KR100778633B1 - Glp-1 derivative linked biotin and biotin-polyethylene glycol, method for the preparation thereof and pharmaceutical composition comprising the same - Google Patents

Abstract

A biotin and biotin polyethylene glycol-linked GLP-1 derivative is provided to be usefully used for preventing or treating diseases caused by excessive secretion of insulin, lowering of glucose in plasma, inhibition of stomach or intestine motion, inhibition of an empty stomach or intestine, or inhibition of food intake, type 2 diabetes, obesity and irritable colon syndrome. A GLP-1 derivative in which biotin and biotin-polyethylene glycol are selectively linked to No. 26 lysine residue and No. 34 lysine residue is represented by the formula(2), wherein the biotin-polyethylene glycol is represented by the formula(1) having a molecular weight of 3,400. A method for preparing the GLP-1 derivative comprises the steps of: (a) adding biotin, biotin-polyethylene glycol and GLP to a buffer solution and an organic solvent to be subject to reaction; (b) storing the reaction mixture obtained from the step(a) under a certain temperature for a certain period of time; (c) removing non-reacted reaction products after the reaction completion of the step(b); and (d) isolating GLP-1 in which the biotin and biotin-polyethylene glycol are linked from the non-reacted products-removed products and purifying them. A pharmaceutical composition for preventing or treating diabetes or obesity comprises the biotin and biotin polyethylene glycol-linked GLP-1 derivative.

Description

GLP-1 derivative linked biotin and biotin-polyethylene glycol, method for the preparation about and pharmaceutical composition comprising the same}

Figure 1 is a ^{GLP-1, Lys 34 -mono-} biotin-GLP-1 (MB-GLP-1) , and ^{Lys 26, 34 -di-biotin-} GLP-1 (DB-GLP-1) mixture of HPLC chromatograms The figure shown.

2 shows MALDI-TOF mass spectrometry spectra of GLP-1, MB-GLP-1 and DB-GLP-1.

FIG. 3 shows MALDI-TOF mass spectrometry of GLP-1 derivatives (Lys ²⁶ -Biotin-Lys ³⁴ -Biotin-PEG-GLP-1; DBP-GLP-1) conjugated to biotin and biotin-polyethylene glycol of the present invention. The figure shown.

4 is a diagram showing a mass spectrometry spectrum of DBP-GLP-1 measured after digesting DBP-GLP-1 of the present invention with lysine-C, a protease.

Figure 5 is a diagram showing the time-residual analysis results of GLP-1, DB-GLP-1 and DBP-GLP-1 in trypsin enzyme.

FIG. 6 shows the results of time-remaining analysis of GLP-1, DB-GLP-1 and DBP-GLP-1 in BBMV enzymes.

Figure 7 is a diagram showing the time-residual analysis results of GLP-1, DB-GLP-1 and DBP-GLP-1 in the DPP IV enzyme.

Figure 8 is a diagram showing the results of insulin secretion promoting experiments of GLP-1, DB-GLP-1 and DBP-GLP-1 in the rat pancreatic diagram.

9 is a diagram showing the glucose tolerance change in the control group (placebo, saline), GLP-1 and DB-GLP-1 administration group.

10 is a diagram illustrating the area under the curve of glucose concentration in the control group, GLP-1 and DB-GLP-1 administration group based on the result of FIG. 9.

11 is a diagram showing the change in glucose tolerance in the GLP-1, DB-GLP-1 and DBP-GLP-1 administration group.

12 is a diagram illustrating the area under the glucose concentration curve in the GLP-1, DB-GLP-1, and DBP-GLP-1 administration groups based on the result of FIG. 11.

Figure 13 shows the changes in blood concentrations after oral administration of GLP-1 and DBP-GLP-1 in SD rats.

The present invention relates to a GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol, a preparation method thereof, and a pharmaceutical composition comprising the same.

Glucagon-like peptide-1 (GLP-1) is a variety of biological agents, such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric fasting, inhibiting gastric or intestinal locomotion, enhancing glucose use and inducing weight loss. Induce effect. GLP-1 also prevents pancreatic β-cell degeneration caused by the progression of type II diabetes, Type II diabetes, and non-insulin dependence diabetes mellitus (NIDDM). It is known to be able to restore the insulin secretion ability by the production promotion. In particular, a salient feature of GLP-1 lies in its ability to stimulate insulin secretion without the risks associated with insulin therapy, oral use that acts by increased insulin expression, and the risks associated with hypoglycemia with some types. In addition, it is known that no side effects such as death and necrosis of β-cells in the pancreas following long-term administration of a blood sugar lowering agent sulfone urea (sulfonylurea). Therefore, it is considered to be a very effective substance in the treatment of type 2 diabetes.

