KR20110007520A - Exendin-4 derivative conjugated with taurodioxycholic acid, preparation method thereof and use thereof - Google Patents

Abstract

The present invention relates to an exendin-4 derivative conjugated with taurodioxycholic acid, a preparation method thereof, and a pharmaceutical composition comprising the same as an active ingredient.

These exendin-4 derivatives not only exert insulin secretion function similar to exendin-4 but also significantly prevent vesicle stress, a major cause of beta cell death in the pancreas, and act as a very useful substance for maintaining the endocrine function of the pancreas. It can be used as a better treatment for diabetes and related diseases.

Taurodioxycholic acid, exendin-4, endoplasmic reticulum stress, beta cells

Description

Exendin-4 derivatives conjugated with taurodioxycholic acid, preparation method thereof and use thereof {Taurodeoxycholic acid-modified exedin-4, preparation method and usage approx}

The present invention relates to an exendin-4 derivative conjugated with taurodioxycholic acid, a preparation method thereof, and a pharmaceutical composition comprising the same as an active ingredient.

Glucagon-like peptide-1 (GLP-1) has various biological effects, such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric fasting, inhibiting gastric or intestinal motility, enhancing glucose use and inducing weight loss. Induce. In addition, GLP-1 prevents the degeneration of pancreatic beta cells caused by the progression of type 2 diabetes, type II diabetes, non-insulin dependence diabetes mellitus (NIDDM), and production of new beta cells. It is known to promote insulin secretion. 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 to not cause side effects such as death and necrosis of beta cells in the pancreas following long-term administration of blood sugar lowering 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 eliminated in vivo and thus in vivo. The very short half-life has limited the usefulness of the therapy involving GLP-1 peptides. 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 the major cause of short in vivo half-life (O 'Harte et al., Biochim Biophys Acta. 2000, vol. 1474, pp. 13-22).

Thus, DPP-IV inhibitors (P93 / 01, NVP-LAF237, NVP-DPP728, 815541A, 823093, MK-0431, etc.) may be used to inhibit degradation of GLP-1, or to inhibit GLP-1 receptor agonists or GLP-1. Various approaches have been attempted to extend the half-life of the GLP-1 peptide's disappearance or to reduce peptide removal from the body while maintaining biological activity using derivatives (exendin, liraglutide, GLP-1 / CJC-1131, etc.) It is becoming.

Exendin, another group of polypeptides that lower blood sugar levels, was first presented by John Eng (US Pat. No. 5,424,286). Exendin-4 has the following sequence structure and GLP-1 (7-36) NH. ₂ shows some sequence similarity (53% similarity) to 2 (Goke et al., J Biol Chem. 1993, vol. 268, pp. 19650-19655).

His ¹ -Gly-Glu-Gly-The-Phe-The-Ser-Asp-Leu-Ser-Lys ¹² -Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu- Trp-Leu-Lys ²⁷ -Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 1)

Exendin is found in the venom or bead venom; exendin-3 is present in the venom of the Mexican beaded lizard Heloderma horridum, and exendin-4 is the american poison lizard It is present in the poison of Heloderma suspectum. Exendin-4 differs from exendin-3 only at positions 2 and 3 and is more resistant to degradation by DPP-IV in mammals and more than GLP-1 having a half-life of less than 2 minutes for DPP-IV. It has a long half-life (Kieffer TJ et al., Endocrinology. 1995, vol. 136, pp.3585-3596). In addition, in vivo experiments have been shown to have a half-life of 2 to 4 hours, and to achieve sufficient blood levels by intraperitoneal administration 2 to 3 times a day (Fineman MS et al., Diabetes Care. 2003, vol. 26, pp.2370-2377). Exendin-4 is also known to regulate gastrointestinal motility, reduce food intake and inhibit plasma glucagon (US Pat. Nos. 6,858,576, 6,956,026, 6,872,700). In addition, HbAlc levels within 1 range after 28 days of administration in both exendin-4 monotherapy as well as in combination therapy with anti-diabetic agents such as sulfonylurea and / or metformin in relation to the glycemic control action of exendin-4 (Egan JM et al., Am J Physiol Endocrinol Metab. 2003. vol. 284, pp. E1072-E1079). Recently, synthetic exendin-4 under the trademark Byetta ™ has been licensed for sale from the US Food and Drug Administration.

