WO2021229479A1 - 엠피레귤린 특이적 이중가닥 올리고뉴클레오티드 구조체를 포함하는 비만 관련 질환의 예방 및 치료용 조성물 - Google Patents
엠피레귤린 특이적 이중가닥 올리고뉴클레오티드 구조체를 포함하는 비만 관련 질환의 예방 및 치료용 조성물 Download PDFInfo
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Definitions
- the present invention relates to an obesity-related disease comprising the Empiregulin-specific double-stranded oligonucleotide construct. It relates to a composition for prevention and treatment, and more particularly, a double-stranded oligonucleotide capable of inhibiting the expression of ampiregulin with very specific and high efficiency, a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, and nanoparticles It relates to a composition for preventing and treating obesity-related diseases, including. background
- RNAi double-stranded RNA
- RNA therapy targeting such RNA is a method that removes the function of the gene using oligonucleotides directed against the target RNA.
- ASOs antisense oligonucleotides
- Antisense oligonucleotide is a short-length synthetic DNA designed to bind to a target gene according to the Watson-Crick base reaction, and can specifically inhibit the expression of a specific nucleotide sequence of a gene. It has been used to study the function of genes and develop therapeutics that can treat diseases such as cancer at the molecular level.
- Such ASO has the advantage that it can be easily manufactured by setting various targets for suppressing gene expression, and there have been studies to use it to suppress the expression of oncogenic genes and the growth of cancer cells.
- the process by which ASO suppresses the expression of a specific gene combines with the complementary [ 1 sequence and induces [ 1356 H activity] 2021/229479 ? €1/162021/054077
- ASO binds to genomic DNA and inhibits gene transcription by forming a triple-helix structure.
- ASO has the above potential, but in order to be used in clinical practice, it must be efficiently delivered into a target tissue or cell to improve stability to nucleases and specifically bind to the nucleotide sequence of a target gene.
- the secondary and tertiary structure of gene mRNA is an important factor for specific binding of ASO, and since the part with less secondary structure of mRNA is very advantageous for ASO to access, less secondary structure of mRNA is generated before synthesizing ASO Efforts have been made to effectively achieve gene-specific inhibition in vivo as well as in vitro by systematically analyzing the target region.
- RNA interference (hereinafter referred to as 'RNAi') has been found to act on sequence-specific mRNA in various types of mammalian cells (Barik, S., J Mol.
- 'siRNA' short interfering RNA
- bp base pair
- the gene expression suppression technology using SiRNA suppresses the expression of the target gene in the target cell and observes the resulting change, which is useful for research to identify the function of the target gene in the target cell.
- infectious virus or cancer cells Suppressing the function of a target gene in the back will be useful for developing a treatment method for the disease. It has been reported that it is possible to suppress the expression of a target gene by siRNA.
- siRNA directed to the same target gene has an excellent inhibitory effect on mRNA expression in vitro and in 1//1/0) compared to antisense oligonucleotide (ASO). It has been found that the effect has a long lasting effect.
- siRNA binds to the target mRNA and regulates the expression of the target gene in a sequence-specific manner, so it is an innovative target compared to existing antibody-based drugs or small molecule drugs. It has the advantage that it can be expanded to Despite siRNA's excellent effect and wide range of uses, for siRNA to be developed as a therapeutic agent, siRNA must be effectively delivered to target cells by improving the stability of siRNA in the body and improving cell delivery efficiency.
- nuclease resistance or a viral vector In order to improve in vivo stability and solve the problem of innate immune stimulation of siRNA, some nucleotides or backbone of siRNA are modified to have nuclease resistance or a viral vector, Research on this, such as using a carrier such as liposomes or nanoparticles (nanopartide), is actively 2021/229479 ? €1/162021/054077
- a delivery system using a viral vector such as an adenovirus or a retrovirus has high transfection efficacy, but high immunogenicity and oncogenicity.
- a non-viral delivery system containing nanoparticles has lower cell delivery efficiency than a viral delivery system, but has high stability in vivo (1//1/1/ ⁇ ) , target-specific delivery, uptake and internalization of RNAi oligonucleotides into cells or tissues, and little cytotoxicity and immune stimulation. is evaluated as a powerful delivery method compared to the viral delivery system.
- the nanocarrier is used to form nanoparticles by using various polymers such as liposomes and cationic polymer complexes, and siRNA This is a method of delivering to cells by carrying them on such nanoparticles, that is, a nanocarrier.
- the main methods used among the methods using a nanocarrier are polymeric nanoparticles, pdymer micelles, and liposomes.
- lipoplex there are flex (lipoplex), etc., among which the method using lipoplex is composed of cationic lipids and interacts with anionic lipids of endosomes of cells to induce a destabilizing effect of endosomes and deliver them into cells
- flex lipoplex
- the method using lipoplex is composed of cationic lipids and interacts with anionic lipids of endosomes of cells to induce a destabilizing effect of endosomes and deliver them into cells
- siRNA table H passenger (sense) strand it has enhanced pharmacokinetics characteristics in vivo ( ⁇ ). 2021/229479 ? 1/162021/054077
- siRNA in the form of a polymer compound conjugated with polyethylene glycol PEG
- PEG polyethylene glycol
- micelles composed of polymer complexes are extremely small in size compared to other systems used as drug delivery vehicles, such as microspheres or nanoparticles, but their distribution is very constant and spontaneously formed. It has the advantage of being easy to control the quality of the formulation and secure the reproducibility.
- a hydrophilic material e.g., polyethylene glycol (PEG)
- PEG polyethylene glycol
- SAMiRNAä self assembled micelle inhibitory RNA
- PEG polyethyleneglycol
- HEG Hexaethylenglycol
- PEG polyethyleneglycol
- HEG Hexaethylenglycol
- PEG is a synthetic polymer and is often used in pharmaceuticals, especially proteins, for increasing solubility and pharmacokinetics. used for control.
- PEG is a polydisperse material, and one batch of polymer is composed of the sum of different numbers of monomers, so the molecular weight is in the form of a Gaussian curve, and the polydisperse value (Mw/Mn) is It expresses the degree of homogeneity of a substance.
- PEG has a low molecular weight (3 ⁇ 5kDa), it shows a polydispersity index of about 1.()1, and when it has a high molecular weight (20kDa), it shows a high polydispersity index of about 1.2. relatively low characteristics. Therefore, when PEG is combined with pharmaceuticals, the polydispersity of PEG is reflected in the conjugate, making it difficult to verify a single substance.
- the present inventors used a DNA sequence that is ASO as a guide and an RNA sequence as an antisense strand that is a passenger.
- a double-stranded oligonucleotide in the -RNA hybrid form it was attempted to enhance the expression inhibitory effect and stability of the target gene.
- obesity is an important health problem worldwide, and a number of complications such as heart disease, type 2 diabetes, and certain cancers may increase.
- One of the main causes of obesity is the excessive accumulation of visceral fat in the body [Carr DB, Diabetes.
- Visceral fat refers to the fat surrounding internal organs. Visceral fat is mainly a genetic factor [Rosenberg B, Panminerva Med 2005; 47:229-44], race, physical activity, lifestyle and inflammatory factors [Deurenberg P, Int J Obes Relat Metab Disord 1998;22:1164-71]. In addition, according to race, the accumulation of visceral fat was higher in Asians according to WHO Expert Consultation. Lancet 2004;363:157 - 2021/229479 ? €1/162021/054077
- Visceral fat causes metabolic abnormalities and cardiovascular disease [Matsuzawa Y, Obes Res 1995;3 Suppl 5:645-7] The more visceral fat accumulates, the higher the insulin resistance, and the more subcutaneous fat In more cases, heart function is reduced and hypertension and circulatory disease develop [Schaffler, Nat Clin Pract Gastroenterol Hepatol 005;2:273-80].
- Visceral fat also causes digestive disorders, including fatty liver and non-alcoholic It is known as a factor of steatohepatitis [Busetto L, Diabetes Obes Metab 2005;7:301-6] These factors cause adiponectin to decrease, reduce anti-inflammatory mechanisms, and promote fatty liver formation in the liver, resulting in non-alcoholic fatty liver.
- visceral fat causes many problems in respiratory-related diseases [Schapira DV, Cancer 1994;74:632-9] The more visceral fat than subcutaneous fat, the lower the expiratory reserve, which can lead to restrictive pulmonary ventilation. Visceral fat is also known to increase the rate of breast cancer.
- the present invention provides (I) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and an antisense strand comprising a sequence complementary thereto -sense strand) containing an empiregulin-specific double-stranded oligonucleotide;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the empiregulin of (i) refers to a specific double-stranded oligonucleotide
- (iii) nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii) above; provides a pharmaceutical composition for preventing or treating obesity, characterized in that it is any one selected from the group consisting of.
