KR100746658B1 - Exendin-4 derivative conjugated with hydrophobic bile acid, preparation method thereof, and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to an exendin-4 derivative conjugated with a hydrophobic bile acid, a method for preparing the same, and a pharmaceutical composition comprising the same. A bile acid is selectively conjugated to lysine residues 12 and 27, preferably lysine residues 27.

Description

Exendin-4 derivatives conjugated with hydrophobic bile acids, methods for preparing the same, and pharmaceutical compositions comprising same {Exendin-4 derivative linked hydrophobic bile acid, method for the preparation of pharmaceutical and pharmaceutical composition comprising the same}

1 is a view schematically showing a process for preparing an exendin-4 derivative conjugated with a hydrophobic bile acid of the present invention.

Figure 2 is a diagram showing the change in the degree of reaction according to the reaction ratio of bile acid / exendin-4.

Figure 3 is a view showing the relative ratio of each product for deriving the optimum reaction ratio for the optimum yield of the bile acid conjugated exendin-4 derivatives 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 unreacted exendin-4 and three isomers (LCA-M1, LCA-M2, and LCA-DI) using a MALDI-TOF mass spectrometer.

FIG. 6 shows MALDI-TOF results confirming bile acid substitution sites in unreacted exendin-4 and three isomers (LCA-M1, LCA-M2, and LCA-DI) through digestion with lysine-C. to be.

7 is a view showing the nanoparticle formation behavior according to the concentration change of the LCA-M1 of the present invention in an aqueous solution.

8 is a diagram showing albumin adsorption behavior of LCA-M1 of the present invention.

9 is a diagram showing the amount of albumin binding quantified the albumin adsorption of LCA-M1 of the present invention.

Figure 10 is a diagram showing the results of insulin secretion promoting experiments through pancreatic islet stimulation of exendin-4 derivatives (LCA-M1, LCA-M2, and LCA-DI) conjugated to exendin-4 and lysocolic acid.

11 is a diagram showing the change in oral glucose tolerance behavior according to subcutaneous administration of exendin-4 and LCA-M1 in type 2 diabetic animals.

12 is a diagram showing the area under the blood concentration curve (glucose AUC) according to each experimental animal in order to quantify the change in oral glucose tolerance behavior of FIG.

Figure 13 is a diagram showing the active duration of the type 2 diabetes animal model of exendin-4 (LCA-M1) conjugated to lysoholic acid of the present invention.

FIG. 14 is a diagram showing an area under a curve (glucose AUC) using the result of FIG. 13.

Figure 15 is a diagram showing the food intake over time of the experimental animals in the control group, exendin-4 and LCA-M1 (10 nmole / kg administration group, respectively).

Figure 16 shows the pharmacokinetic behavior of exendin-4.

17 shows the pharmacokinetic behavior of LCA-M1 of the present invention.

Figure 18 is a diagram showing the change in blood glucose of the experimental animals during the experimental period through the administration of LCA-M1 of the present invention.

Figure 19 is a diagram showing the weight change of the experimental animal during the experimental period through the administration of LCA-M1 of the present invention.

20 is a diagram showing the food intake of experimental animals during the experimental period through the administration of LCA-M1 of the present invention.

Figure 21 is a diagram showing the change in oral glucose tolerance of experimental animals before administration of LCA-M1 of the present invention.

Figure 22 is a diagram showing the oral glucose tolerance change of the experimental animals 28 days after the administration of LCA-M1 of the present invention.

FIG. 23 is a diagram showing an area under a curve (glucose AUC) using the result of FIG. 22.

The present invention relates to an exendin-4 derivative conjugated with a hydrophobic bile acid, 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 promoting the production of. 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.). .

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

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

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 resistant to degradation by DPP-IV in mammals and has a half-life of less than 2 minutes to DPP-IV in mammals. Has a longer half-life (Kieffer TJ et al., 1995). In addition, in vivo experiments showed a half-life of 2 to 4 hours, it has been found that sufficient blood concentration can be reached by intraperitoneal administration 2-3 times a day (Fineman MS et al., 2003). In addition, exendin-4 is 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 connection with exendin-4 monotherapy as well as combination therapy with antidiabetic drugs such as sulfonylurea and / or metformin, HbAlc levels have been reported to be lowered to within 1% after 28 days of administration (Egan JM). et al., 2003). Recently, synthetic exendin-4 under the trademark Byetta ™ has been licensed for sale from the US Food and Drug Administration.

On the other hand, various attempts are being made to make sustained drugs by sustained release of protein and peptide drug release and by changing the physicochemical properties of the drug. The most active techniques for introducing sustained release without applying chemical conjugation to the drug include those that introduce the drug into microspheres, hydrogels and various devices and then allow the drug to be released from this medium at a constant rate.