Insulin resistance is one of the most frequent symptoms in type 2 diabetes, and one of the biological problems that have been discussed in connection with insulin resistance is ER stress of pancreatic beta cells. . The mechanism interrelationship between the endoplasmic reticulum and insulin secretion has emerged as an important factor in biologically applied therapies, and one of the ways to address these factors is chemical or pharmacological saffron that can fundamentally resolve vesicular stress. chaperone, etc., among them, bile acid and its derivatives such as ursodeoxycholic acid, and taurine-dioxycholic acid conjugated with taurine to ursoic acid are important substances that can control the improvement of endoplasmic reticulum. (Umut Ozcan et al., Science, 2006, vol. 313, pp. 1137-1140).

However, the above-mentioned exendin-4 not only increases beta cell insulin secretion but also directly acts on beta cells, thereby enhancing insulin biosynthesis and overcoming insulin resistance. Research has been shown to prevent degradation and prevent cell necrosis (Daniel J. Drucker et al., Cell Metabolism 2006, vol. 4, 391-406).

Thus, the inventors of the present invention while trying to further improve the therapeutic effect of exendin-4 exercin-4 conjugated with taulorsodioxycholic acid is to prevent the vesicle stress which is the main cause of pancreatic beta cell death more dramatically It confirmed that it is more useful for the treatment of diabetes, etc., and came to complete this invention.

One object of the present invention is to provide an exendin-4 derivative conjugated with taulorsodioxycholic acid.

Another object of the present invention is the step of (1) activating the carboxylic acid group of taurusodioxycholic acid; (2) reacting the activated taurusordioxycholic acid with exendin-4; And (3) separating the exendin-4 derivative conjugated with taulorsodioxycholic acid from the reactant.

Still another object of the present invention is to provide a pharmaceutical composition comprising as an active ingredient an exendin-4 derivative conjugated with taurusodioxycholic acid.

The present invention relates to an exendin-4 derivative in which taurodeoxycholic acid is conjugated.

Here, the exendin-4 may be natural or recombinant exendin-4. Exendin-4 has three sites to which taulorsodioxycholic acid can be conjugated. Specifically, the N-terminal histidine residues, lysine residues 12 and 27. However, when all three sites are conjugated with taurusordioxycholic acid, there is a problem in that the instability and biological activity of the target molecule are reduced to an unsuitable use for pharmaceutical use. In the present invention, it is preferable that taurusodioxycholic acid be conjugated to at least one lysine residue of lysine residues 12 and 27 of exendin-4 to increase biological stability and activity as desired. In the present invention, it is most preferable that the taurusodioxycholic acid is conjugated to the lysine residue 27 of the exendin-4.

In addition, the present invention comprises the steps of (1) activating the carboxylic acid group of taurusodioxycholic acid; (2) reacting the activated taurusordioxycholic acid with exendin-4; And (3) separating the exendin-4 derivative conjugated with taulorsodioxycholic acid from the reactant. Hereinafter, each step will be described in detail.

In step (1) the taurusordioxycholic acid may be obtained commercially or chemically synthesized. In one embodiment, it may be prepared by reacting taurusodioxycholic acid carbonate with ε-amino-n-caproic acid (EACA) (FIG. 1 and Example 1). See 1). Any compound that activates -OH to facilitate the reaction with amine (-NH ₂ ) in place of the taurusordioxycholic acid carbonate can be used. In addition, instead of ε-amino-n-caproic acid, one end is an amine (-NH ₂ ), the other end is -COOH, and the number of intermediate alkyl (-CH _2- ) is not limited, but about 2 to 6 Any compound can be used as long as it is a phosphorus compound.

