptom, age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating a renal toxic disease caused by an anticancer agent according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, The composition for preventing or treating a nephrotoxic disease caused by an anticancer agent according to the present invention may be selected according to various factors such as age, sex, weight and severity of a patient, and the composition for preventing or treating a nephrotoxic disease In combination with known compounds having the effect of preventing, ameliorating or treating the symptoms of the disease.

In addition, in the present invention, the SIRT2 inhibitor includes antisense oligonucleotides, RNAi, siRNA, shRNA, aptamer, antibody, single chain variable region fragment, low molecular weight compound or natural extract specific to the SIRT2, , Preferably AGK2 or AK-I.

The AK-1 may include a compound represented by the following general formula (1).

The AGK2 may include a compound represented by the following formula (2).

In addition, AK-1 and / or AGK2 can inhibit SIRT2 activity by targeting the SIRT2 nicotinamide binding site through cell permeation of benzylsulfonamide.

The SIRT2 inhibitor of the present invention provides a cytoprotective effect and a cell proliferation effect by the anticancer agent cisplatin.

Specifically, as shown in Example 5 of the present invention, the effect of SIRT2 inhibitors AGK2 and AK-1 when cisplatin was treated with mouse proximal tubular cells was examined in order to confirm cytotoxicity inhibition effect of cisplatin As a result, it was confirmed that adherent cells were decreased in the cells treated with cisplatin, but it was confirmed that adhered cells were significantly increased in the control group using SIRT2 inhibitor (see FIGS. 8 and 9).

In addition, as shown in FIG. 10, when the cell proliferation test of the SIRT2 inhibitor was confirmed by the cell proliferation test, XTT test showed that the decrease of cell proliferation upon treatment with cisplatin resulted in the inhibition of the SIRT2 inhibitor AGK2 and AK-1 , Respectively.

The results showed that the inhibition of renal damage by cisplatin when the SIRT2 gene was absent was found to be effective for the protection of kidney, and it was confirmed that the regulation of SIRT2 gene expression was effective for acute renal injury treatment.

In the present invention, the term "antisense oligonucleotide" means a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA. The term " antisense oligonucleotide " . ≪ / RTI > The antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the gene and capable of binding to the mRNA, and the translation, mRNA translocation, maturation, or all other overall biological It is possible to inhibit the essential activity for the function.

In addition, the antisense nucleic acid can be modified at one or more bases, sugar or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5, 3, 343-55, 1995 ). The nucleic acid backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic biantennary bond, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. The antisense nucleic acid may comprise a modified base. Modified bases include hypoxanthane, 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, genentioyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine . In addition, the antisense nucleic acid of the present invention may be chemically combined with one or more moieties or conjugates that enhance the activity and cytotoxicity of the antisense nucleic acid. A cholesterol moiety, a cholesteryl moiety, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, hexylamino- And liposoluble moieties such as Cole sterol moieties. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nuclease and increase the binding affinity of the antisense nucleic acid with the target mRNA.

The antisense oligonucleotides may be synthesized in vitro in a conventional manner and administered in vivo or may allow the synthesis of antisense oligonucleotides in vivo. An example of synthesizing an antisense oligonucleotide in a test tube is to use RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

In the present invention, "RNAi" refers to RNA interference, and in Korean, it means RNA interference. RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. It is thought to be a type of gene monitoring mechanism used by cells to suppress defenses against transfection, to inhibit transposon, or to remove abnormal mRNA. In particular, gene suppression by small RNA is referred to as RNA interference in a broad sense, and RNA interference in the narrow sense implies mRNA degradation by siRNA. RNA interference also implies a technique for gene silencing using siRNA.

The term "siRNA" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Publication Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409 and 00/44914). siRNA is provided as an efficient gene knock-down method or gene therapy method since it can inhibit the expression of a target gene.

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to the mRNA sequence of the marker gene) and an antisense strand (a sequence complementary to the mRNA sequence) are positioned on opposite sides to form a double strand, , The siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to the complete pairing of double-stranded RNA moieties in RNA pairs, but may be mated by mismatch (corresponding bases are not complementary), bulge (no bases corresponding to one strand) And a portion that is not achieved may be included. In addition, the siRNA terminal structure can be blunt or cohesive at both ends, if the expression of the marker gene can be inhibited by the RNAi effect, and the adhesive terminal structure has a 3'-terminal protruding structure and a 5'- Both end protruding structures are possible.

In addition, the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence is Thereby forming a hairpin structure, which in turn forms a stem-and-loop structure. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The siRNA of the present invention can be prepared by directly synthesizing siRNA in a test tube, introducing the siRNA into a cell through a transformation process, and a siRNA expression vector or PCR-derived siRNA expression cassette, There is a method of transforming or infecting into a cell.