Since chemical modification of drugs changes the intrinsic properties of drugs, especially physicochemical properties, various attempts to prepare sustained or sustained drugs through this process are also made. The most representative examples thereof include chemically conjugating polyethylene glycol to a protein or peptide (Harris JM. Et al. Nature Reviews Drug Discovery 2003).

Another approach is to chemically conjugate fatty acids to protein and peptide drugs. Such fatty acid conjugation is known to impart reversible albumin binding capacity to drugs to confer sustained drug efficacy. It uses the unique properties of albumin, albumin is the protein that occupies the largest part of the blood is known to reversibly bind to a variety of blood substances, a representative substance is a fatty acid. One molecule of albumin is known to have more than eight fatty acid binding sites, and since these binding sites are above the amount of fatty acids in the blood, a certain portion is always present in the blood without fatty acid binding. In addition, albumin has a high molecular weight and circulates in the blood for a long time without loss in the kidneys.

Thus, various attempts have been reported to confer sustained properties of protein and peptide drugs using these properties. Kurtzhals et al. Have developed long-acting insulin by conjugating fatty acids to insulin (Kurtzhals P. et al. Biochem. J., 1995). Similar trials are also underway to develop long-acting formulations by developing new GLP-1 derivatives that incorporate fatty acids into the side branches of GLP-1 analogs (Rolin B. et al., Am. J. Physiol. Endocrinol. Metab). , 2002; Korean Patent No. 10-0539193).

However, the fatty acid as well as the bile acid is known to have an albumin binding capacity, and has been reported for protein drugs that give the lasting characteristics by using these properties. Jonassen et al. Developed persistent insulin derivatives by chemically binding bile acid derivatives to insulin by aggregate formation and albumin binding (Jonassen I. et al., Pharm. Res., 2006).

The various approaches above can be used to obtain exendin compounds that have an extended half-life or enhanced potency compared to natural exendin. On the other hand, if two or more fatty acids and bile acids are covalently bound to exendin, there is a risk of developing serious and adverse properties that are unsuitable for use as therapeutic agents, such as reduced instability and biological activity to molecules.

Thus, the present inventors are studying hydrophobicity at a specific position of exendin-4 while studying the exendin-4 derivative which can exhibit excellent biological stability by increasing the half-life of the drug while showing similar biological activity as natural exendin-4. By selectively conjugating bile acids, mixed forms of bile acids have higher purity, form nanoparticles, exhibit very strong albumin adsorption behavior compared to conjugated exendin-4 derivatives, and exhibit biological stability, biological activity, and diabetes in It was confirmed that having an improved pharmacokinetic profile to increase the glycemic strengthening effect of the drug, and to increase the body remaining time of the drug to reduce the dose or frequency required for the therapeutic effect, and completed the present invention.

The present invention is to provide an exendin-4 derivative conjugated with a hydrophobic bile acid, a preparation method thereof, and a pharmaceutical composition comprising the same.

The present invention provides exendin-4 derivatives conjugated to hydrophobic bile acids.

The hydrophobic bile acid conjugated exendin-4 derivative is characterized in that the bile acid is selectively conjugated to lysine residues 12, 27 or 12 and 27, preferably lysine residues of exendin-4. .

Exendin-4 used in the present invention may be any of natural or recombinant exendin-4. In the case of the exendin-4, there are three sites to which the hydrophobic bile acid can be bound (N-terminal histidine, lysine residues 12 and 27). However, when the three sites are conjugated with the bile acid, there is a problem that the instability and biological activity of the target molecule is reduced to an extent that is unsuitable for use in pharmaceutical use. In the present invention, bile acids selectively conjugate to lysine residues 12, 27 or 12 and 27 of exendin-4, thereby increasing biological stability and biological activity. Thus, the bile acid is preferably conjugated to lysine residues 12, 27 or 12 and 27 of exendin-4, and most preferably conjugated to lysine residue 27 of exendin-4.

The bile acid used in the present invention is not particularly limited, but is preferably cholic acid, deoxycholic acid, chenodeoxycholic acid, lysocolic acid, ursoxic acid, ursodeoxycholic acid, iso-sodeoxycholic Natural or synthetic, including acids, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycodenodeoxycholic acid, dihydrocholic acid, hyocholic acid and aiodeoxycholic acid Steroids, derivatives of colonic acid. More preferably, they are lysoholic acid, deoxycholic acid, collic acid, and colonic acid.

In addition, the present invention

1) reacting the bile acid with N-hydroxysuccinimide (NHS) to activate the carboxylic acid group of the bile acid,

2) reacting bile acid-NHS and exendin-4 activated in step 1), and

3) to provide a method for producing a bile acid conjugated exendin-4 derivative comprising the step of separating the bile acid conjugated exendin-4 from the reaction mixture.