- the present invention also provides a formulation in a lyophilized form comprising the pharmaceutical composition.
- the present invention also includes a sense strand (sense strand) comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and a sequence complementary thereto to a subject in need of prevention or treatment of obesity an empiregulin-specific double-stranded oligonucleotide comprising an anti-sense strand; 2021/229479 ? 1/162021/054077
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the above (i) means an empiregulin-specific double-stranded oligonucleotide
- nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii); provides a method for preventing or treating obesity, comprising administering any one selected from the group consisting of.
- the present invention also provides (i) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 for use in a method for preventing or treating obesity and a sequence complementary thereto an empiregulin-specific double-stranded oligonucleotide comprising an anti-sense strand;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the empiregulin-specific of (i) double-stranded oligonucleotide; and 2021/229479 ? €1/162021/054077
- nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii) above; provides a pharmaceutical composition, characterized in that it is any one selected from the group consisting of.
- the present invention also provides a sense strand comprising any one sequence selected from the group consisting of 0) SEQ ID NOs: 10, 11 and 12 for the preparation of a drug for the prevention or treatment of obesity and a sequence complementary thereto an empiregulin-specific double-stranded oligonucleotide comprising an anti-sense strand;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the empiregulin-specific in (i) double-stranded oligonucleotide
- FIG. 1 shows the results of screening 1,257 SAMiRNAs targeting human ampiregulin.
- Figure 2 shows the selected empiregulin-specific double-stranded oligonucleotide comprising 2021/229479 ? €1/162021/054077
- Double-stranded oligo 0 / ⁇ /[ 1 represents your nanoparticle size distribution.
- the lung cancer cell line 549 by varying the concentration (200 or 600 nM) of $ 1 ⁇ hour [small 1 / ⁇ having the sequences of SEQ ID NOs: 1 to 14 of the present invention as the sense strand, respectively.
- This is a graph showing the relative expression level (%) of empiregulin mi [ ⁇ 1/ ⁇ treated with .
- Example 4 is empiregulin of Example 5
- BMP LES gyulrin this by analyzing the relative expression level (%) of the 1 / ⁇ (ratio $ [buy ⁇ 11 (a / ⁇ ; is a graph confirming the value 50.
- Example 5 is empiregulin of Example 5 As a result of quantitative analysis of the expression level, by varying the concentration of $ 1 ⁇ /11 [ ⁇ / ⁇ having the sequence of SEQ ID NO: 11 as the sense strand of the present invention (12.5 25 50 100 200 nM, 600 nM or 1200 nM) ) treated with lung cancer cell line 549, (3) by analyzing the relative expression level (%) of 1 (; 50 This is a graph confirming the value.
- Example 6 is empiregulin of Example 5 As a result of quantitative analysis of the expression level, by varying the concentration of £ 1 ⁇ /11 [ ⁇ / ⁇ having the sequence of SEQ ID NO: 12 as the sense strand of the present invention (12.5 25 50 100 200 nM, 600 nM or 1200 nM) ) treated with lung cancer cell line 549, (3) by analyzing the relative expression level (%) of 1 (; 50 This is a graph confirming the value. 2021/229479 ? €1/162021/054077
- FIG. 7 shows the results of an innate immune response test analysis for the empiregulin candidate sequence of Example 6, and SEQ ID NOs: 10 (AR-1), 11 (AR-2) of the present invention, Empiregulin SAMiRNA 2.5 MM having 12(AR-3) as the sense strand was treated in human peripheral blood mononuclear cells (PBMC), respectively, and the intrinsic immunity-related cytokines by the empiregulin-specific SAMiRNA were It is a graph evaluating the in vitro cytotoxicity using human peripheral blood mononuclear cells by analyzing the relative increase in mRNA.
- PBMC peripheral blood mononuclear cells
- RNA/RNA hybrid SAMiRNA This is the analysis result comparing the relative expression level (%) of Mpiregulin mRNA by oligo DNA/RNA hybrid and RNA/RNA hybrid.
- a lung cancer cell line by varying the concentration of SAMiRNA having the sequence of SEQ ID NOs: 10 (AR-1), 11 (AR-2), and 12 (AR-3) as the sense strand of the present invention (200 nM, 600 or 1200 nM)
- 10A shows the results of quantitative analysis of the mouse empiregulin mRNA expression level of Example 8, by varying concentrations of SAMiRNA having the sequences of SEQ ID NOs: 19, 20, and 21 as sense strands of the present invention (200 or 500). nM) It is a graph showing the relative expression level (%) of MPregulin mRNA after treatment in MLg, a mouse lung fibroblast cell line. 2021/229479 ? €1/162021/054077
- FIG. 10B shows the results of quantitative analysis of the mouse empiregulin mRNA expression level of Example 8.
- the SAMiRNA having the sequences of SEQ ID NOs: 19, 20, and 21 of the present invention as the sense strand was used at different concentrations (200, 500 or 1000 nM) treated in LA-4, a mouse Lung epithelial cell line, and a graph showing the relative expression level ( ⁇ / ⁇ ) of empiregulin mRNA.
- 11 is a graph showing the change in body weight of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 12 is a graph showing the change in feed intake of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 13 is a graph showing the change in drinking water intake of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 14 is a result showing the subcutaneous fat ratio of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 15 is a result showing the visceral fat ratio (visceral fat ratio) of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 16 is a photograph showing the effect of reducing visceral fat of the construct according to the present invention in a mouse animal model experiment according to the present invention.
- 17 is a result showing the fasting blood glucose level in whole blood 8 weeks before sacrifice of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 18 is a result showing the glucose level in the serum 8 weeks before sacrifice of the control group and the experimental group in the mouse animal model experiment according to the present invention.
- 17 is a high-fat diet in the high-fat diet obese mouse model experiment according to the present invention.
- 20 is a graph showing the change in body weight of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 21a is a graph showing the average feed intake of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 21b is a graph showing the average negative water intake of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 22 is a graph showing the dietary efficiency (Food efficiency ratio (%)) of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 23a is a high-fat diet obese mouse model experiment according to the present invention in the control group and experimental group subcutaneous fat (subcutaneous fat pad, SFP), epididymal fat (epididymal fat pad, EFP), peripheral fat (Perirenal fat pad, PFP), mesentery It is a result showing the weight of fat (mesenteric fat pad, MFP).
- subcutaneous fat subcutaneous fat pad, SFP
- epididymal fat epididymal fat
- EFP epididymal fat
- PFP peripheral fat
- mesentery It is a result showing the weight of fat (mesenteric fat pad, MFP).
- 23b is a high-fat diet obese mouse model experiment according to the present invention in the control group and the experimental group subcutaneous fat (subcutaneous fat pad, SFP), epididymal fat (epididymal fat pad, EFP), peripheral fat (Perirenal fat pad, PFP), mesentery This is the result showing the mtio (%) of the fat (mesenteric fat pad, MFP) weight divided by the body weight.
- Figure 24a shows the subcutaneous fat weight and visceral fat weight of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention. 2021/229479 ? €1/162021/054077
- 24B is a result showing the subcutaneous fat ratio and visceral fat ratio to the total weight of the mouse in the control group and the experimental group, respectively, in the high-fat diet obese mouse model experiment according to the present invention.
- 25 is a graph showing the Mkro-CT photograph and volume comparing the effect of reducing subcutaneous fat and visceral fat in the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 26 is a subcutaneous fat pad (SFP), epididymal fat pad (EFP), and peripheral fat (PFP) of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- SFP subcutaneous fat pad
- EFP epididymal fat pad
- PFP peripheral fat
- 27A is a photograph comparing the effect of reducing fat accumulation in liver tissues of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 27B is a quantitative graph comparing the effect of reducing fat accumulation in the liver tissue of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- 28 is a subcutaneous fat pad (SFP), epididymal fat pad (EFP), and peripheral fat (perirenal fat pad, PFP) of the control group and the experimental group in the high-fat diet obese mouse model experiment according to the present invention.
- SFP subcutaneous fat pad
- EFP epididymal fat pad
- PFP peripheral fat
- the present invention provides (i) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 14, preferably SEQ ID NOs: 10, 11 and 12 an empiregulin-specific double-stranded oligonucleotide comprising an anti-sense strand comprising a sequence complementary thereto;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each 2021/229479 ? 1/162021/054077
- R refers to the empiregulin-specific double-stranded oligonucleotide of (i) above;
- (iii) nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii); relates to a pharmaceutical composition for the prevention or treatment of obesity, characterized in that any one selected from the group consisting of.
