Adenosine diphosphate ribosyl cyclase and its inhibitors

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a disease mediated by adenosine diphosphate ribose cyclase, comprising an expression inhibitor or an activity inhibitor of adenosine diphosphate ribose cyclase or a naturally occurring variant thereof as an effective ingredient. In addition, the present invention relates to a composition for diagnosing a disease mediated by adenosine diphosphate ribose cyclase, comprising a preparation for detecting the gene expression level of the above-mentioned adenosine diphosphate ribose cyclase or a naturally occurring variant thereof or a preparation for detecting the protein level. The composition of the present invention has the effect of inhibiting the increase in calcium in kidney cells caused by the increase in the expression or activity of adenosine diphosphate ribose cyclase through angiotensin II, and therefore, can be effectively used as a therapeutic agent for adenosine diphosphate ribose cyclase-mediated diseases, especially kidney diseases.

Description

Technical Field

[0001] The present invention relates to the use of nicotinamide adenine dinucleotide (NAD + A novel adenosine diphosphate ribose cyclase that converts 2-amino-4-adenosine diphosphate into cyclic adenosine diphosphate ribose and its inhibitors. Background Art

[0002] Adenosine diphosphate ribosyl cyclase (ADPRC) is a cytosine cyclase that converts nicotinamide adenine dinucleotide (NAD + ) synthesizes cyclic ADP-ribose (cADPR) from nicotinamide adenine dinucleotide phosphate (NADP + ) is an enzyme that synthesizes nicotinic acid adenine dinucletide phosphate (NAADP) from ribonucleotides, and is widely found in organisms from plants to mammals. It is known that the above-mentioned adenosine diphosphate ribosyl cyclase regulates various cellular functions by releasing calcium from intracellular calcium stores to the cytoplasm [Berridge MJ, Bootman MD, Roderick HL. NatRev Mol Cell Biol. 4, 517-529, 2003; Lee HC, Mol. Med. 12 317-323, 2006].

[0003] In the steady state, the intracellular calcium concentration is maintained at 10 -7 However, when cells are activated, the intracellular calcium concentration increases to about 10 times the basal value. Under pathological conditions, the continued high calcium concentration will affect many functions of the cells, resulting in the disappearance of normal cell functions.

[0004] In hypertension or diabetes mellitus complicated by hypertension, obesity, dementia, ischemia, and diseases complicated by cell proliferation, intracellular calcium metabolism is abnormally regulated and the intracellular calcium concentration is higher than normal [Resnick LM. Am. J. Hypertens. 6:123S-134, 1993].

[0005] For example, when observing patients with chronic diseases such as hypertension, it was found that due to various genetic, environmental or secondary causes of certain diseases, the calcium concentration in vascular smooth muscle cells continued to show values ​​higher than normal, which resulted in increased resistance of peripheral blood vessels inducing contraction of vascular smooth muscle, thus causing blood pressure to continue to remain higher than normal blood pressure, thereby causing hypertension.

[0006] It is known that the resulting increase in blood pressure secondarily causes proliferation and hypertrophy of vascular smooth muscle cells and fibrous tissue, which in turn further increases peripheral vascular resistance and blood pressure, inhibits the function of organs such as the cardiovascular system, brain and kidneys, and may even cause death due to myocardial infarction, angina pectoris, stroke and chronic heart failure [Cowley AW Jr. Physiol Rev. 72:231-300, 1992].

[0007] It is known that in the process of insulin secretion from the pancreas, CD38 activity, a type of ADP-ribosyl cyclase, plays an important role through calcium increase that cyclizes ADP-ribosyl. In addition, it is known that the onset of diabetes increases the production of angiotensin, a peptide hormone that not only induces kidney disease, hypertension, and heart disease, but also activates ADP-ribosyl cyclase.

[0008] Therefore, ADP-ribosyl cyclase exists in immune cells, cardiomyocytes, pancreatic β cells, extensor cells and nerve cells, and is associated with receptors of multiple hormones, increasing intracellular calcium concentration and regulating multiple physiological activities. If the expression or activity regulation of the above-mentioned ADP-ribosyl cyclase is abnormal, it will cause abnormal regulation of physiological activities (immunity, kidney function, insulin secretion, cardiovascular function).

[0009] The above-mentioned ADP-ribose cyclase can be activated by various hormones, and the enzyme generates cyclic ADP-ribose as a product from nicotinamide adenine dinucleotide as a substrate, and the above-mentioned cyclic ADP-ribose increases intracellular calcium in almost all organs. Furthermore, it is known that the abnormal increase of calcium caused by the activity of ADP-ribosyl cyclase is a factor of various in vivo pathologies such as insulin secretion [Kim BJ, Park KH, Yim CY, Takasawa S, Okamoto H, Im MJ, Kim UH. Diabetes. 57: 868-878, 2008], cell hypertrophy [GulR, ParkJH, Kim SY, Jang KY, Chea JK, Ko JK, Kim UH. Cardiovasc Res. 81: 582-591, 2009], and cell proliferation [Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, Kim UH. Am J Physiol Renal Physiol. 294: F989-F989, 2008].

[0010] To date, the most thoroughly studied ADP-ribosyl cyclase is CD38, a T-cell surface antigen. This molecule is expressed not only on immune cells but also widely in various organs.

[0011] However, it has been found that adenosine diphosphate ribosyl cyclase, which is different from CD38, is expressed in the heart, kidney, and brain [Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL, Oppenheimer N, Garvy B, Kusser K, Goodrich S, Howard M, Harmsen A, Randall TD, Lund FE. Nat Med 7:1209-16, 2001].

[0012] Therefore, regulating the expression of tissue-specific ADP-ribosyl cyclase will help treat diseases induced by increased intracellular calcium. In addition, the mechanism of ADP-ribosyl cyclase activity is not yet fully understood.

[0013] The description in the above background technology is only used to enhance the understanding of the background of the present invention and cannot be recognized as equivalent to the prior art known to ordinary technicians in the technical field to which the present invention belongs. Summary of the invention

[0014] Technical issues

[0015] The present inventors have discovered a new type of ADP-ribosyl cyclase that is different from the previously known ADP-ribosyl cyclases (CD38 and CD157) in mammals and confirmed its activity, thereby completing the present invention.

[0016] Therefore, an object of the present invention is to provide a novel adenosine diphosphate ribosylcyclase (ADP-ribosylcyclase; ADPRC) or a naturally occurring variant thereof.

[0017] Another object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned ADP-ribose cyclase or a naturally occurring variant thereof.

[0018] Another object of the present invention is to provide a vector comprising the above nucleic acid molecule.

[0019] Another object of the present invention is to provide a host cell comprising the above vector.

[0020] Another object of the present invention is to provide a catalyst composition for converting nicotinamide adenine dinucleotide into cyclized adenosine diphosphate ribose, comprising: the above-mentioned adenosine diphosphate ribose cyclase or its naturally occurring variant, a nucleic acid molecule encoding the same, or a vector comprising the above-mentioned nucleic acid molecule.

[0021] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases mediated by ADP-ribose cyclase, comprising an expression inhibitor or activity inhibitor of the above-mentioned ADP-ribose cyclase or its naturally occurring variant as an active ingredient.

[0022] Another object of the present invention is to provide a food composition for preventing or improving diseases mediated by ADP-ribose cyclase, comprising an expression inhibitor or activity inhibitor of the above-mentioned ADP-ribose cyclase or its naturally occurring variant as an effective ingredient.

