KR100890989B1 - Mono modified exendin with polyethylene glycol or its derivatives and uses thereof - Google Patents

Abstract

The present invention relates to a single modified exendin with polyethylene glycol or a derivative thereof, a method for preparing the same, and a use thereof. More specifically, the present invention relates to polyethylene glycol at the lysine residue of exendin 27 to provide biological activity similar to that of natural exendin. Inhibition of insulin hypersecretion, or lowering plasma glucose, suppressing gastric or intestinal motility, by producing a single modified exendin, which increases the body's half-life of the drug and minimizes the side effects of the polyethylene glycol binding sites and the number of bonds. It may be useful for the treatment of diseases that may be caused by suppressing stomach or intestinal fasting, or inhibiting food intake, and particularly useful in diabetes, obesity and irritable bowel syndrome.

Description

Monomodified exendin with polyethylene glycol or its derivatives and uses thereof

The present invention relates to a single modified exendin with polyethylene glycol or a derivative thereof, a method for preparing the same, and a use thereof.

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 motility, 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, such as 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., 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.).

In addition, exendin, another group of polypeptides that lower blood sugar levels, was first presented by John Eng (US Pat. No. 5,542,286). Exendin-4 has the following sequence structure and GLP-1 (7). -36) shows some sequence similarity (53%) with NH ₂ (Goke et al., 1993).

His1 -Gly -Glu-Gly-The-Phe-The-Ser-Asp-Leu-Ser- Lys12 -Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu- Lys27 -Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

On the other hand, exendin is found in the venom or bead venom, and exendin-3 is present in the venom of the Mexican beaded lizard Heloderma horridum, and exendin-4 is the american venom lizard It is present in the poison of Heloderma suspectum. Exendin-4 differs from exendin-3 only at positions 2 and 3. It has a longer half-life than GLP-1 in mammals that is resistant to degradation by DPP-IV and has a half-life of less than 2 minutes for DPP-IV (Kieffer TJ et al., 1995). In vivo experiments have shown that it has a half-life of 2 to 4 hours and can reach sufficient blood levels 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. 6858576, 6956026, and 6872700). In relation to exendin-4 monotherapy as well as combination therapy with antidiabetic agents such as sulfonylurea or metformin, the amount of hemoglobin bound to glucose in the blood within 1% after 28 days of administration. Has been reported to lower levels of glycated hemoglobin (HbA1C) (Egan JM et al., 2003). Recently, Synthetic Exendin-4 under the trademark Byetta ™ was licensed for sale by the US Food and Drug Administration.

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 while maintaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration, protecting the protein from cells and antibodies that recognize external antigens, and by protease Can also reduce the decomposition. 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, toxicity is known to be quite low. When the molecular weight range of the polyethylene glycol is 1,000 ~ 6,000 is distributed throughout the body and metabolized through the kidney, especially polyethylene glycol having a molecular weight of 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. 4179337 discloses that the use of proteins and enzymes combined with polyethylene glycol as therapeutic agents provides the advantages of reducing the antigenicity, increasing water solubility, and increasing the retention period of the body, which are advantages of polyethylene glycol. Doing. Since this patent, attempts have been made to combine the bioactive proteins with polyethylene glycol to overcome their disadvantages. For example, Veronese et al. Combined ribonuclease and superoxide dismutase with PEG (Veronese et al., 1985), US Patent No. 4766106 and US Patent No. No. 4917888 discloses binding of polymers containing PEG to proteins to increase the water solubility of the protein. U.S. Patent No. 44902502 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-glycoglycolated peptides. Is described. Incorporation of one or two Cysteine (Cys) residues into specific amino acid sites of the peptide provides a thiol group, which is covalently bonded to polyethylene glycol or its derivatives (especially polyethylene glycol-maleimide) to a GLP that is polyethylene glycolated. The -1 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. Polyethyleneglycol or derivatives thereof in International 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 as 20,000-40,000 daltons. Polyethyleneglycolated GLP-1 compounds prepared by International Patent Publication No. 04/093823 have increased half-lives and reduced removal rates 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.

US Pat. No. 6,264,264 describes another form of modified exendin and exendin agonist. Exendin-4 has three sites capable of binding to polyethylene glycol (N-terminal histidine, lysine groups 12 and 27). The molecular weight of polyethylene glycol used in US Pat. No. 6,264,264 ranges from 5,000 to 12,000 Daltons, and is capable of covalent bonding to two lysine epsilon-amino groups present in exendin-4. Unlike GLP-1, which is primarily hydrolyzed, it is said to provide the benefit of being removed from the plasma by almost all renal filtration.