- the sequences of SEQ ID NOs: 10, 11 and 12 included in the preferred double-stranded oligonucleotide provided according to the present invention are as follows.
- the double-stranded oligonucleotide according to the present invention is a concept including all substances having a general RNAKRNA interference) action, and an mRNA-specific double-stranded oligo that encodes the empiregulin protein. It is apparent to those of ordinary skill in the art that the nucleotide includes empiregulin-specific shRNA and the like. That is, the oligonucleotide may be characterized in that it is siRNA, shRNA or miRNA.
- one or more bases are substituted or deleted in the sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 or in the antisense strand complementary thereto , or the inserted sequence 2021/229479 ? €1/162021/054077
- Ampiregulin specific comprising sense strand and antisense strand comprising 21 It is apparent to those skilled in the art that antisense oligonucleotides are also included in the scope of the present invention.
- the sense or antisense strand may be independently di or [ ⁇ , and the sense strand may antisense strands Hybrid 0 u) form can be used.
- the SEQ ID NOs: 10, 11 and Although it is described in the form, when the form is used, the sequences of SEQ ID NOs: 10, 11 and 12 may use the corresponding [sequence, that is, a sequence changed to Gani.
- the double-stranded oligonucleotide according to the present invention is 100% complementary to the binding site of the Ampiregulin gene, that is, if the sense strand of the sequence is 100% complementary as long as specificity for Empiregulin is maintained. In addition to this), if some nucleotide sequences do not match, include
- the double-stranded oligonucleotide according to the present invention may include one or more unbonded (6! ⁇ 3) overhangs (6! ⁇ 3) at the 3' end of one or both strands.
- the sense strand or the antisense strand may preferably consist of 19 to 31 nucleotides, but is not limited thereto. 2021/229479 ? €1/162021/054077
- the double-stranded oligonucleotide comprising a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and an antisense strand comprising a sequence complementary thereto is amphiregulin (amphiregulin). ) may be characterized as being specific, but is not limited thereto.
- the sense strand or antisense strand of the double-stranded oligonucleotide includes various chemical modifications to improve in vivo stability, or to confer nuclease resistance and reduce nonspecific immune response.
- the chemical modification is a hydroxyl group (-OH) at the 2' carbon position of the sugar structure in the nucleotide is a methyl group (-[bur), a methoxy group (-OCH 3 ), an amine group ( -NH 2 ), fluorine ), 0-2 -methoxyethyl group, 0-propyl group, 0-2 -methylthioethyl group, 0-3-aminopropyl group, 0-3 -dimethylaminopropyl group, 0-N-methylacetami a modification substituted with any one selected from the group consisting of doggy and 0-dimethylamidooxyethyl; a modification in which the oxygen of the sugar structure in the nucleotide is replaced with sulfur; a modification in which the nucleotide bond is any one bond selected from the group consisting of a phosphorothioate bond, a boranophosphate bond and a methyl phosphon
- At least one phosphate group at the 5' end of the antisense strand of the double-stranded oligonucleotide It may be characterized in that it is bonded, preferably, it may be characterized in that 1 to 3 phosphate groups are bonded.
- the present invention relates to a double-stranded oligonucleotide structure comprising the structure of the following Structural Formula (1), wherein A is a hydrophilic material, B is a hydrophobic material, and X and Y are each independently simple It refers to a covalent bond or a linker-mediated covalent bond, and R refers to a double-stranded oligonucleotide.
- the double-stranded oligonucleotide structure comprising the empiregulin-specific sequence according to the present invention preferably has a structure as shown in Structural Formula (1).
- AXRYB Structural Formula (1) In Structural Formula (1), A is a hydrophilic material, B is a hydrophobic material, X and Y are each independently a simple covalent bond or a linker-mediated covalent bond, and R is an empiregulin-specific double bond.
- strand oligonucleotide In the form of the double-stranded oligonucleotide according to the present invention, DNA-RNA hybrid, siRNA (short interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), etc. 2021/229479 ? €1/162021/054077
- the double-stranded oligonucleotide according to the present invention is Although mainly described, it will be apparent to those skilled in the art that it can be applied to other double-stranded oligonucleotides having the same properties as the double-stranded oligonucleotides of the present invention. More preferably, the double-stranded oligonucleotide structure comprising the Empiregulin-specific double-stranded oligonucleotide according to the present invention has the structure of the following structural formula (2).
- Structural Formula (2) In Structural Formula (2), ⁇ ⁇ X and V are the same as defined in Structural Formula (1), £ is the sense strand of the empiregulin-specific nucleotide sequence, and ⁇ is the empiregulin refers to the antisense strand of a specific double-stranded oligonucleotide. More preferably, the double-stranded oligonucleotide structure comprising the empiregulin-specific double-stranded oligonucleotide has the structure of the following structural formula (3) or (4). 2021/229479 ? €1/162021/054077
- the hydrophilic material may be characterized in that polyethylene glycol is selected from the group consisting of polyvinylpyrrolidone and polyoxazoline, but is not limited thereto.
- the double-stranded oligonucleotide construct comprising the Empiregulin-specific double-stranded oligonucleotide in Structural Formulas (1) to (4) has a phosphate group at the end of the antisense strand.
- One to three may be combined, and it will be apparent to those of ordinary skill in the art that [ 1/ ⁇ may be used instead of [ ⁇ 1].
- the hydrophilic material in Structural Formulas (1) to (4) is preferably a polymer material having a molecular weight of 200 to 10,000, and more preferably, a polymer material having a molecular weight of 1,000 to 2,000.
- hydrophilic polymer material it is preferable to use a nonionic hydrophilic polymer compound such as polyethylene glycol, polyvinylpyrrolidone, and polyoxazoline, but is not limited thereto. 2021/229479 ? €1/162021/054077
- the hydrophilic material (/ ⁇ ) in Structural Formulas (1) to (4) may be used in the form of a hydrophilic material block example 0) having the form of the following Structural Formula (5) or Structural Formula (6).
- a hydrophilic material block example 0 having the form of the following Structural Formula (5) or Structural Formula (6).
- (/ ⁇ - ⁇ Structural Formula (5) A linker connecting between the hydrophilic material monomers or between the two hydrophilic material monomers and the oligonucleotide, which is an integer from 1 to 15, n is an integer from 1 to 10, and is represented by (/ ⁇ ) or / ⁇ '
- the repeating unit corresponds to the basic unit of the hydrophilic material block.
- the double-stranded oligonucleotide structure comprising the empiregulin-specific double-stranded oligonucleotide according to the present invention is the following Structural Formula (7) or Structural Formula (8) ) can have the same structure.
- X, (I and 8 are the same as defined in Structural Formula (1), / V, ⁇ , and n are in Structural Formulas (5) and (6)
- the hydrophilic material monomer (/V) in the structural formulas (5) and (6) can be used without limitation as long as it meets the purpose of the present invention among the monomers of the nonionic hydrophilic polymer, preferably A monomer selected from compounds (1) to (3) described in Table 1, more preferably a monomer of compound (1) may be used, and 6 in compound (1) is preferably 0, £ can be selected.
- the monomer represented by compound (1) can introduce various functional groups, has good in vivo compatibility and induces less immune response, etc. Rather, it has the advantage of increasing the in vivo stability of the double-stranded oligonucleotide contained in the construct according to the structural formula (7) or (8) and increasing the delivery efficiency, so it is very suitable for the preparation of the construct according to the present invention do.
- the hydrophilic substance in the structural formulas (5) to (8) has a total molecular weight in the range of 1,000 to 2,000.
- hexaethylene glycol 6 ⁇ 36 6116 according to compound (1) in structural formulas (7) and (8) in structural formulas (7) and (8) is used, i. Since the molecular weight of the glycol spacer is 344, it is preferable that the number of repeating characters is 3 to 5.
- the present invention provides structural formula (5) and structural formula It is characterized in that the repeating unit of the indicated hydrophilic group, that is, the hydrophilic material block ⁇ 10 ⁇ ) can be used in an appropriate number represented by n.
- the same linker may be used for each block of the hydrophilic material, or a different linker may be used for each block of the hydrophilic material as a linker for mediating the binding of the hydrophilic material monomer.
- the same number of hydrophilic material monomers may be used in the material block.
- the linker (J) is preferably selected from the group consisting of -PCV-, -S0 3 - and -C0 2 -, but is not limited thereto, and according to the monomer of the hydrophilic material used, the present invention It is apparent to those skilled in the art that any linker may be used as long as it meets the purpose of
- the hydrophobic material (B) in Structural Formulas (1) to (4), Structural Formula (7) and Structural Formula (8) is formed through hydrophobic interaction between Structural Formulas (1) to Structural Formulas (4), Structural Formulas (7) and Structural Formulas ( It serves to form nanoparticles composed of the double-stranded oligonucleotide structure according to 8).
