KR102208542B1 - An oral anti-obesity composition comprising GLP-1 and a gene carrier - Google Patents

Abstract

The present invention relates to an oral composition for the prevention, treatment, or improvement of obesity, by binding Arginine 9 amino acids having cationic properties to the Fc portion C-terminal and condensing it with the GLP-1 gene having anionic properties in the body. It can effectively induce the expression of the GLP-1 gene, and when administered orally, the gene can be transferred to the small intestine by protecting the gene from degradation caused by the immune function of gastric acid and leukocytes, and when the gene is expressed in the small intestine Since the half-life was relatively long, the possibility of long-term therapeutic effect was confirmed. Through effective delivery of GLP-1, it is expected to be useful for preventing, treating, or improving obesity.

Description

An oral anti-obesity composition comprising GLP-1 and a gene carrier}

The present invention relates to an anti-obesity composition for oral administration, and more specifically, GLP-1 delivery system comprising a linker made of cationic arginine capable of aggregating anionic genes at the C-terminus of GLP-1, and immunoglobulin Fc region It relates to an oral composition for preventing, treating or improving obesity comprising as an active ingredient.

Obesity occurs when the amount of calories consumed is greater than the amount of calories consumed due to excessive adipose tissue in the body, and also occurs due to various causes such as mental and social factors, inheritance, disease, and drugs. According to the World Health Organization (WHO) in 2010, the global population of overweight adults is about 1.6 billion, and the number of obese people is about 400 million, and 2.6 million people die each year from obesity and overweight. In the case of Korea, according to reports from the Ministry of Health and Welfare and the Centers for Disease Control and Prevention, the adult obesity rate is continuously increasing, and as of 2010, it was 30.8% (male 36.3%, female 24.8%). As such, the obese population continues to increase worldwide, and the burden of medical expenses is also increasing.

Obesity is perceived to be more serious than its own risk because of various complications that can be caused by obesity. Obesity is known to increase the risk of hypertension, hyperlipidemia, metabolic syndrome such as diabetes, fatty liver, joint abnormalities, and cancer. According to the World Health Organization's announcement in 2010, the risk of hypertension, diabetes, and dyslipidemia is more than twice as high in obese people compared to those of normal weight (2.5 times for hypertension, 2 times for diabetes, 2.3 times for hypercholesterolemia, Hypertriglyceridemia 2.4 times) was higher. In addition to the above diseases, it is known that in the case of women, if the body fat is excessive, the balance of sex hormones is broken, and in severe cases, infertility may occur, and the risk of endometrial cancer and breast cancer is increased. In addition, since obesity can cause not only physical diseases, but also mental diseases such as social isolation, alienation, lack of confidence, and depression, the need for prevention and treatment of obesity is recognized as very important.

Obesity can be treated through diet, regular exercise and lifestyle improvements such as behavioral therapy, and drugs such as appetite suppressants and fat absorption suppressants. Since obesity is a chronic disease, long-term use is required when attempting drug treatment. Currently, products approved for long-term use for more than 3 months in Korea include sibutramine, an appetite suppressant, and orlistat, a lipolytic enzyme inhibitor. ). However, these obesity treatment drugs are mostly psychotropic drugs that act on the central nervous system to control appetite, so they have side effects such as headache and vomiting, and there are problems such as abuse concerns. Therefore, research has been actively conducted to develop a material having high stability and excellent anti-obesity effect that can solve the side effects of the commercially available anti-obesity agents.

On the other hand, GLP-1 and its analogs and derivatives, which have recently emerged as the next-generation diabetes treatment, show good potential in clinical trials for the treatment of type 2 diabetes, and stimulate insulin secretion, inhibit glucagon secretion, inhibit gastric fasting, gastric motility or intestinal tract It induces numerous biological effects, such as inhibition of motility and induction of weight loss. In addition, it has a pancreatic protection function even when taken for a long time, there is no risk of hypoglycemia, and adequate blood sugar can be maintained for a long time.

The secretion of GLP-1 is known to be promoted by activation of TGR5 and GPR119, a kind of G protein-coupled receptors (GPCRs), or α-gustducin. In particular, activation of G protein-coupled receptor (GPCR) TGR5 (GPR131) expressed in brown adipose tissue and muscle increases energy expenditure, resulting in an effect of treating obesity, and is related to improvement of liver disease. It is known to be present and has been reported to inhibit arteriosclerosis.

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 as a therapeutic agent. Thus, various approaches have been performed to extend the half-life of GLP-1 or reduce the removal rate of peptides from the body while maintaining biological activity. That is, the development of various GLP-1 analogs is actively progressing, and an approach has been attempted to fuse GLP-1 to an immunoglobulin Fc region (US Patent 7452966), but it is still insufficient.