However, the activity of GLP-1 itself is insufficient, and two cleaved naturally occurring peptides, GLP-1 (7-37) OH and GLP-1 (7-36) NH _2, are rapidly removed in vivo and thus have a half-life in vivo. The very short has been limited the usefulness of the therapy involving the GLP-1 peptide. In particular, endogenously produced dipeptidyl peptidase IV (DPP-IV) inactivates the GLP-1 peptide by removing the N-terminal histidine (No. 7) and alanine residue (No. 8). This is known to be a major cause of short in vivo half-life (O 'Harte et al., 2000).

Thus, DPP-IV inhibitors can be used to inhibit degradation of GLP-1 (P93 / 01, NVP-LAF237, NVP-DPP728, 815541A, 823093, MK-0431, etc.) or GLP-1 receptor agonists or GLP-1 derivatives. Various approaches have been attempted to prolong the loss half-life of GLP-1 peptides or reduce the rate of peptide removal from the body while maintaining biological activity (exendin, liraglutide, GLP-1 / CJC-1131, etc.). .

On the other hand, vitamins are indispensable for various physiological control of the human body, and are directly involved in metabolism and growth of humans, in particular, the production of digestive enzymes, antibodies, and fatty acids, and are accompanied by various diseases in their deficiency. However, since humans and mammals have lost their ability to synthesize these vitamins, it is essential to obtain them from the outside, so the human small intestine has a very well developed transport system that can absorb these vitamins. Or active transport systems, concentration-dependent passive transport systems, intracellular transport systems, receptor-mediated receptor-mediated cells, depending on various environmental conditions such as pH. It is absorbed into the human body by the endocytotic pathway, and its intestinal tract is also very diverse. For example, most of thiamin and niacin are absorbed in the duodenum, and cyanocobalamin occurs throughout the small intestine. Each has its own absorption system, the most well known of which is the Na-dependent multivitamin transport system, a biotin absorption system that is one type of active transport. In addition to the small intestine, the system is widely distributed in various organs of the human body such as the liver, kidneys, and heart. The affinity constant for biotin in the small intestine of this system is known to be about 2.6 nM.

On the other hand, polyethylene glycol (PEG) is a high-molecular compound having a structure of HO-(-CH ₂ CH ₂ O-) _n -H, because of its high hydrophilicity can be coupled to the pharmaceutical protein to increase its solubility. Proper binding also increases the molecular weight of the modified protein and the volume under aqueous solution while retaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration and protecting and degrading proteins from cells and antibodies that recognize external antigens. The degradation of proteins by enzymes can also be reduced. As such, the molecular weight range of polyethylene glycol that can bind to protein is approximately 1,000 to 100,000, and when the molecular weight is 1,000 or more, the toxicity is known to be relatively low. The molecular weight range of the polyethylene glycol is 1,000 ~ 6,000 is distributed throughout the body and metabolized through the kidney, in particular polyethylene glycol of molecular weight 40,000 is known to be distributed in organs including blood and liver and metabolism is made in the liver.

In general, medical and pharmacologically useful proteins administered through the parenteral route are antigenic in vivo, generally have poor water solubility and short duration of life. It is becoming. U.S. Patent No. 4,179,337 discloses that the use of proteins and enzymes conjugated with polyethylene glycol as therapeutic agents can provide the benefits of reduced antigenicity, increased water solubility, and increased body retention, which are advantages of polyethylene glycol. have. Since this patent, attempts have been made to combine the bioactive proteins with polyethylene glycol to overcome their disadvantages. For example, Veronez et al. Combined ribonuclease and superoxide dismutase with PEG (Veronese et al., 1985), U.S. Patent No. 4,766,106 and U.S. Patent No. 4,917,888 discloses an increase in protein solubility by binding a polymer comprising PEG to the proteins. U.S. Patent No. 4,902,502 also discloses the binding of polyethyleneglycol or other polymers to recombinant proteins to reduce antigenicity and increase body persistence.

On the other hand, despite the above advantages, in polyethylene and protein binding, the polyethylene glycol is usually covalently bound to one or more free lysine (lysine) residues of the protein to be bound, wherein If a portion of the surface portion directly related to the activity of the protein is combined with polyethylene glycol, the portion is no longer able to perform a biological function there is a problem that the activity of the protein is reduced. In addition, since the binding of the polyethylene glycol and lysine residues occurs at random, many kinds of polyethylene glycol and protein conjugates exist as a mixture depending on the binding position, which makes the process of purely separating a desired compound complicated. There is.