The various approaches above have yielded exendin compounds with extended half-lives or increased potency compared to natural exendin, but when two or more polyethyleneglycol molecules are covalently bound to a biologically active small peptide such as GLP-1 or exendin There is a risk of developing serious and adverse properties such as instability to the molecule and a decrease in biological activity that are unsuitable for use as a therapeutic agent.

Accordingly, in the present invention, by providing a single modified exyndin-4 modified with polyethylene glycol only at a specific position of the exendin, the purity of the exendin in the body is higher than that of the mixed polyethyleneglycol-exendin in various forms. To find out the exendin monomodified with polyethylene glycol having an improved pharmacokinetic profile and pharmacological properties to reduce the dose or frequency required for the therapeutic effect of the natural exendin and the preparation method thereof Was completed.

It is an object of the present invention to provide a single modified exendin with polyethylene glycol or a derivative thereof.

Another object of the present invention to provide a method for producing a single modified exendin with the polyethylene glycol or a derivative thereof.

Another object of the present invention to provide a use of a single modified exendin with the polyethylene glycol or derivatives thereof.

In order to achieve the above object, an object of the present invention provides a single modified exendin with polyethylene glycol or a derivative thereof.

In addition, another object of the present invention to provide a method for producing a single modified exendin with the polyethylene glycol or a derivative thereof.

Still another object of the present invention is to provide the use of a single modified exendin with the polyethylene glycol or a derivative thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides a single modified exendin or a pharmaceutically acceptable salt thereof with polyethylene glycol or a derivative thereof.

The exendin according to one embodiment of the present invention may be preferably modified with any exendin among natural or recombinant exendin. In this case, however, the exendin is preferably exendin-4.

In addition, the polyethylene glycol or a derivative thereof according to an embodiment of the present invention is preferably modified by a single position at position 27 of exendin-4. In the case of the exendin-4, there are three sites to which the polyethylene glycol or a derivative thereof may be bound (N-terminal histidine, 12 and 27 lysine groups). However, if two or more sites are modified with the polyethyleneglycol or derivatives thereof, the instability and biological activity on the target molecule are reduced to an extent that is unsuitable for use in pharmaceutical applications.

The polyethyleneglycol or derivatives thereof according to an embodiment of the present invention may be a single modification to exendin, both linear and branched. In this case, the branched polyethylene glycol may include a multi-branched branch composed of two or more branches, preferably polyethylene glycol consisting of three branches.

In addition, the molecular weight of the polyethylene glycol or derivatives thereof according to the embodiment of the present invention preferably has a molecular weight of 2 to 60 kDa, more preferably 20 to 45 kDa. If the molecular weight is less than the above range, the increase in half-life of the modified exendin does not increase significantly compared to natural exendin. On the other hand, in the case of exceeding the above range, as described above, instability or biological activity on the target molecule may be reduced.

Examples of the polyethylene glycol derivatives include methoxy polyethylene glycol succinimidyl propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, and multiple kinds of polyethylene glycols. Any one or more may be selected from among others, but is not limited thereto. Preferably, methoxy polyethylene glycol succinimidyl propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, bi-branched methoxy polyethylene glycol N-hydroxysuccinimide or It is a triple branched polyethylene glycol derivative represented by the formula (1).

(In the above formula

n has an integer value of 1 to 1,000, m has an integer value of 10 to 1,000, and the functional group capable of chemically reacting with proteins and peptides including exendin is N-hydroxysuccinimide, and Z is exyndine and polyethylene glycol. A (CH ₂ ) _S or (CH ₂ ) _S NHCO (CH ₂ ) _S group that acts as a linker between _S , where S represents an integer value from 1 to 6.

In addition, the present invention comprises the steps of reacting by adding polyethylene glycol or derivatives thereof, exendin and a reducing agent to a solvent (step 1); Storing the reaction mixture of step 1 under light at a constant temperature for a predetermined time (step 2); After completion of the reaction of step 2, the step of removing unreacted reactants (step 3) and separating and purifying a single modified exendin with polyethylene glycol or a derivative thereof from the product from which the unreacted reactants of step 3 have been removed It provides a method for producing a single modified exendin from the polyethylene glycol or a derivative thereof comprising the step (step 4).

In step 1 according to an embodiment of the present invention, the reaction molar ratio of polyethylene glycol or derivatives thereof to exendin is preferably selected within a range of 1: 1 to 4 depending on the type of polyethylene glycol or derivatives thereof. . For example, methoxy polyethylene glycol propionaldehyde is preferably used in a reaction molar ratio of 1 to 2, and methoxy polyethylene glycol succinimidyl propionate is preferably used in a reaction molar ratio of 1 to 4. In addition, the solvent may be used a phosphate buffer or an organic solvent, the organic solvent is preferably DMSO. The reaction molar ratio may be preferably selected in consideration of the molecular structure and molecular weight of the polyethylene glycol or a derivative thereof modified, as well as the pH of the reaction solution, the reaction temperature, the reaction time, and the like.