- the hydrophobic material preferably has a molecular weight of 250 to 1,000, a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C 12 to Cso unsaturated or saturated Hydrocarbons, diacyl phosphatidylcholine, fatty acids, phospholipids, lipopolyamines, etc. may be used, but are not limited thereto, and any It is obvious to those of ordinary skill in the art that a hydrophobic material can also be used.
- the steroid derivatives include cholesterol, cholestanol, cholic acid, and cholesteryl. 2021/229479 ? €1/162021/054077
- the glyceride derivative may be selected from mono-, di- and tri-glycerides, wherein the fatty acid of the glyceride is c 12 to Unsaturated or saturated fatty acids are preferred.
- the hydrophobic material is bound to the distal end of the hydrophilic material, and may be bound to any position of the sense strand or the antisense strand of the siRNA.
- the hydrophilic material or hydrophobic material in Structural Formulas (1) to (4), Structural Formulas (7) and Structural Formula (8) according to the present invention and the empiregulin-specific double-stranded oligonucleotide have a simple covalent bond or linker-mediated It is bonded by a covalent bond (X or .
- the linker mediating the covalent bond is covalently bonded to a hydrophilic material or a hydrophobic material at the end of the empiregulin-specific double-stranded oligonucleotide, and can be degraded in a specific environment if necessary. Therefore, the linker activates the empiregulin-specific double-stranded oligonucleotide and/or the hydrophilic material (or the hydrophobic material) during the preparation of the double-stranded oligonucleotide construct according to the present invention. Any compound can be used to bind to the above.
- the covalent bond may be either a non-cleavable bond or a degradable bond. 2021/229479 ? 1/162021/054077
- the non-degradable bond includes an amide bond or a phosphorylation bond
- the degradable bond includes a disulfide bond, an acid-decomposable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzyme-degradable bond, but is not limited thereto.
- the empiregulin-specific double-stranded oligonucleotides represented by R (or S and AS) in the structural formulas (1) to (4), structural formulas (J) and (8) are specific to the mRNA of empiregulin.
- any double-stranded oligonucleotide having properties capable of binding positively can be used without limitation, and preferably, in the present invention, the sense strand comprising any one sequence selected from SEQ ID NOs: 10, 11 and 12 and complementary thereto consists of the antisense strand comprising the sequence.
- the present invention also relates to a double-stranded oligonucleotide construct comprising the Empiregulin-specific double-stranded oligonucleotide according to the present invention, wherein an amine group or polyhistidine (polyhistidine) ) groups may be additionally introduced.
- histidine formula can be used to increase the endosomes escape efficiency in such a liposome non-viral gene delivery system (non-viral gene carrier)
- ⁇ Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer in human hepatoma HepG2 cells.
- Controlled Release 118, pp262-270 The amine group or polyhistidine group may be connected to the hydrophilic material or the hydrophilic material block through one or more linkers.
- Structural Formula (1) When an amine group or a polyhistidine group is introduced into the hydrophilic material of the double-stranded oligonucleotide structure according to Structural Formula (1) of the present invention, it may have a structure as shown in Structural Formula (9).
- - ⁇ - ⁇ Structural Formula (9) ⁇ X in Structural Formula (9) is the same as the definition in Structural Formula (1), 2021/229479 1 ⁇ (:1 ⁇ 2021/054077
- ⁇ ) 2 is a simple covalent bond or 0 3 , is preferably an alkyl, but is not limited thereto.
- _) 2 is a simple covalent bond or 0 3 -
- the silver compound (4) is preferable, but is not limited thereto.
- Unity when the hydrophilic material of the double-stranded oligonucleotide structure according to Structural Formula (9) is a hydrophilic material block according to Structural Formula (5) or (6), and an amine group or polyhistidine group is introduced thereto It may have a structure such as Structural Formula (10) or Structural Formula (11). 2021/229479 ? €1/162021/054077
- the hydrophilic material is preferably in a form bound to the 3' end of the empiregulin-specific double-stranded oligonucleotide sense strand, and in this case, the Structural Formulas (9) to Structural formula (11) may have the form of the following structural formulas (12) to (14).
- Structural formula (12) ⁇ 4 ) ( ⁇ 3, 5 5 ⁇ 8 ⁇ Structural formula (13)
- the amine group that can be introduced in the present invention refers to the 5' end and 3' end of the specific double-stranded oligonucleotide sense strand.
- the amine group that can be introduced in the present invention primary to tertiary amine groups may be used, and it is particularly preferable that primary amine groups are used.
- the introduced amine group may exist as an amine salt, for example, the salt of the primary amine group may exist in the form of 1 ⁇ 11_1 3 + .
- the polyhistidine group that can be introduced in the present invention may include 3 to 10 histidines, particularly preferably 5 to 8, and most preferably 6 histidines. Additionally, one or more cysteines may be included in addition to histidine.
- a targeting moiety is provided in the double-stranded oligonucleotide construct comprising the Empiregulin-specific double-stranded oligonucleotide according to the present invention and the nanoparticles formed therefrom, it effectively promotes delivery to the target cell, resulting in a relatively low It can be delivered to a target cell even at a dose of a high concentration, thereby exhibiting a high target gene expression control function, and preventing the non-specific transfer of the non-specific empiregulin-specific double-stranded oligonucleotide to other organs and cells.
- the present invention provides a ligand (1_), particularly a receptor-mediated
- a double-stranded ligand (1 ⁇ ratio additionally bound to a ligand that specifically binds to a receptor that promotes target cell internalization 0
- oligo RNA and a structure, for example, a form in which a ligand is bound to a double-stranded oligo RNA structure according to Structural Formula (1) has a structure as shown in Structural Formula (15) below.
- (Li-Z)-AXRYB Structural Formula (15) In Structural Formula (15), A,min )( and ⁇ are the same as defined in Structural Formula (1) above, and L is receptor-mediated endocytosis, RME) refers to a ligand having a property of specifically binding to a receptor that promotes internalization of a target cell, and i is an integer of 1 to 5, preferably an integer of 1 to 3.
- the ligand is preferably a target receptor-specific antibody, aptamer, or peptide having RME properties for enhancing target cell-specific cell internalization; or folate (generally folate and foHc acid cross each other)
- folate generally folate and foHc acid cross each other
- folic acid refers to folate in a natural state or an activated state in the human body
- hexoamine such as N-acetyl galactosamine (NAG), and glucose (glucose) , mannose
- the hydrophilic substance A in Structural Formula (15) is Structural Formula (5) and Structural Formula (6) ) according to the hydrophilic material block. 2021/229479 ? €1/162021/054077
- the present invention provides a method for preparing a double-stranded oligonucleotide construct comprising an empiregulin-specific double-stranded oligonucleotide.
- the process of preparing a double-stranded oligonucleotide construct comprising an empiregulin-specific double-stranded oligonucleotide according to the present invention is, for example,
- the solid support in the present invention is preferably Controlled Pore Glass (CPG), but is not limited thereto, and polystyrene (PS), polymethyl methacrylate 1 1 / ⁇ ) silica gel, cellulose paper, etc. may be used.
- CPG Controlled Pore Glass
- PS polystyrene
- PS polymethyl methacrylate 1 1 / ⁇ silica gel
- cellulose paper etc.
- step (5) when the production is completed, the purified -Polymer constructs and oligonucleotide single-stranded
- step (4) of synthesizing an oligonucleotide single-stranded sequence complementary to the sequence of the oligonucleotide single-strand synthesized in step (2) is performed before step (1) or steps (1) to (5) It may be performed during any one of the processes.
- the oligonucleotide single strand synthesized in step (2) and the oligonucleotide single strand comprising the complementary sequence may be a production method characterized in that a phosphate group is attached to the end thereof.
- a method for preparing a double-stranded oligonucleotide construct in which a ligand is additionally bound to the double-stranded oligonucleotide construct comprising the empiregulin-specific double-stranded oligonucleotide of the present invention is, for example,
- (7) preparing a ligand-double-stranded oligonucleotide structure through annealing of the prepared ligand-oligonucleotide-polymer structure and an oligonucleotide single-stranded complementary sequence; may be included.
- step (6) when the preparation is completed, the ligand-oligonucleotide-polymer structure and the oligonucleotide single-strand of the complementary sequence are separated and purified, and the molecular weight is measured with a / ⁇ 1_- mass spectrometer to measure the desired ligand-oligo. It can be confirmed whether the nucleotide-polymer structure and the complementary oligonucleotide have been prepared.