It is preferable to activate the carboxylic acid group of taurusordioxycholic acid for the smooth reaction between the carboxylic acid group of taurousodioxycholic acid and the primary amine in exendin-4. . To this end, in one embodiment, it can be activated by reacting taurusodioxycholic acid with N-hydroxysuccinimide (NHS) or sulfo-NHS (see Example 1 2). In the case of the prepared taurusordioxycholic acid-NHS, more than 99% of the terminal carboxylic acid group can be confirmed by 1H-NMR analysis (see FIG. 1B).

The reaction molar ratio of exendin-4 and taurousodioxycholic acid in step (2) may be appropriately selected within the range of 1 to 4 molar ratios of taurousodioxycholic acid per 1 mole of exendin-4. Preferred (see FIG. 3).

In step (3), taurusodioxycholic acid can be separated from the reactants by methods known in the art. For example, methods such as chromatography (eg, hydrophobicity and size exclusion) or electrophoresis (eg, SDS-PAGE) may be used, but are not limited thereto. In one embodiment, separation can be accomplished using high performance liquid chromatography. Through this separation step, exendin-4 derivatives selectively conjugated with taurousodioxycholic acid to 12 or 27 or 12 and 27 lysine porcelain of exendin-4 can be obtained with a purity of 98% or more. (See FIG. 4 and Experimental Example 1).

In the present invention, it was demonstrated that the exendin-4 derivative conjugated with the taurusordioxycholic acid of the present invention exhibited a superior therapeutic effect than exendin-4. It will be described in detail below.

The exendin-4 derivative conjugated with taulorsodioxycholic acid according to the present invention shows a stronger binding force than exendin-4 to the glucagon-like peptide-1 (GLP-1) receptor on the surface of the insulin secreting cell line (FIG. 7). And Experimental Example 2). That is, the activity of exendin-4 is not reduced, but rather increased, because tauurosodioxycholic acid is conjugated to exendin-4.

In addition, the exendin-4 derivative conjugated with taulorsodioxycholic acid according to the present invention significantly inhibits the increase in blood glucose than exendin-4, and the increased blood sugar is also lowered quickly (FIG. 8 and Experimental Example 3). Reference). That is, the exendin-4 derivative conjugated with taulorsodioxycholic acid according to the present invention can normalize blood glucose in a shorter time than exendin-4.

In addition, the survival rate of the cell line was examined after treatment with the vesicle stress inducer and exendin-4 or the exendin-4 derivative conjugated with the taurousodioxycholic acid of the present invention. As a result, exendin-4 showed high cell viability at low concentration, but as the concentration increased, the derivatives conjugated with taurusodioxycholic acid showed better survival than exendin-4 (Fig. 9 and Experimental Example 4). Reference). As a result, it can be seen that the exendin-4 derivative conjugated with taurusoledioxycholic acid according to the present invention can defend against vesicle stress.

In addition, the effect of taulorsodioxycholic acid, exendin-4 and exendin-4 derivatives conjugated with taurousodioxycholic acid of the present invention on the expression of GRP78 protein under vesicle stress conditions was investigated. GRP78 is the first molecule to increase in the vesicle stress conditions of pancreatic beta cells, and decreases when it blocks fundamentally the vesicle stress. Exendin-4 is known to reduce endoplasmic reticulum stress but not GRP78. On the other hand, taurusorbodioxycholic acid and the exendin-4 derivative conjugated by it could reduce GRP78, and the exendin-4 derivative conjugated by it rather than taurousodioxycholic acid significantly reduced GRP78. (See FIG. 10 and Experimental Example 5).