The term "aptamer" in the present invention refers to an oligonucleotide molecule having a binding activity to a given target molecule. An aptamer can inhibit the activity of a given target molecule by binding to a predetermined target molecule.

The aptamers of the present invention may be RNA, DNA, modified oligonucleotides or mixtures thereof. The aptamers of the present invention may also be in the form of straight chain or cyclic. The length of the aptamer of the present invention is not particularly limited and may be usually 15 to 200 nucleotides, but is, for example, 15 to 100 nucleotides, preferably 18 to 80 nucleotides, more preferably 20 to 60 nucleotides, Lt; RTI ID = 0.0 > nucleotides. ≪ / RTI > When the total number of nucleotides is small, the chemical synthesis, chemical modification and mass production are easier, economical, and in vivo, the toxicity is low because of high stability.

In addition, the SIRT2 inhibitor of the present invention includes a SIRT2 protein activity inhibitor. Examples of such a protein activity inhibitor include an antibody, a single chain variable region fragment, a peptide, a low molecular weight compound or a natural extract that specifically binds to SIRT2 can do.

The antibody that specifically binds to and inhibits the activity of the SIRT2 protein that can be used in the present invention is a polyclonal or monoclonal antibody. Antibodies against the SIRT2 protein can be generated using methods conventionally practiced in the art such as the fusion method (Kohler et al., European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method 4,816,56) or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 . ≪ / RTI > A general procedure for antibody preparation is described in Harlow, E. et al., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art . Polyclonal antibodies can be obtained by injecting a SIRT2 protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

In the present invention, the antibody may comprise a single chain variable region segment (scFv). The single-stranded variable region fragment may be composed of "variable-region (VL) of light chain-variable-region (VH) of linker-double-stranded ". The linker refers to a constant-length amino acid sequence that artificially connects the variable region of the heavy chain and the light chain.

According to another preferred embodiment of the present invention, an anticancer adjuvant containing an SIRT2 inhibitor having an inhibitory activity against renal toxicity induced by an anticancer agent as an active ingredient is provided, and the above-mentioned problem is solved.

According to another preferred embodiment of the present invention, there is provided a health functional food for preventing or ameliorating a renal toxic disease caused by an anticancer agent comprising SIRT2 inhibitor as an active ingredient, thereby solving the above-mentioned problem.

The present invention provides a health functional food for preventing and improving a renal toxic disease caused by an anticancer agent including a SIRT2 inhibitor and a pharmaceutically acceptable food additive. Foods to which the SIRT2 inhibitor of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

&Quot; Health functional food "as defined in the present invention means food prepared and processed by using a raw material or ingredient having functionality useful to the human body according to Law No. 6727 on Health Functional Food, and" Of the structure and function of the nutrient to control the physiological effects, such as to obtain a beneficial effect for health is intended to eat.

Furthermore, it can be added to foods or beverages for the purpose of preventing the nephrotoxic diseases caused by anticancer drugs. At this time, the amount of the extract in the food or beverage may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, have.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.

For example, the health functional food of the tablet form may be prepared as it is, or it may be prepared by putting an excipient, a binder, a disintegrant, or other additives into the granules by an appropriate method, Functional food may be prepared by directly compressing the mixture of the food or by mixing directly with an excipient, binder, disintegrant or other suitable additive, or by mixing the preformed granules with the proper food additives or the appropriate additives, Add an excipient, binder or other suitable additive to the functional food, wet the powder evenly with a solvent, mold the wet powder into a low-pressure mold, and dry it by a suitable method. In addition, a mating agent or the like may be added to the health functional food in the form of tablets, if necessary.

The hard capsule of the capsule type health functional food is usually prepared by mixing the capsule with an appropriate excipient such as a health functional food or a health functional food or by granulating it in a suitable manner or by granulating it into a granular form suitable for granulation, The soft capsules are usually capsules containing an excipient suitable for a health functional food or a health functional food. The capsules are encapsulated in a suitable capsule base such as gelatin or the like with glycerin or sorbitol, And if necessary, a coloring agent preservative or the like may be added to the capsule base.

The ring type health functional food is usually prepared by mixing an excipient, a binder and a disintegrant in an ordinary healthy functional food, shaping it into a spherical form by an appropriate method, and then adding a gelatin to starch or other suitable skin care agent, It may also be an objectionable substance.

The granular health functional foods are usually prepared by mixing the healthy functional food as it is or by adding the excipient, binder, disintegrant, etc. to the health functional food and then making it into granular form by proper method and evenly grinding the granular material as needed. , A mating agent, and the like. In the next granularity test using granules 12 (1680 ㎛), 14 (1410 ㎛) and 45 (350 ㎛) sieve, the whole of the 12th trough passed through the granule, And not more than 5.0% of the total amount and not more than 15.0% of the total amount.