- a ligand-double-stranded oligonucleotide structure can be prepared by annealing the prepared ligand-oligonucleotide-polymer structure and an oligonucleotide single-stranded complementary sequence.
- the step (4) of synthesizing an oligonucleotide single-stranded sequence complementary to the sequence of the oligonucleotide single-strand synthesized in step (3) is an independent synthesis process before step (1) or step (1). to (6) may be performed during any one of the steps. 2021/229479 ? €1/162021/054077
- the present invention relates to nanoparticles comprising the double-stranded oligonucleotide construct according to the present invention.
- self-assembled nanoparticles are formed by the hydrophobic interaction of hydrophobic substances (Korea Patent Publication No. 1224828). Not only the stability of the drug is very good, but also the uniformity of the particle size is excellent, so that the preparation process as a drug is simple because it is easy to divide.
- the nanoparticles may be characterized in that a double-stranded oligonucleotide structure comprising a double-stranded oligonucleotide comprising different sequences is mixed, for example, the nanoparticles are SEQ ID NO: 10 to It may include one type of empiregulin-specific double-stranded oligonucleotide comprising a sense strand comprising any one sequence selected from 12 and an antisense strand comprising a sequence complementary thereto, but in another embodiment, the sequence It may include different types of empiregulin-specific double-stranded oligonucleotides including a sense strand comprising any one sequence selected from Nos.
- composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.
- a pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, saline, sterile water, 2021/229479 ? €1/162021/054077
- Ringer's solution buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one component or a mixture of two or more of these components can be used.
- diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc.
- a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for freeze-drying may be added.
- composition of the present invention can be determined by one of ordinary skill in the art based on the symptoms of the ordinary patient and the severity of the disease. In addition, it can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc. .
- the composition of the present invention can be administered orally or parenterally.
- the route of administration of the composition according to the present invention is not limited thereto, but for example, oral, inhalational, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, Enteral, sublingual or topical administration is possible.
- the dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, method, excretion rate or severity of disease, etc. can decide In addition, 2021/229479 ? €1/162021/054077
- compositions of the present invention can be formulated into suitable dosage forms using known techniques.
- the present invention relates to a formulation in a lyophilized form comprising the pharmaceutical composition according to the present invention.
- the present invention provides an object in need of prevention or treatment of obesity (i) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and complementary thereto an empiregulin-specific double-stranded oligonucleotide comprising an anti-sense strand comprising a sequence;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the empiregulin-specific double bond of (i). strand oligonucleotide
- nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii) above; it relates to a method for preventing or treating obesity, comprising administering any one selected from the group consisting of.
- the present invention for use in a method for preventing or treating obesity (i) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and complementary thereto An antisense strand containing a specific sequence (anti-sense 2021/229479 ? €1/162021/054077
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is the empiregulin specific in (i) red double-stranded oligonucleotides
- nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of (ii) above; relates to a pharmaceutical composition, characterized in that any one selected from the group consisting of.
- the present invention provides (i) a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11 and 12 and an antisense strand comprising a sequence complementary thereto (anti- sense strand) containing an empiregulin-specific double-stranded oligonucleotide;
- A is a hydrophilic material
- B is a hydrophobic material
- X and Y are each independently a simple covalent bond or a linker-mediated covalent bond
- R is (i) 2021/229479 ? €1/162021/054077
- Nanoparticles comprising the empiregulin-specific double-stranded oligonucleotide structure of 01) above; relates to a use for the manufacture of a drug for the prevention or treatment of obesity in any one selected from the group consisting of.
- the obesity may be characterized as visceral fat type obesity due to diabetes, but is not limited thereto.
- the empiregulin-specific double-stranded oligonucleotide construct according to the present invention may be characterized by exhibiting one or more of the following effects, but is not limited thereto:
- SAMiRNA-based drug high-throughput screening is possible by applying a 1-base or 2-base sliding window algorithm to the entire mRNA. This is a method of generating a candidate sequence, removing unnecessary candidate sequences by performing homology filtering, and confirming the degree of inhibition of the corresponding gene expression for all SAMiRNAs finally selected.
- the design process for the SAMiRNA candidate sequence for Empiregulin is composed of 19 bases by applying a 1-base sliding window algorithm to NM_001657.3 (1290bp), a human Ampiregulin mRNA.
- NM_001657.3 (1290bp
- an experiment on the degree of inhibition of empiregulin was performed. 2021/229479 ? €1/162021/054077
- RNA or DNA synthesis is performed, and then C 24 (C 6 -SSC 18 ) containing a disulfide bond, which is a hydrophobic material, is additionally bound to the 5' end, and NAG-hexaethylene glycol- at the 3' end.
- RNA single-stranded and oligo synthesized by treatment with 28% (v/v) ammonia in a water bath - Two I, deprotection reaction in which the polymeric structure is separated from CPG
- the protective residue was removed through RNA single-stranded and oligo (DNA or RNA) with protective residues removed - 2021/229479 ? €1/162021/054077
- RNA single-stranded, oligo (DNA or RNA)-polymer structures and ligand-bound oligo (DNA or RNA)-polymer structures were separated from the reactants by High Performance Liquid Chromatography (HPLC), and MALDI The molecular weight was measured with a -TOF mass spectrometer (MALDI TOF-MS, SHIMADZU, Japan), and it was confirmed whether the nucleotide sequence and the oligo-polymer structure to be synthesized matched.
- HPLC High Performance Liquid Chromatography
- each double-stranded oligo structure equal amounts of the sense strand and the antisense strand were mixed, followed by 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate), 2 mM magnesium acetate (Magnesium acetate), pH. 7.0 ⁇ 7.5), reacted at 90°C for 3 minutes in a constant temperature water bath, and then reacted again at 37°C to prepare the desired SAMiRNA. Annealing of the prepared double-stranded oligo RNA constructs was confirmed through electrophoresis.
- Example 3 High-Speed Bulk Screening (HTS) of SAMiRNA Nanoparticles Inducing RNAi by Targeting Human Empiregulin
- a human lung cancer cell line A549 (ATCC ® CCL-185ä, Manassas, VA, USA) was used, and the A549 cell line was 10% Fetal.
- GibcoäHam's F-12K (Kaighn's) medium (Thermo, US) containing Bovine Serum (Hyclone, US) and 1% Penicillin-Streptomycin (Hyclone, US) was used and cultured at 37 °C, 5% CO 2 conditions.
- the A549 cell line was aliquoted in a 96-well plate (Costar, US) at 2X10 4 cells/well, and the next day, SAMiRNA homogenized with deionized distilled water in Example 3.1 was mixed with 1X DPBS. was diluted to 500 nM or 1000 nM to the cells. SAMiRNA was treated once every 12 hours, a total of 4 times, and cultured at 37 °C, 5% C0 2 conditions.
- the performance of the primer and hydrolysis probe sequence X used was determined by PCR amplification efficiency (Table 3) by creating a calibration curve using A549 cell total RNA.
- RT-qPCR was performed at 95 °C, 5 min, (95 °C). C, 5 sec, 58 °C, 15 sec) x 45 cycles and a protocol in which the fluorescence value is detected at each cycle was used
- SAMiRNA-treated 96-well plate (Costar, US) is an automated equipment from total RNA extraction to RT-qPCR, ExiStationTM HTKorea, HT DNA/RNA extraction kit (Bioneer, Korea) and primers and probes for empiregulin analysis
- Total RNA extraction and one-step RT-qPCR were performed according to the automation program using the separately manufactured AccuPower 13 Dual-HotStart RT-qPCR kit (Bioneer, Korea).
- 2(-Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. Dec;25(4):4 02-8], the relative amount of empiregulin mRNA in the experimental group compared to the control group ( ⁇ / ⁇ ) was calculated through relative quantitative analysis. 2021/229479 ? €1/162021/054077
- Ampiregulin-specific SAMiRNA candidate sequences selected by 1-base sliding window screening and high-throughput screening (HTS)
- Example 4 Screening of SAMiRNA nanoparticles inducing RNAi by targeting human (Human) Empiregulin Using SAMiRNA having the sequences of SEQ ID NOs: 1 to 14 selected in Example 3 as sense strands, respectively, the lung cancer cell line A549 was treated, and the expression pattern of empiregulin mRNA in the cell line was analyzed.
- the A549 cell line was aliquoted in a 12-well plate (Costar, US) at a condition of 8X10 4 cells/well, and the next day, SAMiRNA homogenized with deionized distilled water in Example 3.1 was mixed with 1X DPBS. was diluted to 200 nM or 600 nM in cells. SAMiRNA was treated once every 12 hours, a total of 4 times, and cultured at 37 °C, 5% C0 2 conditions. 2021/229479 ? €1/162021/054077
- RNA reverse transcriptase e.g., RNA reverse transcriptase (AccuPower ® RocketScriptTM Cycle).