[0023] Another object of the present invention is to provide an animal model lacking the above-mentioned heterozygous gene of adenosine diphosphate ribose cyclase or its naturally occurring variant.

[0024] Another object of the present invention is to provide a method for providing information related to the diagnosis of diseases mediated by ADP-ribosylcyclase.

[0025] Another object of the present invention is to provide a composition for diagnosing diseases mediated by ADP-ribose cyclase, comprising a preparation for detecting the gene expression level of the above-mentioned ADP-ribose cyclase or its naturally occurring variants or a preparation for detecting the protein level.

[0026] Another object of the present invention is to provide a diagnostic kit for diseases mediated by ADP-ribose cyclase, comprising a preparation for detecting the gene expression level of the ADP-ribose cyclase or its naturally occurring variants or a preparation for detecting the protein level.

[0027] Another object of the present invention is to provide a method for screening substances for preventing or treating the above-mentioned diseases mediated by ADP-ribose cyclase.

[0028] Other objects and advantages of the present invention will be further clarified through the following detailed description of the present invention, the scope of protection of the invention and the accompanying drawings.

[0029] Solutions to the problem

[0030] According to one embodiment of the present invention, the present invention provides an ADP-ribose cyclase or a naturally occurring variant thereof, comprising the amino acid sequence of sequence 1 in the sequence list.

[0031] The present inventors have devoted themselves to discovering an enzyme that converts nicotinamide adenine dinucleotide into cyclized adenosine diphosphate ribose, that is, a novel adenosine diphosphate ribose cyclase different from the currently known adenosine diphosphate ribose cyclase, thereby completing the present invention.

[0032] In the present specification, the term "adenosine diphosphate ribose cyclase" or "ADP-ribosyl cyclase (ADPRC)" refers to an enzyme that converts nicotinamide adenine dinucleotide into cyclized adenosine diphosphate ribose.

[0033] In the present specification, the term "variant of ADP-ribose cyclase" refers to a variant in which a part of the amino acid sequence of the above-mentioned ADP-ribose cyclase is naturally or artificially substituted, deleted, or added, and refers to a variant of ADP-ribose cyclase that retains the catalytic activity of converting nicotinamide adenine dinucleotide into cyclized ADP-ribose.

[0034] In the present specification, the term "naturally occurring variant of ADP-ribose cyclase" refers to a naturally occurring variant of ADP-ribose cyclase comprising the amino acid sequence of Sequence 1 in the above sequence list, and refers to a variant of ADP-ribose cyclase that retains the catalytic activity of converting nicotinamide adenine dinucleotide into cyclized ADP-ribose.

[0035] According to a preferred embodiment of the present invention, the naturally occurring variants of the adenosine diphosphate ribose cyclase of the present invention are naturally occurring variants selected from the group consisting of species variants, species homologs, isoforms, allelic variants, conformation variants, splice variants and point mutant variants.

[0036] Preferably, the naturally occurring variant of the ADP-ribose cyclase of the present invention has more than 50% homology with the ADP-ribose cyclase comprising the amino acid sequence of sequence 1 in the sequence list, more preferably, more than 60% homology, even more preferably, more than 70% homology, further more preferably, more than 80% homology, and most preferably, more than 90% homology.

[0037] According to a preferred embodiment of the present invention, the naturally occurring variant of the ADP-ribose cyclase of the present invention occurs in an organism selected from the group consisting of mammals, birds, reptiles, amphibians and fish.

[0038] According to one embodiment of the present invention, it is confirmed that the naturally occurring variant of the ADP-ribose cyclase of the present invention has more than 70% homology in mammals, more than 60% homology in birds, reptiles and amphibians, and more than 50% homology in fish compared to the ADP-ribose cyclase comprising the amino acid sequence of sequence 1 in the sequence table.

[0039] According to one embodiment of the present invention, the naturally occurring variant of the adenosine diphosphate ribose cyclase of the present invention shows 96% homology with humans and rats, 93% homology with chimpanzees, 91% homology with guinea pigs and horses, 90% homology with dogs, goats and sheep, 89% homology with rabbits, 88% homology with pigs, 78% homology with cattle, 70% homology with chickens, 63% homology with frogs, and 62% homology with turkeys compared with the adenosine diphosphate ribose cyclase comprising the amino acid sequence of sequence 1 in the sequence table, and thus it is confirmed that it is an enzyme with a high conserved sequence between species ( Figure 4 ).

[0040] According to a preferred embodiment of the present invention, the naturally occurring variant of the ADP-ribose cyclase of the present invention comprises an amino acid sequence selected from the group consisting of Sequence 2 to Sequence 21 in the sequence list.

[0041] According to another embodiment of the present invention, the present invention provides a nucleic acid molecule encoding the above-mentioned ADP-ribose cyclase or a naturally occurring variant thereof, a vector comprising the above-mentioned nucleic acid molecule, or a host cell comprising the above-mentioned vector.

[0042] The nucleic acid molecules of the present invention may be isolated or recombinant, and include not only deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in single-stranded and double-stranded form, but also corresponding complementary sequences. In the case where an "isolated nucleic acid" is a nucleic acid isolated from a natural source, the nucleic acid is a nucleic acid isolated from surrounding gene sequences present in the genome of the individual from which the nucleic acid is isolated. In the case of nucleic acids synthesized from a template by enzymatic or chemical methods, such as polymerase chain reaction (PCR) products, complementary DNA (cDNA) molecules or oligonucleotides, nucleic acids generated by such processes may also be understood as isolated nucleic acid molecules. An isolated nucleic acid molecule is a nucleic acid molecule that appears in the form of a separate fragment or as a component of a larger nucleic acid construct. When a nucleic acid forms a functional relationship with other nucleic acid sequences, an "operably linked" is formed. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein in the form of a secreted polypeptide, it is operably linked to the DNA of the polypeptide, if a promoter or enhancer affects the transcription of the polypeptide sequence, it is operably linked to the coding sequence, or when a ribosome binding site is configured to promote translation, it is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequences being linked are adjacently configured, and in the case of a secretory leader, it means that they are adjacent and present in the same reading frame. However, enhancers do not necessarily have to be adjacently configured. The connection is achieved by connecting at a convenient restriction enzyme site. If such a site does not exist, a synthetic oligonucleotide linker or adapter can be used according to the usual method.

[0043] According to a preferred embodiment of the present invention, the nucleic acid molecule is complementary DNA.

[0044] In this specification, the term "vector" refers to a transfer body into which a nucleic acid sequence can be inserted in order to be introduced into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence can be exogenous or heterologous. The vector can be a plasmid, a cosmid, and a virus (e.g., a bacteriophage), but is not limited thereto. A person of ordinary skill in the art to which the present invention belongs can construct a vector by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0045] In this specification, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. An expression vector may contain a variety of regulatory sequences. Along with regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.

[0046] In this specification, the term "host cell" includes eukaryotic organisms and prokaryotes, and refers to any transformable organism that can replicate the above-mentioned vector or can express the gene encoded by the vector. The host cell can be transfected or transformed by the above-mentioned vector, which refers to the process of transferring or introducing exogenous nucleic acid molecules into the host cell.

[0047] Preferably, the host cell of the present invention can be a bacterial cell, a yeast, an animal or a human cell (Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, NIH3T3 cell, etc.), but is not limited thereto.

[0048] According to another embodiment of the present invention, the present invention provides a catalyst composition for converting nicotinamide adenine dinucleotide into cyclized adenosine diphosphate ribose, comprising: the above-mentioned adenosine diphosphate ribose cyclase or its naturally occurring variant, a nucleic acid molecule encoding the same, or a vector comprising the above-mentioned nucleic acid molecule.

