KR20190076126A - An oral antidiabetic composition comprising GLP-1 and a gene carrier - Google Patents

Abstract

The present invention relates to an oral composition for preventing, treating or alleviating obesity. It is confirmed that the oral composition can effectively induce the expression of GLP-1 genes in vivo by combining nine amino acids Arginine having positive ion properties to Fc part C-terminal to condense the same with GLP-1 genes having negative ion properties, and also protects genes from disintegration due to immune action of gastric acid and leukocytes during oral administration, thereby enabling gene delivery to a small intestine. In addition, the oral composition is confirmed to have a possibility of long-term treatment effect since a half-life is relatively long when genes are expressed in the small intestine. Therefore, by effectively delivery of GLP-1, the oral composition is expected to be useful for preventing, treating, or alleviating obesity.

Description

An oral antidiabetic composition comprising GLP-1 and a gene carrier and an antidiabetic composition comprising GLP-1 and a gene carrier,

The present invention relates to an antidiabetic composition for oral administration, and more particularly, to a GLP-1 receptor antagonist composition comprising GLP-1 and a GLP-1 transporter comprising a linker consisting of a cationic arginine capable of aggregating an anionic gene at the end of the immunoglobulin Fc region C As an active ingredient, to an oral composition for the prevention, treatment or amelioration of diabetes.

Diabetes is a metabolic disease caused by the absence of insulin secretion or action, characterized by hyperglycemia. Diabetes mellitus can be classified into Type 1 and Type 2 diabetes. Type 1 diabetes is caused by the destruction of pancreatic β-cells by autoimmune mechanisms and viral infections. In the case of type 2 diabetes (abbreviation: T2DM) Insulin resistance is the leading cause of decreased insulin secretion in β-cells and insulin action in target organs such as liver, adipose tissue and muscle. Type 2 diabetes can be improved through exercise therapy and weight loss, but in the case of type 1 diabetes, it is difficult to improve due to a decrease in insulin secretory ability, so means for supplementing insulin from the outside are being used.

Currently, drugs used in the treatment of type 2 diabetes include insulin preparations and hypoglycemic agents, and the most popular oral hypoglycemic agents are biguanide, which inhibits glucose synthesis in the liver, sulfonylurea, which promotes insulin secretion in β-cells And glinide, and thiazolidinedione, an insulin sensitizer. Despite the combined treatment of these drugs, about one-third of patients whose blood sugar is regulated below the regulatory target, these hypoglycemic agents and insulin can not cure or cure diabetes. In addition, current diabetes medicines have problems such as hypoglycemic effect, gastrointestinal disorder, diarrhea, side effects such as liver and cardiac toxicity, and therefore, there is a problem that strict management is required as it is a therapeutic agent for continuous administration.

There is a therapeutic method using nucleic acid transfer, which is a typical method that can be used to solve such a problem. As a method for this, there are a method using a viral vector and a method using a non-viral vector. However, viral vectors have limitations in vivo applications because they use carriers derived from viruses as raw materials, and research is currently under way to use vectors based on nonviral vectors, which are nonviral vectors. This method basically involves cationic compounds such as polymers, lipids or inorganic materials capable of binding or adsorbing negatively charged DNA to nano-sized particles. In addition, gene therapy using these nucleic acid transporters is attracting interest as a promising method for the prevention and treatment of T2MD by treating the source of T2MD.

On the other hand, in gene therapy, it has been known that a method using a gene for oral delivery can provide various advantages. First, convenience can be provided because it is possible to avoid an injection for a patient. Secondly, , It is possible to maintain the stability of the gene and to express the gene after transduction. In addition, an effective oral gene delivery system can be used for systemic delivery of protein drugs because it can induce protein circulation through the expression and secretion of therapeutic proteins of epithelial cells. Although the concept of oral gene therapy has already been proven, nonviral gene delivery through the intestinal tract is suffering from a lot of egg yolk due to the problem of low expression. There is also a difficulty in effectively controlling intestinal enzymes, microorganisms, and digestive enzymes.

References to the guidelines of the American Diabetes Association (ADA) suggest that the primary drug of choice is metformin, the secondary and tertiary drugs are sulfonylurea, glinide, thiazolidinedione ) And DPP-4 inhibitors, and thereafter injections such as GLP-1 (glucagon like peptide-1) agonist or insulin injections are used. However, in the case of existing oral diabetic medicines currently used in clinical practice, in addition to the positive aspect of maintaining normalization of blood glucose continuously, long-term use of diabetic medicines causes various side effects such as hypoglycemia, diarrhea, weight gain, cardiovascular problems, Eventually, the pancreatic beta cells are destroyed and finally injected with insulin. In addition, insulin administration, which is the last treatment method, is also inconvenient because it has to be subcutaneously injected 2-3 times a day, and the most serious side effect, hypoglycemia,

In order to overcome these problems, GLP-1 has recently emerged as a next-generation diabetes remedy. GLP-1 and its analogs and derivatives have shown good potential in clinical trials for the treatment of type 2 diabetes and have been shown to be effective in the treatment of type 2 diabetes, including insulin secretion stimulation, glucagon secretion inhibition, gastric emptying inhibition, gastric motility or intestinal motility inhibition, It induces numerous biological effects. In addition, even when taking a long-term pancreas protection function, there is no risk of hypoglycemia and can maintain good blood sugar for a long time.

However, in vivo, the GLP-1 is cleaved and inactivated by an enzyme called DPP-4, and thus has a very short in vivo half-life, making it difficult to develop it as a therapeutic agent. Thus, a variety of approaches have been performed to prolong the half-life of GLP-1 while maintaining biological activity or to reduce the rate of peptide removal from the body. In other words, various GLP-1 analogues have been actively developed, and GLP-1 fusion to the immunoglobulin Fc region has also been attempted (U.S. Pat. No. 7452966).