If the bile acid conjugated exendin-4 derivative according to the present invention will be described in detail step by step.

In step 1), bile acid-NHS is prepared by activating the carboxylic acid group of the bile acid with N-hydroxysuccinimide for smooth reaction between the carboxylic acid group of the bile acid and the primary amine in exendin-4. In the case of the bile acid-NHS prepared above, it can be confirmed through ¹ H-NMR analysis that at least 99% of the terminal carboxylic acid groups are converted to NHS-ester form.

In step 2), the reaction molar ratio of bile acid is preferably selected within a molar ratio range of 0.5 to 2, depending on the type of bile acid with respect to 1 mole of exendin-4. For example, when the bile acid is lisocholic acid, the reaction ratio of lysoholic acid / exendin-4 is preferably 1: 1 to 1.5: 1. Selection of the appropriate molar ratio of the reaction may be preferably selected in consideration of the molecular structure and molecular weight of the bile acid to be conjugated, as well as the pH of the reaction solution, the reaction temperature, the reaction time and the like.

In step 3), the separation of the exendin-4 derivatives conjugated with bile acids from the reaction mixture may be performed using high performance liquid chromatography, but is not limited thereto. By performing the above separation step, an exendin-4 derivative obtained by selectively conjugating bile acid to lysine residues 12, 27 or 12 and 27 of exendin-4 can be obtained with a purity of 98% or more.

The present invention also relates to diabetes involving bile acid conjugated exendin-4 derivatives, particularly type 2 diabetes, or diseases caused by oversecretion of insulin, such as obesity, or lowering plasma glucose, such as irritable bowel syndrome, stomach Or it provides a pharmaceutical composition for the prevention or treatment of diseases caused by inhibition of intestinal movement, inhibition of stomach or intestinal fasting or inhibition of food intake.

Exendin-4 derivatives conjugated with bile acids of the present invention may be prepared by selectively conjugating bile acids to lysine residues 12, 27, or 12 and 27, preferably lysine residues, of exendin-4. Forms, exhibits very strong albumin adsorption behavior, exhibits biological activity equivalent to or similar to natural exendin-4, and increases the half-life of the drug, resulting in very good biological stability. In addition, by selectively limiting the binding sites and the number of binding of bile acids, the side effects caused by the diversity of these factors are minimized to increase the intestinal absorption rate using the intestinal active transport system. Thus, the exendin-4 derivatives conjugated to the bile acids of the present invention may be used for the prevention of diseases that may be caused by insulin oversecretion, or lowering plasma glucose, inhibiting stomach or intestinal motility, inhibiting stomach or intestinal fasting, or inhibiting food intake or It can be usefully used for the treatment, especially for diabetes mellitus, preferably type 2 diabetes, obesity and irritable bowel syndrome.

Pharmaceutical compositions comprising bislic acid conjugated exendin-4 derivatives of the present invention may be formulated in oral or parenteral dosage forms for clinical administration, but are not limited thereto.

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 the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the active ingredient. Mix is prepared. 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 the exendin-4 derivative conjugated with bile acids 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.

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  : Lysocolic acid Spliced Exendin Manufacture of -4

1. Activated Lysolic acid  Produce

Bile acid-NHS was prepared by activating the carboxylic acid group of the bile acid with N-hydroxysuccinimide (NHS) for a smooth reaction between the carboxylic acid group of the bile acid and the primary amine in exendin-4.

3 g of N-hydroxysuccinimide (NHS) (2.1 mole excess for bile acid) and 5.3 g of 3-dicyclohexyl carbodimide (DCC) (2 mole excess for bile acid) ) Was dissolved in 100 ml of anhydrous tetrahydrofuran (THF). The solution was allowed to react with sufficient stirring for 24 hours under a stream of nitrogen. The dicyclohexylurea (DCU) produced after the reaction was removed using a paper filter, the reaction solution was concentrated under reduced pressure, and then precipitated in excess n-hexane (more than 10 vol. Of the concentrate). Phosphorous lysicolic acid-NHS and other lysicolic acid-NHS were obtained. In order to improve the purity of the obtained product and to remove impurities, the product was further dried in benzene after vacuum drying and then precipitated in n-hexane. The final product was dried in vacuo for 48 hours to obtain a powder, the reaction and purity was confirmed using ¹ H NMR.

In the case of the prepared lysolic acid-NHS showed a purity relative to the weight of about 85 ~ 90%, the impurities contained in the final product was confirmed that the majority of the unreacted NHS. Because unreacted NHS did not affect the reaction of activated lysocholic acid-NHS with exendin-4, the final lysocholic acid-NHS was obtained without further purification. In the case of lysoholic acid-NHS, more than 99% of the terminal carboxylic acid group was converted into NHS-ester form was confirmed by ¹ H-NMR analysis.