The present invention was devised to solve the above problems, and as a result of intensive research on a delivery system for efficiently delivering the GLP-1 gene into cells in the body, in order to effectively condense the GLP-1 gene having anionic properties It was confirmed that the gene delivery system in which nine amino acids Arginine having cationic properties were bound to the C-terminal of the immunoglobulin Fc region had stability against pH and enzymes in the body, and the possibility of use as a gene delivery system for oral administration was confirmed. Based on this, the present invention has been completed.

An object of the present invention is GLP (glucagon like peptide)-1; And

As an oral composition for the prevention or treatment of obesity, comprising a GLP-1 delivery system,

The GLP-1 transporter is

Immunoglobulin Fc region; And

It is characterized in that it comprises a linker linked to the C-terminus of the immunoglobulin Fc region, to provide a composition.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention

Glucagon like peptide (GLP)-1; And

As an oral composition for the prevention or treatment of obesity, comprising a GLP-1 delivery system,

The GLP-1 transporter is

Immunoglobulin Fc region; And

It provides a composition comprising a linker connected to the C-terminus of the immunoglobulin Fc region.

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

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

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

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

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

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

In addition, the present invention provides a method for preventing or treating obesity, comprising administering the composition to an individual.

In addition, the present invention provides the use of the composition for preventing or treating obesity.

The oral composition for preventing or treating obesity of the present invention binds 9 amino acids having cationic properties to the C-terminal of the Fc portion and condenses them with the GLP-1 gene having anionic properties. It can effectively induce expression, and when administered orally, it protects the gene from degradation caused by the immune function of gastric acid and leukocytes, allowing gene transfer to the small intestine, and when the gene is expressed in the small intestine, the half-life is relatively long. As the possibility of long-term therapeutic effect was confirmed, it is expected that it can be usefully used for prevention, treatment, or improvement of obesity through effective delivery of GLP-1.

Figure 1a shows a process of forming a complex of antibody hIgG1-Fc-9Arg and plasmid DNA (pDNA), and a process in which the antibody-therapy gene complex is delivered in the body through a cell receptor.
Figure 2 shows the results of SDS-PAGE of 9 Arginine-bound antibodies to Human IgG1-Fc ((850bp), Lane 1.pcr annealing TM = 55°C, Lane 2.pcr annealing TM = 52°C).
3 shows the nucleotide sequence of hIgG1-Fc-9Arg.
Figure 4 is a molecular weight SDS-PAGE result of purified antibody hIgG1-Fc-9Arg ((Protein Ladder 10-245 kDa). Lane 2. 0.1 μg, Lane 3. 1 μg, Lane 4. 10 μg antibody used, Lane 5.) and It shows the molecular weight result of hIgG1-Fc-9Arg maintaining SS bond under non-reducing conditions.
Figure 5 shows the results of hIgG1-Fc-9Arg and pDNA complex agarose gel electrophoresis (0.8% agarose gel).
6 shows the results of TM-AFM imaging of the hIgG1-Fc-9Arg/pDNA complex, (a) hIgG1-Fc-9Arg, (b) 20/1 ratio (w/w) of hIgG1-Fc-9Arg/pDNA Complex, (cd) 50/1 ratio (w/w) hIgG1-Fc-9Arg/pDNA complex, (e) 100/1 ratio (w/w) hIgG1-Fc-9Arg/pDNA complex (Scale bars 200 nm).
7 shows the TEM imaging results of the hIgG1-Fc-9Arg/pDNA complex, (a) the hIgG1-Fc-9Arg/pDNA complex at a 20/1 ratio (w/w), (b) a 50/1 ratio (w /w) hIgG1-Fc-9Arg/pDNA complex, (C) 100/1 ratio (w/w) of the hIgG1-Fc-9Arg/pDNA complex (Scale bars 100 nm).
FIG. 8 shows the results of confirming the stability of the antibody hIgG1-Fc-9Arg over time in various pH (pH 1.5, 5.0, 6.0, 7.4) conditions by SDS-PAGE.
9 shows the results of confirming the stability of the plasmid DNA and hIgG1-Fc-9Arg/pDNA complex by agarose gel (0.8%) electrophoresis at pH 2.0 under gastric acid conditions.
Figure 10 shows the results of the serum protein stability assay of the 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 of the complex, (d) 100/1 ratio of the complex stability confirmation results are shown.
11 shows the results of western blots confirming the FcRN receptor protein present in various cell lines.
12 shows the fluorescence imaging results of FITC-hIgG1-Fc-9Arg absorbed through the FcRN receptor (1 hour incubation).
13 shows the results of endosome formation and endosomal escape fluorescence imaging of FITC-hIgG1-Fc-9Arg (reaction conditions for 30 minutes and 4 hours).
14 shows the results of gene expression fluorescence imaging by treating the hIgG1-Fc-9Arg/bobo-3-pDNA complex in a Caco-2 cell line for 24 hours, and as a control lipofectamine.
15 shows the results of a single layer membrane permeation experiment of the Caco-2 cell line of the hIgG1-Fc-9Arg/bobo-3-pDNA complex.
FIG. 16 shows the results of fluorescence imaging through monolayer membranes of the Caco-2 cell line of the hIgG1-Fc-9Arg/bobo-3-pDNA complex (cells are fixed with 4% paraformaldehyde solution).
Figure 17 shows the results of fluorescence imaging of pGFP gene expression in Caco-2 cells (cells are fixed with 4% paraformaldehyde solution).
18 is a cytotoxicity test result of hIgG1-Fc-9Arg and hIgG1-Fc-9Arg/pDNA, (a) cytotoxicity test result of delivery system, (b) cytotoxicity test of hIgG1-Fc-9Arg/pDNA complex It shows the results (confirmed by reacting to Caco-2 cell line for 24 hours, MTT assay, ±SD (n=10)).
Figure 19 shows the results of confirming the absorption of FITC-hIgG1-Fc-9Arg by Ex vivo fluorescence imaging through the animal model and the fluorescence intensity absorbed in the animal model organs. The fluorescence intensity result of Fc-9Arg, (b) shows the fluorescence intensity quantification result absorbed in each organ (balb/c mouse model).