[0049] The catalyst composition of the present invention effectively catalyzes the process of converting nicotinamide adenine dinucleotide into cyclic adenosine diphosphate ribose.

[0050] According to another embodiment of the present invention, the present invention provides a composition comprising the above-mentioned expression inhibitor or activity inhibitor of adenosine diphosphate ribose cyclase or its naturally occurring variant.

[0051] According to a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition for preventing or treating diseases mediated by ADP-ribose cyclase.

[0052] The pharmaceutical composition of the present invention may comprise: (a) the above-mentioned expression inhibitor or activity inhibitor; and (b) a pharmaceutically acceptable carrier.

[0053] According to a preferred embodiment of the present invention, the composition of the present invention is a food composition for preventing or improving diseases mediated by ADP-ribose cyclase.

[0054] According to another embodiment of the present invention, the present invention provides a method for preventing or treating a disease mediated by ATP-ribose cyclase, comprising the step of administering a pharmaceutically effective amount of the above-mentioned pharmaceutical composition to a subject.

[0055] According to another embodiment of the present invention, the present invention provides an expression inhibitor or activity inhibitor of the above-mentioned ADP-ribose cyclase or its naturally occurring variant for use in therapy.

[0056] The types of ADP-ribose cyclase-mediated diseases to be prevented or treated by the present invention are not limited, and are preferably diabetes or kidney disease, and more preferably renal failure, nephropathy, nephritis, renal fibrosis or nephrosclerosis.

[0057] According to a preferred embodiment of the present invention, the renal failure is chronic renal failure, acute renal failure or mild renal failure before dialysis.

[0058] According to a preferred embodiment of the present invention, the renal disease is nephropathy syndrome, lipoid nephropathy, diabetic nephropathy, IgA nephropathy, analgesic nephropathy or hypertensive nephropathy.

[0059] According to one embodiment of the present invention, the composition of the present invention is confirmed to be a candidate for a therapeutic agent for kidney diseases including chronic renal failure or diabetic nephropathy by significantly reducing kidney weight relative to body weight, significantly increasing creatinine clearance, and significantly reducing urine albumin ( FIG. 6 ); and reducing the activity of adenosine diphosphate ribose cyclase and the concentration of cyclized adenosine diphosphate ribose to normal levels ( Figure 7 ); reduce the expression of transforming growth factor-β1 (TGF-β1), fibronectin (Fibronectin), and collagen IV (Collagen IV) to normal levels ( Figure 8 ); It also retains the ability to restore the histopathological changes of the kidney by restoring glomerular hypertrophy, infiltration of inflammatory cells, and formation of transitional epithelial cells to normal levels ( Fig. 9); The possibility of being applicable to hypertensive nephropathy was also confirmed by significantly increasing creatinine clearance in a hypertension model ( Fig.11b ), has the potential to be a therapeutic agent for various kidney diseases caused by abnormal kidney function or damage.

[0060] In the present specification, the term "expression inhibitor" refers to a substance that inhibits the expression of the above-mentioned adenosine diphosphate ribose cyclase, and a person skilled in the art of the present invention can easily prepare it according to the structure and function of the above-mentioned adenosine diphosphate ribose cyclase.

[0061] Preferably, the expression inhibitor of the present invention is one selected from the group consisting of antisense oligonucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozyme, deoxyribozyme and peptide nucleic acid (PNA) that complementarily bind to the messenger RNA (mRNA) of the above-mentioned adenosine diphosphate ribose cyclase or its naturally occurring variant gene, but is not limited thereto.

[0062] According to a preferred embodiment of the present invention, the above-mentioned small interfering RNA (siRNA) comprises the nucleotide sequence of sequence 22 in the sequence list.

[0063] In the present specification, the term "activity inhibitor" refers to a substance that inhibits the activity of the above-mentioned expressed ADP-ribose cyclase protein, preferably, one selected from the group consisting of compounds, peptides, peptide analogs (mimetics), aptamers and antibodies that specifically bind to the above-mentioned ADP-ribose cyclase or its naturally occurring variant protein.

[0064] According to a preferred embodiment of the present invention, the compound is a compound selected from the group consisting of 4,4'-dihydroxyazobenzene, 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazoline-4(1H)-one and Dicaffeoylquinic acid (DCQA).

[0065] Preferably, the 4,4′-dihydroxyazobenzene of the present invention may be represented by Chemical Formula 1 below.

[0066] Chemical formula 1

[0067]

[0068] Preferably, the 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazolin-4(1H)-one of the present invention may be represented by the following Chemical Formula 2.

[0069] Chemical formula 2

[0070]

[0071] The dicaffeoylquinic acid of the present invention may be a compound represented by a chemical formula selected from the group consisting of the following Chemical Formulas 3 to 8 (1,4-DCQA, 3,4-DCQA, 3,5-DCQA, 4,5-DCQA, 1,3-DCQA, and 1,5-DCQA).

[0072] Chemical formula 3

[0073]

[0074] Chemical formula 4

[0075]

[0076] Chemical formula 5

[0077]

[0078] Chemical formula 6

[0079]

[0080] Chemical formula 7

[0081]

[0082] Chemical formula 8

[0083]

[0084] According to one embodiment of the present invention, it was confirmed that the novel ADP-ribose cyclase expression inhibitor or activity inhibitor of the present invention plays a role in suppressing kidney diseases through a mechanism of regulating the expression or activity of ADP-ribose cyclase.

[0085] Therefore, the novel inhibitor of expression or activity of adenosine diphosphate ribosyl cyclase of the present invention can be effectively used clinically as a selective inhibitor of each different cell, and further can be used as a preventive, ameliorative and / or therapeutic agent for kidney diseases.

[0086] The composition of the present invention can be used not only with the novel ADP-ribosylcyclase expression inhibitor or activity inhibitor as an active ingredient but also with a conventionally known therapeutic agent for ADP-ribosylcyclase-related diseases.

[0087] The pharmaceutical composition of the present invention can be formulated into an appropriate form together with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a carrier that is physiologically acceptable and generally does not cause digestive tract disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.

[0088] The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention can be a carrier commonly used in preparation, including lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but not limited thereto. In addition to the above-mentioned ingredients, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Appropriate pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0089] The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably, parenterally, for example, by intravenous injection, local injection, intraperitoneal injection, etc.

[0090] The appropriate dosage of the pharmaceutical composition of the present invention can vary according to factors such as the preparation method, administration method, patient's age, weight, sex, disease state, diet, administration time, administration route, metabolic rate and reaction sensitivity, and can be easily determined or prescribed by a doctor with ordinary proficiency for the desired effective dosage for treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 mg / kg to 100 mg / kg.

[0091] The pharmaceutical composition of the present invention can be prepared in a single dose form or in a multi-dose container using pharmaceutically acceptable carriers and / or excipients according to methods that are easily implemented by those of ordinary skill in the art to which the present invention belongs. In this case, the dosage form can be in the form of a solution, suspension or emulsion in an oil or aqueous solvent, and can also be in the form of an extractant, powder, granule, tablet or capsule, and can also contain a dispersant or stabilizer.