As the bile acid, various bile acids such as deoxycholic acid, collic acid and colonic acid can be used in addition to the lysicolic acid used above.

2. Lysocolic acid Spliced Exendin Manufacture of -4

0.1 ml of lysocholic acid-NHS (0.4446-1.778 mg / ml in dimethyl sulfoxide (DMSO)) prepared in 1 was added to 0.1 ml of exendin-4 (8 mg / ml in DMSO), and triethylamine 0.1 ml of DMSO containing 3% of (triethylamine) was added thereto, followed by reaction with moderate stirring at room temperature for 45 minutes. 0.3 ml of distilled water containing 1% (v / v) of trifluoroacetic acid (TFA) was added to the reaction solution to terminate the reaction. At this time, the molar ratio of exendin-4: lysicolic acid-NHS participating in the reaction was changed to 1: 0.5 to 2, followed by analysis using high performance liquid chromatography (HPLC) to establish optimized reaction conditions. HPLC analysis used a 30% to 100% linear increase in fluidized bed change with acetonitrile using water and acetonitrile containing 0.1% (v / v) TFA, respectively.

The schematic diagram of the bioconjugation reaction of lysolic acid and exendin-4 is shown in FIG. 1, and the change in reaction degree according to the reaction ratio of bile acid / exendin-4 is shown in FIG. 2, and the exendin-4 conjugated with bile acid is shown. The relative ratios of each product for deriving the optimum reaction ratio for the optimum yield of the derivatives are shown in Table 1 and FIG. 3.

As shown in FIG. 2, various types of reactants were produced depending on the ratio of exendin-4 and lysoholic acid. Four representative isolates are: 1) unreacted exendin-4 (eluted at 5.5 minutes), 2) exendin-4 (eluted at 12.93 minutes) with lysocolic acid conjugated to lysine residue No. 27, hereinafter referred to as' LCA-M1 3) exendin-4 conjugated to lysine residues at lysine residues 12 (elution at 14.25 min, hereinafter referred to as 'LCA-M2'), and 4) lysophilic at lysine residues 12 and 27; The acid was conjugated exendin-4 (eluted at 22.75 minutes, hereinafter referred to as 'LCA-DI').

In addition, as shown in Table 1, in the case of the exendin-4 derivative (LCA-M1) in which the lysicolic acid conjugated to the lysine residue No. 27 of the present invention, different results depending on the initial dose of the exendin-4 and the lysocolic acid Indicated. Therefore, in order to calculate the maximization condition for the optimum yield of LCA-M1, an optimization graph using the reaction yield ratio and initial input ratio of LCA-M1 / Native and LCA-M1 / LCA-DI was derived. As shown in FIG. 3, the ratio of LCA-M1 / Native showed a sharp increase as the response dose ratio (lysoholic acid / exendin-4) increased, while the ratio of LCA-M1 / LCA-DI was administered. As the ratio increased, it gradually decreased. Using this relative ratio, it can be seen that the initial reaction ratio of lysoholic acid / exendin-4 can be prepared in the maximum amount of LCA-M1 under the reaction conditions of 1: 1 to 1.5: 1.

Experimental Example  One  : Lysocolic acid Spliced Exendin -4 separation and analysis

Exendin-4 (LCA-Exendin-4) conjugated to lysocolic acid prepared in Example 2 of Example 1 using unreacted exendin-4, LCA-M1, LCA-M2 and LCA using high performance liquid chromatography. -Divided into four different materials of -DI. 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 the MALDI-TOF mass spectrometer are shown in Table 2 and Figure 5 It was.

As shown in FIG. 4, each of the unreacted and lysolytic acid conjugated exendin-4 (LCA-M1, LCA-M2, and LCA-DI) separated using HPLC showed very good purity. Exendin-4 conjugated with lysocolic acid having a purity of approximately 98% or higher was obtained.

In addition, as shown in Table 2 and Figure 5, each of the three isomers could be 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. The experiment was carried out with each purified unreacted exendin-4 and three isomers (LCA-M1, LCA-M2, and LCA-DI) in 5 μl triethylamine-hydrochloric acid buffer (50 mmol / L; pH 8.6). After dissolving at a concentration of 50 μg / ml, 5 μl of Lys-C enzyme (10 μg / ml) was added and reacted at 37 ° C. for 1 hour. The conjugation of specific site lysine residues was confirmed by the specific reaction of the lysine binding site cleavage and the bioconjugation to the lysine residue (bile acid conjugation), which is a specific reaction of the Lys-C enzyme.