The definitions of the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents and the like of the present invention are described in documents known in the art, and their functions and the like are the same or similar (Korean Pharmacopoeia, , Council of Korean Pharmacy College, 5th ed., P. 33-48, 1989).

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned SIRT2 inhibitor as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the SIRT2 inhibitor of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the compounds of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

As a result, the SIRT2 inhibitor according to the present invention has the effect of reducing the renal toxicity, which is a side effect of cisplatin when administered in combination with cisplatin, and enhancing the anticancer efficacy. Therefore, the medicines for prevention and treatment of cancer diseases and the prevention and improvement of cancer diseases It can be used as a health functional food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[ Example ]

Example  1: Preparation of experimental animals

The experimental animals used in the present invention were SIRT2 - / - mice (Jackson laboratory, USA), mice lacking SIRT2 protein at 8-10 weeks of age and SIRT2 + / + mice (C57BL / 6, Orient, Korea), standard laboratory feeds and water were arbitrarily provided to the experimental animals, and were maintained in accordance with protocols approved by Chonbuk National University Animal Experimental Ethics Committee.

1-1. Preparation of experimental animals

The experimental animals were divided into four groups as shown in Table 1 below.

group SIRT2 gene Cisplatin (20 [mu] g / kg) Vehicle One + / + - + 2 + / + + - 3 - / - - + 4 - / - + -

2) normal control animals (WT) were treated with cisplatin (20 μg / kg), and 3) control animals without SIRT2 protein Mice treated with vehicle (KO) and control mice treated with cisplatin (20 μg / kg) in mice lacking SIRT2 protein (KO). The control buffer and cisplatin were intraperitoneally injected in sterile physiological saline (100 μl of 0.9% NaCl).

1-2. Preparation of samples

The animals or groups of animals receiving the vehicle or cisplatin were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg) three days after the administration to collect blood and kidney tissues.

More specifically, the blood collection was performed by anesthetizing the experimental animals with 100 mg / kg of ketamine and 10 mg / kg of xylazine, and then collecting blood by cardiac puncture. The kidney tissues were collected after being divided into quarters and quadrants were fixed in 4% paraformaldehyde and fixed periodic acid-Schiff (PAS). The other two fractions were extracted with protein.

Example  2: SIRT2 In gene-deficient mice To cisplatin  Of kidney damage

2-1. Check kidney function

First, in order to examine the effect of SIRT2 gene expression on renal toxicity by cisplatin, renal function and degree of damage were confirmed by administering cisplatin to SIRT2 gene-deficient mice.

As shown in Example 1-2, BUN and creatinine were separated from the mouse using a centrifuge, and then analyzed using an automatic analyzer (Hitachi 7180; Tokyo, Japan). The results are shown in Fig. 1 and Fig.

As shown in Figs. 1 and 2, when cisplatin was administered to mice, kidney damage was induced and BUN and creatinine were increased. In addition, it was confirmed that in mice lacking SIRT2, BUN and creatinine increased significantly by cisplatin.

2-2. Check kidney damage

PAS staining was performed to confirm the histological damage of the kidney due to cisplatin administration.

The damaged kidney tissue was visualized using a Zeiss Z1 microscope through PAS staining. The results are shown in Fig.

As shown in FIG. 3, in the kidney of the control group treated with cisplatin in the SIRT2 gene WT (SIRT2 + / +) mouse, tissue damage such as epithelial cell detachment, loss of brush border and tubular cast formation was observed, (SIRT2 < - > / -) mice significantly reduced these kidney damage.

2-3. Confirm survival rate

As shown in FIG. 4, in the control group, all mice died after 6 days of administration of cisplatin, but survival rate was also increased by confirming death at 8 days in the group treated with cisplatin in the SIRT2 gene-deficient mouse I could.

This suggests that inhibition of renal damage by cisplatin when the SIRT2 gene is absent is suppressed.

Example  3: SIRT2 Cell death by gene expression apoptosis ) Control effect

Generally, the damage of kidney by cisplatin is affected by apoptosis. The acetylation of caspase-3 and p53 was confirmed by western blotting in order to examine the effect of the SIRT2 gene on cell death control by cisplatin.

The expression of caspase-3 and acetylp53 protein was measured using caspase-3 and acetyl p53 antibody (Cell Signaling Technology, Denvers, MA, USA) after obtaining kidney tissues by the method of Example 1-2, . The same blot was peeled off and confirmed by Actin (Sigma Aldrich, St Louis, MO, USA) and p53 (Cell Signaling Technology). The results are shown in Fig. 5 and Fig.