In addition, a reducing agent may be additionally used in the reactant in step 1, and a reducing agent such as, for example, NaCNBH ₃ or triethylamine may be used as a reducing agent, but is not limited thereto.

Furthermore, the buffer solution is not particularly limited and may be appropriately selected and used according to the reaction conditions for the polyethylene glycol used when modified as a buffer solution commonly used in the art.

Storage temperature and time of step 2 according to an embodiment of the present invention, as described for the reaction molar ratio, it is preferable that it is appropriately adjusted according to the type of modified polyethylene glycol or derivatives thereof. It may be left at 4 ° C. for 2 hours, or may be left at room temperature for a shorter time. This relates to the degree of reactivity of the modified polyethylene glycol and its derivatives. The modification reaction is carried out while standing, and after a reasonable time, it can be stopped using a glycine solution or the like.

Removal of the unreacted materials of step 3 according to one embodiment according to the present invention can be removed by methods commonly 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).

Further, the separation purification of step 4 may be performed using size exclusion chromatography, reverse phase high performance liquid chromatography, but is not limited thereto.

In conclusion, by performing the above separation and purification step, it is possible to obtain the exendin-4 of the present invention in which the position 27 is modified.

In addition, the present invention is an excessive secretion or lowering of plasma glucose, inhibiting gastric or intestinal motility, inhibiting stomach or intestinal fasting or food intake of insulin containing a single modified exendin-4 as an active ingredient with the polyethylene glycol or a derivative thereof. Provided are prophylactic and therapeutic agents for diseases caused by inhibition.

Exendin-4 according to one embodiment of the present invention is a single modification to a specific position, preferably lysine position of polyethylene glycol or a derivative thereof, increases the half-life in the body, and may exhibit biological activity similar to natural exendin. . In addition, by selectively limiting the number of binding sites and the number of couplings, it is possible to minimize side effects resulting from the diversity of these elements.

As a result, exendin monomodified with the polyethyleneglycol or derivatives thereof according to the present invention may be used for diseases caused by excessive secretion of insulin, such as diabetes or obesity, or plasma glucose lowering such as irritable bowel syndrome, gastric or intestinal motility. It can be usefully used to prevent and treat diseases caused by inhibition, suppression of gastric or intestinal fasting or suppressing food intake.

The composition containing the polyethylene glycol modified exendin according to the present invention as an active ingredient may be formulated and administered in various oral or parenteral dosage forms as described below, but is not limited thereto.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. 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 compound, for example, starch, calcium carbonate, sucrose. Or it is prepared by mixing lactose (Lactose), gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate 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, PEG, 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, method of administration, excretion rate and severity of the disease, but generally from 1 week to 2 weeks. Effective doses can be administered even several times a week. In addition, it can be divided into once or several times a day within the effective dosage range.

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  1> Methoxypolyethylene Propionaldehyde Combined Exendin Manufacture of -4

To 0.5 ml of exendin-4 (American Peptide, 1 mg / ml in 50 mM sodium acetate buffer at pH 5.5), methoxypolyethylene propionaldehyde (Nektar, mPEG-propionaldehyde, mPEG-ALD, 2 kDa, pH 5.5) 0.95 mg / ml) in 50 mM sodium acetate buffer was added, and 20 mM NaCNBH ₃ was further added as a reducing agent in the reaction. The molar ratio of exendin-4 to mPEG-ALD was set to 1 to 2, and the mixture of mPEG-ALD and exendin-4 was reacted with light shielding at 4 ° C. for 2 hours. The reaction was terminated by adding distilled water solution containing 0.1% trifluoroacetic acid (TFA) to the reaction mixture to obtain PEG-modified exendin at the N-terminus.

< Example  2> Methoxy polyethylene glycol Succinimidyl With propionate  Formulated Exendin Manufacture of -4

Dissolved in Exendin-4 (American Peptide, DMSO) in methoxypolyethylene glycol succinimidyl propionate (Nektar, mPEG-succinimidyl propionate, 2, 5, 10, 20 kDa, respectively) ) Were mixed in the same volume ratio. At this time, 9% triethylamine (dissolved in triethylamine, TEA, DMSO) as a reducing agent was added in the same volume so that the final concentration was 3%, and the mixture was reacted by blocking light at room temperature for about 60 minutes. I was. The molar ratio of exendin-4 to mPEG-SPA is 1 to 4, and the reaction is terminated by adding distilled water containing 0.1% TFA in the same volume as the reaction mixture to give PEG molecular weights of 2, 5, 10 and 20 kDa. This modified exendin-4 (PEG2K-Exendin-4, PEG5K-Exendin-4, PEG10K-Exendin-4 and PEG20K-Exendin-4, respectively) was obtained.