Hereinafter, the present invention will be described in detail.

As a result of intensive research on a delivery system for efficiently delivering the GLP-1 gene into the body's cells, the present inventors condensed the anionic GLP-1 gene with 9 amino acids having cationic properties in the Fc portion. The present invention was completed by confirming that the gene delivery system bound to the C-terminal has stability against pH and enzymes in the body, and the possibility of use as a gene delivery system for oral administration.

Thus, the present invention GLP (glucagon like peptide)-1; And

As an oral composition for the prevention or treatment of obesity, comprising a GLP-1 delivery system,

The GLP-1 transporter is

Immunoglobulin Fc region; And

It provides a composition comprising a linker connected to the C-terminus of the immunoglobulin Fc region.

"Obesity", which is a disease subject to improvement, prevention or treatment of the present invention, refers to a state in which fat cells proliferate and differentiate in the body due to metabolic disorders, and thus fat is accumulated excessively, and hypertension, diabetes, and abnormal lipids It can lead to related complications, including metabolic syndrome accompanied by hyperemia. When the amount of energy absorption increases relative to the amount of consumption, the number and volume of adipocytes increase, resulting in an increase in the mass of adipose tissue.

The term “prevention” used in the present invention means any action that suppresses obesity or delays the onset of obesity by administration of the pharmaceutical composition according to the present invention.

The term "treatment" used in the present invention refers to any action in which symptoms of obesity are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The term “GLP-1 ((glucagon like peptide-1)” used in the present invention belongs to the Incretin family, and is a polypeptide secreted by L cells of the small intestine, and GLP-1-(7-37) And GLP-1-(7-36)-amide. GLP-1 exerts antidiabetic action by binding to a specific receptor, the glucagon-like peptide 1 receptor (GLP1R), whose main physiological function is the pancreas. Improves β cell function, promotes insulin secretion, lowers postprandial blood sugar to maintain a normal blood sugar state, enhances insulin biosynthesis, inhibits glucagon secretion, and inhibits gastrointestinal peristalsis, especially gastric discharge, It is known to control weight by increasing it and reducing appetite.

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

In the present invention, the GLP-1 delivery system may consist of the amino acid sequence of SEQ ID NO: 2, and the gene encoding it may be formed of the nucleotide sequence of SEQ ID NO: 3. At this time, the amino acid sequence represented by SEQ ID NO: 2 or the nucleotide sequence represented by SEQ ID NO: 3 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more It may contain an amino acid or nucleotide sequence having homology.