- RT Premix with oligo (dT)20 RNA reverse transcriptase
- cDNA was prepared in the following manner. Specifically, 1pg of RNA extracted per tube was added to AccuPower ® RocketScriptTMCycle RT Premix with oligo (dT)20 (Bioneer, Korea) contained in a 0.25 mH Eppendorf tube, and DEPC (diethyl pyrocarbonate) treated distilled water was added.
- RNA and primers were hybridized for 30 seconds at 37 °C with a gene amplifier (MyGenie TM 96 Gradient Thermal Block, BIONEER, Korea), and producing cDNA at 48 °C for 4 minutes. , the amplification reaction was terminated by inactivating the enzyme at 95 °C for 5 min.
- a gene amplifier MyGenie TM 96 Gradient Thermal Block, BIONEER, Korea
- the relative expression rate of empiregulin mRNA compared to the SAMiRNA control sample was analyzed as follows.
- the cDNA prepared in Example 4-2-1 was diluted 5-fold with distilled water, and the diluted cDNA 3 and Accu Power ® 2X GreenStarä qPCR MasterMix ( BIONEER, Korea) 25 "2, distilled water 19 "2, empiregulin qPCR primers (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole ⁇ , respectively, BIONEER, Korea) 3/J2 was put in to make a mixed solution.
- Glyceraldehyde-3-Phosphate Dehydrogenase Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
- GPDH Glyceraldehyde-3-Phosphate Dehydrogenase
- the 96-well plate containing the mixture was subjected to the following reaction using an Exicyclerä Real-Time Quantitative Thermal Block (BIONEER, Korea): Reacted at 95 °C for 15 minutes to activate the enzyme and remove the secondary structure of cDNA.
- the Ct (threshold cycle) value of each obtained target gene was treated with two I, which obtained the Ct value of the target gene corrected through the GAPDH gene, and SAMiRNA (SAMiCONT), a control sequence that does not cause gene expression inhibition.
- the difference in ACt values was calculated using the treated experimental group as a control group.
- the expression level of the target gene in the cells treated with the empiregulin-specific SAMiRNA was relative quantified using the ACt value and the formula 2(- ⁇ Ct)x100. 2021/229479 ? €1/162021/054077
- Example 5 Inhibition of Expression of Human Empiregulin Using SAMiRNA Selected in Lung Cancer Cell Line (A549) Using SAMiRNA having the sequences of SEQ ID NOs: 10, 11 and 12 selected in Example 4 as sense strands, respectively It was treated with the lung cancer cell line A549, and the expression pattern of the empiregulin mRNA in the cell line was analyzed to determine the IC 5Q value of SAMiRNA.
- the size and polydispersity index of the SAMiRNA were measured using Zetasizer Nano ZS (Malvern, UK), and the size and polydispersity of nanoparticles for the selected SAMiRNA The index is shown in Table 7 below, and the graph was measured as shown in FIG. 2 .
- A549 a human-derived lung cancer cell line
- the A549 cell line was 10% Fetal Bovine Serum (Hyclone, US) and 1 GibcoäHam's F-12K (Kaighn's) medium (Thermo, US) containing % Penicillin- Streptomycin (Hyclone, US) was used and cultured at 37 °C, 5% C0 2 conditions.
- the A549 cell line was aliquoted in a 12-well plate (Costar, US) under the condition of 8X10 4 cells veil, and the next day, SAMiRNA homogenized with deionized distilled water in Example 5.1 was mixed with 1X DPBS. was diluted to 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 600 nM or 1200 nM cells. SAMiRNA was treated once every 12 hours, a total of 4 times, and cultured at 37 °C, 5% C0 2 conditions.
- Example 5-2 the whole was extracted from the cell line treated with £ 1 ⁇ /11[ ⁇ 1 2021/229479 ? €1/162021/054077
- the mRNA expression level of the MPIregulin gene was relative quantified using two I, real-time PCR, in which 60 cDNA was prepared.
- RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea)
- RNA reverse transcriptase (AccuPower ® RocketScriptTM Cycle).
- cDNA was prepared in the following manner. Specifically, add 1pg of RNA extracted per tube to 0.25 (AccuPower ® RocketScriptTMCycle RT Premix with oligo (dT)20 (Bioneer, Korea) contained in an Eppendorf tube and DEPC (diethyl) so that the total volume is 20.
- pyrocarbonate pyrocarbonate-treated distilled water was added.
- This is a gene amplifier (MyGenie TM 96 Gradient Thermal Block, BIONEER, Korea) that hybridizes RNA and primers at 37°C for 30 seconds, and cDNA is prepared at 48°C for 4 minutes.
- the amplification reaction was terminated by inactivating the enzyme for 5 minutes at both I and 95, where the process was repeated 12 times.
- Example 5-3-1 5-3-2 Relative quantitative analysis of human (Human) Empiregulin mRNA
- the real-time qPCR of the SYBR green method was performed on the ampiregulin mRNA compared to the SAMiRNA control sample.
- the relative expression rate was analyzed as follows. In each well of a 96-well plate, the cDNA prepared in Example 5-3-1 was diluted 5-fold with distilled water, and the expression level of Empiregulin mRNA 2021/229479 ? €1/162021/054077
- diluted cDNA 3 and Accu Power ® 2XGreenStarä qPCR MasterMix (BIONEER, Korea) were mixed with 25"2, distilled water 19"2, Ampiregulin qPCR primers (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole each) , BIONEER, Korea) was added to 3/J2 to make a mixed solution.
- Ampiregulin qPCR primers SEQ ID NOs: 17 and 18 (Table 5); 10 pmole each) , BIONEER, Korea
- Glyceraldehyde-3-Phosphate Dehydrogenase Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
- the 96-well plate containing the mixture was subjected to the following reaction using an ExicycleräReal-Time Quantitative Thermal Block (BIONEER, Korea): Reacted at 95 °C for 15 minutes to activate the enzyme and remove the secondary structure of cDNA. Then, 42 cycles of 4 processes: denaturing at 94 °C for 30 sec, annealing at 58 °C for 30 sec, extension at 72 °C for 30 sec, and SYBR green scan Repeatedly, after performing a final extension at 72 °C for 3 minutes, the temperature was maintained at 55 °C for 1 minute, and the melting curve from 55 °C to 95 °C was analyzed.
- BIONEER ExicycleräReal-Time Quantitative Thermal Block
- the Ct (threshold cycle) value of each obtained target gene was treated with two I, the Ct value of the target gene corrected through the GAPDH gene, and SAMiRNA (SAMiCONT), a control sequence that does not inhibit gene expression.
- the difference in ⁇ Ct was calculated using the experimental group as a control group.
- the expression level of the target gene in the cells treated with the empiregulin-specific SAMiRNA was relative quantified using the ⁇ Ct value and the formula 2 (- ⁇ Ct) X 100.
- ePBMC ® Cryopreserved Human PBMC (human peripheral blood mononuclear cells, Cellular Technology Limited) in RPMI1640 (Hycloneä) medium containing 10% FBS (fetal bivine serum; Hyclone). , USA) was dispensed in a 12-well plate (Costar ® USA) so that 5X10 5 cells per well were incubated for 1 hour at 37°C, 5% C0 2 incubator to stabilize the cells, and then SAMi was added to the dispensed PBMCs.
- RNA extraction kit RNeasy Mini Kit, Qiagen, Germany
- RNase-Free DNase Set Qiagen, Germany
- 200ng of extracted RNA, deionized sterile DW (Bioneer, Korea), and RNA reverse transcriptase AccuPower ® RocketScriptTMCycle RT Premix with oligo (dT)20, Bioneer, Korea
- a gene amplifier MyGenie TM 96 Gradient
- a total of 20 Ml cDNA was synthesized using the same conditions as (37°C 30 sec, 48°C 4 min, 55°C 30 sec) x 12 cycle, 95°C 5 min using Thermal Block, BIONEER, Korea). did.
- the synthesized cDNA was mixed with qPCR primers for RPL13A, I L1 B, IL6, IFNG, TNF, and IL12B genes, and then using ExicyclerTM96 Real-Time Quantitative Thermal Block (Bioneer, Korea) at 95°C for 5min, (95 °C 5 sec, 58 °C 15 sec) x 45 cycle conditions.
- 2(-Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001.