DISCLOSURE OF THE INVENTION An object of the present invention is to solve the above problems and to provide a method for efficiently delivering GLP-1 gene into an organism cell by efficiently conducting an anion- It was confirmed that the gene carrier having 9 amino acid Arginine having cationic properties bound to the C-terminal of the immunoglobulin Fc region has stability against pH and enzymes in the body and confirmed the possibility of use as a gene carrier for oral administration. The present invention has been completed.

It is an object of the present invention to provide GLP (glucagon like peptide) -1; And

An oral composition for the prevention or treatment of diabetes comprising a GLP-1 carrier,

The GLP-1 delivery vehicle

Immunoglobulin Fc region; And

Wherein the immunoglobulin Fc region comprises a linker linked to the C-terminus of the immunoglobulin Fc region.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object,

GLP (glucagon like peptide) -1; And

An oral composition for the prevention or treatment of diabetes comprising a GLP-1 carrier,

The GLP-1 delivery vehicle

Immunoglobulin Fc region; And

Wherein the immunoglobulin Fc region comprises a linker linked to the C-terminus of the immunoglobulin Fc region.

In one embodiment of the present invention, the GLP-1 may comprise the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the GLP-1 transporter may comprise the amino acid sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the GLP-1 transporter may comprise the nucleotide sequence of SEQ ID NO: 3.

In another embodiment of the present invention, the immunoglobulin Fc region may comprise the amino acid sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the immunoglobulin Fc region may comprise the nucleotide sequence of SEQ ID NO: 5.

In another embodiment of the present invention, the immunoglobulin Fc region may be derived from any one selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

In another embodiment of the present invention, the immunoglobulin Fc region can be derived from IgG.

In another embodiment of the present invention, the linker may comprise an amino acid sequence of Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg.

The present invention also provides a method of preventing or treating diabetes, comprising the step of administering the composition to a subject.

In addition, the present invention provides a composition for preventing or treating diabetes mellitus.

The oral composition for the prevention or treatment of diabetes according to the present invention is characterized in that 9 amino acid arginines having cationic properties are bound to the C-terminal of the Fc portion and the GLP-1 gene having an anionic property is condensed, In addition, it is possible to protect the gene from the degradative action due to the immunological action of gastric acid and leukocyte upon oral administration, so that it is possible to transfer the gene to the small intestine. Further, when the gene is expressed in the small intestine, By confirming the possibility of therapeutic effect, it is expected that GLP-1 can be effectively used for prevention, treatment or improvement of diabetes through effective delivery of GLP-1.

FIG. 1A shows the process of complex formation of the antibody hIgG1-Fc-9Arg with plasmid DNA (pDNA), and the process of transferring the antibody-therapeutic gene complex through the cell receptor.
FIG. 2 shows SDS-PAGE results of (850 bp) Lane 1.pcr annealing TM = 55 ° C and Lane 2.pcr annealing TM = 52 ° C for 9 antibodies bound to Arginine in human IgG1-Fc region.
Fig. 3 shows the nucleotide sequence of hIgG1-Fc-9Arg.
Figure 4 shows the molecular weight SDS-PAGE results of the purified antibody hIgG1-Fc-9Arg (Protein Ladder 10-245 kDa), Lane 2. 0.1 μg, Lane 3.1 μg, Lane 4. 10 μg antibody, Lane 5.) and The molecular weight results of hIgG1-Fc-9Arg maintaining SS binding under non-reducing conditions are shown.
FIG. 5 shows the results of electrophoresis of hIgG1-Fc-9Arg and pDNA complex agarose gel (0.8% agarose gel).
Figure 6 shows the results of TM-AFM imaging of the hIgG1-Fc-9Arg / pDNA complex, (a) hIgG1-Fc-9Arg, (b) hIgG1-Fc-9Arg / pDNA at 20/1 ratio Fc-9Arg / pDNA complex at a 50/1 ratio (w / w), (e) a hIgGl-Fc-9Arg / pDNA complex at a 100/1 ratio (w / w) (Scale bars 200 nm).
Figure 7 shows the TEM imaging results of the hIgG1-Fc-9Arg / pDNA complex, (a) hIgG1-Fc-9Arg / pDNA complex of 20/1 ratio (w / w), (b) 50/1 ratio (w / w) hIgGl-Fc-9Arg / pDNA complex (Scale bars 100 nm).
FIG. 8 shows the results of SDS-PAGE for the stability of the antibody hIgG1-Fc-9Arg over time under various pH conditions (pH 1.5, 5.0, 6.0, 7.4).
FIG. 9 shows the stability of plasmid DNA and hIgG1-Fc-9Arg / pDNA complex at pH 2.0 in gastric acid condition by agarose gel (0.8%) electrophoresis.
FIG. 10 shows the results of a serum protein stability test of a serum protein stability assay of hIgG1-Fc-9Arg / pDNA complex (10% Fetal bovine serum), (a) plasmid DNA, (b) 20/1 ratio complex c) 50/1 ratio, and (d) 100/1 ratio composite stability.
FIG. 11 shows western blots of FcRN receptor protein in various cell lines.
Figure 12 shows the fluorescence imaging results of FITC-hIgGl-Fc-9Arg absorbed through the FcRN receptor (1 hour incubation).
Figure 13 shows endosome formation and endosomal escape fluorescence imaging results of FITC-hIgG1-Fc-9Arg (30 min, 4 hr reaction conditions).
Fig. 14 shows gene expression fluorescence imaging results of the hIgG1-Fc-9Arg / bobo-3-pDNA complex treated with Caco-2 cell line for 24 hours as a control lipofectamine.
15 shows the results of the Caco-2 cell line monolayer permeation experiment of hIgG1-Fc-9Arg / bobo-3-pDNA complex.
Figure 16 shows the fluorescence imaging results of Caco-2 cell line monolayer transmission of hIgG1-Fc-9Arg / bobo-3-pDNA complexes (cells fixed in 4% paraformaldehyde solution).
Figure 17 shows fluorescence imaging results of pGFP gene expression in Caco-2 cells (cells fixed in 4% paraformaldehyde solution).
FIG. 18 shows cytotoxicity results of hIgG1-Fc-9Arg and hIgG1-Fc-9Arg / pDNA, (a) cytotoxicity test results of carrier, (b) cytotoxicity test of hIgG1-Fc-9Arg / pDNA complex (Caco-2 cell line reacted for 24 hrs, confirmed by MTT assay, ± SD (n = 10)).
FIG. 19 shows fluorescence intensity results obtained by examining the FITC-hIgG1-Fc-9Arg uptake by an ex vivo fluorescence imaging and an animal model organ through an animal model. (A) An animal model The FITC-hIgG1- Fc-9Arg, and (b) fluorescence intensities measured by each organ (balb / c mouse model).