The term “immunoglobulin Fc region” as used herein refers to the heavy and light chain variable regions of immunoglobulins, heavy chain constant region 2 (CH2) and heavy chain constant region excluding heavy chain constant region 1 (CH1) and light chain constant region (CL1). It means a 3(CH3) moiety, and may include a hinge moiety in the heavy chain constant region. In addition, as long as the immunoglobulin Fc region of the present invention has substantially the same or improved effect with the native type, part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region except for the heavy and light chain variable regions of the immunoglobulin It may be an extended Fc region including 1 (CL1). In addition, it may be a region from which some considerably 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 native amino acid sequence as well as a sequence derivative (mutant) thereof. An amino acid sequence derivative means that one or more amino acid residues in a natural amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

In the present invention, the immunoglobulin Fc region may consist of the amino acid sequence of SEQ ID NO: 4, and the gene encoding it may be formed of the nucleotide sequence of SEQ ID NO: 5. At this time, the amino acid sequence represented by SEQ ID NO: 4 or the nucleotide sequence represented by SEQ ID NO: 5 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more It may contain an amino acid or nucleotide sequence having homology.

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

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain compared to the natural type, a reduced sugar chain compared to the natural type, or a form in which sugar chains are removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used for the increase or decrease of such immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region from which the sugar chain has been removed from the Fc significantly decreases the binding ability with complement (c1q), and the antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so it does not cause unnecessary immune responses in vivo. Does not. In this regard, a form more suitable for the original purpose as a drug carrier would be referred to as an immunoglobulin Fc region in which sugar chains are removed or non-glycosylated.

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 binds the most abundant ligand to human blood. It is derived from IgG, which is known to enhance the half-life of proteins.

In the present invention, in order to effectively condense genes having anionic properties, an amino acid as a linker having cationic properties was bonded to the C-terminal of the immunoglobulin portion of the Fc region, and the linker is Arg-Arg-Arg-Arg-Arg- It may consist of the amino acid sequence of Arg-Arg-Arg-Arg, but is not limited thereto.

In one embodiment of the present invention, the gene delivery system of hIgG1-Fc-9Arg consisting of the linker 9Arg and the immunoglobulin Fc region was recombined, and the characteristics of the recombined gene delivery system were confirmed (see Example 2).

In addition, in another embodiment of the present invention, it was attempted to confirm the characteristics of the complex in which the gene delivery system of hIgG1-Fc-9Arg and DNA were fused. More specifically, to confirm whether hIgG1-Fc-9Arg can be used as an efficient gene carrier, as a result of confirming agarose gel electrophoresis, AFM, and TEM image, the cationic properties increased due to the mass ratio of hIgG1-Fc-9Arg. While, it was specifically confirmed that the anionic gene was condensed efficiently, and stability against acids and enzymes was specifically confirmed (see Example 3).

In addition, in another embodiment of the present invention, it was attempted to confirm the intracellular gene transfer efficiency of the hIgG1-Fc-9Arg/pDNA complex. More specifically, the cell lines for confirming the expression of the intracellular FcRN receptor were selected from Caco-2, HT-29, HEK 293, HEK 293-FcRN, and HeLa cell lines, and the Caco-2 and HT-29 cell lines were selected, and hIgG1-Fc- Whether the 9Arg/pDNA complex was delivered into the cell to form an endosome was confirmed through a confocal microscope. In addition, the antibody-derived carrier hIgG1-Fc-9Arg efficiently transferred the gene into the cell by binding Bobo-3 (Ex = 570 nm, Em = 602 nm) with pDNA to confirm the expression of the gene in the cell. Since it is bound to FcRN receptor and passes through the epithelial cell membrane, the gene transfer effect can be exhibited. Therefore, the cell membrane permeability test of Caco-2 cell monolayer was conducted to confirm the cell permeability, and the antibody-derived carrier can be used as a safe carrier. In order to confirm whether the cell viability for the Caco-2 cell line was checked, cell viability of 90% or more was confirmed to confirm the safety as a delivery vehicle.

In another embodiment of the present invention, to confirm the organ specificity of the gene delivery system of the present invention in an animal model, hIgG1-Fc-9Arg was bound to FITC, a fluorescent substance, orally administered to a mouse, and then fluorescence imaging was measured. As a result, it was confirmed that IgG1-Fc-9Arg was delivered through binding to the FcRN receptor expressed in the organ, and hIgG1-Fc-9Arg was confirmed to be absorbed a lot in kidney, liver, stomach, duodenum, jejunum and colon (Example 4).

The above results confirmed the pH and enzyme stability of the oral gene delivery system according to the present invention, confirmed that it is absorbed into various organs, and has a high absorption rate in the intestinal organs, so hIgG1-Fc-9Arg has excellent binding power with various organs. Based on this, it is expected to show an efficient ability when used as a gene delivery system. It also suggests that it can be applied 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 obesity, comprising administering the composition to an individual.