[0092] When the composition of the present invention is prepared as a food composition, it not only contains the above-mentioned expression inhibitor or activity inhibitor of adenosine diphosphate ribose cyclase as an effective ingredient, but also contains ingredients usually added when preparing food, such as protein, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates include: monosaccharides, such as glucose, fructose, etc.; disaccharides, such as lactose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as maltodextrin and cyclodextrin, and sugar alcohols such as sorbitol and erythritol. Flavoring agents can use natural flavoring agents (thaumatin, stevia leaf extract (such as rebaudioside A, glycyrrhizic acid, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0093] For example, when the food composition of the present invention is prepared as a drink, in addition to the expression inhibitor or activity inhibitor of the adenosine diphosphate ribose cyclase of the present invention, it can also contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, eucommia extract, jujube extract, liquorice extract, etc.

[0094] The food composition of the present invention plays an excellent role in improving kidney diseases by regulating the relevant factors of various pathological forms related to the kidney (significant reduction of kidney weight relative to body weight, significant increase of creatinine clearance, significant reduction of urinary albumin; reducing the expression of transforming growth factor-β1, fibronectin, and collagen IV to normal levels; restoring glomerular hypertrophy, infiltration of inflammatory cells, and formation of transitional epithelial cells to normal levels).

[0095] According to another embodiment of the present invention, the present invention provides an animal model lacking an isoform of adenosine diphosphate ribose cyclase or a naturally occurring variant thereof.

[0096] According to a preferred embodiment of the present invention, the above-mentioned animal is a mammal, bird, reptile, amphibian or fish other than human.

[0097] According to another embodiment of the present invention, the present invention provides a method for identifying a disease mediated by ADP-ribose cyclase, comprising the following steps: step (a), inducing a specific disease in an animal model lacking an ADP-ribose cyclase or a naturally occurring variant thereof and a wild-type animal model; and step (b), confirming the differences between the above-mentioned animal models.

[0098] When using the above-mentioned animal models, the differences between the above-mentioned animal models (for example, differences in body weight, kidney weight, blood pressure, creatinine clearance, blood sugar, inflammation, degree of phage hypertrophy, etc.) can be used to confirm whether a specific disease (for example, diabetes, kidney disease, hypertension, obesity, etc.) is mediated by ADP-ribosylcyclase or occurs independently of ADP-ribosylcyclase.

[0099] According to another embodiment of the present invention, the present invention provides a method for providing information on the diagnosis of diseases mediated by ADP-ribose cyclase, comprising: step 1), detecting the expression or activity level of ADP-ribose cyclase or its naturally occurring variants containing the amino acid sequence of sequence 1 in the sequence list in a sample separated from a subject; and step 2), judging whether an individual has a risk of developing a cyclase-mediated disease by comparing the expression or activity level of ADP-ribose cyclase or its naturally occurring variants in the above step 1) with that of a normal control group.

[0100] In this specification, the term "diagnosis" refers to the act of confirming the presence or characteristics of a pathological state. For the purpose of the present invention, diagnosis refers to the act of confirming the occurrence or possibility of the occurrence of a disease related to or mediated by ADP-ribosylcyclase.

[0101] Adenosine diphosphate ribose cyclase is activated by various hormones, and the enzyme generates cyclic ADP ribose as a product from nicotinamide adenine dinucleotide as a substrate, and the cyclic ADP ribose increases intracellular calcium in almost all organs. In addition, it is known that the abnormal increase in calcium caused by the activity of ADP ribose cyclase is a factor of various living body pathologies such as insulin secretion, cell hypertrophy, and cell proliferation. Therefore, the expression or activity level of the above-mentioned ADP ribose cyclase can be detected to provide useful information for the diagnosis of ADP ribose cyclase-mediated diseases caused by increased intracellular calcium.

[0102] According to another embodiment of the present invention, the present invention provides a composition for diagnosing diseases mediated by adenosine diphosphate ribose cyclase, comprising a preparation for detecting the gene expression level of adenosine diphosphate ribose cyclase or its naturally occurring variants or a preparation for detecting the protein level, wherein the adenosine diphosphate ribose cyclase or its naturally occurring variants comprises the amino acid sequence of sequence 1 in the sequence table.

[0103] According to a preferred embodiment of the present invention, the preparation of gene expression level of the present invention is a primer or a probe that specifically binds to a gene encoding adenosine diphosphate ribose cyclase or a naturally occurring variant thereof.

[0104] According to a preferred embodiment of the present invention, the agent for detecting protein level of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to ADP-ribose cyclase or its naturally occurring variant.

[0105] Since the above-mentioned antibody or antigen-binding fragment thereof specifically binds to ADP-ribose cyclase or its naturally occurring variant, the amount of ADP-ribose cyclase or its naturally occurring variant contained in a sample can be accurately detected by using the antibody or its antigen-binding fragment.

[0106] If the diagnostic composition of the present invention is used, the antigen to the above-mentioned antibody can be analyzed by an antigen-antibody binding reaction, thereby quantifying the amount of adenosine diphosphate ribose cyclase or its naturally occurring variant. Preferably, the above-mentioned antigen-antibody binding reaction is selected from the group consisting of conventional enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, polyacrylamide gel protein blotting (Western Blot), immunoblot assay and immunohistochemical staining, but is not limited to this.

[0107] The immobilized body used for the antigen-antibody binding reaction may be a immobilized body selected from the group consisting of nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, a well plate synthesized from polyethylene (Polyvinyl) resin or polystyrene (Polystyrene) resin, and a slide glass (Slide glass), but is not limited thereto.

[0108] Preferably, the second antibody is labeled with a common color developer that produces a color development reaction, and a label selected from the group consisting of fluorescent substances and dyes such as horseradish peroxidase (HRP), alkaline phosphatase, colloidal gold, poly L-lysine-fluoresceinisothiocyanate (FITC), and rhodamine-B-isothiocyanate (RITC) can be used. Preferably, the substrate inducing color development is used according to the marker that undergoes the color development reaction, preferably, one selected from the group consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD), but not limited thereto.

[0109] According to another embodiment of the present invention, the present invention provides a diagnostic kit for diseases mediated by adenosine diphosphate ribose cyclase, comprising a preparation for detecting the gene expression level of adenosine diphosphate ribose cyclase or its naturally occurring variants or a preparation for detecting the protein level, wherein the adenosine diphosphate ribose cyclase or its naturally occurring variants comprises the amino acid sequence of sequence 1 in the sequence table.

[0110] The diagnostic kit of the present invention may further include one or more other structural component compositions, solutions or devices suitable for the analytical method, or instructions for use thereof.

[0111] According to another embodiment of the present invention, the present invention provides a method for screening substances for preventing or treating diseases mediated by adenosine diphosphate ribose cyclase: step 1), using a candidate substance to treat cells expressing adenosine diphosphate ribose cyclase or a naturally occurring variant thereof that contains an amino acid sequence of sequence 1 in a sequence list; step 2), detecting the gene expression level or protein level of the adenosine diphosphate ribose cyclase or a naturally occurring variant thereof by treatment with the above-mentioned candidate substance; and step 3), comparing the above-mentioned gene expression level or protein level with that of a control group that has not been treated with the candidate substance, and if it is reduced, the above-mentioned candidate substance is screened as a substance for preventing or treating diseases mediated by adenosine diphosphate ribose cyclase.

[0112] Preferably, the gene expression level of the above-mentioned adenosine diphosphate ribose cyclase or its naturally occurring variant is detected by one or more methods selected from the group consisting of immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, real-time polymerase chain reaction (RT-PCR), Western blotting and flow cytometry fluorescence sorting (FACS), but is not limited thereto.

[0113] Preferably, the protein level of the above-mentioned adenosine diphosphate ribose cyclase or its naturally occurring variant is detected by one or more methods selected from the group consisting of SDS polyacrylamide gel electrophoresis (SDS-PAGE), immunofluorescence, enzyme-linked immunosorbent assay, mass spectrometry and protein chip, but is not limited thereto.