- Example 7 Comparative analysis of inhibition of expression of human ampiregulin by DNA/RNA hybrid and RNA/RNA hybrid SAMiRNA containing the selected sequences of SEQ ID NOs: 10, 11 and 12 as sense strands Selection in Example 4
- a lung cancer cell line A 549 was treated using double-stranded oligo DNA/RNA hybrid and RNA/RNA hybrid containing the empiregulin-specific SAMiRNA having the sequences of SEQ ID NOs: 10, 11 and 12 as sense strands, respectively, and The relative expression level ( ⁇ / ⁇ ) of empiregulin mRNA in the cell line was comparatively analyzed.
- the A549 cell line was aliquoted in a 12-well plate (Costar, US) at a condition of 8X10 4 cells/well, and the next day, SAMiRNA homogenized with deionized distilled water in Example 3.1 was mixed with 1X DPBS. was diluted to 200 nM, 600 nM or 1200 nM to the cells. SAMiRNA was treated once every 12 hours, a total of 4 times, and cultured at 37 °C, 5% C0 2 conditions. 2021/229479 ? €1/162021/054077
- Example 7-1 SAMiRNA Screening by Analysis of Efficacy of Human Ampiregulin mRNA Expression Inhibition
- , real-time PCR real-time PCR
- RNA extracted per tube was added to Accu Power ® RocketScriptTM Cycle RT Premix with oligo (dT)20 (Bioneer, Korea), and DEPC (diethyl pyrocarbonate)-treated distilled water was added so that the total volume was 20). .
- This is a gene amplifier (MyGenie TM 96 Gradient Thermal Block, BIONEER, Korea) that hybridizes RNA and primers at 37 °C for 30 seconds, and repeats the two processes of producing cDNA at 48 °C for 4 minutes 12 times. The amplification reaction was terminated by inactivating the enzyme at 95 °C for 5 min.
- the relative expression rate of ampiregulin mRNA was analyzed in the following way, compared to the SAMiRNA control sample, through real-time qPCR using the SYBR green method.
- the cDNA prepared in Example 7-2-1 was diluted 5-fold with distilled water, and the diluted cDNA 3 and Accu Power ® 2X GreenStaräqPCR MasterMix (BIONEER) , Korea) 25 "2, distilled water 19 "2, empiregulin qPCR primers (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole ⁇ each, BIONEER, Korea) was added to 3 to make a mixed solution.
- Glyceraldehyde-3-Phosphate Dehydrogenase Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
- GPDH Glyceraldehyde-3-Phosphate Dehydrogenase
- the 96-well plate containing the mixture was subjected to the following reaction using an ExicycleräReal-Time Quantitative Thermal Block (BIONEER, Korea): Reacted at 95 °C for 15 minutes to activate the enzyme and remove the secondary structure of cDNA.
- the Ct (threshold cycle) value of each obtained target gene was treated with two I, the Ct value of the target gene corrected through the GAPDH gene, and SAMiRNA (SAMiCONT), a control sequence that does not inhibit gene expression.
- the difference in ⁇ Ct was calculated using the experimental group as a control group.
- the expression level of the target gene in the cells treated with the empiregulin-specific SAMiRNA was relative quantified using the ⁇ Ct value and the formula 2 (- ⁇ Ct) x 100. 2021/229479 ? €1/162021/054077
- Table 8 shows the in vitro expression inhibitory effect of treatment at a concentration of 1 uM in cell culture conditions in the presence of
- Primary screening was performed using primers.
- FIG. 9 two sequences (SEQ ID NOs: 19 and 20) selected from the NIH3T3 cell line through additional screening and mouse SAMiRNA-MP of SEQ ID NO: 21 discovered through prior milestone studies For Regulin, the mouse lung fibroblast cell line MLg cell line (ATCC® CCL-206ä, Manassas, VA, USA) was treated with 200 nM and 500 nM treatment concentrations in cell culture conditions in the presence of 10% FBS. Results of additional screening SEQ ID NO: 20 confirmed the best expression inhibitory effect.
- FIG. 10a In addition, two sequences selected in the mouse Lung epithelial cell line LA-4 cell line (ATCC® CCL-196ä, Manassas, VA, USA) No. 19, 20) and mouse SAMiRNA-empiregulin of SEQ ID NO: 21 discovered through previous milestone studies were added by treatment with 200 nM, 500 nM and 1000 nM treatment concentrations in cell culture conditions in the presence of 10% FBS 2021/229479 ? €1/162021/054077
- Example 9 Confirmation of Obesity Inhibitory Effect Using a Type 2 Diabetes Model
- the sense strand represented by the following sequence and the complementary antisense strand were included as the empiregulin expression inhibitor.
- SAMiRNA-AREG SAMi-mAREG#20
- SAMi-mAREG#20 SAMiRNA-AREG was administered to a type 2 diabetes model and the effect on fat reduction was observed. After administering the drug 3 times a week for 8 weeks, the mice were sacrificed and various indicators were observed.
- mice BKS.Cg- m ++/+ /e> ⁇ /(KRIBB; Korea Research Institute of Bioscience and Biotechnology) 5 weeks old mice were sold and the experiment was carried out.
- the obese diabetic group (db/db mice) was acclimatized for 3 weeks after distribution, and rodent diet (Rodent diet, 2918C, Harlan, USA) was induced for 8 weeks, and the experiment was carried out according to the experimental conditions below.
- rodent diet Rodent diet, 2918C, Harlan, USA
- the normal control group was given 100 ul of PBS (C-9011, Bioneer, Korea), and the obese and diabetic group was divided into 3 groups and each given 100 ul of PBS, 100 ul of SAMiRNA-Cont 5 mg/kg, and 100 ul of SAMiRNA-AREG 5 mg/kg. It was administered by subcutaneous injection three times. Breeding environment temperature is 21 ⁇ 2°C, humidity 55 ⁇ 5%, 15 ni 7/hour, illumination 150 300 Lux, 12 hours cycle (light on: 06:00, off: 18:00). 2021/229479 ? €1/162021/054077
- the intake of 72 was also about 5 mL/day in the normal control mice, whereas in the obese and diabetic mice, the intake was significantly higher at 20-35 mL/day ( FIG. 13 ).
- Serum glucose level ⁇ was 149.6 ⁇ 20.4 in the normal control group, 689.1 ⁇ 185.4 in the obese and diabetic group on average, and £1 ⁇ /11[ ⁇ 1/ ⁇ -(;The 0 administration group averages 755.5 ⁇ 89.4, $ 1 ⁇ hour [ ⁇ / ⁇ - [ 3 administration group recorded an average of 874.3 ⁇ 119.4 (FIG. 18)). Therefore, it was confirmed that the decrease in the expression level of empiregulin according to the present invention in obese diabetic mice had no significant effect on regulating fasting blood glucose or serum glucose levels in diabetic animal models.
- Example 10 Confirmation of obesity suppression effect using high fat diet obesity model Pears [ (3#20) were administered to a high-fat diet-induced obesity model, and the effect on fat reduction was observed. After administration of the drug 3 times a week for 5 weeks of diet induction, the mice were sacrificed and various indicators were observed.
- mice [57 mi_/6 (Nara Biotech) 5 weeks old mice were sold and the experiment was carried out. After acclimatization for 1 week after distribution, a 60 kcal% Fat diet (D12492, Research Diets, Inc.) was induced for 6 weeks. 2021/229479 ? 1/162021/054077
- RNA extraction from adipose tissue was performed with a combination of QIAzol Lysis Reagent (QIAZEN., GER) and RNeasy Mini Kit (QIAZEN., GER). 100 mg of adipose tissue was put into QIAzol Lysis Reagent, and after homogenization, incubated for 5 minutes at room temperature. After centrifugation at 4 °C and 12000 g for 10 minutes, the lower part of the sample was recovered by avoiding the upper fat monolayer.
- QIAzol Lysis Reagent QIAZEN., GER
- RNeasy Mini Kit QIAZEN., GER
- [Table 9] [ ⁇ for primer sequence information is the forward direction and the non-primer, and is the reverse direction (meaning “geunyi primer, respectively).”
- the expression level of ampiregulin increased 12-fold compared to the normal control group by induction of a high-fat diet (Fig. 13 ⁇ 4.
- PBS normal control group
- HFD+PBS negative control group
- the temperature of the breeding environment is 21 ⁇ 2°C, humidity 55 ⁇ 5%, 15 ni 7/hour, illumination 150 300 Lux, 12 hours cycle (light: 06:00, off: 18:00) constant during the experiment period. kept. After drug administration for 5 weeks, they were fasted for 16 hours and sacrificed.
- Body weight, feed and drinking water intake were measured at a certain time for each measurement, and were measured 2 hi/week.
- the weight of 76 mice at sacrifice was 37.3 ⁇ 1.20.