As shown in FIG. 5 and FIG. 6, cleaved caspase-3 increased in the group treated with cisplatin in WT mice, and it was confirmed that the expression of acetyl p53 was also decreased in SIRT2 gene-deficient mice I could.

This suggests that the lack of SIRT2 gene reduces apoptosis in mouse kidney tissue, which is regulated by the p53 acetylation pathway.

Example  4: SIRT2 By gene expression Inflammatory molecule  Moderate effect

We confirmed the expression of the ICAM-1 and VCAM-1 genes, which are involved in the inflammatory response, in the experimental group treated with cisplatin by the SIRT2 gene.

Western blotting was performed using ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody in the same manner as in Example 3 above. The results are shown in Fig.

As shown in FIG. 7, it was confirmed that the expression of ICAM-1 and VCAM-1 genes, which were increased when cisplatin was administered to WT mice, decreased when cisplatin was administered to mice lacking SIRT2 gene.

Example  5: SIRT2 Effect of Inhibitor on Cell Damage

The effects of SIRT2 inhibitors AGK2 and AK-1 on cisplatin-treated mouse proximal tubular cells were examined to confirm cytotoxic inhibitory effects of cisplatin.

Specifically, mouse proximal tubular cells were treated with SIRT2 inhibitor AGK2 (10 μM) and AK-1 (10 μM) 30 minutes before treatment with cisplatin, treated with cisplatin (20 μg / . The results are shown in Fig. 8 and Fig.

As shown in FIGS. 8 and 9, it was confirmed that adherent cells were decreased in the cells treated with cisplatin, but it was confirmed that adhered cells were significantly increased in the control group using SIRT2 inhibitor.

In addition, cell proliferation test confirmed the cell proliferation effect of SIRT2 inhibitor.

Cell proliferation was examined by trypsinization of mouse proximal tubular cells and 1 × 10 3 cells were added to each well of a 96-well plate. After culturing for 24 hours at 37 ° C., the culture solution was removed, and the cells were incubated with AGK2, AK -1, cisplatin, cisplatin and AGK2 and AK-1, respectively. Treatment After 24 hours of incubation at 37 ° C, cell proliferation was measured using Cell Proliferation Kit II (XTT, Roche, Mannheim, Germany). The proliferation of mouse proximal tubular cells was measured by absorbance, and the result is shown in FIG. 10, since the absorbance increases with increase of chromogenic material in intracellular mitochondria as the cells proliferate.

As shown in FIG. 10, when XTT test was performed, it was confirmed that decreased cell proliferation when treated with cisplatin significantly increased when SIRT2 inhibitors AGK2 and AK-1 were treated together.

As a result, it was confirmed that when the SIRT2 gene is absent, the kidney protective effect is suppressed by inhibiting the cisplatin-induced kidney damage. Thus, it is confirmed that the regulation of SIRT2 gene expression is effective for acute renal injury treatment.

Statistical analysis

Data are presented as mean ± standard deviation. Anova (ANOVA) was used for statistical significance (p <0.05).

Therefore, the SIRT2 inhibitor according to the present invention suppresses apoptosis and regulates the expression of ICAM-1 and VCAM-1, which are inflammatory reaction related factors, thereby suppressing renal damage caused by the anticancer drug cisplatin and reducing renal toxicity And by enhancing the anticancer efficacy of an anticancer agent when the anticancer agent is used in combination with an anticancer agent, it is useful as a pharmaceutical composition or a health functional food for preventing and treating a renal toxic disease caused by an anticancer agent Can be used.

Claims (7)

A pharmaceutical composition for the prevention and treatment of renal toxic diseases caused by an anticancer agent comprising SIRT2 inhibitor as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the anticancer agent is cisplatin. 2. The pharmaceutical composition according to claim 1, wherein the anticancer agent is cisplatin. The method according to claim 1, wherein the SIRT2 inhibitor comprises at least one member selected from the group consisting of an antisense oligonucleotide, an siRNA, an aptamer or an antibody specific to the SIRT2 gene. And a pharmaceutical composition for therapeutic use. The pharmaceutical composition according to claim 1, wherein the SIRT2 inhibitor comprises at least one member selected from the group consisting of AGK2 or AK-1. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits renal injury by inhibiting the expression of ICAM-1 and VCAM-1, molecules involved in apoptosis and inflammatory response. A pharmaceutical composition for the prevention and treatment of toxic diseases. An anticancer adjuvant comprising an SIRT2 inhibitor having an inhibitory activity against renal toxicity induced by an anticancer drug as an active ingredient. A health functional food for preventing or ameliorating a renal toxic disease caused by an anticancer agent containing SIRT2 inhibitor as an active ingredient.

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