[0114] Effects of the Invention

[0115] The features and advantages of the present invention are summarized as follows:

[0116] (i) The present invention provides a pharmaceutical composition for preventing or treating a disease mediated by ADP-ribose cyclase, comprising a novel expression inhibitor or activity inhibitor of ADP-ribose cyclase or a naturally occurring variant thereof as an active ingredient.

[0117] (ii) Furthermore, the present invention provides a composition for diagnosing a disease mediated by ADP-ribose cyclase, comprising a preparation for detecting the gene expression level of the ADP-ribose cyclase or a naturally occurring variant thereof, or a preparation for detecting the protein level.

[0118] (iii) The composition of the present invention has the effect of inhibiting the increase in calcium in kidney cells caused by the increased expression or activity of the novel ADP-ribosyl cyclase of angiotensin II, and therefore can be effectively used as a therapeutic agent for ADP-ribosyl cyclase-mediated diseases, especially kidney diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The figure shows the detection of nicotinamide adenine dinucleotide glycosidase (NADase) activity of a novel ADP-ribosyl cyclase in HEK293 cells and MES13 cells.

[0120] Figure 2 Figure 2 is a graph evaluating the ability of purified FLAG-ADP-ribose cyclase (FLAG-ADPRC) to cyclize ADP-ribose.

[0121] Figure 3The figure is a graph showing the detection of cyclized ADP-ribose generated in MES13 cells by a novel ADP-ribose cyclase when stimulated with angiotensin II.

[0122] Figure 4 The results of comparing the interspecies sequence homology of the adenosine diphosphate ribose cyclase of the present invention by sequence alignment (based on mice, humans (96%), rats (96%), dogs (90%), pigs (88%), rabbits (89%), sheep (90%), chickens (70%), cattle (78%), chimpanzees (93%), horses (91%), frogs (63%), goats (90%), turkeys (62%), guinea pigs (91%), Echinococcus granulosus (27%), Schistosoma haematobium (22%), Trichinella spiralis (22%), Drosophila (19%), zebrafish (56%)).

[0123] Figure 5 The figure is a graph showing the effect of detecting a novel inhibitor of nicotinamide adenine dinucleotide glycosidase activity of a novel adenosine diphosphate ribosyl cyclase.

[0124] Figures 6a to 6d To investigate the effect of dicaffeoylquinine on blood glucose in a mouse model of diabetic nephropathy. Figure 6a ), the ratio of kidney weight to body weight ( Figure 6b ), creatinine clearance ( Figure 6c ) and urine albumin content ( Figure 6d ) effect.

[0125] Figure 7 To detect the activity of dicaffeoylquinic acid on adenosine diphosphate ribosyl cyclase in the kidney tissue of a diabetic nephropathy mouse model ( Figure 7 of the a part) and the concentration of cyclic ATP-ribose ( Figure 7 Part b) of the diagram shows the effect of

[0126] Figure 8 The figure shows the effect of dicaffeoylquinic acid on the expression changes of transforming growth factor-β1, fibronectin and collagen IV in the kidney tissue of a diabetic nephropathy mouse model.

[0127] Fig. 9 The figure shows the histopathological changes observed in the kidney tissue of the diabetic nephropathy mouse model by hematoxylin and eosin (H&E) staining.

[0128] FIG10 is a graph showing the blood glucose level of normal mice and ADPRC hetero (ADPRC (+ / -)) mice in the diabetic nephropathy model ( Fig.10a ), the ratio of kidney weight to body weight ( Fig.10b ) and creatinine clearance ( Fig.10c )’s picture.

[0129] Figure 11a to Figure 11b To examine the effects of dicaffeoylquinic acid on blood pressure ( Fig.11a ) and creatinine clearance ( Fig.11b ) effect. DETAILED DESCRIPTION

[0130] The present invention is described in more detail below by way of examples. These examples are only used to describe the present invention in more detail, and those skilled in the art should understand that, according to the gist of the present invention, the scope of the present invention is not limited to these examples.

[0131] Example

[0132] Example 1. Enzyme activity of the novel ADP-ribosyl cyclase nicotinamide adenine dinucleotide glycosidase

[0133] In order to confirm the activity of the adenosine diphosphate ribose cyclase of sequence 1 in the sequence list, the complementary DNA sequence encoding the adenosine diphosphate ribose cyclase was connected to the FLAG-CMV-2 vector to prepare the FLAG-ADPRC plasmid, and the overexpression of the novel adenosine diphosphate ribose cyclase was induced in HEK293 cells or MES13 cells using a transfection reagent. The overexpressed adenosine diphosphate ribose cyclase was lysed using a lysis buffer. 45 μl of the lysed sample was treated with 5 μl of 2 mM ε-NAD (Nicotinamide 1, N6-ethenoadenine dinucleotide) and reacted at 37°C for 1 hour. After the reaction, 50 μl of 10% trichloroacetic acid was used to stop the enzyme-substrate reaction. After 10 minutes of centrifugation, 80 μl of the supernatant was added to 720 μl of 0.1 M sodium phosphate buffer, and the absorbance was measured using a fluorescence analyzer under the conditions of excitation wavelength of 297 nm and emission wavelength of 410 nm. Figure 1 shown.

[0134] Example 2. Evaluation of the ability of a novel ADP-ribose cyclase to synthesize ADP-ribose

[0135] The evaluation of the ability of the above-mentioned ADP-ribose cyclase to cyclize ADP-ribose was carried out by the method reported by Graeff R et al [Graeff R, Lee HC. Biochem. J. 361:379-384, 2002].

[0136] Specifically, after overexpressing FLAG-ADPRC plasmid in HEK293 cells using a transfection reagent, the cells were lysed using a lysis buffer. The lysed sample was purified using a FLAG-agarose column to purify the overexpressed FLAG-ADPRC. The purified sample was treated with 100 μM β-NAD and reacted at 37°C for 1 hour. After the reaction, cyclic adenosine diphosphate ribose was extracted using trichloroacetic acid to a final concentration of 0.6 M, and 50 μl of a mixed solution of adenosine diphosphate ribose cyclase (ADPR cyclase) (0.3 μg / ml), nicotinamide (nicotinaminde, 30 mM), and sodium phosphate (sodium phosphate, 100 mM) was added to 0.1 ml of the extract and 0.1 ml of the cyclic adenosine diphosphate ribose standard solution, and the reaction was allowed to proceed at room temperature for 30 minutes.

[0137] Add ethanol (2%), alcohol dehydrogenase (100μg / ml), resazurin (20μM), diaphorase (10μg / ml), flavin mononucleotide (FMN, 10μM), nicotinamide (10mM), bovine serum albumin (BSA, 0.1mg / ml), sodium phosphate (100mM) to the mixture and react for 2 to 4 hours. Measure the absorbance of the reactant using a fluorescence spectrophotometer between 544nm and 590nm. The results are as follows Figure 2 shown.

[0138] Example 3. Changes in intracellular cyclized ADP-ribose concentration by ADP-ribose cyclase in MES13 cells

[0139] The above-mentioned changes in the intracellular concentration of adenosine diphosphate ribose cyclized by adenosine diphosphate ribose cyclase were carried out by the method reported by Graeff R et al [Graeff R, Lee HC. Biochem. J. 361:379-384, 2002].