< Example  3> linear, Double Branch  And Triple branch type With polyethylene glycol  Formulated Exendin Manufacture of -4

Example 2 except for using linear (20 kDa), bi-branched (20 kDa) or triple-branched (23 or 40 kDa) methoxypolyethylene glycol N-hydroxysuccinimide (Nektar) Exendin modified with PEG at the N-terminus was obtained in the same manner as.

< Experimental Example  1> Polyethylene glycol  Single formula Exendin Separation purification of the 4-position isomer and identification of the isomer

The following experiment was conducted to isolate PEG-modified exendin-4.

In order to separate the PEG-modified exendin-4 prepared through Examples 1 to 3, Lichrospher RP-8 (250 × 4.0 mm, 5 μm, Merk, Germany) was used as a column, and TFA 0.1 was used as the mobile phase, respectively. Acetonitrile and distilled water containing% were used. Mobile phase conditions were varied linearly with 35-45% solvent B (acetonitrile with 0.1% TFA) and 65-55% solvent A (distilled water with 0.1% TFA). Each peak was quantified using a UV absorbometer 215 nm. The PEG-modified exendin-4 isolated by the above method was confirmed the number of PEG conjugated by mass spectrometry using a MALDI-TOF mass spectrometer (Matrix assisted laser desorption / ionization time-of-flight mass spectrometry). In addition, each position isomer was digested with lysine-C, a protease, and confirmed by MALDI-TOF mass spectrometry. The experiment was carried out by dissolving each purified PEG conjugate in 50 μl of triethylamine-hydrochloric acid buffer (10 mmol / L; pH 7.4) at a concentration of 1 mg / mL, and then adding 50 μl of enzyme (1 mg / mL) to 37 ° C. It was reacted for 1 hour at. 5 μl of 10% (v / v) TFA was added to complete the lysine-C digestion reaction, and then the reaction mixture was mass analyzed with the MALDI-TOF mass spectrometer. The results are shown in FIGS. 1 to 3 and Table 1 below.

PEG single formula position Expected mass MALDI-TOF Analysis Results Exendin-4 mass PEG mass Exendin-4 mass PEG mass Lys-27 1278.3 5127.4 1282.5 5120.5 N-terminal 1921.2, 1024.1 3478.3 1988.4, 1055.9 3488.4 Lys-12 1024.1 5381.5 1064.4 5399.5

Figure 1 shows an HPLC chromatogram of N-terminal PEG modified exendin. The final product of Example 1 was separated from two different materials, and analysis confirmed that each material was unreacted exendin-4 and PEG-modified exendin-4 at the N-terminus. Also, the isolated N-terminal PEG-modified exendin-4 had a purity of 98% or more.

Figure 2 shows an HPLC chromatogram of PEG reaction with exendin-4 lysine residues and the final isolated material. The reaction mixtures of Examples 2 and 3 consisted of a mixture of four different substances, and as shown in FIG. 3 and Table 1 above, they were each reacted to unreacted natural exendin-4 and lysine-27. PEG-modified exendin-4 (Lys ²⁷ -PEG2k-Exendin-4), Lysine-12 PEG-modified exendin-4 (Lys ¹² -PEG2k-Exendin-4), Lysine-12,27 it was confirmed that the PEG-modified exendin-4 (Lys ^{12, 27} -PEG2k- exendin-4). It was also confirmed that the final isolates were at least 98% high purity.

As shown in FIG. 4, PEG is a single-modified exendin-4 MALDI-TOF assay at the lysine position 27 according to the present invention. As shown in Example 2, as the molecular weight of PEG was increased, exendin-4 modified with various molecular weights of PEG was produced.

< Experimental Example  2> PEG According to a single qualified position Exendin Determination of Biological Activity of -4 Conjugates

In order to determine the biological activity of natural exendin-4 and PEG-modified exendin-4, the following experiment was performed.