Accordingly, various attempts have been made to polyethyleneglycol GLP-1 or its analogs and exendin, which have a short half-life and have therapeutic utility. International Publication No. 04/093823 discloses a GLP-1 compound or derivative thereof coupled to one or more polyethyleneglycol molecules that produce biologically active peptides having extended half-lives and reduced removal rates compared to non-polyglycolated peptides. Is described. Incorporation of one or two Cysteine (Cys) residues into a specific amino acid site of the peptide provides a thiol group, wherein the thiol group is covalently bonded to polyethylene glycol or a derivative thereof, in particular polyethylene glycol-maleimide, to form a polyethylene glycolated GLP- One compound is produced or a lysine residue or carboxy terminus of a GLP-1 peptide, analog or fragment thereof is covalently linked to one or more polyethyleneglycol molecules or polyethyleneglycol derivatives to produce molecules with extended half-life and reduced removal rates. Polyethylene glycol or derivatives thereof in International Patent Publication No. 04/093823 refer to 1 to 3 cysteines at positions 26 and 34 of GLP-1 or GLP-1 analogue peptide compounds, 1 to 3 at positions 18, 22 and 26. It is disclosed that it may be attached at the lysine residue, or at the carboxy terminal amino acid of the peptide. In addition, one to six polyethyleneglycols may be attached in one peptide molecule, and preferred polyethyleneglycol molecular weights are shown in 20,000-40,000 daltons. Polyethyleneglycolated GLP-1 compounds prepared by International Patent Publication No. 04/093823 have a half-life increase and a reduction in removal rate compared to natural GLP-1 compounds or Val ₈ -GLP-1 (7-37) OH. It is disclosed that there is an effect of maintaining all or part of the biological activity of native GLP-1.

The various approaches described above yield GLP compounds with extended half-life compared to native GLP, but if two or more polyethyleneglycol molecules are covalently bound to a biologically less active peptide, such as GLP-1, the molecule would be unsuitable for therapeutic use. There is a risk of developing severe and adverse properties such as instability to molecules and a decrease in biological activity.

Accordingly, the present inventors have studied bioactive GLP-1 derivatives having excellent biological stability by increasing the half-life of the drug while exhibiting biological activities equivalent or similar to those of natural GLP-1. A GLP-1 derivative in which polyethylene glycol was selectively conjugated was prepared, and the GLP-1 derivative in which the biotin and biotin-polyethylene glycol were selectively conjugated was mixed in biotin-GLP-1 or polyethylene glycol-GLP-1 in which various forms were mixed. Compared with high purity, excellent biological stability, biological activity, and glycemic effect in diabetic animals, particularly confirmed by absorption oral administration and thereby effective pharmacological properties, and completed the present invention.

The present invention provides a GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol, a preparation method thereof, and a pharmaceutical composition comprising the same.

The present invention provides a GLP-1 derivative conjugated with biotin and biotin-polyethyleneglycol.

The GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol of the present invention is characterized by selectively conjugating biotin to lysine residue 26 of GLP-1 and biotin-polyethylene glycol to lysine residue 34 respectively.

In the GLP-1 derivative in which the biotin and biotin-polyethylene glycol are conjugated, the GLP-1 may be either natural or recombinant GLP-1.

In the case of GLP-1, there are three sites to which biotin and biotin-polyethyleneglycol can be combined (N-terminal histidine, lysine residues 26 and 34). However, when the three sites are conjugated with the biotin and biotin-polyethylene glycol, there is a problem that the instability and biological activity of the target molecule is reduced to an unsuitable use for pharmaceutical use. In the present invention, biotin and biotin-polyethyleneglycol are selectively conjugated to lysine residues 26 and 34 of GLP-1, respectively, to increase biological stability and biological activity. Thus, the biotin and biotin-polyethyleneglycol are preferably conjugated to lysine residues 26 and 34 of GLP-1.

The biotin-polyethylene glycol may be optionally selected from biotin-polyethylene glycol having a molecular weight of 3400 or 5000, but is not limited thereto. Preferably, the biotin-polyethylene glycol is biotin-polyethylene glycol (molecular weight 3400) represented by the following Chemical Formula 1.