In the present invention, 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, mouse, rat, dog, cat , It means mammals such as horses and cattle.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experiment preparation and experiment method

1-1. Preparation of experimental materials

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

In addition, Lipofectamin™ plus was purchased from Thermo Fisher scientific, and 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), Dulbecco's Phosphate Buffered Saline (PBS) and trypsin were purchased from Sigma Aldrich (Taufkirchen, Germany), and fetal bovine serum (FBS) was purchased from EMD milipore (USA). Cell culture plastics and other disposable plastics were manufactured by SPL Life Sciences (Korea).

In addition, human epithelial colon adenocarcinoma cell lines (Caco-2 and HT-29), human embryonic kidney cell lines (HEK-293), and human cervical cancer cell lines (HeLa) were purchased and used from Korea Cell Bank (KCLB, Korea), and GLP The -1 ELA kit was purchased from Sigma-Aldrich (USA), and the pAcGFP-N1 expression vector was provided by Konkuk University (Chungju, Korea). Finally, the pβ-sp-GLP-1 expression vector was obtained from Hanyang University (Seoul, Korea).

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

The expression vector for the Fc region of Human IgG1 was provided by KRIBB (Korea Institute of Biotechnology), and the 9-Arginine tail was engineered at the C-terminus of the peptide to facilitate purification.

At this time, the sequence of the kidney peptide containing amino acids is as follows: RRRRRRRRRGGGSRRRRRRRRR (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

In order to isolate the recombinant protein from the culture supernatant, the supernatant of HEK293F cell culture infected with ExpiFectamine™ 293 Transformation Reagent (Thermo Fisher Scientific) was collected by 0.22 μm microfiltration. Thereafter, 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 to obtain the final product.

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

The formation of the complex between higG1-Fc-9Arg and pDNA was analyzed using 1% agarose gel electrophoresis. At this time, the preparation was prepared as shown in Table 1 below, and 1 μl of a pDNA aqueous solution (1 mg / 1 ml in TE buffer) in 1 ul of hIgG1-Fc-9Arg (9 µg / 1 mL in PBS) and 18 ul PBS (pH7.4) It was prepared by mixing in a 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, and ethidium bromide (0.1 μg/mL), Tris-acetate-EDTA (TAE) buffer solution in 1% agarose gel as a background. It was reacted at 100V for 30 minutes to analyze whether the final complex was formed.

1-5. Atomic and Transmission Electron Microscopy

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

More specifically, the AFM volumes of the hIgG1-Fc-9Arg and hIgG1-Fc-9Arg / pDNA complexes were measured using Gwyddion software, and then the hIgG1-Fc-9Arg / pDNA complex was imaged using FE-EM and after binding. The shape and size were confirmed. At this time, as a pretreatment process, nanoparticles were spread on a silicon wafer substrate and then stained with 2% uranyl acetate, and observation was performed in a high vacuum mode and under 20 kV conditions.

1-6. Confirmation of stability against pH and serum of complex

The hIgG1-Fc-9Arg / pDNA complex was evaluated by treating the culture medium three times after formation of the complex to form the complex. More specifically, hIgG1-Fc-9Arg / pDNA nanoparticles were diluted with FBS (fetal bovine serum) protein, incubated at 37° C., and their stability was confirmed over time. After EDTA was added to the complex according to the corresponding analysis time, 1% agarose gel electrophoresis was performed.

1-7. Cell absorption and endosome escape study

To confirm cellular uptake of hIgG1-Fc-9Arg, a confocal microscope was used. In this case, Fluorescein (FITC) was used in hIgG1-Fc-9Arg to observe the FcRn target delivery of the hIgG1-Fc-9Arg / pDNA complex. It was used as a label.

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

Next, the cells were washed three times with PBS at the time of each analysis, fixed with 4% paraformaldehyde (PFA), and treated with LysoTracker® for 10 minutes for Endosomal Trafficking studies to stain late endosomes and lysosomes. , The image was observed with a confocal laser scanning microscope.

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

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

More specifically, cells were first cultured in a Transwell plate (Corning® Transwell® polyester membrane pore size 0.4 μm, diameter 12 mm cell culture insert, Millipore) to form a monolayer, and then the cell layer was serum-starved, and pDNA 1 μg The bobo-3-labeled-pDNA encapsulated complex applied to the apical side in (HBSS, pH 6.0) was added to the serum. Its apical-basolateral translocation was observed in the presence of hIgG1-Fc-9Arg or bobo-3-labeled-pDNA in basolateral medium (HBSS, pH 7.4) at 10, 30 and 60 minutes, and at 2, 4, 10, 12 and 24 hours. It was determined by measuring. Thereafter, bobo-3-labeled-pDNA was quantified by microplate spectroscopy using a 96 well plate, and images were taken after fixing with 4% PFA cells.