Experimental Example  2-1: Insulin Secretion Promotion Experiment

Pancreatic islet cells were isolated from Sprague Dawley rats using collagenase digestion and Ficoll density difference separation. The isolated pancreatic islet cells were cultured in a cell culture for 2 to 3 days, then divided into 20 wells and divided into 24 well plates containing 1 ml of KRH buffer (containing 16.7 mM glucose), followed by natural exendin-4 and N-. Terminals, Lysine-12 and Lysine-27 are exendin-4 isomers modified with PEG (N ^ter -PEG2k-Exendin-4, Lys ¹² -PEG2k-Exendin-4, Lys ²⁷ -PEG2k-Exendin- 4) is added to 0.1, 1, 10, 100 nM, respectively, and incubated for 2 hours in a cell incubator. After incubation, 200 μl of the culture solution was taken, and the insulin concentration in the obtained sample was measured using an insulin enzyme immunoassay kit. The results are shown in FIG.

As shown in Figure 5, Lys ²⁷ -PEG2k-Exendin-4 showed the best insulin secretion ability, this effect did not show a significant difference from the natural exendin-4. On the other hand, N ^ter -PEG2k-Exendin-4 and Lys ¹² -PEG2k-Exendin-4 showed low effect, and N ^ter -PEG2k-Exendin-4 showed the lowest insulin secretion ability. . In addition, the EC ₅₀ values of natural exendin-4, Lys ^27- PEG2k-exendin-4, Lys ^12- PEG2k-exendin-4 and N ^ter -PEG2k-exendin-4 were 2.94, 3.11, 9.65 and 135.5, respectively. nM.

Experimental Example  2-2: receptor binding test

In order to examine the interaction of the exendin-4 and PEG modified exendin-4 treatment with the GLP-1 receptor, a receptor of insulin secreting cells, the following experiment was conducted.

Insulin-secreting cell lines INS-1 cells were dispensed 2.5 × 10 ⁵ cells in each well of 12 well plates and cultured for about 2 days to stably attach the cells to the culture vessel. After cell attachment, the cell culture solution was exchanged with a binding buffer, and ¹²⁵ I-Exendin-4 (exendin derivatives having amino acids 9 to 39) was added to the final 30 pM. The final concentrations of natural exendin-4 and Lys ²⁷ -PEG2k-exendin-4, Lys ¹² -PEG2k-exendin-4 and N ^ter -PEG2k-exendin-4 were added in a range of 0.001 to 1000 nM. Competitive receptor binding was performed for 2 hours at room temperature. After the end of the experiment, cells were washed three times with cold phosphate buffer to remove unbound ¹²⁵ I-Exendin-4. Finally, the cells were disrupted using a lysis buffer, and then the solution was taken to measure the amount of cell binding ¹²⁵ I-Exendin-4 by using a gamma detector. The results are shown in FIG.

As shown in FIG. 6, as the concentration of the sample increased, the binding of competitive ¹²⁵ I-exendin-4 decreased. In addition, in the case of positional isomers, the bond strength was shown to be different according to the modified position. IC ₅₀ (50% ¹²⁵ I-Exendin-4 binding of native Exendin-4, Lys ^27- PEG2k-Exendin-4, Lys ^12- PEG2k-Exendin-4 and N ^ter -PEG2k-Exendin-4 Concentrations) were 0.1, 1.8, 3.9 and 36.5 nM, respectively.

In this regard, the biological effects and efficacy of PEG-modified exendin-4 isomers through the effects of cell culture conditions and receptor binding experiments were determined by Lys ²⁷ -PEG2k-Exendin-4> Lys ¹² -PEG2k-Exendin-4> N. ^ter -PEG2k-exendin-4 was found in the order.

Experimental Example  2-3: Animal Model Experiment

Six-week-old male db / db mice (diabetic disease model mice) (C57 / BLKS / J-db / db, Korea research institute of bioscience and biotechnology to examine the activity of native exendin-4 and PEG conjugates in animal models) Saline (control), natural exendin-4, and PEG modified exendin-4s (Lys ²⁷ -PEG2k-exendin-4, Lys ¹² -PEG2k-exendin-4 and N ^ter -PEG2k-exendin). -4) subcutaneously at a dose of 1 nmol / kg (100 μl injection) at -30 min, followed by oral administration of 200 mg / ml (200 μl dose) of glucose and -30, 0, 15, 30, 60 Blood glucose changes in blood from tail veins were observed at 120 and 180 minutes. The results are shown in FIGS. 7 and 8.

As shown in FIG. 7, the saline-only control group showed a rapid blood sugar rise and a slow blood sugar drop behavior according to glucose administration. On the other hand, natural exendin-4 and PEG modified exendin-4 showed relatively low blood sugar elevation and relatively fast blood sugar dropping behavior. There is also a slight blood glucose difference between exendin-4 and PEG modified exendin-4, which is thought to be a difference in activity and receptor binding capacity between PEG conjugates. In addition, based on the results of FIG. 7, a diagram illustrating an area under a glucose concentration curve between 0 and 180 minutes is shown in FIG. 8.