The results are shown in FIG.

As shown in FIG. 6, in the case of unreacted exendin-4, LCA-M1 and LCA-M2 are degraded into three segments by the enzymatic reaction of Lys-C depending on the presence of two lysine residues in the amino acid sequence. In the case of, lysine residues were decomposed into two different segments due to the bioconjugation reaction. In addition, when both lysine residues reacted, it was observed that one peak showing the original molecular weight was observed without degradation by Lys-C. Through this analysis, each substance isolated by HPLC was LCA-M1 conjugated with lysonic acid at lysine residue 27, LCA-M2 conjugated with lysine 12, and LCA conjugated with lysine 12 and 27. -DI could be confirmed.

Experimental Example  2  : Lysocolic acid Spliced Exendin -4( LCA - M1 Physicochemical Properties of analysis

Bile acids, especially terminal carboxylic acid groups, exhibit mostly hydrophobic character when they participate in removal and reaction. Therefore, in the case of exendin-4 conjugated with bile acids, micelles or nanoparticles are formed under aqueous solution through the hydrophobicity of bile acids participating in the conjugation and the interaction between these hydrophobic bile acids. The formation of these micelles and nanoparticles also tends to depend on the concentration under aqueous solution. In order to observe the formation of such nanoparticles, the size of the nanoparticles of LCA-M1 at different concentrations was observed using dynamic light scattering.

The nanoparticle formation behavior according to the concentration change of LCA-M1 in aqueous solution is shown in FIG. 7.

As shown in FIG. 7, nanoparticle formation behavior was not observed under low concentrations (0-30 μg / ml) of lysoclinic acid conjugated exendin-4 (LCA-M1), whereas 50 μg / ml or more. LCA-M1 at the concentration was confirmed that the particles of about 200nm or more formed.

Experimental Example  3  : Lysocolic acid Spliced Exendin -4( LCA - M1 Albumin Adsorption Behavior of Research

In order to confirm the albumin adsorption behavior of the lysicolic acid conjugated exendin-4 (LCA-M1), the interaction between albumin-coupled column packing and LCA-M1 was observed.

NHS-activated Sepharose resin (4ml) and 30mg HSA (Human Serum Albumin, human plasma albumin) were dissolved in 10mM pH 7.4 phosphate buffer solution and reacted at room temperature for 2 hours or more. Conjugation. In addition, a control without NHS was prepared by reacting in the same manner as above without adding HSA. The resins thus terminated were thoroughly washed with 10 mM pH 7.4 phosphate buffer solution. 80 μl of the resin thus prepared was put in a tube, and 155 μl of 10 mM pH 7.4 phosphate buffer solution was added thereto. 50 μl (100 μg / ml) of exendin-4 (LCA-MA) conjugated with unreacted exendin-4 and lysocolic acid was added thereto, and reacted with shaking at room temperature for 2 hours. After the reaction was terminated, the supernatant was collected by centrifugation at 10,000 g for 15 minutes, and the concentration of the sample remaining without adsorption to albumin was measured using micro BCA protein assay.

The albumin adsorption behavior of LCA-M1 of the present invention is shown in Figure 8, the albumin binding amount quantified albumin adsorption is shown in Figure 9.

As shown in Figure 8, LCA-M1 of the present invention shows a very strong albumin adsorption behavior shows a large concentration difference between the albumin binding resin and the general resin. On the other hand, exendin-4 showed low albumin adsorption behavior by itself. This indicates that lysolic acid conjugated to exendin-4 plays a very important role in albumin adsorption.

In addition, as shown in Figure 9, the LCA-M1 of the present invention showed 83% albumin adsorption, exendin-4 showed 30% albumin adsorption behavior. Therefore, LCA-M1 prepared according to the present invention is believed to exhibit a longer biological half-life and slower disappearance through binding to albumin in the human body after administration to the human body.

Experimental Example  4  : Lysocolic acid Spliced Exendin -4 Pancreatic diagram  Insulin secretion promoting activity measurement

In order to confirm the insulin secretion promoting activity of the lysicolic acid conjugated exendin-4 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. Insulin secretion stimulation experiments were performed using exendin-4 derivatives (LCA-M1, LCA-M2, and LCA-DI) conjugated with exendin-4 and lysolic acid to isolated pancreatic islets. The experiment was carried out by dispersing 20 pancreatic sites with an average size of 150 μm in 1 ml of Craps-Ringer Buffer (KRB, containing 16.7 mM glucose), and then conjugating each exendin-4 and lysocolic acid. Exendin-4 derivatives were administered at concentrations of 0.1, 1, 10, 100 nM and insulin secretion was measured using an ELISA kit for 2 hours.