In addition, the present invention

1) adding biotin, biotin-polyethylene glycol and GLP to the buffer and the organic solvent to react (step 1);

2) storing the reaction mixture of step 1 under a constant temperature for a predetermined time (step 2);

3) after completion of the reaction of step 2, removing unreacted reactants (step 3); And

4) the biotin and biotin-polyethylene glycol comprising the step of separating and purifying GLP-1 conjugated with biotin and biotin-polyethylene glycol from the product from which the unreacted reactant of step 3 is removed (step 4) Provided are methods for preparing the conjugated GLP-1 derivatives.

Biotin and biotin-polyethylene glycol according to the present invention will be described in detail step by step for the preparation of GLP-1 conjugated as follows.

In step 1, the reaction molar ratio of biotin and biotin-polyethylene glycol is preferably selected within a molar ratio of 1 to 4 depending on the type of biotin and biotin-polyethylene glycol with respect to 1 mol of GLP-1. The appropriate molar ratio of the reaction may be preferably selected in consideration of the molecular structure and molecular weight of the biotin and biotin-polyethylene glycol to be conjugated, as well as the pH, reaction temperature, reaction time, and the like of the reaction solution.

The buffer solution and the organic solvent are not particularly limited, and a buffer solution commonly used in the art may be used, and may be appropriately selected and used depending on the biotin and biotin-polyethylene glycol used.

In step 2, the storage temperature and the storage time, as described for the reaction molar ratio, it is preferable to be appropriately adjusted according to the type of biotin and biotin-polyethylene glycol to be conjugated. Typically, it can be left at 4 ° C. for 2 hours, or it can be left at room temperature for a shorter time. This relates to the degree of reactivity of the biotin and biotin-polyethyleneglycol to which it is conjugated. The conjugation reaction is carried out while standing, and after a reasonable time, it can be stopped using a glycine solution or the like.

In step 3, the removal of the unreacted material may be removed by a method conventionally used in the art. For example, it may be removed by dialysis or the like using a suitable buffer, for example, a solution such as phosphate buffered saline (PBS).

In step 4, separation and purification may be performed using size exclusion chromatography, reversed phase high performance liquid chromatography, and the like, but is not limited thereto.

In conclusion, by performing the above separation and purification steps, biotin and biotin-polyethylene glycol are selectively conjugated by biotin to the lysine residue 26 of GLP-1 and biotin-polyethylene glycol to the lysine residue 34. GLP-1 derivatives can be obtained with a purity of at least 98%.

The present invention also relates to diabetes, including GLP-1 derivatives in which biotin and biotin-polyethylene glycol are conjugated, particularly those caused by hypersecretion of insulin, such as type 2 diabetes, or obesity, or plasma such as irritable bowel syndrome. It provides a pharmaceutical composition for the prevention or treatment of diseases caused by glucose lowering, inhibition of gastric or intestinal movement, inhibition of gastric or intestinal fasting or inhibition of food intake.

In the present invention, the GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol is selectively conjugated with natural GLP-1 by selectively conjugating biotin to lysine residue 26 of GLP-1 and biotin-polyethylene glycol to 34 lysine residue. The biological stability is very good by increasing the half-life of the drug while exhibiting equivalent or similar biological activity. In addition, by selectively limiting the binding sites and the number of binding of biotin and biotin-polyethylene glycol, the side effects caused by the diversity of these elements are minimized to increase the intestinal absorption rate using the intestinal active transport system. Thus, the GLP-1 derivatives conjugated with biotin and biotin-polyethyleneglycol of the present invention may be caused by insulin oversecretion, or by lowering plasma glucose, inhibiting gastric or intestinal motility, inhibiting gastric or intestinal fasting, or inhibiting food intake. It can be usefully used for the prevention or treatment of diseases, and particularly useful for diabetes mellitus, preferably type 2 diabetes, obesity and irritable bowel syndrome.

The pharmaceutical composition comprising the GLP-1 derivative conjugated with the biotin and biotin-polyethyleneglycol of the present invention may be formulated in oral or parenteral dosage form during clinical administration, and oral administration is more preferred. However, it is not limited to this.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants commonly used are prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used.

The dosage of the composition of the present invention may vary depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, etc. Multiple doses also allow for effective dose administration. The daily dose of DBP-GLP-1 of the present invention is 0.01 to 50 mg / kg, preferably 0.1 to 10 mg / kg may be administered once to several times daily.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Example  One  Biotin Spliced GLP Manufacture of -1

20 μl of Biotin-NHS (biotin-N-hydroxysuccinimide, Sigma, 2 mg / ml in 0.3% TEA (triethyamine) added DMSO solution) was added 380 μl of GLP-1 (Bachem, 0.3% TEA) 5 mg / ml) in DMSO solution. At this time, the molar ratio of GLP-1: Biotin-NHS was 1: 2. The mixture was allowed to react for 60 minutes at room temperature. The reaction was terminated with distilled water to which 100 μl of 1% trifluoroacetic acid (TFA) was added.