As shown in FIG. 8, Lys ²⁷ -PEG2k-Exendin-4 showed similar activity as natural Exendin-4, and Lys ¹² -PEG2k-Exendin-4 and N ^ter -PEG2k-Exendin. -4 showed poor effect. This difference in drug efficacy is believed to be due to the difference in biological activity as shown in Experiment 2.

Therefore, the experimental results of Experimental Example 1, it can be seen that the PEG conjugate with the best biological activity and hypoglycemic effect in the animal model is a single modified PEG molecule at position 27.

< Experimental Example  3> At Lysine 27 PEG Has a single formula Exendin -4 PEG  Determination of Biological Activity of Exendin-4 Binders by Molecular Weight

In order to determine the biological activity according to the PEG molecular weight of PEG-modified exendin-4 in lysine No. 27 by a method similar to Experimental Example 1 was carried out as follows.

Experimental Example  3-1: Insulin Secretion Promotion Experiment

Pancreatic islet cells isolated from mice were also exendin-4 (PEG2K-Exendin-4, PEG5K-Exendin-4, PEG10K-Exen) modified with PEG of 2, 5, 10 and 20 kDa prepared according to Example 2. The experiment was conducted in the same manner as in Experimental Example 1-1, except that Dean-4 and PEG20K-Exendin-4) were administered at a concentration of 10 nM. The results are shown in FIG.

As shown in FIG. 9, PEG showed no biological activity difference below 5 kDa compared to native exendin-4. On the other hand, as the molecular weight of PEG increased, the gradual decrease in activity was observed, and it was found that exendin-4, which has a very large PEG of 20 kDa, exhibited more than 40% of residual activity.

Experimental Example  3-2: receptor binding test

Insulin-secreting cell lines INS-1 cells are administered PEG2K-Ex-4, PEG5K-Ex-4, PEG10K-Ex-4 and PEG20K-Ex-4 prepared according to Example 2 at a concentration of 0.001 to 1000 nM, respectively. Except that, the experiment was carried out in the same manner as in Experiment 1-2. The results are shown in FIG.

As shown in Figure 10, it can be seen that the relatively low receptor binding capacity as the modified PEG molecular weight increases. IC ₅₀ values of exendin-4 and PEG-modified exendin-4 obtained through experiments were 0.1 nM (natural exendin-4), 1.8 nM (PEG2k-exendin-4) and 2.4 nM (PEG5k-exene), respectively. Dean-4), 5.6 nM (PEG10k-Exendin-4) and 10.7 nM (PEG20k-Exendin-4). Therefore, it can be seen from the above experiment that the interaction with the receptor decreases as PEG molecular weight modified to exen-4 is increased, which leads to a decrease in biologically active insulin secretion. However, even when the high molecular weight PEG of 20 kDa is modified (IC ₅₀ = 10.7 nM) compared to the result of the exendin-4 modified molecular weight of 2 kDa shown in FIG. It was found that the receptor binding ability was better than conjugated exendin-4 (IC ₅₀ = 36.5 nM).

In conclusion, it can be inferred that exendin-4 modified with PEG at lysine 27 retains its biological activity even when high molecular weight PEG is conjugated.

Experimental Example  3-3: in animals Antidiabetic  Efficacy Measurement

To investigate the activity of native exendin-4 and PEG modified exenden-4 in animal models, 6-7 week old male db / db mice (with an average blood glucose of around 250 mg / dL) (C57 / BLKS / J- saline (control), exendin-4 and PEG2K-Ex-4, PEG5K-Ex-4, PEG10K-Ex-4 and PEG20K prepared according to Example 2 After subcutaneous injection of Ex-4, changes in blood glucose in blood collected from the tail vein were observed. The results are shown in FIG.