Results of insulin secretion promoting experiments through pancreatic islet stimulation of exendin-4 and lysoholic acid conjugated exendin-4 derivatives (LCA-M1, LCA-M2, and LCA-DI) are shown in FIG. 10.

As shown in FIG. 10, it was found that exendin-4 derivatives (LCA-M1, LCA-M2, and LCA-DI) conjugated with exendin-4 and lysocolic acid enhance insulin secretion of pancreatic islets in a concentration-dependent manner. Could. At 1 nM, which is relatively low concentration, there was no particular insulin secretion effect, but as the concentration increased to 10 nM or more, it showed a function of promoting rapid improvement of insulin secretion. Different locations showed different trends. The most noticeable difference in efficacy was observed at a concentration of 10 nM. LCA-M1 of the present invention showed a slight decrease in activity compared to natural exendin-4, but did not show a significant difference statistically (p value by Student t-test = 0.14), and LCA-M2 statistically There was a significant decrease in activity (p = 0.01). In addition, LCA-DI showed relatively low activity. Therefore, it could be confirmed that LCA-M1 of the present invention is the best bioconjugate.

Experimental Example  5  Of the present invention Lysocolic acid Spliced Exendin -4( LCA - M1 Of biological activity in animal models

In order to measure oral glucose tolerance (OGTT) in an animal model of lysicolic acid conjugated exendin-4 (LCA-M1), the following experiment was performed.

200 μl of glucose (200 mg / ml) and drug (exendin-4 and LCA) 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) -M1, 1 nmole / kg body, dose volume 100ml, injection 30 minutes prior to glucose administration) by oral and subcutaneous administration, respectively, at 0, 15, 30, 45, 60, 90, 120, 180 minutes. Blood glucose changes were observed in blood samples taken from the tail vein.

The change in oral glucose tolerance behavior following subcutaneous administration of exendin-4 and LCA-M1 in type 2 diabetic animals is shown in FIG. 11, and in order to quantify the change in oral glucose tolerance behavior, The area under the concentration curve (glucose AUC) is shown in FIG. 12.

As shown in FIG. 11, the control group (physiological saline injection) showed a rapid hypoglycemic trend and a very slow hypoglycemic behavior according to the administration of glucose, whereas the experimental group to which exendin-4 and LCA-M1 were administered glucose was administered. In addition, hypoglycemia and rapid hypoglycemic hypoglycemia were found to be normalized.

In addition, as shown in Figure 12, the control group showed a glucose AUC value of about 41000 ng.min / ㎖, whereas for exendin-4 and LCA-M1 glucose 1UC and 12000 ng.min / ㎖ respectively Seemed. In addition, the exendin-4 and LCA-M1 did not show statistical difference as the p-value of 0.47 by Student t-test.

Experimental Example  6  Of the present invention Lysocolic acid Spliced Exendin -4( LCA - M1 Activity duration in animal models

In order to measure the activation duration in the animal model of the lysicolic acid conjugated exendin-4 (LCA-M1), the following experiment was performed.

In 6-week-old male db / db mice (C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology), 0.1 ml of exendin-4 (10 nmole / kg) and LCA-M1 (5, 10, 15 nmole / Kg) was administered subcutaneously to mice, and blood glucose was measured using blood samples in the tail vein at 0.5, 1, 2, 4, 6, 8, 10, 12, 24 hours. In addition, in order to determine the efficacy under normal circumstances, water and food can be freely ingested and food intake was measured after 12 and 24 hours.

In the type 2 diabetic animal model of the lysicolic acid conjugated exendin-4 (LCA-M1), the activity duration results are shown in FIG. 13 and the area under the curve (glucose AUC) using the results of FIG. 13. Figure 14 is shown in Figure 14, the food intake over time in the control group, exendin-4 and LCA-M1 (10 nmole / kg administration group, respectively) is shown in Figure 15.

As shown in FIG. 13, the blood sugar of the control group (physiological saline administration) in the diabetic animal model was maintained at a level of 250mg / ㎖ or more. On the other hand, in the exendin-4 group (10 nmole / kg), there was a marked decrease in blood glucose after 30 minutes after administration, and this reduction was maintained until 4 hours after the administration, after which a gradual increase in blood glucose was observed. After time, hyperglycemic levels (200 mg / ml blood glucose) were reached. In the LCA-M1 administration group of the present invention showed a similar hypoglycemic tendency to exendin-4, but showed a longer duration of normal blood glucose retention time, it was proportional to the dose in the case of normal blood glucose maintenance time. When the LCA-M1 of the present invention was administered in amounts of 5 and 10 nmole / kg, the normal blood glucose retention time was measured at about 8 hours and 12 hours, respectively. It was found that the value was maintained at 108 mg / ml.