[0140] Specifically, a small interfering RNA (small interfering RNA, sequence 22 in the sequence list) of a novel ADP ribose cyclase was used as a transfection reagent to inhibit the expression of ADP ribose cyclase in MES13 cells. After the cells whose expression of ADP ribose cyclase was inhibited were treated with 150nM angiotensin II for 60 seconds, cyclized ADP ribose was extracted with 0.6M trichloroacetic acid, and 50μl of ADP ribose cyclase (0.3μg / ml), nicotinamide, and sodium phosphate mixed solution were added to 0.1ml of the extract and 0.1ml of the cyclized ADP ribose standard solution, and reacted at room temperature for 30 minutes.

[0141] To the mixture, add ethanol (2%), alcohol dehydrogenase (100 μg / ml), resazurin (20 μM), diaphorase (10 μg / ml), flavin mononucleotide (10 μM), nicotinamide (10 mM), bovine serum albumin (0.1 mg / ml), and sodium phosphate (100 mM) and react for 2 to 4 hours. Measure the absorbance of the reactant using a fluorescence spectrophotometer between 544 nm and 590 nm. The results are shown in Figure 3 shown.

[0142] Example 4. Effect of novel inhibitors of nicotinamide adenine dinucleotide glycosidase activity of novel adenosine diphosphate ribosyl cyclase

[0143] In order to find an inhibitor of the activity of the novel adenosine diphosphate ribose cyclase of sequence 1 in the sequence list, 4,4'-dihydroxyazobenzene (4-DHAB, TCI (Japan)), 2,2'-dihydroxyazobenzene (2-DAB, Sigma-Aldrich (USA)), 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (Quercetin, Sigma-Aldrich (USA)), San4825 (kannt A et al [Kannt A, Sicka K, Kroll K, Kadereit D, Gogelein H. Naunyn.Schmiedebergs.Arch. Pharmacol. 385:717-727, 2012], Synthesis (China)) and Compound Screening Newly discovered 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazoline-4(1H)-one (2-BMQ, Synthesis (China)), 1,4-Dicaffeoylquinic acid (1,4-DCQA, Biopurify (China), hereinafter referred to as DCQA) were reacted with 40μl of novel adenosine diphosphate ribose cyclase (ADPRC) and 5μl of inhibitor in ice water for 15 minutes. After the reaction, 5μl of 2mM ε-NAD was used for treatment and the reaction was carried out at 37℃ for 1 hour. After the reaction, 50μl of 10% trichloroacetic acid was used to stop the enzyme-substrate reaction. After 10 minutes of centrifugation, 80 μl of the supernatant was added to 720 μl of 0.1 M sodium phosphate buffer and the absorbance was measured using a fluorescence analyzer under the conditions of an excitation wavelength of 297 nm and an emission wavelength of 410 nm. Figure 5 As shown. Figure 5 As shown, it was confirmed that 4-DHAB inhibited the activity of nicotinamide adenine dinucleotide glycosidase by 42.40%, 2-BMQ by 36.49%, and DCQA by a surprising 79.64% relative to the control group.

[0144] Example 5. Effect of DCQA on blood glucose, kidney weight to body weight ratio, creatinine clearance and urinary albumin in a mouse model of renal disease

[0145] The blood glucose, kidney-to-body weight ratio, creatinine clearance, and urinary albumin content in the renal disease mouse model were measured according to the method reported by Kim SY et al [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am. J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0146] Specifically, C57BL / 6J mice were divided into groups (Control group and DCQA group) in which 0.2 ml of 50 mM citrate buffer (pH 4.8) was intraperitoneally injected into each mouse, and groups (STZ group and STZ+DCQA group) in which 200 μl of streptozotocin (STZ, Sigma-Aldrich, USA) dissolved in 50 mM citrate buffer at a concentration of 5 mg / ml was intraperitoneally injected into each mouse. Blood glucose was measured from the second day after STZ injection, and mice with blood glucose values ​​of 300 mg / dL were divided into STZ group and STZ+DCQA group for later use. DCQA, which has the most prominent inhibitory effect on the novel adenosine diphosphate ribose cyclase in Example 5, was dissolved in dimethyl sulfoxide to a concentration of 9 mg / ml, and then diluted with physiological saline to a concentration of 9 μg / ml. 100 μl was intraperitoneally injected into mice with elevated blood sugar every day for 6 weeks. On the last day of the 6th week, the mice were placed in a metabolic cage to collect urine for 24 hours.

[0147] After 24 hours of urine collection, the mice were weighed and their blood glucose levels were tested. Figure 6a ). In order to confirm the possibility of using it as a therapeutic agent for kidney disease, serum was obtained after euthanasia using anesthesia and the kidneys were removed. The ratio of kidney weight to body weight was measured using the removed kidneys ( Figure 6b ), part of the kidney was fixed in 10% formalin solution for fluorescent staining and hematoxylin and eosin staining, and the remaining kidney was divided into multiple pieces to detect the activity of adenosine diphosphate ribose cyclase and the concentration of cyclic adenosine diphosphate ribose. The creatinine in urine and serum was measured using a creatinine detection kit (BioAssay System, USA) to calculate the creatinine clearance ( Figure 6c ), using the albumin detection kit (BioAssay System, USA) to detect the amount of urinary albumin ( Figure 6d ). The results are shown in Figure 6.

[0148] As shown in FIG6 , in the STZ-induced diabetic mouse model, in the STZ+DCQA group, blood glucose did not decrease compared with the control group (STZ) ( Figure 6a ), but by a significant reduction in the ratio of kidney weight to body weight ( Figure 6b ), a significant increase in creatinine clearance ( Figure 6c ), a significant reduction in urinary albumin ( Figure 6d ) has been confirmed to have the potential to be a candidate for the treatment of kidney diseases such as chronic renal failure and diabetic nephropathy.

[0149] Example 6. Effect of DCQA on the activity of adenosine diphosphate ribose cyclase (ADPRC) and the concentration of cyclic adenosine diphosphate ribose in kidney tissue of a nephrotic mouse model

[0150] The detection of ADP-ribose cyclase activity and cyclic ADP-ribose concentration in the renal tissue of the renal mouse model was carried out according to the method reported by Kim SY et al [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am. J.Physiol. RenalPhysiol. 296:F291-F297, 2009].

[0151] Specifically, each C57BL / 6J mouse was intraperitoneally injected with 0.2 ml of 50 mM citrate buffer (pH 4.8) (Control group and DCQA group) and each mouse was intraperitoneally injected with 200 μl of streptozotocin (Sigma-Aldrich Company (USA)) dissolved in 50 mM citrate buffer at a concentration of 5 mg / ml (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with blood glucose values ​​of 300 mg / dL were divided into STZ group and STZ+DCQA group for later use. DCQA, which has the most prominent inhibitory effect on the novel adenosine diphosphate ribose cyclase in Example 5 above, was dissolved in dimethyl sulfoxide to a concentration of 9 mg / ml, and then diluted with physiological saline to a concentration of 9 μg / ml, and 100 μl was intraperitoneally injected into mice with elevated blood glucose every day for 6 weeks. In order to confirm its potential as a therapeutic agent for kidney disease, serum was obtained and kidneys were removed after euthanasia using anesthesia.

[0152] After a portion of the kidney tissue removed from each group was lysed using a lysis buffer, 5 μl of 2 mM nicotinamide guanine dinucleotide (NGD) was treated with 45 μl of the lysed sample and reacted at 37°C for 1 hour. After the reaction, 50 μl of 10% trichloroacetic acid was used to stop the enzyme-substrate reaction. After 10 minutes of centrifugation, 80 μl of the supernatant was added to 720 μl of 0.1 M sodium phosphate buffer, and the absorbance was measured using a fluorescence analyzer under the conditions of an excitation wavelength of 297 nm and an emission wavelength of 410 nm.