As shown in FIG. 11, when exendin-4 and PEG-modified exendin-4 are injected into a diabetic-induced experimental animal, the blood glucose level and blood glucose normalization (blood sugar <150) are accelerated by promoting insulin secretion in the body of the experimental animal. mg / dL) was induced. In the case of the control group (physiological saline infusion), it was found to maintain high hyperglycemia. On the other hand, in the group injected with exendin-4 and PEG-modified exendin-4, the normal blood glucose maintenance period tended to be different in exendin-4 and PEG-modified exendin-4. Exendin-4 and PEG2k-Exendin-4 did not show any difference in hypoglycemic behavior, and returned to hyperglycemic state after maintaining normal blood glucose for approximately 6 hours after infusion. Exendin-4 modified with PEG5k and PEG10k showed improved hypoglycemic behavior compared to native exendin-4, and normal blood glucose retention time was 12 hours and 18 hours, respectively. More pronounced changes in hypoglycemic behavior were observed in PEG20k-exendin-4 with a molecular weight of 20 kDa modified PEG. Through administration of the same dose, PEG20k-Exendin-4 exhibited a normal blood sugar maintenance function of approximately 36 hours or more, which was more than 2 times more than PEG10k-Exendin-4 and 6 times more than natural exendin-4. It represents. Therefore, it can be seen that exendin-4 modified with high molecular weight PEG at lysine 27 can be used as a long-acting antidiabetic drug that maintains long-term efficacy.

Experimental Example  3-4: in animals Pharmacokinetics  Behavior investigation

Through the above experiments, it was confirmed that when PEG of high molecular weight was modified at lysine position 27 of exendin-4, the characteristics of the sustained peptide drug were shown. Therefore, the pharmacokinetic behavior of exendin-4, PEG2k-exendin-4, and PEG20k-exendin-4 was investigated to investigate the cause of these persistent properties.

Blood samples over time were obtained by subcutaneously injecting a sample in an amount of 1 nmol / rat (4.18 ㎍ / rat) to an experimental rat (SD rat) weighing about 200 g and taking a blood sample from a tube inserted into the rat jugular vein. The change in concentration was measured using an ELISA kit. The results are shown in FIG.

As shown in Figure 12, natural exendin-4 and PEG2k-exendin-4 showed similar pharmacological behavior, and a rapid decrease in blood concentration rapidly after reaching the maximum blood concentration 30 to 45 minutes after administration After 4 hours, basal concentrations (<2 ng / mL) were reached, and the half-life values calculated using these changes in blood concentrations were 1.6 ± 0.4 hours for exendin-4 and PEG2k. Exendin-4 did not show a difference of 1.5 ± 0.3 hours. On the other hand, PEG20k-Exendin-4 showed a tendency to decrease gradually after reaching the maximum blood drug concentration after about 12 hours with drug concentration, followed by a continuous, moderate increase in concentration. The calculated half-life of PEG20k-Exendin-4 was 22.0 ± 1.7 hours.

Therefore, it can be seen that the sustained drug efficacy of PEG20k-exendin-4 observed in the above experimental example is due to a change in pharmacokinetic behavior, particularly a very long half-life.

< Experimental Example  4> A single modified position at Lysine 27 PEG According to the molecular structure of Exendin Determination of Biological Activity of -4 Conjugates

Through the process of Experimental Examples 1 to 3 it was confirmed that the exendin-4 modified high molecular weight PEG of molecular weight 20 kDa or more in lysine 27 shows the characteristics of the sustained drug. Furthermore, in order to investigate the effect of modified PEG molecular structure on the efficacy and effect of the drug, the effect of the PEG having the same structure of about 20 kDa with different exendin-4 in lysine 27 was investigated.

Experimental Example  4-1: Receptor Binding Experiment

Receptor binding capacity in the same manner as Experimental Example 1-2 except that the bi-branched PEG (20 kDa) and the tri-branched PEG (23 kDa) prepared according to Example 3 were treated with modified exendin-4. Was measured. The results are shown in FIG.

As shown in FIG. 13, the binding capacity of the PEG-modified exendin-4 according to the present invention showed that the triple branched form was larger than the double branched form, and the values of IC ₅₀ obtained through the calculation were 80.6 nM (triple branched), respectively. Type) and 280.8 nM (bibranched).

Experimental Example  4-2: in animals Antidiabetic  Efficacy Measurement

Except for treating the exendin-4 modified bi-branched PEG and tri-branched PEG prepared according to Example 3 in the same manner as in Experiment 3-3. The results are shown in FIG.

As shown in FIG. 14, the bi-branched PEG conjugate showed a tendency to return to hyperglycemic state after maintaining normal blood glucose for about 8 hours, which is considered to be because the bi-branched PEG conjugate shows very low receptor binding ability. (IC ₅₀ = 280 nM, see figure 13). On the other hand, all three branched PEG conjugates showed sustained efficacy, and the triple branched form showed long-term hypoglycemic effect of about 48 hours. The difference in efficacy of these tribranched forms seems to be due to the pharmacokinetic behaviors mentioned above, in particular the differences in dissipation rate and distribution volume.

Therefore, it was confirmed through the observation process that the triple branched PEG modified exendin-4 shows the best sustained drug properties.