In addition, the effect of taulorsodioxycholic acid, exendin-4, and exendin-4 derivatives conjugated with taurousodioxycholic acid of the present invention on the expression of phospho-elf2α protein under vesicle stress conditions were investigated. . Phospho-elf2α, a molecule that increases as a lower mechanism than GRP78 in the endoplasmic reticulum stress environment, affects beta-cell inhibition by inhibiting the translation of proteins when phospholation increases. Exendin-4 reduced saturation and exendin-4 derivatives conjugated to tauurosodioxycholic acid of the present invention reduced saturation more than exendin-4, while tauurosodioxycholic acid exhibited vesicle stress It has been confirmed that it has an effect on the upper mechanism in the environment but does not affect the lower mechanism (see FIG. 10 and Experimental Example 5).

In conclusion, exendin-4 derivatives conjugated to tauurosodioxycholic acid according to the present invention can exert a superior therapeutic effect than exendin-4 and tauurosodioxycholic acid.

Accordingly, the present invention is directed to diabetics, particularly type 2 diabetes, or diseases caused by undersecretion of insulin, such as obesity, or irritable bowel syndrome, including exendin-4 derivatives conjugated with taurusodioxycholic acid Provided are pharmaceutical compositions for the prevention or treatment of diseases caused by lowering plasma glucose, inhibiting gastric or intestinal motility, inhibiting gastric or intestinal fasting or inhibiting food intake.

Pharmaceutical compositions containing the exendin-4 derivatives conjugated to taulorsodioxycholic acid of the present invention may be formulated for oral administration such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. It can be formulated into sterile injectable solutions, suppositories, and preparations for transdermal administration. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, it is formulated with diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like.

In one embodiment, the exendin-4 derivatives conjugated to the taurusordioxycholic acid of the present invention may be formulated as a solid preparation for oral administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the extract. Formulated by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

In another embodiment, the pharmaceutical composition containing the exendin-4 derivative conjugated with the taurusordioxycholic acid of the present invention may be formulated into a liquid formulation for oral administration. Liquid preparations for oral administration include suspensions, solvents, emulsions, syrups, and the like. In addition to the inert diluents commonly used (e.g., purified water, ethanol, liquid paraffin), various excipients may be used, for example. Wetting agents, sweetening agents, fragrances, preservatives and the like can be included.

In another embodiment, pharmaceutical compositions containing exendin-4 derivatives conjugated to taurusodioxycholic acid of the present invention may be formulated into a formulation for parenteral administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As a sterile aqueous solution, suitable buffer solutions such as Hanks' solution, Ringer's solution, or physically buffered saline can be used.For non-aqueous solvents, suspensions include propylene glycol, polyethylene glycol, olive oil, Same vegetable oils, injectable esters such as ethyloleate, and the like can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts for controlling osmotic pressure and / or buffers may also be used. Meanwhile, in the case of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like, which are conventional bases thereof, may 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 dosage of the taurousodioxycholic acid conjugated exendin-4 derivative of the present invention is 0.01 to 50 mg / kg, and preferably 0.1 to 10 mg / kg may be administered once to several times daily. have.

It is an object of the present invention to prepare exendin-4 derivatives having a better therapeutic effect than exendin-4.

According to the present invention, the activity of exendin-4 is increased as taurousodioxycholic acid is conjugated to exendin-4. In addition, the exendin-4 derivatives conjugated to taulorsodioxycholic acid of the present invention can normalize blood glucose within a faster time than exendin-4. In addition, the exendin-4 derivatives conjugated to taurusoledioxycholic acid of the present invention may act both on the upper and lower mechanisms in the endoplasmic reticulum stress conditions to prevent endoplasmic reticulum stress more dramatically than exendin-4.