- the average body weight was 26.57 ⁇ 0.36 when the normal control group was administered with PBS, and the average weight was 33.21 ⁇ 1.16 when administered with SAMiRNA-AREG to the test substance group.
- body weight was observed at 4 and 5 weeks, a statistically significant difference was observed in the test substance administration group compared to the negative control group.
- the weight of the mice in the high-fat diet-induced negative control group increased significantly, while the weight of the mice was decreased in the test substance administration group, and a statistically significant difference was observed ( FIG. 20 ).
- the normal control group was 0.02 ⁇ 0.001
- the negative control group was 0.12 ⁇ 0.007
- the test substance administration group showed 0.08 ⁇ 0.011.
- a significantly lower dietary efficiency was observed compared to the control group (FIG. 22).
- Fat weight change Subcutaneous fat in sacrificed mice for fat weight measurement pad), epididymal fat (6
- the mouse subcutaneous fat weight in this case, the normal control group showed 0.38 ⁇ 0.02, the negative control group showed 1.74 ⁇ 0.12, the test substance administration group showed 1.05 ⁇ 0.14, and the epididymal fat weight (in this case, the normal control group showed 0.52 ⁇ 0.03, the negative control group showed 2.32 ⁇ 0.09, the test substance administration group showed 1.79 ⁇ 0.16, and the weight of peripheral fat (in this case, the normal control group showed 0.20 ⁇ 0.02, the negative control group showed 0.86 ⁇ 0.04, the test substance administration group showed 0.67 ⁇ 0.08, and mesenteric fat Weight (in this case, the normal control group showed 0.25 ⁇ 0.01, the negative control group showed 0.66 ⁇ 0.09, and the test substance administration group showed 0.45 ⁇ 0.04 (FIG. 233).
- the normal control group showed 1.39 ⁇ 0.08
- the negative control group showed 4.65 ⁇ 0.23
- the test substance administration group showed 3.11 ⁇ 0.35-1.
- the normal control group was 1.91 ⁇ 0.10
- the negative control group showed 6.23 ⁇ 0.09
- the test substance administration group showed 5.36 ⁇ 0.29
- the weight ratio (%) of peripheral fat was normal.
- the control group showed 0.73 ⁇ 0.06, the negative control group showed 2.33 ⁇ 0.13, and the test substance administration group showed 2.01 ⁇ 0.17, and in the case of the mesenteric fat weight ratio (%), the normal control group showed 0.91 ⁇ 0.02, the negative control group showed 1.76 ⁇ 0.19, and the test The substance administration group showed 1.35 ⁇ 0.07 (Fig. 23 bar, the mouse fat was divided into subcutaneous fat and visceral fat (epididymal fat, Perirenal fat, mesenteric fat) to represent the weight (g) and weight ratio (%).
- subcutaneous fat and visceral fat epididymal fat, Perirenal fat, mesenteric fat
- the normal control group showed 0.38 ⁇ 0.02, the negative control group showed 1.74 ⁇ 0.12, and the test substance administration group showed 1.05 ⁇ 0.14, and the weight ratio was 1.39 ⁇ 0.08 for the normal control group and 4.65 ⁇ 0.23 for the negative control group.
- the test substance administration group showed 3.11 ⁇ 0.35
- the normal control group showed 0.97 ⁇ 0.06, the negative control group showed 3.85 ⁇ 0.18, the test substance administration group showed 2.92 ⁇ 0.27, and the weight ratio was 3.56 for the normal control group.
- the negative control group showed 10.34 ⁇ 0.16
- the test substance administration group showed 8.73 ⁇ 0.50
- the weight and weight ratio of subcutaneous fat and visceral fat significantly increased in the negative control group compared to the normal control group
- the test substance administration group showed a significant increase It was confirmed that the effect of significantly reducing this increase in the control group (FIGS. 24a, 24b).
- the area of 79 (mm 3 ) was 1069 ⁇ 90 in the normal control group, 3782 ⁇ 7 in the negative control group, and 2623 ⁇ 166 in the test substance administration group.
- the area of subcutaneous fat and visceral fat increased in the negative control group compared to the normal control group, and it was confirmed that the area decreased in the test substance administration group compared to the negative control group (Fig. 2).
- the tissue slides hardened with the preservation solution were photographed at 200 magnification using an inverted microscope (INMkon eclipse TS2), and the area of adipocytes was calculated.
- the area (mm 2 ) was 913.8 ⁇ 129.9 in the normal control group, 3575.0 ⁇ 346.7 in the negative control group, and 1337.0 ⁇ 211.1 in the test substance administration group.
- the area (mm 2 ) was 950.6 ⁇ in the normal control group. 73.2, the negative control group showed 1598.0 ⁇ 99.6, the test substance administration group showed 1347.0 ⁇ 100.1, and the area (mm 2 ) was normal for the peripheral fat.
- the control group showed 1734.0 ⁇ 158.8, the negative control group showed 5365.0 ⁇ 190.4, and the test substance administration group showed 2516.0 ⁇ 288.3.
- the area (mm 2 ) was 1552.0 ⁇ 680.3 for the normal control group, 3495.0 ⁇ 425.3 for the negative control group, and the test substance.
- Substance administration group showed 1436.0 ⁇ 163.3.
- the area of adipocytes increased in the negative control group compared to the normal control group, and it was confirmed that the area decreased in the test substance administration group compared to the negative control group (FIG. 23 ⁇ 4.
- liver tissue The liver was removed and fixed in 10% neutral buffered formalin (10% neutral buffered formalin, Sigma, HT50-1-640) for more than 24 hours for H&E staining and Oil red 0 analysis. was carried out.
- H&E staining H&E (Hematoxylin & Eosin) staining was performed to observe the overall shape of the liver tissue obtained after fixation in neutral buffered formalin and the degree of lipid accumulation in the liver tissue.
- Tissue specimens were prepared by paraffin infiltration. After dehydration with ethanol, it was subjected to a three-step transparent process with xylene. Thereafter, a paraffin block was prepared through infiltration and embedding in liquid paraffin. Using a microtome, the tissue block was made into 5 M m-thick sections and dried in a slide dryer for 1 hour, then paraffin was removed with xylene and hydrated through an ethanol step. Harris 2021/229479 ? €1/162021/054077
- Nuclei were first stained for 5 minutes with 81 hematoxylin staining solution, and then counterstained with eosin solution. After staining, the mounting solution was dropped, and then covered with a cover glass to harden. The tissue slides hardened with the preservative solution were observed using a microscope.
- test substance administration group had an inhibitory effect on hepatic adipogenesis (FIG. 27).
- RNA extraction from adipose tissue was performed using a combination of TRI-Reagent (MRC Inc., USA) and Universal RNA extraction kit (BIONEER, Korea). 100 mg of adipose tissue was put into 1 ml of Tri reagent, and after homogenization, incubated at room temperature for 5 minutes. After centrifugation at 4 °C and 12000 g for 10 minutes, the sample at the bottom was recovered by avoiding the fat monolayer at the top.
- the pharmaceutical composition according to the present invention has an excellent effect of reducing visceral fat, especially subcutaneous fat, so that complications due to visceral fat are a problem in the treatment and prevention stage of cardiovascular disease, metabolic disease, diabetes and various other diseases. It may be useful.
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US17/998,662 US20230348912A1 (en) | 2020-05-14 | 2021-05-13 | Composition for preventing or treating obesity-related disease containing amphiregulin-specific double-stranded oligonucleotide structure |
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AU2021270895A AU2021270895A1 (en) | 2020-05-14 | 2021-05-13 | Composition for preventing or treating obesity-related disease containing amphiregulin-specific double-stranded oligonucleotide structure |
EP21803785.1A EP4151236A4 (en) | 2020-05-14 | 2021-05-13 | COMPOSITION FOR PREVENTING OR TREATING A DISEASE ASSOCIATED WITH OBESITY CONTAINING A DOUBLE-STRANDED OLIGONUCLEOTIDE STRUCTURE SPECIFIC TO AMPHIREGULIN |
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Publication number | Publication date |
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EP4151236A4 (en) | 2024-06-26 |
CN116157158A (zh) | 2023-05-23 |
KR102671315B1 (ko) | 2024-06-03 |
JP7554850B2 (ja) | 2024-09-20 |
BR112022022868A2 (pt) | 2023-01-31 |
KR20210141399A (ko) | 2021-11-23 |
EP4151236A1 (en) | 2023-03-22 |
JP2023525168A (ja) | 2023-06-14 |
CA3178609A1 (en) | 2021-11-18 |
US20230348912A1 (en) | 2023-11-02 |
AU2021270895A1 (en) | 2022-12-15 |
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