1-9. hIgG1-Fc-9Arg / pAcGFP-N1 transfection experiment

Caco-2 was incubated at 37° C. with hIgG1-Fc-9Arg / pAcGFP-N1 in FBS-containing culture at pH 6.0 for 1, 2, 5, 10 and 21 days, and then the plate was washed 3 times with PBS, It was fixed with 4% PFA for 5 to 10 minutes, and all results were observed with a confocal laser scanning microscope.

1-10. Plasmid DNA transfection using Lipofectamine

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

1-11. Preparation of animal models

To conduct animal testing, Daehan Bio Link, Inc. Balb / c mice (5-7 weeks old) and db / db mice (males, 7-9 weeks old) were purchased from (Chungbuk, Korea), and all purchased mice were sterilized with 3 mice per cage with a standard diet. Kept in At this time, all animal experiments followed the guidelines established by the Chonnam National University Animal Laboratory Utilization Committee, and appropriate approval was obtained before the study.

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

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

Example 2. Recombination confirmation of antibody hIgG1-Fc-9Arg

In order to form the complex according to the present invention, gene recombination was performed using the nucleotide sequence of the Fc portion, which is a constant region of antibody IgG.

More specifically, as shown in Fig. 1a, since it is difficult to effectively condense genes having anionic properties due to phosphate groups only with the Fc portion of the antibody, 9 amino acids having cationic properties are used in the Fc portion of C-terminal. And inserted into an expression vector for cloning, and transfected into HEK 293F cell line to produce an antibody.

As shown in FIG. 1B, the resulting recombinant complex formed an Fc-series receptor with low immunogenicity, so it was predicted that drug delivery would be possible through oral administration.

In addition, in the case of hIgG1-Fc-9Arg, in order to confirm the binding efficiency of the primers due to the long extension size, the reaction product obtained by performing PCR by changing the annealing temperature was confirmed through electrophoresis.

As a result, as shown in FIG. 2, when the annealing temperature of No. 1 was performed at 55° C. and the annealing temperature of No. 2 was performed at 52° C., annealing of hIgG1-Fc-9Arg was normally performed under two temperature conditions, resulting in 850 bp of annealing. It was confirmed that it was expressed in size. That is, it could be confirmed that the change in the temperature between 52℃ and 55℃ did not affect the PCR process.

In addition, in order to confirm whether the recombinant gene was normally cloned, the recombinant hIgG1-Fc-9Arg was extracted by treatment with a restriction enzyme, and gene sequencing was analyzed.

As a result, as shown in FIG. 3, it was confirmed that the 5'terminal Kozak nucleotide sequence and the start codon were identified, and that the cloning was performed normally.

As a result of expressing and purifying hIgG1-Fc-9Arg and confirming it through SDS-PAGE, as shown in FIG. 4, it was confirmed that the size of hIgG1-Fc-9Arg was 30 to 35 kDa in SDS-PAGE, and the band was in two lines. The reason for this appears is that the Fc portion of the antibody consists of two heavy chains, and it was confirmed that hIgG1-Fc-9Arg in the non-reducing condition forming the SS bond has a size of 70 kDa.

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

3-1. Confirmation of functionality as a gene delivery system

In order to confirm whether the hIgG1-Fc-9Arg can be used as an efficient gene delivery system, agarose gel electrophoresis, AFM, and TEM images were confirmed.

More specifically, whether or not the complex is formed is confirmed through 0.8% agarose gel electrophoresis by inducing the formation of a complex between hIgG1-Fc-9Arg and plasmid DNA by keeping the same amount of pDNA and varying the mass ratio of hIgG1-Fc-9Arg. I did.

As a result, as shown in Fig. 5, it was confirmed that the composite was formed from a ratio of 20/1.

In addition, as shown in FIG. 6, as a result of the TM-AFM image, the structure of DNA was confirmed because the ratio of 20/1 did not form a perfect complex, but hIgG1-Fc- in the ratio of 50/1 and the ratio of 100/1 It was confirmed that the 9Arg and plasmid DNA complex formed a spherical shape.

In addition, as shown in Fig. 7 and Table 2, as a result of confirming whether or not the complex was formed and the size of the complex by the TEM image, it was confirmed that the average diameter size was uniformly formed to less than 100 nm, and at the ratio of 100/1, the increased It was confirmed that the cationic property was increased due to the mass ratio of hIgG1-Fc-9Arg, resulting in a smaller condensation of the plasmid than other mass ratios. Through this, it was confirmed that a perfect composite 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. Confirmation of acid stability of gene delivery system

In oral administration, stability against gastric acid and enzymes in the body was confirmed. More specifically, first, the stability of hIgG1-Fc-9Arg in which hIgG1-Fc-9Arg was reacted for 0 minutes, 30 minutes and 60 minutes at various pHs 1.5, 5.0, 6.0 and 7.4 was confirmed through SDS-PAGE. I did.