Therefore, the exendin-4 derivative conjugated with taulorsodioxycholic acid according to the present invention can be used as a therapeutic agent for diseases such as diabetes, which is superior to exendin-4.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Preparation of Exendin-4 Conjugated to Taurusordioxycholic Acid

1. Preparation of Taurusordioxycholic Acid-COOH

Taurusordie using ε-amino-n-caproic acid (EACA) as a preliminary step for the preparation of activated taurusordioxycholic acid for exendin-4 conjugation Oxycholic acid-COOH was prepared (FIG. 1A).

After dissolving 150 mg (0.226 mmol) of taurusodioxycholic acid tritan salt in 2 ml DMF, 1 ml of distilled water and 46.53 mg (0.46 mmol) of 4-Methylmorpholine were added, and heated at 50 ° C. for 1 hour, followed by ε-amino-n-capro 1 ml of acid (296.4 mg / ml) was added slowly. The reaction mixture was allowed to stand for 16 hours, concentrated in vacuo, eluted with methanol and dichloromethane using column chromatography packed with silica resin, and purified.

2. Preparation of Activated Taurusodioxycholic Acid

Activate the carboxylic acid group of taurusodioxycholic acid with N-hydroxysuccinimide (NHS) for smooth reaction of the carboxylic acid group of taurousodioxycholic acid with the primary amine in exendin-4. Taurusordioxycholic acid-NHS was prepared.

50 moles of taurusordioxycholic acid in two moles relative to 17.53 mg of N-hydroxysuccinimide (NHS) and 31.4 mg of 3-dicyclohexyl carbodiimide (DCC) (tauurosordioxycholic acid) Dissolved in 1 ml of Dimethylformamide (DMF). The solution was allowed to react with sufficient stirring for 24 hours under a stream of nitrogen. Dicyclohexylurea (DCU) produced after the reaction was removed using a paper filter, and the reaction solution was concentrated under reduced pressure, and then precipitated in ether. Sodioxycholic acid-NHS was obtained. The final product was dried in a vacuum for 48 hours to obtain a powder form, the reaction and purity was confirmed by using 1H NMR (Fig. 1b).

3. Preparation of Exendin-4 Conjugated to Taurusordioxycholic Acid

To 0.1 ml of exendin-4 (5 mg / ml in DMSO) was added 0.1 ml of taurusordioxycholic acid-NHS (0.9-3.6 mg / ml in dimethyl sulfoxide (DMSO)) 0.1 ml of DMSO containing 9% triethylamine was added thereto, followed by reaction with moderate stirring at room temperature for 60 minutes. The reaction was terminated by adding 0.3 ml of distilled water containing 1% (v / v) of trifluoroacetic acid (TFA, trifluoracetic acid). At this time, the reaction conditions of exendin-4: taurusordioxycholic acid-NHS participating in the reaction by changing the molar ratio of 1: 1 to 4 and then optimized by analysis using high performance liquid chromatography (HPLC) Was established. HPLC analysis was performed using fluid phase changes of 30 to 100 linear incremental forms based on acetonitrile using acetonitrile and water containing 0.1% (v / v) TFA, respectively.

A schematic diagram of the conjugation reaction of taurusoledioxycholic acid and exendin-4 is shown in FIG. 2, and each product for deriving an optimal reaction ratio for an optimal yield of an exendin-4 derivative conjugated with taurusodioxycholic acid is shown. The relative ratios of these are shown in FIG.

As shown in FIG. 3, the yield of the exendin-4 derivative conjugated with taulorsodioxycholic acid was high when the reaction ratio of taulorsodioxycholic acid and exendin-4 was 1: 2 to 3, and more. In the reaction ratio, exendin- conjugated with tauurosodioxycholic acid other than TUDi-Ex4 (exendin-4 derivatives in which taurousodioxycholic acid is conjugated to lysine residues 12 and 27 of exendin-4) It was confirmed that the yield of 4 derivative is lowered.