[0153] Furthermore, after treating a portion of the kidney tissue removed from each group with 0.2 ml of 0.6 M trichloroacetic acid to extract cyclic adenosine diphosphate ribose, 50 μl of a mixed solution of adenosine diphosphate ribose cyclase (0.3 μg / ml), nicotinamide (30 mM), and sodium phosphate (100 mM) was added to 0.1 ml of the extract and 0.1 ml of the cyclic adenosine diphosphate ribose standard solution, and the mixture was reacted at room temperature for 30 minutes. Ethanol (2%), alcohol dehydrogenase (100 μg / ml), resazurin (20 μM), diaphorase (10 μg / ml), flavin mononucleotide (10 μM), nicotinamide (10 mM), bovine serum albumin (0.1 mg / ml), and sodium phosphate (100 mM) were added to the mixture and reacted for 2 to 4 hours. The absorbance of the reactant was measured using a fluorescence spectrophotometer between 544 nm and 590 nm. The results are as follows. Figure 7 shown.

[0154] like Figure 7 As shown in part a of FIG. 1 , the DCQA of the present invention reduces the activity of ADP-ribosyl cyclase increased by STZ to below the level of the control group, as shown in FIG. Figure 7 As shown in part b, the DCQA of the present invention reduced the concentration of cyclic adenosine diphosphate ribose increased by STZ to the level of the control group, thereby confirming that DCQA has the ability to reduce the activity of adenosine diphosphate ribose cyclase and the concentration of cyclic adenosine diphosphate ribose caused by renal dysfunction or damage associated with renal disease.

[0155] Example 7. Effect of 1,4-DCQA on the expression of transforming growth factor-β1, fibronectin and collagen IV in the kidney tissue of a nephropathy mouse model

[0156] The expression changes of transforming growth factor-β1, fibronectin, and collagen IV in the renal tissue of the nephropathy mouse model were detected by the method reported by Kim SY et al [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am. J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0157] Specifically, each C57BL / 6J mouse was intraperitoneally injected with 0.2 ml of 50 mM citrate buffer (pH 4.8) (Control group and DCQA group) and each mouse was intraperitoneally injected with 200 μl of streptozotocin (Sigma-Aldrich Company (USA)) dissolved in 50 mM citrate buffer at a concentration of 5 mg / ml (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with blood glucose values ​​of 300 mg / dL were divided into STZ group and STZ+DCQA group for later use. DCQA, which has the most prominent inhibitory effect on the novel adenosine diphosphate ribose cyclase in Example 5 above, was dissolved in dimethyl sulfoxide to a concentration of 9 mg / ml, and then diluted with physiological saline to a concentration of 9 μg / ml, and 100 μl was intraperitoneally injected into mice with elevated blood glucose every day for 6 weeks. In order to confirm the possibility of using it as a therapeutic agent for kidney disease, serum was obtained after euthanasia using anesthesia and the kidneys were removed. A part of the removed kidney was fixed in 10% formalin solution. The kidney tissue fixed with 10% formalin solution was cut into kidney tissue sections using a freezing tissue slicer and placed on a slide. After washing with TTBS (Tris-buffered saline (TBS) and 0.1% Tween 20) buffer, it was reacted with TTBS buffer containing 1% bovine serum albumin (BSA) for 1 hour. The primary antibody (transforming growth factor-β1 (Santa Cruz, USA), fibronectin (Santa Cruz, USA), collagen IV (Abcam, UK)) was diluted to 1:200 with TTBS buffer containing 1% bovine serum albumin and reacted at 4°C for more than 12 hours. After washing the tissue reacted with the primary antibody three times with TTBS buffer, the secondary antibody attached with poly-L-lysine-fluorescein isothiocyanate was diluted in TTBS buffer at a ratio of 1:200 and reacted at room temperature for 1 hour in a dark place. After washing three times with TTBS buffer after the secondary antibody reaction, the cover glass was bonded with a sealing solution. The stained kidney tissue was observed using a fluorescence microscope (Carl Zeiss, Germany) with green fluorescence observation. The results are as follows Figure 8 shown.

[0158] like Figure 8 As shown, DCQA of the present invention reduced the expression levels of transforming growth factor-β1, fibronectin, and collagen IV increased by STZ to the level of the control group, thereby confirming that DCQA has the ability to reduce the expression levels of transforming growth factor-β1, fibronectin, and collagen IV caused by renal dysfunction or damage associated with renal disease.

[0159] Example 8. Histopathological changes in kidney tissue of nephrotic mouse model by hematoxylin and eosin staining

[0160] The histopathological changes of renal tissue in the nephrotic mouse model were detected by the method reported by Shu B et al [Shu B,Feng Y, Gui Y, Lu Q, Wei W, Xue X, Sun X, He W, Yang J, Dai C. Cell. Signal. 42:249-258, 2018].

[0161] Specifically, each C57BL / 6J mouse was intraperitoneally injected with 0.2 ml of 50 mM citrate buffer (pH 4.8) (Control group and DCQA group) and each mouse was intraperitoneally injected with 200 μl of streptozotocin (Sigma-Aldrich Company (USA)) dissolved in 50 mM citrate buffer at a concentration of 5 mg / ml (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with blood glucose values ​​of 300 mg / dL were divided into STZ group and STZ+DCQA group for later use. DCQA, which has the most prominent inhibitory effect on the novel adenosine diphosphate ribose cyclase in Example 5 above, was dissolved in dimethyl sulfoxide to a concentration of 9 mg / ml, and then diluted with physiological saline to a concentration of 9 μg / ml, and 100 μl was intraperitoneally injected into mice with elevated blood glucose every day for 6 weeks. In order to confirm the possibility of using it as a therapeutic agent for kidney disease, serum was obtained after euthanasia using anesthesia and the kidneys were removed. A portion of the removed kidney was fixed in a 10% formalin solution. The kidney tissue fixed with a 10% formalin solution was cut into kidney tissue sections using a frozen tissue slicer and placed on a slide. After rinsing with running water for 5 minutes, it was stained with hematoxylin for 5 minutes. After staining, it was rinsed with running water for 5 minutes, and then quickly immersed in 157mM hydrochloric acid twice, taken out, and rinsed with running water for 5 minutes. After quickly soaking in 0.25% ammonia water once and taking it out, rinse it with running water again for 5 minutes, stain it with eosin for about 30 seconds, react in 70% ethanol for 30 seconds, react in 80% ethanol for 30 seconds, react in 90% ethanol for 30 seconds, react in 100% ethanol for the first time for 30 seconds, react in 100% ethanol for the second time for 30 seconds, and react in 100% ethanol for the third time for 30 seconds. Finally, after the first reaction in xylene for 5 minutes and the second reaction in xylene for more than 5 minutes, use the sealing solution to bond the coverslip. The stained kidney tissue was observed using an optical microscope (Lieca, Germany). The results are as follows. Fig. 9 shown.

[0162] like Fig. 9 As shown, DCQA of the present invention restored glomerular hypertrophy, infiltration of inflammatory cells, and formation of transitional epithelial cells increased by STZ to levels similar to those of the control group, thereby confirming that DCQA has the ability to restore renal histopathological changes caused by renal dysfunction or damage associated with renal diseases.