In addition, as shown in FIG. 14, the experimental animals administered with exendin-4 and LCA-M1 showed a marked decrease in glucose AUC values compared to the control group when analyzed using Student t-test. The LCA-M1 administration group showed very low glucose AUC values compared to exendin-4, and the 10 and 15 nmole / kg administration groups showed statistically significantly lower glucose AUC values.

In addition, as shown in Figure 15, 12 hours of food intake showed a tendency to decrease significantly in the exendin-4 and LCA-M1 administration group, there was no statistical difference between exendin-4 and LCA-M1. On the other hand, the LCA-M1 administration group showed significantly lower food intake for 24 hours.

Through these experiments, it was found that exendin-4 (LCA-M1) conjugated with lysicolic acid of the present invention exhibited the property of sustained drug to maintain normal blood glucose for a longer period of time, and also had a long-term effect on food intake. I could see it.

Experimental Example  7  Of the present invention Lysocolic acid Spliced Exendin -4( LCA - M1 Pharmacokinetic behavior in animal models

In order to investigate the drug in vivo in the animal model of the lysicolic acid conjugated exendin-4 (LCA-M1), the following experiment was performed.

To facilitate blood sampling from 6-week-old Sprague-Dawley rats, a PE-10 tube is inserted about 2.5 cm into the animal's right jugular vein, and then sutured. Connected with blood vessels. After about 24 hours of stabilization and recovery period, exendin-4 and LCA-M1 were injected subcutaneously at doses of 4.18 μg / rat and 50 μg / rat, respectively, and then blood was periodically applied for 5 minutes to 24 hours. Was collected (0.2 ml, for each time point). Blood samples were taken only from the plasma using a centrifuge, and then the in vivo kinetics of exendin-4 and LCA-M1 in plasma were measured using an assay kit (Exendin-4 ELISA kit).

The pharmacokinetic behavior of exendin-4 is shown in FIG. 16 and the pharmacokinetic behavior of LCA-M1 is shown in FIG. 17.

As shown in FIG. 16, in the case of exendin-4, the blood peak was reached about 30 minutes after drug administration, followed by a rapid decrease in blood levels after 1 hour, and most of the exendin-4 in the blood after 3 hours. I could see that it disappeared. The half-life of exendin-4 derived from these results was 1.6 ± 0.4 hours. On the other hand, in the case of LCA-M1, the blood peak was reached after about 6-8 hours with continuous increase in blood concentration after LCA-M1 administration, and then gradually decreased. The calculated half-life of LCA-M1 was 9.7 ± 1.4 hours, which is a long half-life about 6 times greater than exendin-4.

Experimental Example  8  Of the present invention Lysocolic acid Spliced Exendin -4( LCA - M1 Long-term administration of)

In order to investigate the therapeutic effect of type 2 diabetes through long-term administration in the animal model of lysicolic acid conjugated exendin-4 (LCA-M1), the following experiment was performed.

LCA-M1 was administered once daily at a dose of 15 nmole / kg for db / db mice, a type 2 diabetic animal model, and blood glucose, food intake, and body weight were used. In addition, the blood glucose removal ability of the test animals was examined by measuring oral glucose tolerance before and after 4 weeks of administration. Blood glucose measurements were performed immediately prior to drug administration to rule out the initial effects immediately after new drug administration.

The blood glucose change of the experimental animals during the experimental period through the administration of LCA-M1 of the present invention is shown in Figure 18, the weight change of the experimental animals is shown in Figure 19, the food intake of the experimental animals is shown in Figure 20. In addition, the oral glucose tolerance change of the experimental animals before administration of LCA-M1 of the present invention is shown in Figure 21, the oral glucose tolerance change of the experimental animals 28 days after the administration of LCA-M1 of the present invention is shown in Figure 22, A schematic of the area under the curve (glucose AUC) using the results of FIG. 22 is shown in FIG. 23.

As shown in FIG. 18, the control group administered only saline showed a high blood glucose of 250 mg / ml or higher after 4 weeks through the gradual increase in the initial blood glucose mean of about 170 mg / ml. In contrast, the experimental group administered LCA-M1 daily maintained normal blood glucose levels of 100-150 mg / ml for the entire experimental period. In addition, after 28 days of drug administration, with the withdrawal of drug administration, hyperglycemia was observed, which was similar to that of the control animals. Therefore, it can be seen that the daily administration of LCA-M1 of the present invention shows effective efficacy for normalizing blood glucose of diabetic animals.

In addition, as shown in FIG. 19, the control experimental animals showed a final obesity state of 40g or more with continuous weight gain, but the LCA-M1 administration group of the present invention showed a tendency to alleviate weight gain according to drug administration. Could.