Experimental Example 1 Isolation and Analysis of Exendin-4 Conjugate to Taurusordioxycholic Acid

The exendin-4 derivatives conjugated to taurousodioxycholic acid prepared in Example 3 of Example 1 were unreacted exendin-4 and TUM1-Ex4 (27 lysine of exendin-4 using high performance liquid chromatography). Exendin-4 derivatives conjugated with taurousodioxycholic acid to residues), TUM2-Ex4 (exendin-4 derivatives conjugated with taurousodioxycholic acid to lysine residue 12 of exendin-4) and TUDi- Four different materials of Ex4 were separated. Each unreacted exendin-4 and three isomers isolated were identified by mass spectrometry using a MALDI-TOF mass spectrometer to identify unreacted and degree of substitution (one and two).

HPLC chromatogram confirming the purity of the final product using HPLC is shown in Figure 4, the molecular weight measurement results of unreacted exendin-4 and three isomers using a MALDI-TOF mass spectrometer are shown in Table 1 and Figure 5 It was.

As shown in Figure 4, for each unreacted exendin-4 and taurousodioxycholic acid conjugated exendin-4 isolated using HPLC (TUM1-Ex4, TUM2-Ex4 and TUDi-Ex4) Very good purity was obtained, and exendin-4 conjugated with taurusordioxycholic acid having a purity of approximately 98% or more was obtained.

In addition, as shown in Table 1 and Figure 5, each of the three isomers was confirmed that the pure material does not contain impurities, and showed a mass similar to the theoretical molecular weight.

In addition, Lys-C digestion method, an endoproteinase, was used to confirm the substitution of a specific site of exendin-4. Purified each unreacted exendin-4 and three isomers (TUM1-Ex4, TUM2-Ex4 and TUDi-Ex4) were added to 5 µl of triethylamine-hydrochloric acid buffer (50 mmol / L; pH 8.6) at 50 µg / After dissolving at a mL concentration, 5 μl of Lys-C enzyme (10 μg / mL) was added thereto and reacted at 37 ° C. for 1 hour. Through this process, the specific reaction of Lys-C enzyme was confirmed by the lack of specificity due to lysine binding site cleavage and bioconjugation to lysine residues (tauurosodioxycholic acid conjugation). . The results are shown in FIG.

As shown in FIG. 6, unreacted exendin-4 is degraded into three segments by the enzymatic reaction of Lys-C according to the presence of two lysine residues in the amino acid sequence, whereas TUM1-Ex4 and TUM2-Ex4 In the case of, lysine residues were decomposed into two different segments due to changes in the conjugation reaction. In addition, when two lysine residues were conjugated, it was observed that no degradation by Lys-C occurred and one peak showing the original molecular weight was seen. Through this analysis, each substance isolated by HPLC was separated into TUM1-Ex4 conjugated with taurusodioxycholic acid at lysine residue 27, TUM2-Ex4 conjugated with lysine 12, and lysine 12 and 27. It was confirmed that all of the bonded TUDi-Ex4.

Experimental Example 2: Receptor Binding Test of Exendin-4 Containing Taurusordioxycholic Acid>

Figure 7 shows the binding behavior of the GLP-1 receptor on the surface of insulin secreting cells (RINm5F). As shown in FIG. 7, the binding of competitive ¹²⁵ I-exendin-4 decreased as the concentration of the derivative was increased. In addition, in the case of positional isomers, even when conjugated with taurousodioxycholic acid to a lysine residue, binding was stronger than unconjugated exendin-4, indicating that there was no decrease in activity in receptor binding capacity.

Experimental Example 3: Determination of Biological Activity in Animal Model of Exendin-4 Conjugated with Taurusordioxycholic Acid

Intraperitoneal glucose tolerance test (IPGTT, Intraperitoneal glucose tolerance test) in an animal model of exendin-4 conjugated with taulorsodioxycholic acid of the present invention was performed as follows.