Hereinafter, the present invention will be described in detail.

As a result of intensive studies on the delivery of GLP-1 gene into body cells, the inventors of the present invention found that 9 amino acid arginines having cationic properties are effectively used as immunoglobulins for the efficient condensation of GLP-1 gene having an anion- Confirming that the gene carrier bound to the Fc region C-terminal has stability against the pH and the body enzyme, and confirming the possibility of use as a gene delivery vehicle for oral administration, thus completing the present invention.

Accordingly, the present invention provides a method for producing GLP (glucagon like peptide) -1; And

An oral composition for the prevention or treatment of diabetes comprising a GLP-1 carrier,

The GLP-1 delivery vehicle

Immunoglobulin Fc region; And

Wherein the immunoglobulin Fc region comprises a linker linked to the C-terminus of the immunoglobulin Fc region.

The term " diabetes mellitus " used in the present invention is a type of metabolic disorder such as insufficient secretion of insulin or a failure to function normally. It is characterized by hyperglycemia with high blood glucose concentration, It causes symptoms and signs and excretes glucose from the urine. Diabetes mellitus includes type 1 diabetes, which is caused by insulin secretion due to the breakdown of pancreatic beta cells, type 2 diabetes caused by insufficient insulin secretion in the body or by insulin resistance that cells do not respond to insulin . In the present invention, diabetes includes both type 1 diabetes and type 2 diabetes.

As used herein, the term " prophylactic " means any action that inhibits or slows the onset of diabetes by administration of the composition of the present invention.

As used herein, the term " treatment " means any action that improves or alters the symptoms of diabetes by administration of the composition according to the present invention.

The term " glucagon like peptide-1 " used in the present invention belongs to the Incretin family, is a polypeptide secreted by small intestinal mucosal L cells, and GLP-1- (7-37) GLP-1 binds to the glucagon-like peptide 1 receptor (GLP1R), which is a specific receptor, and exhibits an anti-diabetic effect. The main physiological function is to inhibit the pancreas improve insulin secretion, promote insulin secretion, lower postprandial glucose levels to maintain normal blood glucose levels, enhance insulin biosynthesis, inhibit glucagon secretion, and inhibit gastrointestinal motility, especially gastric emptying, , And it is known to control weight by reducing appetite.

In the present invention, the GLP-1 may be a nucleotide sequence of SEQ ID NO: 1.

In the present invention, the GLP-1 transporter may comprise the amino acid sequence of SEQ ID NO: 2, and the gene encoding the GLP-1 transporter may be the nucleotide sequence of SEQ ID NO: 3. At this time, the nucleotide sequence of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 is preferably 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% Or an amino acid or base sequence having homology.

As used herein, the term " immunoglobulin Fc region " refers to the heavy and light chain variable region, the heavy chain constant region 2 (CH2) and the heavy chain constant region (CH1) except for the heavy chain constant region 1 (CH1) 3 (CH3) moiety, and may include a hinge portion in the heavy chain constant region. Also, as long as the immunoglobulin Fc region of the present invention has a substantially equivalent or improved effect as compared with the wild type, the immunoglobulin Fc region may contain a heavy chain constant region 1 (CH1) and / or a heavy chain constant region 1 < / RTI > (CL1). It may also be a region in which some long amino acid sequences corresponding to CH2 and / or CH3 have been removed.

In addition, the immunoglobulin Fc region of the present invention includes a naturally occurring amino acid sequence as well as its sequence derivative (mutant). An amino acid sequence derivative means that at least one amino acid residue in the native amino acid sequence has a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

In the present invention, the immunoglobulin Fc region may be composed of the amino acid sequence of SEQ ID NO: 4, and the gene encoding the immunoglobulin Fc region may be the nucleotide sequence of SEQ ID NO: 5. At this time, the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% Or an amino acid or base sequence having homology.

The Fc region may also be obtained from natural forms isolated in vivo in animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, and may be obtained from transformed animal cells or microorganisms Recombinant or derivative thereof. Here, the method of obtaining from the native form may be a method of separating the whole immunoglobulin from the living body of human or animal, and then obtaining the protein by treating the proteolytic enzyme. When papain is treated, it is cleaved into Fab and Fc, and when pepsin is treated, it is cleaved into pF'c and F (ab) 2. Fc or pF'c can be isolated using size-exclusion chromatography or the like. In the present invention, the immunoglobulin Fc region is preferably a recombinant immunoglobulin Fc region obtained from a microorganism from a human-derived Fc region.