Example  2  Biotin and Biotin Polyethylene glycol Spliced GLP -1's My article

100 μl of Bio-PEG (Nektar, MW 3400, 6 mg / ml in DMF solution with 0.3% TEA) was added 100 μl of Fmoc-GLP-1 (Fluorenylmethoxycarbonyl-GLP-1, American Peptide Company, 0.3%). 2 mg / ml in DMF solution with TEA added). At this time, the molar ratio of GLP-1: Bio-PEG was 1: 1.5. The mixture was allowed to react for 60 minutes at room temperature. The reaction was terminated with distilled water added with 100 μl of 1% TFA. The mixture was then lyophilized to add DMF solution with 0.3% TEA and excess Biotin-NHS and mixed. 5% piperidine solution was further added to remove Fmoc. After 10-20 minutes, the reaction was terminated with distilled water to which 100 μl of 1% TFA was added.

Example  3  Biotin and Biotin Polyethylene glycol Spliced GLP -1 separation, purification and analysis

Biotin or biotin and biotin-polyethylene glycol conjugated GLP-1 prepared in Examples 1 and 2 were separated by high performance liquid chromatography (hereinafter referred to as “RP-HPLC”). -18 (250 × 10 mm, 5 μm, Phenomenex, USA) and Capcell-pak RP-18 (250 × 4 mm, 5 μm, Shiseido, Japan) were used, and the mobile phase conditions were 35-45% solvent B (0.1 Acetonitrile with% TFA) and 65-55% solvent A (distilled water with 0.1% TFA) were linearly changed while maintaining a flow rate of 1 ml / min, each peak using a UV absorbance 215 nm. Biotin separated from the above-described method or GLP-1 conjugated with biotin and biotin-polyethylene glycol were identified by mass spectrometry using MALDI-TOF mass spectrometry. .

In addition, 1 mg / ml of GLP-1 conjugated with purified biotin and biotin-polyethylene glycol was dissolved in 50 µl of triethylamine-hydrochloric acid buffer (10 mmol / L; pH 7.4), followed by 50 µl of protease. Lysine-C (1 mg / ml) was added and reacted at 37 ° C. for 1 hour. 5 μl of 10% (v / v) TFA was added to complete the lysine-C digestion reaction and then the reaction mixture was analyzed by MALDI-TOF mass spectrometer.

The HPLC chromatogram of GLP-1 conjugated with the mixture and finally separated biotin or biotin and biotin-polyethylene glycol after completion of the reaction is shown in FIG. 1, and their MALDI-TOF mass spectra are shown in FIG. 2.

As shown in Figures 1 and 2, three different substances were separated from the reaction mixture, and these substances were unreacted GLP-1, Lys ³⁴ -mono-biotin-GLP-1 (MB-GLP-1) and Lys ^{26, 34} -di-biotin-GLP-1 (DB-GLP-1) was confirmed. In addition, the purity of the separated and purified biotin and biotin-polyethylene glycol conjugated GLP-1 derivative was 98% or more.

The MALDI-TOF mass spectrometry of the GLP-1 derivative (Lys ²⁶ -Biotin-Lys ³⁴ -Biotin-PEG-GLP-1; DBP-GLP-1) conjugated with the purified biotin and biotin-polyethylene glycol is shown in FIG. 3, the mass spectra of DBP-GLP-1 measured after digesting DBP-GLP-1 with lysine-C, a protease, are shown in FIG. 4.

Experimental Example  One  : Biotin and Biotin of the Present Invention Polyethylene glycol Spliced GLP -1 derivatives ( DBP - GLP -1) biological stability analysis

In order to confirm the biological stability of the biotin and biotin-polyethylene glycol conjugated GLP-1 derivative (DBP-GLP-1), time using trypsin, BBMV, DPP IV, enzymes that degrade GLP-1- Residual analysis was performed.