As a result, as shown in FIGS. 8 and 9, stability was maintained at a pH of 1.5 similar to gastric acid for 60 minutes, showing a size of 30 to 35 kDa in the SDS-PAGE gel, and 1 hour in various pH ranges of 5.0 to 7.4 It was confirmed to maintain stability until, and it was confirmed through electrophoresis that the stability of hIgG1-Fc-9Arg/pDNA was maintained until 30 minutes when the stability of hIgG1-Fc-9Arg/pDNA was confirmed by reacting at pH 2.0. In addition, it was confirmed that the stability was maintained for up to 6 hours under strong acid conditions with only plasmid DNA. Through this, it was confirmed that the complex showed some instability under strong acid conditions, but prevented the plasmid DNA from being degraded.

3-1. Enzyme stability confirmation of gene delivery system

In order to confirm the enzyme stability of the gene delivery system, the stability of the serum protein (10% fetal bovine serum) present in the blood was confirmed by treatment on a cell basis.

More specifically, naked DNA, 20/1, 50/1, 100/1 ratio of complexes in serum from at least 0 minutes, 30 minutes, 2 hours, 4 hours, 7 hours, 10 hours, 12 hours, and up to 24 hours. It reacted with the protein and confirmed by electrophoresis.

As a result, as shown in FIG. 10, it was confirmed that the naked DNA started to be degraded after 2 hours and completely degraded at 24 hours, and the genes encapsulated in the complexes of 20/1, 50/1, and 100/1 ratios were 24 It was confirmed that it was not degraded until time, and through this, it was confirmed that the stability of the 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. Confirmation of FcRN receptor expression in cells

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

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

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

In order to confirm whether the antibody hIgG1-Fc-9Arg is absorbed into the cell through the FcRN receptor, the fluorescent substance FITC (Ex=490nm, Em=525nm) is bound to the antibody hIgG1-Fc-9Arg, and Caco-2, HEK 293, HeLa The cells were treated and confirmed through a confocal image after 1 hour.

As a result, as shown in Fig. 12, the cell lines Caco-2 and HEK 293, which are cell lines having an FcRN receptor, were absorbed into cells and exhibited fluorescence. On the contrary, it was confirmed that the HeLa cell line without the FcRN receptor did not show fluorescence.

In addition, Caco-2 and HT-29 cell lines were selected as cell lines to be applied to the cell experiment through the above results.

In addition, in order to check whether the hIgG1-Fc-9Arg/pDNA complex was delivered into the cell to form an endosome, the antibody FITC-hIgG1-Fc-9Arg bound with a fluorescent substance was treated with Caco-2 cell line and reacted for 30 minutes and 4 hours. After staining with Lysotracker, it was confirmed through confocal image.

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

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

In order to confirm the gene expression, it was confirmed whether or not the hIgG1-Fc-9Arg/pDNA complex was delivered by combining the red fluorescent Bobo-3 (Ex=570nm, Em=602nm) with pDNA.

As a result, as shown in FIG. 14, the confocal image was confirmed 24 hours after the complex of 0/1, 50/1, and 100/1 was treated with the Caco-2 cell line. As a result, 20/1, 50/1, All 100/1 showed red fluorescence, and the 50/1 composite showed the highest fluorescence intensity. In addition, it was confirmed that the developed antibody-derived carrier hIgG1-Fc-9Arg efficiently delivered genes into cells because the complex of 50/1 ratio showed higher fluorescence intensity than the lipofectamine used as a positive control.

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

As an orally administered gene delivery system, the cell membrane permeability test of the Caco-2 cell monolayer was performed because it had to pass through the epithelial cell membrane by binding with the FcRN receptor of the small intestine epithelial cells to exhibit the gene transfer effect.

More specifically, to confirm the change of the Caco-2 cell monolayer, hIgG1-Fc-9Arg/bobo-3- in various ratios of 20/1, 50/1, and 100/1 using pDNA bound to bobo-3 The result was confirmed by reacting the pDNA complex to the Caco-2 cell monolayer for 24 hours and 36 hours.