200 μl of glucose (100 mg / ml) and drug (exendin-4, TUM1) in 6-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) 10 mgole / kg body of Ex4 and TUDi-Ex4, 200 μl of injection volume, injected 30 minutes prior to glucose administration by oral and subcutaneous administration, respectively, at tails at 0, 15, 30, 60, 90, and 120 minutes. Blood glucose changes were observed in blood samples taken from veins.

The change in oral glucose tolerance behavior following subcutaneous administration of exendin-4 and its derivatives in type 2 diabetic animals is shown in FIG. 8.

As shown in Figure 8, the control group (physiological saline injection) showed a rapid hypoglycemic tendency and very slow hypoglycemic behavior according to the administration of glucose, while the experimental group administered exendin-4, TUM1-Ex4 and TUDi-Ex4 In the blood glucose administration, blood glucose normalization through low blood sugar increase and fast blood sugar drop was confirmed. In particular, blood glucose increase was more effectively suppressed in the experimental group administered TUM1-Ex4 and TUDi-Ex4 than exendin-4, and blood glucose normalized quickly.

Experimental Example 4 Intracellular Viability Test of Exendin-4 Conjugated with Taurusordioxycholic Acid

In order to determine the cell viability of exendin-4 and taurusodioxycholic acid conjugated exendin-4 against endoplasmic reticulum stress, insulin-secreting cell line (RINm5F) was incubated at 37 ° C. for 24 hours, followed by unconjugated exendin-4 and TUM1-Ex4 was injected at a concentration of 0.01-100 nM, and an hour later, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5 was treated with Thapsigargin, a vesicle stress inducer, at a concentration of 0.3 μM. -diphenyl tetrazolium bromide) test.

As shown in FIG. 9, the group treated with Thapsigargin alone showed a significant drop in cell viability, whereas the cell viability of the group treated with exendin-4 and TUM1-Ex4 was improved. Cell viability was shown, but its viability increased with concentration, showing a slight increase than exendin-4 at 10 nM. As a result, the exendin-4 conjugated with taurousodioxycholic acid was able to confirm the protective ability of the intracellular vesicle stress.

Experimental Example 5: Western Blot Test of Exendin-4 Containing Taurusordioxycholic Acid

Exendin-4 conjugated with taulorsodioxycholic acid of the present invention was designed as a type 2 diabetes therapeutic substance with biological function, and the molecule that is increased or decreased in an endoplasmic reticulum stress environment to examine its effect in detail is shown in FIG. As shown in, it was confirmed by Western blot test.

Western blot sample preparation method incubated insulin secreting cells (INS-1) for 48 hours at 37 ℃ after exendin-4, taurusodioxycholic acid (TUDCA) and TUM1-Ex4 500μM, 50nM and Injected at 50 nM, and treated with 0.5 μM Thapsigargin in the same manner and left in a 37 ° C. incubator for 2 hours and 4 hours to obtain intracellular protein with Lysis buffer and 90 ° C. with the sample buffer. After heating, Western blot was performed after SDS-PAGE electrophoresis.

GRP78 is the first molecule to increase in the vesicle stress of pancreatic beta cells, and it is decreased when blocking the vesicle stress fundamentally. In TUDCA, GRP78 was slightly decreased, and TUM1-Ex4 decreased GRP78 more than TUDCA. Exendin-4 is also known to reduce endoplasmic reticulum stress, but did not show a reduction effect of GRP78, the highest increase.

On the other hand, phospho-eIF2α (peIF2α), a molecule that increases by a lower mechanism than GRP78 in endoplasmic reticulum stress environment, inhibits protein translation when phosphorylation increases, thereby affecting function inhibition in beta cells. After 4 hours of Thapsigargin treatment, the same amount of unsaturated eIF2α was present in all groups, but in the Thapsigargin-only group, peIF2α was increased, exendin-4 reduced saturation, and TUM1-Ex4 It can be seen that phosphosylation was reduced more than exendin-4, and in the case of TUDCA, it acts as a molecular chaperon in the endoplasmic reticulum stress environment and has an effect on the ER vesicle stress upper mechanism, but does not affect the vesicle stress reduction. It can be seen that it does not appear.