In addition, as shown in Figure 20, the LCA-M1 administration group of the present invention showed a sharp decrease in food intake for the first 1-2 weeks, after 2 weeks it was found that does not show a significant difference compared to the control group.

In addition, as shown in Figure 21 and 22, using the experimental animal showing the same oral glucose tolerance initially, oral glucose tolerance before and after the administration of LCA-M1 of the present invention for 4 weeks was confirmed to show a significant difference. .

In addition, as shown in Figure 23, the administration of LCA-M1 of the present invention for 4 weeks was found to have a significant effect on the increase in oral glucose tolerance of the diabetic experimental animals.

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

Formulation example  : Pharmaceutical Formulations

One. Powder  Produce

2 g of exendin-4 derivatives conjugated with bile acids

1g lactose

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

2. Preparation of Tablets

100 mg of exendin-4 derivatives conjugated with bile acids

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 exendin-4 derivatives conjugated with bile acids

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.

4. Preparation of Injection Solution

10 μg / ml of exendin-4 derivative conjugated with bile acids

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the exendin-4 derivative conjugated with the bile acid of the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP, and use the volume of sodium chloride BP for injection. Was adjusted and mixed well. The solution was filled into a clear 5 ml Type I ampoule of glass and dissolved under glass by dissolving the glass, and autoclaved at 120 ° C. for at least 15 minutes to prepare an injection solution.

Exendin-4 derivatives conjugated with bile acids of the present invention may be prepared by selectively conjugating bile acids to lysine residues 12, 27, or 12 and 27, preferably lysine residues, of exendin-4. Forms, exhibits very strong albumin adsorption behavior, exhibits biological activity equivalent to or similar to natural exendin-4, and increases the half-life of the drug, resulting in very good biological stability. In addition, by selectively limiting the binding sites and the number of binding of bile acids, the side effects caused by the diversity of these factors are minimized to increase the intestinal absorption rate using the intestinal active transport system. Thus, the exendin-4 derivatives conjugated to the bile acids of the present invention may be used for the prevention of diseases that may be caused by insulin oversecretion, or lowering plasma glucose, inhibiting stomach or intestinal motility, inhibiting stomach or intestinal fasting, or inhibiting food intake or It can be usefully used for the treatment, especially for diabetes mellitus, preferably type 2 diabetes, obesity and irritable bowel syndrome.

Claims (12)

Exendin-4 derivatives conjugated with hydrophobic bile acids. The exendin-4 derivative according to claim 1, wherein the exendin-4 derivative is selectively conjugated with bile acid to lysine residues 12, 27 or 12 and 27 of exendin-4. The exendin-4 derivative according to claim 2, wherein the exendin-4 derivative is selectively conjugated with a bile acid to a lysine residue of exendin-4. The exendin-4 derivative according to claim 1, wherein the exendin-4 is a natural or recombinant exendin-4. The method according to claim 1, wherein the bile acid is cholic acid, deoxycholic acid, chenodeoxycholic acid, lysocholic acid, ursocholic acid, ursodeoxycholic acid, iso-sodeoxycholic acid, lagodeoxycholic acid, Natural or synthetic steroids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycenodeoxycholic acid, dihydrocholic acid, thiocholic acid and aiodeoxycholic acid, and derivatives of cholic acid Exendin-4 derivatives, characterized in that one selected from the group consisting of. 1) reacting the bile acid with N-hydroxysuccinimide (NHS) to activate the carboxylic acid group of the bile acid, 2) reacting bile acid-NHS and exendin-4 activated in step 1), and 3) A method for producing a bile acid conjugated exendin-4 derivative of claim 1 comprising the step of separating the bile acid conjugated exendin-4 from the reaction mixture. The method according to claim 6, wherein the reaction molar ratio of the bile acid in step 2) is a mole ratio of 0.5 to 2 with respect to 1 mole of exendin-4, the method for producing a bile acid conjugated exendin-4 derivative of claim 1. A pharmaceutical composition for preventing or treating a disease caused by excessive secretion of insulin comprising an exendin-4 derivative conjugated with a bile acid of any one of claims 1 to 5. The pharmaceutical composition for preventing or treating a disease caused by the excessive secretion of insulin, characterized in that the disease is diabetes or obesity. The pharmaceutical composition for preventing or treating a disease caused by the excessive secretion of insulin, characterized in that the diabetes is type 2 diabetes. The prevention or treatment of diseases caused by plasma glucose lowering, inhibition of gastric or intestinal motility, inhibition of gastric or intestinal fasting or inhibition of food intake, comprising exendin-4 derivatives conjugated to the bile acids of claim 1. Pharmaceutical composition for. The pharmaceutical composition of claim 11, wherein the disease is irritable bowel syndrome.

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