1-1: Stability Assay in Trypsin Enzyme

20 μl of GLP-1 and DB-GLP-1 and DBP-GLP-1 prepared and isolated in Examples 1 to 3 were added to 20 μl of 2 mM trypsin (25 mM Phosphate Buffer, pH 6.5), respectively. After reaction at 37 ℃ aqueous solution. The reaction was terminated with distilled water to which 100 μl of 1% TFA was added and the mixture for each hour was analyzed using HPLC. The HPLC method was as mentioned in Example 3 above, and the mobile phase conditions were run for 10 minutes while maintaining a flow rate of 1 ml / min with 36% to 42% of solvent B.

Time-resistance analysis of GLP-1, DB-GLP-1 and DBP-GLP-1 in trypsin enzyme is shown in Figure 5, GLP-1, DB-GLP-1 and DBP-GLP-1 in trypsin enzyme The stability analysis results are shown in Table 1.

Stability against Trypsin GLP-1 type Half-life (seconds) Drainage GLP-1 11.6 - DB-GLP-1 85.6 7.38 DBP-GLP-1 210.0 18.10

As shown in FIG. 5, it can be seen that DBP-GLP-1 and DB-GLP-1 have much stronger degradation resistance against trypsin than GLP-1.

In addition, as shown in Table 1, the half-lives of GLP-1, DB-GLP-1, and DBP-GLP-1 are 11.6, 85.6, and 210.0 seconds, respectively, and the half-life of DB-GLP-1 and DBP-GLP-1. Were 7.38 and 18.10 times higher than GLP-1, respectively.

1-2: Microvesicle vesicles ( BBMV ; Brush Border Membrane Vesicle Stability Assay in Enzymes

20 μl of GLP-1 and DB-GLP-1 and DBP-GLP-1 prepared and isolated in Examples 1 to 3 were added to 20 μl of 2 mM BBMV (25 mM Phosphate Buffer, pH 6.5), respectively. After reaction at 37 ℃ aqueous solution. The reaction was terminated with distilled water to which 100 μl of 1% TFA was added and the mixture for each hour was analyzed using HPLC. The HPLC method was as mentioned in Example 3 above, and the mobile phase conditions were run for 15 minutes while maintaining a 1 ml / min flow rate with 36% to 45% solvent B.

Time-resistance analysis of GLP-1, DB-GLP-1 and DBP-GLP-1 in BBMV enzymes is shown in FIG. 6, and GLP-1, DB-GLP-1 and DBP-GLP-1 in BBMV enzymes. The stability analysis results of Table 2 are shown.

Stability against BBMV GLP-1 type Half-life (seconds) Drainage GLP-1 0.57 - DB-GLP-1 4.38 7.68 DBP-GLP-1 13.94 24.46

As shown in FIG. 6, it can be seen that DBP-GLP-1 and DB-GLP-1 have much stronger degradation resistance against trypsin than GLP-1.

In addition, as shown in Table 2, the half-lives of GLP-1, DB-GLP-1, and DBP-GLP-1 are 0.57 seconds, 4.38 seconds, and 13.94 seconds, respectively, and the half-life of DB-GLP-1 and DBP-GLP-1. Were 7.68 and 24.46 times higher than GLP-1, respectively.

1-3: Dipeptidyl Peptidase - IV ( dipeptidyl peptidase - IV ; DPP IV Stability Assay in Enzymes

20 μl of GLP-1 and DB-GLP-1 and DBP-GLP-1 prepared and isolated through Examples 1 to 3 were respectively 20 μl of 400 mg / ml dipeptidyl peptidase-IV (50 mM Tris). -HCl Buffer, pH 7.4) and reacted in an aqueous solution of 37 ℃. The reaction was terminated with distilled water to which 100 μl of 1% TFA was added and the mixture for each hour was analyzed using HPLC. The HPLC method was as mentioned in Example 3 above, and the mobile phase conditions were run for 15 minutes while maintaining a 1 ml / min flow rate with 36% to 45% solvent B.

Time-resistance analysis of GLP-1, DB-GLP-1 and DBP-GLP-1 in DPP IV enzyme is shown in FIG. 7, and GLP-1, DB-GLP-1 and DBP-GLP in DPP IV enzyme. The stability analysis results of -1 are shown in Table 3.

Stability against DPP IV GLP-1 type Half-life (seconds) Drainage GLP-1 49.8 - DB-GLP-1 117.4 2.36 DBP-GLP-1 495 9.94

As shown in FIG. 7, it can be seen that DBP-GLP-1 and DB-GLP-1 have much stronger degradation resistance to trypsin than GLP-1.

In addition, as shown in Table 3, the half-lives of GLP-1, DB-GLP-1, and DBP-GLP-1 are 49.8 seconds, 117.4 seconds, and 495 seconds, respectively, and the half-life of DB-GLP-1 and DBP-GLP-1. Were 2.36 and 9.94 times higher than GLP-1, respectively.