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain as compared to the native type, and a reduced sugar chain or sugar chain as compared with the native type. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region in which sugar chains are removed from Fc is significantly reduced in binding force with complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, resulting in unnecessary immune response in vivo Do not. In this regard, forms that are more consistent with the original purpose of the drug as a carrier will be referred to as immunoglobulin Fc regions in which the sugar chain is removed or unglycosylated.

In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof, and most preferably the most abundant ligand binding Derived IgG known to enhance the half-life of the protein.

In the present invention, an amino acid as a linker having a cationic property is bound to an immunoglobulin Fc region C-terminal in order to effectively condense an anionic gene, and the linker is Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg. ≪ / RTI >

In one embodiment of the present invention, the gene carrier of hIgG1-Fc-9Arg consisting of the linker 9Arg and the immunoglobulin Fc region was recombined to confirm the characteristics of the recombinant gene transporter (see Example 2).

In another embodiment of the present invention, the characteristics of the higG1-Fc-9Arg fusion gene and the DNA fusion were examined. More specifically, in order to confirm whether hIgG1-Fc-9Arg can be used as an efficient gene transporter, agarose gel electrophoresis, AFM, TEM image were confirmed, and cation property was increased due to a mass ratio of hIgG1-Fc-9Arg In addition to confirming the efficient condensation of the anionic gene, the stability to the acid and the enzyme was specifically confirmed (see Example 3).

In another embodiment of the present invention, intracellular gene transfer efficiency of the hIgG1-Fc-9Arg / pDNA complex was examined. More specifically, Caco-2 and HT-29 cell lines were selected from Caco-2, HT-29, HEK 293, HEK 293-FcRN, and HeLa cell lines to select for expression of FcRN receptor in cells. HIgG1- The 9Arg / pDNA complex was transferred into the cells and the formation of endosome was confirmed by confocal microscopy. In addition, the gene expression of higG1-Fc-9Arg, an antibody-derived transporter, was efficiently detected by binding Bobo-3 (Ex = 570 nm, Em = 602 nm) to pDNA and confirming gene expression in the cells. FcRN receptor to pass through the epithelial cell membrane, but it can exhibit gene transfer effect. Therefore, it is possible to confirm cell permeability by conducting cell membrane permeability test of Caco-2 cell monolayer and to use antibody-derived carrier as a safe carrier. The cell viability of Caco-2 cell line was checked to confirm whether or not the cell viability was 90% or more.

In another embodiment of the present invention, the organ specificity of the gene transporter of the present invention in an animal model was investigated. Oral administration of hIgG1-Fc-9Arg was performed by orally administering FITC, a fluorescent substance, to mice and then fluorescence imaging As a result, it was confirmed that IgG1-Fc-9Arg was transferred through binding to FcRN receptor expressed in this organ, and it was confirmed that hIgG1-Fc-9Arg was highly absorbed in kidney, liver, stomach, duodenum, jejunum and colon.

The above results confirmed that the oral gene carrier according to the present invention had pH and enzyme stability and was absorbed into various organs. Since the absorption rate was high in the intestinal organs, hIgG1-Fc-9Arg was excellent in binding ability to various organs Based on this, it is expected that it will show an efficient ability to be used as a gene carrier. Suggesting that it may be applicable as a carrier of various genes in the future.

In another aspect of the present invention, the present invention provides a method for preventing, controlling or treating diabetes comprising administering the composition to a subject.

As used herein, the term "individual" means a subject in need of a method for preventing, controlling or treating a disease, and more specifically, a human or non-human primate, a mouse, a rat, , Horses, and cows.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Preparation of experimental material

FITC (Fluorescein isothiocyanate) and Bobo-3 Iodide (570/602) chemically bonded dyes were ordered from Thermo Fisher scientific. Oragnic solvents were purchased from several suppliers.

In addition, Lipofectamin ™ plus was purchased from Thermo Fisher Scientific, Exfection ™ LE mini was purchased from GeneAll Biotechnology (Korea), and cell culture medium Minimum Essential Medium (MEM), RPMI 1640, Dulbecco's Modified Eagle's Medium (DMEM) Phosphate buffered saline (PBS) and trypsin were purchased from Sigma Aldrich (Taufkirchen, Germany) and fetal bovine serum (FBS) from EMD milipore (USA). Cell culture plastics and other disposable plastics were produced from SPL Life Science (Korea).

Human embryonic kidney cell line (HEK-293) and human cervical cancer cell line (HeLa) were purchased from Korean Cell Bank (KCLB, Korea) and used for GLP -1 ELA kit was purchased from Sigma-Aldrich (USA), and the pAcGFP-N1 expression vector was provided by Konkuk University (Chungju, Korea). Finally, pβ-sp-GLP-1 expression vector was obtained from Hanyang University (Seoul, Korea).

1-2. Production of hIgG1-Fc-9Arg expression vector

The expression vector for the Fc region of human IgG1 was obtained from KRIBB (Korea Biotechnology Institute), and the 9-Arginine tail was manipulated at the C-terminus of the peptide to facilitate purification.

Herein, the sequence of the amino acid-containing kidney peptide is as follows: RRRRRRRRRGGGSRRRRRRRR (9 Arg-GGGS-9 Arg)

The sequence is located downstream of hIgG1-Fc and has a 9-Arg chain at the C-terminus.

1-3. Expression and purification of hIgG1-Fc-9Arg in HEK293F cells

To isolate the recombinant protein from the culture supernatant, the supernatant of HEK293F cell culture infected with ExpiFectamine ™ 293 transfection reagent (Thermo Fisher Scientific) was collected by 0.22 μm microfiltration. Subsequently, the culture supernatant of the transformed HEK293F cells was subjected to Protein A-HiTrap Mabselect SuRe column (GE Lifesciences, Buckinghamshire, England) for antibody purification according to the supplier's instructions.