FIG. 1A is a chemical scheme of a substance modified with taurusordioxycholic acid-COOH to prepare taurusordioxycholic acid in activated form for exendin-4 conjugation.

FIG. 1B is a diagram of the 1H-NMR results showing that the carboxylic acid group of taurusodioxycholic acid was converted to the NHS-ester form.

Figure 2 is a schematic diagram showing the manufacturing process of the exendin-4 derivative conjugated taurousodioxycholic acid of the present invention.

Figure 3 is a view showing the relative ratio of each product for deriving the optimum reaction ratio of the exurodine dioxycholic acid conjugated exendin-4 derivative of the present invention.

4 is a diagram showing an HPLC chromatogram confirming the purity of the final products of the present invention using HPLC.

5 is a diagram showing the molecular weight measurement results of exendin-4 and three isomers (TUM1-Ex4, TUM2-Ex4 and TUDI-Ex4) using a MALDI-TOF mass spectrometer.

FIG. 6 shows the results of MALDI-TOF confirming the position of taurusodioxycholic acid substitution in exendin-4 and three isomers (TUM1-Ex4, TUM2-Ex4 and TUDI-Ex4) by digestion with lysine-C. Is a diagram showing.

7 is a diagram showing the results of GLP-1 receptor binding capacity analysis of exendin-4 and three isomers (TUM1-Ex4, TUM2-Ex4 and TUDI-Ex4) using the insulin secreting cell line (RINm5F).

8 is a diagram showing the change in oral glucose tolerance behavior following subcutaneous administration of exendin-4 and three isomers (TUM1-Ex4, and TUDI-Ex4) in type 2 diabetic animals.

9 is a diagram showing the cell viability according to the concentration of exendin-4 and taurusodioxycholic acid conjugated exendin-4 (TUM1-Ex4) for endoplasmic reticulum stress using insulin secreting cell line (RINm5F).

10 Western blot of taulorsodioxycholic acid, exendin-4 and taurousodioxycholic acid conjugated exendin-4 (TUM1-Ex4) for endoplasmic reticulum stress using insulin secreting cell line (INS-1). (western blot).

Claims (11)

Exendin-4 derivatives conjugated with taulorsodioxycholic acid. The method of claim 1, A derivative characterized in that tauurosodioxycholic acid is conjugated to at least one lysine residue of lysine residues 12 or 27 of exendin-4. 3. The method of claim 2, Derivative, characterized in that taurousodioxycholic acid is conjugated to residue number 27 of exendin-4. The method of claim 1, The exendin-4 is a derivative, characterized in that natural or recombinant exendin-4. (1) activating a carboxylic acid group of taurusodioxycholic acid; (2) reacting the activated taurusordioxycholic acid with exendin-4; And (3) separating the exendin-4 derivative conjugated with taulorsodioxycholic acid from the reactant. The method of claim 5, Reaction molar ratio of exendin-4 and taurusordioxycholic acid in step (2) is in the range of 1 to 4 molar ratios of taurosordioxycholic acid per 1 mole of exendin-4. . A pharmaceutical composition for the prophylaxis or treatment of a disease caused by excessive secretion of insulin, including exendin-4 derivatives conjugated to taurusoledioxycholic acid according to any one of claims 1 to 4. The method of claim 7, wherein The disease is characterized in that diabetes or obesity. The method of claim 8, The diabetes is a composition, characterized in that the type 2 diabetes. 5. Depression of plasma glucose, inhibition of gastric or intestinal motility, inhibition of gastric or intestinal fasting or inhibition of food intake, comprising exendin-4 derivatives conjugated to taulorsodioxycholic acid according to claim 1. Pharmaceutical compositions for the prevention or treatment of diseases caused by. The method of claim 10, The disease is characterized in that the irritable bowel syndrome.

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