[0163] Example 9. Comparison of blood glucose, kidney-to-body weight ratio, and creatinine clearance in normal mice and ADPRC hetero (ADPRC (+ / -)) mice with renal disease

[0164] The blood glucose, kidney-to-body weight ratio, and creatinine clearance in the renal disease mouse model were measured by the method reported by Kim SY et al [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am. J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0165] Specifically, streptozotocin was dissolved in 50 mM citrate buffer (pH 4.8) to a concentration of 5 mg / ml and 200 μl was intraperitoneally injected into each 129S1 / SvImJ mouse (wild type (WT)) and ADPRC hetero knockout (ADPRC (+ / -)) mouse prepared by The Jackson Laboratory (USA). Blood glucose was measured from the second day after injection, and mice with a blood glucose value of 300 mg / dL were used. Six weeks after the blood glucose rise, the mice were placed in a metabolic cage and urine was collected for 24 hours.

[0166] 24 hours after urine collection, the mice were weighed and their blood glucose levels were tested. Fig.10a ). After euthanasia using anesthesia, serum was obtained and the kidneys were removed. The kidney weight and body weight ratio were measured using the removed kidneys ( Fig.10b The creatinine clearance was calculated by measuring the creatinine in urine and serum using a creatinine test kit (BioAssay System, USA). Fig.10c ). The results are shown in Figure 10.

[0167] As shown in Figure 10, in the STZ-induced diabetic mouse model, blood glucose levels in ADPRC (+ / -) were not reduced compared with those in the control group ( Fig.10a ), the ratio of kidney weight to body weight in the ADPRC (+ / -) + STZ group of the present invention was significantly reduced compared with the WT + STZ group ( Fig.10b Furthermore, the decrease in creatinine clearance by STZ was not reduced in the ADPRC (+ / -) + STZ group ( Fig.10c ), thus confirming the importance of the new ADP-ribosylcyclase in kidney diseases such as chronic renal failure or diabetic nephropathy.

[0168] Example 10. Effect of DCQA on blood pressure and creatinine clearance in normal mice and hypertensive mouse models

[0169] The blood pressure and creatinine clearance of the hypertensive mouse model were measured by the methods reported by Allagnat et al [AllagnatF, Haefliger JA, Lambelet M, Longchamp A, Berard X, Mazzolai L,Corpataux JM,Deglise S. Eur. J. Vasc. Endovasc. Surg. 51:733-742, 2016] and Kim SY et al [KimSY, Park KH, Gul R, Jang KY, Kim UH.Am. J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0170] Specifically, 8 mg of L-NAME (Nω-nitro-l-arginine-methyl-ester, Sigma-Aldrich, USA) was dissolved in 1 ml of saline and 0.2 ml was orally administered to each C57BL / 6J mouse every day. Blood pressure was measured at the tail of the mouse on the 7th and 14th day of oral administration. After confirming that the blood pressure was elevated, DCQA, which has the most prominent inhibitory effect on the novel adenosine diphosphate ribose cyclase in Example 5 above, was dissolved in dimethyl sulfoxide to a concentration of 9 mg / ml, and diluted with saline to a concentration of 9 μg / ml. L-NAME was orally administered by intraperitoneal injection of 100 μl every day for 7 days. After 6 days of treatment with DCQA, the mice were placed in a metabolic cage to collect urine for 24 hours.

[0171] The blood pressure of mice was measured 24 hours after urine collection ( Fig.11a ). Serum was obtained after euthanasia using anesthesia.

[0172] The creatinine clearance was calculated by measuring the creatinine in urine and serum using a creatinine test kit (BioAssay System, USA). Fig.11b ). The results are as follows Fig.11a and Fig.11b shown.

[0173] like Fig.11a As shown in Figure 2, it was confirmed that DCQA had no effect on lowering blood pressure in the hypertension model. However, creatinine clearance increased under the action of DCQA, confirming its potential as a candidate therapeutic agent for kidney diseases such as hypertensive nephropathy ( Fig.11b ).

[0174] References

[0175] 1. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 4:517-529,2003.

[0176] 2. Lee HC. Structure and enzymatic functions of human CD38. Mol Med.12:317-323, 2006.

[0177] 3. Resnick LM. Ionic basis of hypertension, insulin resistance,vascular disease, and related disorders. The mechanism of “SyndromeX”. Am. J.Hypertens. 6:123S-134S, 1993.

[0178] 4. Cowley AW Jr. Long-term control of arterial blood pressure.Physiol. Rev. 72:231-300, 1992.

[0179] 5. Kim BJ, Park KH, Yim CY, Takasawa S, Okamoto H, Im MJ, Kim UH.Generation of nicotininc acid adenine dinucleotide phosphate and cyclicADP-ribose by glucagon-like peptide-1 evokes Ca 2+ signal that is essential forinsuline secreation in mouse pancreatic islets. Diabetes. 57:868-878, 2008.

[0180] 6. Gul R, Park JH, Kim SY, Jang KY, Chea JK, Ko JK, Kim UH.Inhibition of ADP-ribosyl cyclase attenuates antiotensin II-induced cardiachypertrophy.Cardiovasc. Res. 81: 582-591, 2009.

[0181] 7. Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, Kim UH.Molecular mechanism of ADP-ribosyl cyclase activation in angiotensinIIsignaling in murine mesangial cells. Am. J. Physiol. Renal. Physiol. 294:F989-F989, 2008.

[0182] 8. Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL, OppenheimerN, Garvy B, Kusser K, Goodrich S, Howard M, Harmsen A, Randall TD, Lund FE.CyclicADP-ribose production by CD38 regulates intracellular calcium release,extracellular calcium influx and chemotaxis in neutrophils and is requiredfor bacterialclearance in vivo. Nat Med 7:1209-1216, 2001.

[0183] 9. Graeff R, Lee HC. A novel cycling assay for cellular cADP-ribosewith nanomolar sensitivity. Biochem. J. 361:379-384, 2002.

[0184] 10. Kim SY, Park KH, Gul R, Jang KY, Kim UH. Role of kidney ADP-ribosyl cyclase in diabetic nephropathy. Am. J. Physiol. Renal Physiol. 296:F291-F297,2009.

[0185] 11. Shu B, Feng Y, Gui Y, Lu Q, Wei W, Xue X, Sun X, He W, Yang J,Dai C. Blockade of CD38 diminishes lipopolysaccharide-induced macrophageclassicalactivation and acute kidney injury involving NF-κB signalingsuppression. Cell. Signal. 42:249-258, 2018.

[0186] 12. Allagnat F, Haefliger JA, Lambelet M, Longchamp A, Berard

[0187] 13. Kannt A, Sicka K, Kroll K, Kadereit D, Gogelein H. Naunyn.Schmiedebergs. Arch. Pharmacol. 385:717-727, 2012.

[0188] The above detailed description of the specific parts of the present invention, but it should be understood by those skilled in the art that these detailed descriptions are only preferred examples, and are not intended to limit the scope of the present invention. Therefore, the essential scope of the present invention should be defined by the equivalents of the attached invention claims.

Claims

1. Use of a composition comprising an active inhibitor of adenosine diphosphate ribose cyclase or a naturally occurring variant thereof as an active ingredient in the preparation of a medicament for preventing or treating chronic renal failure, characterized in that: The above-mentioned adenosine diphosphate ribose cyclase is composed of the amino acid sequence of sequence 1 in the sequence list, The above-mentioned activity inhibitor is dicaffeoylquinic acid, The above naturally occurring variant consists of the amino acid sequence of sequence 2 in the sequence listing, The above-mentioned chronic renal failure includes diabetic nephropathy and hypertensive nephropathy.

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