1-4. Preparation of hIgG1-Fc-9Arg / pDNA complex

Complex formation between higGl-Fc-9Arg and pDNA was analyzed using 1% agarose gel electrophoresis. The preparation was carried out as shown in Table 1 below, and 1 μl of pDNA aqueous solution (1 mg / 1 ml in TE buffer) was added to 1 μl of hIgG1-Fc-9Arg (9 μg / 1 mL in PBS) and 18 μl of PBS micro-centrifuge tube.

hIgG1-Fc-9Arg / pAcGFP-N1 (weight ratio) 5/1 20/1 50/1 80/1 100/1 200/1 pDNA (ul) 5 5 5 5 5 5 hIgG1-Fc-9Arg (ul) 1.10 4.30 10.80 17.30 21.60 43.20 PBS (ul) 198.90 195.70 189.20 182.70 173.40 151.80 Total volume 200ul (9ng / ul)

Next, the hIgG1-Fc-9Arg / pDNA complex product was allowed to stand at room temperature for 30 minutes to promote complexation. To the 1% agarose gel was added ethidium bromide (0.1 μg / mL) and Tris-acetate- EDTA The reaction was conducted at 100 V for 30 minutes to analyze the formation of the final complex.

1-5. Atomic and transmission electron microscopy

AFM (Multimode-N3-AM, Bruker, Germany) and FE-EM (Field Emission Electron Microscope, JEM-2100F, JEOL) were used to confirm the formation of the composite prepared in Example 1-4.

More specifically, AFM volumes of hIgG1-Fc-9Arg and hIgG1-Fc-9Arg / pDNA complexes were measured using Gwyddion software, and hIgG1-Fc-9Arg / pDNA complexes were imaged using FE- Shape and size. At this time, the nanoparticles were spread on a silicon wafer substrate as pretreatment and stained with 2% uranyl acetate. The observation was carried out in a high vacuum mode, 20 kV.

1-6. Determination of pH and Serum Stability of Complex

The hIgG1-Fc-9Arg / pDNA complex was evaluated by treating the culture medium after the formation of the complex for 3 times the complex formation. More specifically, the hIgG1-Fc-9Arg / pDNA nanoparticles were diluted with fetal bovine serum (FBS) protein and incubated at 37 DEG C to confirm its stability over time. EDTA was added to the complex according to the analysis time, followed by 1% agarose gel electrophoresis.

1-7. Cellular uptake and endosome escape studies

To observe the cellular uptake of hIgG1-Fc-9Arg, confocal microscopy was used. Fluorescein (FITC) was added to hIgG1-Fc-9Arg to observe FcRn target transfer of hIgG1-Fc-9Arg / pDNA complex Respectively.

More specifically, the cells were divided into 24-well culture slides for 3 days, and the culture medium was replaced with FBS-excluded MEM medium 1 hour before the treatment of the complex, and then 10 의 of FITC-labeled-hIgG1-Fc-9Arg And then cultured in a time-dependent manner.

The cells were then washed three times with PBS and fixed with 4% paraformaldehyde (PFA). For endosomal trafficking studies, LysoTracker® was treated for 10 minutes to stain late endosomes and lysosomes , And images were observed with a confocal laser scanning microscope.

1-8. Confirmation of transport of complex through Caco-2 intracellular permeation

Caco-2 cells were used in a monolayer transwell permeability assay to determine the endosomal trafficking of the hIgG1-Fc-9Arg / pDNA complex via FcRn.

More specifically, the cells were first cultured in a Transwell plate (Corning Transwell® polyester membrane pore size 0.4 μm, 12 mm diameter cell culture insert, Millipore) to form a monolayer, then the cell layer was serum-starved, The bobo-3-labeled-pDNA encapsulated complex applied to the apical side in serum (HBSS, pH 6.0) was put into serum. Its apical-basolateral translocation was detected in the presence of hIgG1-Fc-9Arg or bobo-3-labeled pDNA in basolateral medium (HBSS, pH 7.4) at 10, 30 and 60 min and at 2, 4, 10, 12 and 24 h Respectively. After that, bobo-3-labeled-pDNA was quantitated by microplate spectrophotometry using a 96-well plate and fixed with 4% PFA cells and images were taken.

1-9. Transfection experiments of hIgG1-Fc-9Arg / pAcGFP-N1

Caco-2 was incubated with hIgG1-Fc-9Arg / pAcGFP-N1 at 37 ° C in FBS containing pH 6.0 for 1, 2, 5, 10 and 21 days after which the plate was washed three times with PBS, 4% PFA for 5 ~ 10 min. All results were observed with a confocal laser scanning microscope.

1-10. Plasmid DNA transfection using Lipofectamine

Lipofectamine ™ plus (LF) complexes were prepared according to the manufacturer's instructions. More specifically, the DNA was diluted with 75 [mu] l of Opti-MEM and the plus reagent was added at a concentration of 1.2 [mu] l per 1 [mu] g DNA. After this, LF was diluted with 75 Opt Opti-MEM and incubated for 5 minutes, after which the LF solution was added to the mixture containing DNA and plus reagent. After incubation for 5 minutes, the complexes were added to the cells.

1-11. Animal model preparation

To conduct animal experiments, Daehan Bio Link, Inc. Balb / c mice (5-7 weeks old) and db / db mice (male, 7-9 weeks old) were purchased from Chungbuk National University (Chungbuk, Korea). Three mice Respectively. At this time, the entire animal experiment was followed by the guidelines established by the Chonnam National University Animal Laboratory Utilization Committee and gained proper approval before the study.