As described above, DBP-GLP-1 of the present invention can be seen that the biological stability is very excellent by increasing the half-life in the body.

Experimental Example  2  : Biotin and Biotin of the Present Invention Polyethylene glycol Spliced GLP -1 derivatives ( DBP - GLP -1) biological activity measurement: insulin secretion promoting experiment

In order to confirm the promotion of insulin secretion of the GLP-1 derivative (DBP-GLP-1) conjugated with the biotin and biotin-polyethylene glycol of the present invention, the following experiment was performed.

Sprague Dawley rats were used as experimental animals, and pancreatic islets were isolated by using collagenase digestion and Ficoll density difference separation. The isolated pancreatic islets were cultured in a cell incubator for 2 to 3 days, then divided into 20 wells and dispensed into a 24-well plate containing 1 ml of KRH buffer solution containing 16.7 mM glucose, followed by GLP-1 and DB-GLP-1. And DBP-GLP-1 were added to 0.1, 1, 10, 100 nM, respectively, and then incubated for 2 hours in a cell incubator. After the incubation, 200 μl of the culture sample was taken, and the insulin concentration in the obtained sample was measured using an insulin enzyme immunoassay kit.

Insulin secretion promoting experiment results of GLP-1, DB-GLP-1 and DBP-GLP-1 are shown in FIG.

As shown in FIG. 8, GLP-1, DB-GLP-1 and DBP-GLP-1 were observed to promote insulin secretion in a concentration dependent manner. These effects do not show a significant difference between GLP-1, DB-GLP-1 and DBP-GLP-1, but all show a concentration-dependent stable increase.

Experimental Example  3  : Biotin and Biotin of the Present Invention Polyethylene glycol Spliced GLP -1 derivatives ( DBP - GLP Measurement of biological activity in animal models of -1): Glucose tolerance  Measure

In order to determine the intraperitoneal glucose tolerance (IPGTT) in the animal model of the biotin and biotin-polyethylene glycol conjugated GLP-1 derivative (DBP-GLP-1), the following experiment was performed. .

Twenty six-week-old male db / db mice (C57 / BLKS / J-db / db, Korea research institute of bioscience and biotechnology) were divided into four groups. Five animals were assigned to each group, and 100 µl of GLP-1, DB-GLP-1, and DBP-GLP-1 (1 nmole / kg) were orally administered to -30 minutes for each group of mice, followed by 200 µl. Glucose (200 mg / ml) was intraperitoneally administered and blood glucose change in blood collected from the tail vein was observed at -30, 0, 15, 30, 60, 120 and 180 minutes.

Changes in glucose tolerance in the control group (placebo, saline), GLP-1 and DB-GLP-1 administration groups are shown in FIG. 9, and in the control group, GLP-1 and DB-GLP-1 administration groups based on the results of FIG. 9. The result of plotting the area under the glucose concentration curve of 0 to 180 minutes of is shown in FIG.

In addition, the glucose tolerance change in the GLP-1, DB-GLP-1 and DBP-GLP-1 administration group is shown in Figure 11, based on the results of Figure 11 GLP-1, DB-GLP-1 and DBP-GLP The area under the curve of glucose concentration between 0 and 180 minutes in the -1 administration group is shown in FIG. 12.

As shown in Figures 9 and 11, the change in glucose tolerance in the DB-GLP-1 and DBP-GLP-1 administration group showed a marked increase in glucose tolerance compared to the control. In the case of the control group, there was a rapid increase in blood glucose and a slow drop in blood glucose with administration of glucose. On the other hand, the administration of DB-GLP-1 and DBP-GLP-1 showed relatively low blood sugar rise and relatively fast blood sugar dropping behavior. This difference in glucose tolerance between native GLP-1 and bioconjugated GLP-1 derivatives (DBP-GLP-1) is believed to be due to good absorption properties between biotin and biotin-polyethyleneglycol conjugates.

In addition, as shown in FIGS. 10 and 12, DBP-GLP-1 and DB-GLP-1 showed a very good glucose tolerance increase compared to GLP-1, and also DB-GLP- in the case of DBP-GLP-1. Compared to 1, it showed an excellent glucose tolerance. This is believed to be due to the dramatically increased biological stability and absorbency due to the introduction of polyethylene glycol in GLP-1.