As a result, as shown in FIG. 15, as a result of measuring the fluorescence intensity of the hIgG1-Fc-9Arg/bobo-3-pDNA complex that permeated into the cell membrane of the Caco-2 cell monolayer, the result showed that the ratio of 50/1 and 100/ The fluorescence intensity at a ratio of 1 increased after 6 hours, and relatively high fluorescence intensity could be confirmed at various complex ratios. Through this, it was confirmed that the ratio of 50/1 and 100/1 complexes had relatively high cell membrane penetration ability of the Caco-2 cell monolayer.

In addition, as shown in FIG. 16, the fluorescence imaging results of hIgG1-Fc-9Arg/bo- bo-3-pDNA absorbed by the Caco-2 cell monolayer, 50/1 ratio of hIgG1-Fc-9Arg/bobo-3- The pDNA complex showed a higher absorption rate than the 100/1 ratio of hIgG1-Fc-9Arg/bobo-3-pDNA complex.

From the above, it was confirmed that the 100/1 ratio of the complex had high fluorescence intensity because the transmittance was relatively higher than the absorption rate bound to the Caco-2 cell monolayer. In contrast, it was confirmed that the 50/1 ratio composite showed a high absorption rate in the Caco-2 cell monolayer and the transmittance increased with time.

Therefore, it could be inferred that the 50/1 ratio complex can deliver genes into the small intestine more efficiently than other ratio complexes.

In addition, as a result of confirming the gene expression by introducing the pGFP gene, as shown in FIG. 17, the doubling time of the Caco-2 cell line was 37 hours, so the cells were treated with the hIgG1-Fc-9Arg/pGFP complex and reacted for 48 hours. As a result of measuring the confocal image, it was confirmed that the pGFP gene expression proceeded in the complex of 50/1 and the fluorescence intensity increased, indicating that the developed antibody-derived delivery system efficiently delivered the gene into the cell.

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

The present inventors sought to confirm whether the antibody-derived delivery system according to the present invention can be used as a safe delivery system.

More specifically, only the carrier was treated with the Caco-2 cell line at a concentration 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 Figure 18a, bPEI at a concentration of 15 μg has a cell viability of 20%, whereas the developed hIgG1-Fc-9Arg has a cell viability of 70% or more, which is a safe carrier when applied to cells. Was confirmed.

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

4-6. Confirmation of the binding effect in the organ of hIgG1-Fc-9Arg in an animal model

In an animal model, to check the delivery process of hIgG1-Fc-9Arg and whether it binds to a specific organ, it was administered orally to a balb/c mouse model, reacted for 1 hour and 4 hours, anesthetized with diethyl ether, and then all organs were extracted. Then, it was washed three times with PBS buffer to measure fluorescence imaging.

As a result, as shown in FIG. 19A, hIgG1-Fc-9Arg was bound to FITC, a fluorescent substance, and orally administered to mice, and then fluorescence imaging was measured. As a result, IgG1-Fc-9Arg was associated with the FcRN receptor expressed in the organ. It was confirmed that it was delivered through binding.

In addition, as shown in FIG. 19B, it was confirmed that hIgG1-Fc-9Arg was absorbed a lot in kidney, liver, stomach, duodenum, jejunum, and colon, and in particular, after 3 hours, the absorption rate specifically increased in the colon. I could confirm.

From the above results, it was confirmed that the gene delivery system according to the present invention is absorbed by various organs, and since the absorption rate in the intestinal organs is high, hIgG1-Fc-9Arg has excellent binding power with various organs. It is expected to show ability. Therefore, it was confirmed that when GLP-1 and the gene delivery system form a complex, it can be usefully used for preventing or treating obesity.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

<110> KB BIOMED <120> An oral anti-obesity composition comprising GLP-1 and a gene carrier <130> PD17-186 <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)

Glucagon like peptide (GLP)-1 gene; And
As an oral composition for the prevention or treatment of obesity, comprising a GLP-1 delivery system,
The GLP-1 transporter is
Immunoglobulin Fc region; And
It comprises a linker linked to the C-terminus of the immunoglobulin Fc region,
The linker is characterized in that consisting of the amino acid sequence of Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg, composition. The composition of claim 1, wherein the GLP-1 gene consists of the nucleotide sequence of SEQ ID NO: 1. The composition of claim 1, wherein the GLP-1 delivery system consists of the amino acid sequence of SEQ ID NO: 2. The composition of claim 1, wherein the gene encoding the GLP-1 transporter comprises a nucleotide sequence of SEQ ID NO: 3. The composition of claim 1, wherein the immunoglobulin Fc region consists of the amino acid sequence of SEQ ID NO: 4. The composition of claim 1, wherein the gene encoding the immunoglobulin Fc region comprises a 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. The composition of claim 7, wherein the immunoglobulin Fc region is derived from IgG. delete

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