1-12. Confirmation of binding affinity of hIGg1-Fc-9Arg with mouse FcRn in the organ of Balb / c mice model

The mice prepared from Examples 1-11 were orally administered with 10 占 퐂 of FITC-hIgG1-Fc-9Arg, and sacrificed after 1 hour and 3 hours. Mouse organs were collected by PBS washing (3 times) and fluorescence images were taken with a Kodak Digital Science ™ Image Station 440CF (IS440CF) system. The fluorescence intensity in the organs was measured by microplate spectrophotometry after grinding organ samples (n = 3).

Example 2. Confirmation of recombination of antibody hIgG1-Fc-9Arg

In order to form the complex according to the present invention, recombination was carried out using the base sequence of the Fc portion, which is the constant region of the antibody IgG.

More specifically, as shown in FIG. 1A, it is difficult to effectively condense an anion-like gene due to a phosphate group due to only the Fc portion of the antibody. To overcome this problem, nine amino acid arginines having cationic properties are added to the Fc portion C-terminal And inserted into an expression vector for cloning. The HEK 293F cell line was transfected to produce an antibody.

The resultant recombinant complexes, as shown in FIG. 1B, form receptors of the Fc family with low immunogenicity, so that oral delivery can effectively deliver the drug.

In addition, in order to confirm the binding efficiency of the primer, it was confirmed by electrophoresis that the amplification product of hIgG1-Fc-9Arg was PCR amplified by changing the annealing temperature.

As a result, annealing of hIgG1-Fc-9Arg was proceeded normally at two temperature conditions when annealing temperature of 1 was 55 ° C and annealing temperature of 2 was 52 ° C as shown in FIG. 2, and 850bp Size. ≪ / RTI > That is, it was confirmed that the change of temperature from 52 ° C to 55 ° C did not affect the progress of the PCR.

In addition, in order to confirm whether the recombinant gene was cloned normally, the recombinant hIgG1-Fc-9Arg was extracted by treating the restriction enzyme to analyze the gene sequencing.

As a result, as shown in Fig. 3, it was confirmed that the Kozak nucleotide sequence at the 5 'end and the initiation codon were confirmed and cloned normally.

HIgG1-Fc-9Arg was expressed and purified and confirmed by SDS-PAGE. As shown in FIG. 4, the size of hIgG1-Fc-9Arg was 30-35 kDa in SDS-PAGE, The reason for this is that the Fc portion of the antibody is composed of two heavy chains, and that hIgG1-Fc-9Arg in the non-reducing condition of SS binding has a size of 70 kDa.

Example 3. Characterization of hIgG1-Fc-9Arg / pDNA complex

3-1. Functional confirmation as a gene carrier

In order to confirm whether hIgG1-Fc-9Arg can be used as an efficient gene carrier, agarose gel electrophoresis, AFM, TEM image were confirmed.

More specifically, the formation of the complex was determined by 0.8% agarose gel electrophoresis to induce the complex formation of hIgG1-Fc-9Arg with plasmid DNA by maintaining the same amount of pDNA and different mass ratios of hIgG1-Fc-9Arg Respectively.

As a result, it was confirmed that a composite was formed from a ratio of 20/1, as shown in Fig.

As shown in FIG. 6, the result of the TM-AFM image showed that the DNA structure was confirmed because the ratio of 20/1 did not form a perfect complex. However, at a ratio of 50/1 to 100/1, hIgG1-Fc- 9Arg and the plasmid DNA complex were found to be spherical.

As shown in FIG. 7 and Table 2, when the composite was formed and the size of the composite was observed by a TEM image, it was confirmed that the average diameter size was uniformly formed to 100 nm or less. At 100/1 ratio, hIgG1-Fc-9Arg mass ratio, it was confirmed that the plasmid was condensed much smaller than other mass ratio. As a result, it was confirmed that a perfect complex was formed at a ratio of 50/1 or more.

Ration (w / w) Mean size ± SD (nm) 20/1 25.71 + - 7.40 50/1 43.31 ± 17.98 100/1 19.23 + - 4.80

3-1. Identification of the acid stability of the gene delivery system

In oral administration, the stability to gastric acid and the body enzymes was confirmed. More specifically, the stability of hIgG1-Fc-9Arg obtained by reacting hIgG1-Fc-9Arg at 0, 30 and 60 minutes at various pHs of 1.5, 5.0, 6.0 and 7.4 was determined by SDS-PAGE Respectively.

As a result, as shown in FIG. 8 and FIG. 9, the stability was maintained for 60 minutes at a pH of 1.5 similar to that of gastric acid, and the size was 30 to 35 kDa in SDS-PAGE gel. . When the stability of hIgG1-Fc-9Arg / pDNA was confirmed by the reaction at pH 2.0, it was confirmed by electrophoresis that the stability was maintained for up to 30 minutes. In addition, it was confirmed that the plasmid DNA alone retained its stability up to 6 hours under strong acid conditions. This confirms that the complex shows some instability under strong acid conditions but prevents the degradation of the plasmid DNA.

3-1. Identification of enzyme stability of gene transporter

To confirm the enzyme stability of the gene transporter, the stability of the serum protein (10% fetal bovine serum) present in the blood was checked by treating it on a cell basis.

More specifically, the complexes of Naked DNA, 20/1, 50/1, 100/1 ratio were incubated for at least 0 minutes, 30 minutes, 2 hours, 4 hours, 7 hours, 10 hours, 12 hours, Protein was reacted with and confirmed by electrophoresis.

As a result, as shown in Fig. 10, it was confirmed that the naked DNA was completely decomposed at 24 hours after decomposition started after 2 hours, and the gene encapsulated in the complex at the ratio of 20/1, 50/1, and 100/1 was 24 It was confirmed that the stability of hIgG1-Fc-9Arg / pDNA complex was maintained even in the presence of serum proteins.