Experimental Example  4  : Biotin and Biotin of the Present Invention Polyethylene glycol Spliced GLP -1 derivatives ( DBP - GLP -1) body Pharmacokinetics  Behavior investigation

By Experimental Examples 1 to 3, it was confirmed that DBP-GLP-1 of the present invention has the best biological stability, biological activity, and hypoglycemic effect in diabetic animals, indicating the characteristics of the sustained peptide drug. Therefore, the pharmacokinetic behavior of GLP-1 and DBP-GLP-1 was observed to investigate the cause of these persistent properties. In the experiment, the sample was orally administered to the experimental rat (SD rat) weighing about 200 g in the amount of 1 nmole / rat (4.18 ㎍ / rat), and the change in blood drug concentration over time was measured using the ELISA kit. . Blood samples were taken from intubated jugular veins.

Blood concentration changes after oral administration of GLP-1 and DBP-GLP-1 to SD rats are shown in FIG. 13.

As shown in Figure 13, DBP-GLP-1 of the present invention showed a tendency to gradually decrease the blood concentration after reaching the maximum blood concentration at 15-30 minutes after injection. However, GLP-1 showed a rapid decrease in blood concentration after the drug administration, and reached a baseline concentration (<2 ng / ml) after 3 hours. Therefore, it was confirmed that the DBP-GLP-1 of the present invention is effectively absorbed through oral administration to the effect.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  : Pharmaceutical Formulations

One. Powder  Produce

2 g of GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

100 mg of GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of GLP-1 derivative conjugated with biotin and biotin-polyethylene glycol

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

The GLP-1 derivative conjugated with the biotin and biotin-polyethylene glycol of the present invention exhibits biological activity similar to that of natural GLP-1, and increases the half-life of the drug so that the biological stability is very excellent, and the binding position of biotin and biotin-polyethylene glycol And by selectively limiting the number of bindings to minimize the side effects caused by the diversity of these factors, thereby increasing the small intestine absorption rate using the active transport system. Thus, the GLP-1 derivatives conjugated with biotin and biotin-polyethyleneglycol of the present invention may be caused by insulin oversecretion, or by lowering plasma glucose, inhibiting gastric or intestinal motility, inhibiting gastric or intestinal fasting, or inhibiting food intake. It can be usefully used for the prevention or treatment of diseases, and particularly useful for diabetes mellitus, preferably type 2 diabetes, obesity and irritable bowel syndrome.

Claims (13)

A biotin and a biotin-polyethylene glycol conjugated GLP-1 derivative represented by the following formula.

The conjugated GLP-1 derivative of claim 1, wherein the biotin and biotin-polyethylene glycol are selectively conjugated to lysine residues 26 and 34, respectively, of GLP-1. The conjugated GLP-1 derivative of claim 1, wherein the GLP-1 is a natural or recombinant GLP-1. The conjugated GLP-1 derivative according to claim 1, wherein the biotin-polyethylene glycol is a biotin-polyethylene glycol (molecular weight 3400) represented by the following Chemical Formula 1. <Formula 1>

1) adding biotin, biotin-polyethylene glycol and GLP to the buffer and the organic solvent to react (step 1); 2) storing the reaction mixture of step 1 under a constant temperature for a predetermined time (step 2); 3) after completion of the reaction of step 2, removing unreacted reactants (step 3); And 4) The biotin and biotin-polyethylene of claim 1 comprising the step of separating and purifying GLP-1 conjugated with biotin and biotin-polyethylene glycol from the product from which the unreacted reactant of step 3 is removed (step 4). Method for preparing a GLP-1 derivative conjugated with glycol. The method of claim 5, wherein the reaction molar ratio of biotin and biotin-polyethylene glycol in step 1 is in the range of 1 to 4 molar ratios with respect to 1 mole of GLP-1, respectively. A pharmaceutical composition for preventing or treating diabetes or obesity, comprising the GLP-1 derivative conjugated with the biotin and the biotin-polyethylene glycol of any one of claims 1 to 4. delete The pharmaceutical composition for preventing or treating diabetes or obesity according to claim 7, wherein the diabetes is type 2 diabetes. The pharmaceutical composition for preventing or treating diabetes or obesity of claim 7, wherein the composition is formulated as a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution, an emulsion, or a syrup. A pharmaceutical composition for the prevention or treatment of irritable bowel syndrome, comprising the GLP-1 derivative conjugated with the biotin and the biotin-polyethylene glycol of any one of claims 1 to 4. delete The pharmaceutical composition for the prevention or treatment of irritable bowel syndrome according to claim 11, wherein the composition is formulated as tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions or syrups.

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