Example 4. Measurement of intracellular gene transfer efficiency of hIgG1-Fc-9Arg / pDNA complex

4-1. Expression of intracellular FcRN receptor

Prior to delivery of the hIgG1-Fc-9Arg / pDNA complex into cells, intracellular FcRN receptors were identified via western blots for various cell lines such as Caco-2, HT-29, HEK 293, HEK 293-FcRN and HeLa.

As a result, as shown in Fig. 11, it was confirmed that the FcRN receptor did not appear in the HeLa cell line, and that the HEK 293-FcRN cell line was expressed relatively lower than the Caco-2, HT-29 and HEK 293 cell lines.

4-2. Confirmation of intracellular absorption of antibody hIgG1-Fc-9Arg

FITC (Ex = 490nm, Em = 525nm) was conjugated to the antibody hIgG1-Fc-9Arg to detect whether the antibody hIgG1-Fc-9Arg was absorbed into the cells via the FcRN receptor, and Caco-2, HEK 293, HeLa Cell lines were treated with Confocal image after 1 hour.

As a result, as shown in Fig. 12, Caco-2 and HEK 293 cell lines having the FcRN receptor were absorbed into the cells and fluoresced. In contrast, the HeLa cell line lacking the FcRN receptor did not show fluorescence.

In addition, Caco-2 and HT-29 cell lines were selected as the cell lines to be used for cell experiments.

In addition, the antibody FITC-hIgG1-Fc-9Arg conjugated with a fluorescent substance was treated with a Caco-2 cell line to react with the hIgG1-Fc-9Arg / pDNA complex for 30 minutes and 4 hours to confirm the formation of endosome After staining with Lysotracker, confocal image was confirmed.

As a result, as shown in FIG. 13, it was confirmed that the antibody FITC-hIgG1-Fc-9Arg was transferred into cells through the appearance of green fluorescence in the cells. In the merged image, the red fluorescence image of Lysotracker and the antibody FITC-hIgG1 -Fc-9Arg green fluorescence imaging was transferred into the cells via overlapping to form endo / lysosome.

4-3. Confirmation of delivery of hIgG1-Fc-9Arg / pDNA complex

In order to confirm gene expression, Bobo-3 (Ex = 570nm, Em = 602nm), which shows red fluorescence, was bound to pDNA to confirm the delivery of hIgG1-Fc-9Arg / pDNA complex.

As a result, as shown in Fig. 14, confocal images were observed 24 hours after the Caco-2 cell line was treated with 0/1, 50/1, and 100/1 complexes. As a result, 20/1, 50/1, 100/1 all showed red fluorescence, and 50/1 ratio complex showed the highest fluorescence intensity. In addition, a 50/1 ratio complex showed a higher fluorescence intensity than lipofectamine used as a positive control, confirming that the antibody-derived transporter hIgG1-Fc-9Arg efficiently transduced genes into cells.

4-4. Examination of cell membrane permeability of Caco-2 cell monolayer

The cell membrane permeability test of Caco-2 cell monolayer was carried out because it could bind with FcRN receptor of small intestinal epithelium as an orally administered gene transporter and pass the epithelial cell membrane but have gene transfer effect.

More specifically, in order to confirm the change of the Caco-2 cell monolayer, bobo-3-conjugated pDNA was used to express hIgG1-Fc-9Arg / bobo-3- pDNA complexes were reacted with Caco-2 cell monolayer for 24 hours and 36 hours.

As a result, as shown in FIG. 15, fluorescence intensities of hIgG1-Fc-9Arg / bobo-3-pDNA complex penetrating into the cell membrane of Caco-2 cell monolayer were measured. As a result, 1 fluorescence intensities were increased after 6 hours and relatively high fluorescence intensities were observed at various complex ratios. These results indicate that the 50/1 and 100/1 complexes have relatively high permeability to Caco-2 cell monolayer.

16, fluorescence imaging of hIgG1-Fc-9Arg / bo-bo-3-pDNA absorbed in a Caco-2 cell monolayer resulted in a 50/1 ratio of hIgG1-Fc-9Arg / bobo-3- The pDNA complex showed higher absorption rate than the hIgG1-Fc-9Arg / bobo-3-pDNA complex at 100/1 ratio.

From the above, it was confirmed that the fluorescence intensity was high because the transmittance ratio of the 100/1 ratio complex was higher than that of the Caco-2 cell monolayer. On the other hand, the 50/1 ratio of the complex showed a high absorption rate in the Caco-2 cell monolayer, and the transmittance increased with time.

Thus, it was possible to infer that the 50/1 ratio of the complexes is able to efficiently transfer genes into the intestines more efficiently than the other proportions of the complexes.

As shown in FIG. 17, since the doubling time of the Caco-2 cell line was 37 hours, the hIgG1-Fc-9Arg / pGFP complex was treated with the cells and the cells were reacted for 48 hours After confocal image analysis, the pGFP gene was expressed in a 50/1 ratio complex and the fluorescence intensity was increased. As a result, it was found that the antibody - derived transporter efficiently transferred the gene into the cell.

4-5. Confirmation of the safety of hIgG1-Fc-9Arg gene transporter

The present inventors have determined whether the antibody-derived carrier according to the present invention can be used as a safe carrier.

More specifically, only the transporter was treated with Caco-2 cell lines at concentrations of 3.75, 7.5, and 15 μg, and reacted for 24 hours, and the absorbance was measured at 570 nm.

As a result, as shown in FIG. 18A, bPEI had a cell viability of 20% at a concentration of 15 μg, whereas the developed hIgG1-Fc-9Arg showed cell viability of 70% or more, Respectively.

Further, as shown in FIG. 18B, the cytotoxicity of the formed carrier was confirmed, and it was found that hIgG1-Fc-9Arg / pDNA complex had cell viability of 90% or more in both sample groups with relatively higher cell viability than bPEI / DAN complex Confirming the safety of hIgG1-Fc-9Arg as a carrier.

4-6. Confirmation of the binding effect of hIgG1-Fc-9Arg organs in animal models

In order to confirm the transfer of hIgG1-Fc-9Arg in animal models and to determine whether it binds to specific organs, the animals were orally administered to balb / c mouse model for 1 hour and 4 hours and then anesthetized with diethyl ether. And washed three times with PBS buffer to measure fluorescence imaging.

As a result, as shown in FIG. 19 (a), hIgG1-Fc-9Arg was orally administered to a mouse to which FITC, which is a fluorescent substance, was bound and then subjected to fluorescence imaging. As a result, IgG1-Fc-9Arg It is confirmed that the signal is transmitted through the coupling.

In addition, as shown in FIG. 19B, it was confirmed that hIgG1-Fc-9Arg was abundantly absorbed in kidney, liver, stomach, duodenum, jejunum and colon. Especially, after 3 hours, I could confirm.

From the above results, it has been confirmed that the gene carrier according to the present invention is absorbed by various organs, and hIgG1-Fc-9Arg is excellent in binding ability to various organs because of its high absorption rate in intestinal organs, It is expected to show ability. Therefore, it has been confirmed that GLP-1 and the gene carrier form a complex, which can be usefully used for the prevention or treatment of diabetes.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> KB BIOMED An oral antidiabetic composition comprising GLP-1 and a gene          carrier <130> PD17-185 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 114 <212> DNA <213> GLP-1 <400> 1 atgcgtcaac gtcgtcatgc tgaagggacc tttaccagtg atgtaagttc ttatttggaa 60 ggccaagctg ccaaggaatt cattgcttgg ctggtgaaag gccgatagtc taga 114 <210> 2 <211> 95 <212> PRT <213> Artificial Sequence <220> <223> hIgG1-Fc-9Arg <400> 2 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Tyr Ser   1 5 10 15 Met Phe Trp Cys Gly Gly His Asp Leu Arg Leu Pro Arg Lys Lys Glu              20 25 30 Met Ser Phe Glu Trp His Leu Phe Ser Ser Thr Val Val Pro Leu Gln          35 40 45 Lys Ser Thr Arg His Tyr Val Leu Arg Asp Val Leu Val Met Cys Val      50 55 60 Phe Ser Glu Arg Asp Arg Gly Pro Phe Arg Arg Arg Arg Arg Arg Arg  65 70 75 80 Arg Arg Gly Gly Gly Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg                  85 90 95 <210> 3 <211> 288 <212> DNA <213> Artificial Sequence <220> <223> hIgG1-Fc-9Arg <400> 3 atgaagtggg tgaccttcat ctccctgctg tttctgttct cctactccat gttctggtgc 60 ggagggcacg acctgaggct gccgaggaag aaggagatgt cgttcgagtg gcacctgttc 120 tcgtccaccg tcgtcccctt gcagaagagt acgaggcact acgtactccg agacgtgttg 180 gtgatgtgcg tcttctcgga gagggacaga ggcccatttc ggcggagaag aagaaggcgc 240 agaagaggcg gcggaagcag acgcaggcgg cgcagacggc ggagataa 288 <210> 4 <211> 73 <212> PRT <213> hIgG1-Fc <400> 4 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Tyr Ser   1 5 10 15 Met Phe Trp Cys Gly Gly His Asp Leu Arg Leu Pro Arg Lys Lys Glu              20 25 30 Met Ser Phe Glu Trp His Leu Phe Ser Ser Thr Val Val Pro Leu Gln          35 40 45 Lys Ser Thr Arg His Tyr Val Leu Arg Asp Val Leu Val Met Cys Val      50 55 60 Phe Ser Glu Arg Asp Arg Gly Pro Phe  65 70 <210> 5 <211> 219 <212> DNA <213> hIgG1-Fc <400> 5 atgaagtggg tgaccttcat ctccctgctg tttctgttct cctactccat gttctggtgc 60 ggagggcacg acctgaggct gccgaggaag aaggagatgt cgttcgagtg gcacctgttc 120 tcgtccaccg tcgtcccctt gcagaagagt acgaggcact acgtactccg agacgtgttg 180 gtgatgtgcg tcttctcgga gagggacaga ggcccattt 219

Claims (9)

GLP (glucagon like peptide) -1; And
An oral composition for the prevention or treatment of obesity comprising a GLP-1 delivery,
The GLP-1 delivery vehicle
Immunoglobulin Fc region; And
Wherein the immunoglobulin Fc region comprises a linker linked to the C-terminus. 2. The composition of claim 1, wherein the GLP-1 comprises the nucleotide sequence of SEQ ID NO: 1. 2. The composition of claim 1, wherein the GLP-1 delivery comprises an amino acid sequence of SEQ ID NO: 2. 2. The composition of claim 1, wherein the GLP-1 delivery comprises a nucleotide sequence of SEQ ID NO: 3. 2. The composition of claim 1, wherein the immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 4. The composition of claim 1, wherein the immunoglobulin Fc region comprises the nucleotide sequence of SEQ ID NO: 5. The composition of claim 1, wherein the immunoglobulin Fc region is derived from any one selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. 8. The composition of claim 7, wherein the immunoglobulin Fc region is derived from IgG. 2. The composition of claim 1, wherein the linker is comprised of an amino acid sequence of Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg.

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