Experimental Example  4-3: Pharmacokinetic Behavior

Except for intravenously injecting modified exendin-4 with bi-branched PEG (20 kDa) and tri-branched PEG (23 kDa) prepared according to Example 3, the same procedure as in Experimental Example 3-3 was performed. Was carried out. The results are shown in Table 2 and FIG. 15.

PEG23k-Exendin-4 (Triple Branch) PEG20k-Exendin-4 (double branch) AUC (ng hr / ml) 7659 ± 1365 * 5717 ± 1015 * Dissipation Half Life (hr) 25.1 ± 9.1 28.0 ± 10.4 Distribution volume (ml) 9.41 ± 1.57 * 25.45 ± 16.84 Distribution volume (L / kg) 0.05 ± 0.01 * 0.13 ± 0.08 Dissipation rate (ml / hr) 0.54 ± 0.09 * 0.63 ± 0.08 * Dissipation rate (ml / hr / kg) 2.69 ± 0.45 * 3.16 ± 0.39 *

As shown in Table 2, the extinction rate showed the lowest extinction rate in the case of exendin-4 modified tri-branched PEG. In addition, the volume of distribution of exendin-4 modified with a tri-branched PEG was much lower than that of exendin-4 modified with a double-branched PEG. It can be said that exendin-4 is most stably present in the blood. In addition, there was no significant difference in the half-life of the two forms of PEG-modified exendin-4, but the triple branched PEG showed the lowest loss rate and stable blood propensity in modified exendin-4. Could know.

As shown in FIG. 15, the exendin-4 modified with the bi-branched PEG and the tri-branched PEG prepared according to Example 3 showed a continuous decrease in blood concentration, according to the PEG structure modified with the exendin-4. A slight difference was shown.

Experimental Example  4-4: Insulin Secretion Promotion Experiment

Natural exendin-4 and tri-branched PEG modified exendin-4 were treated in the same manner as in Experiment 1-1, except that 0.1, 1, 10 and 100 nM were treated. The results are shown in FIG.

As shown in FIG. 16, both exendin-4 and trigated PEG-modified exendin-4 promoted insulin secretion in a concentration-dependent manner.

< Experimental Example  5> A single modified position at Lysine 27 Triple branch type PEG Determination of Biological Activity of Exendin-4 by Molecular Weight

Experimental Example  5-1: in animals Antidiabetic  Efficacy Measurement

Trivalent PEG of 23 or 43 kDa was the same as Experimental Example 3-3 except that the modified exen-4 (PEG23k-Ex-4 or PEG43k-Ex-4) was treated at a concentration of 10 nmol / kg. It was carried out by the method. The results are shown in FIG. 17.

As shown in FIG. 17, the difference in the molecular weights of 23 and 43 kDa of PEG did not affect the substantial persistence, and both materials showed similar persistence. Through this process it can be seen that the tri-branch type has a similar effect in the case of more than 20 kDa.

Experimental Example  5-2: Lysine 27 Triple branch type PEG Is formulated Accented According to treatment concentration of -4 Antidiabetic  Efficacy Measurement

Excepted-4 (PEG23k-Ex-4) modified with 23 kDa triple branched PEG was carried out in the same manner as in Experiment 3-3, except that the concentration was 10 nmol / kg. The results are shown in FIG. 18.

As shown in FIG. 18, the 5 nmol / kg dose showed a duration of about 12-18 hours, and the 10 nmol / kg dose showed a duration of 36-48 hours in diabetic animals. The higher doses of 20 and 50 nmol / kg showed very long drug durations of 72 to 96 and 120 hours, respectively.

Hereinafter, the formulation example for the composition of this invention is illustrated.

Preparation Example Preparation of Pharmaceutical Formulation

Pharmaceutical formulations comprising a single modified exendin-4 PEG according to the invention were prepared as follows.

One. Powder  Produce

2 g of exendin-4 with a single modified PEG according to the invention

1 g lactose

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

2. Preparation of Tablets

100 mg of exendin-4 with single modified PEG according to the present invention

Corn starch 100 mg

Lactose 100 mg

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 with single modified PEG according to the present invention

Corn starch 100 mg

Lactose 100 mg

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 PEG-modified exendin-4 according to the invention

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

PEG in accordance with the present invention dissolves a single modified exendin-4 in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution is adjusted to pH 3.5 using dilute hydrochloric acid BP, and with sodium chloride BP for injection. The volume was adjusted and mixed well. The solution was filled into a 5 ml Type I ampoule of clear glass and filled under the upper grid of air by dissolving the glass to prepare an injection solution.

Polyethyleneglycol, which binds polyethylene glycol to residue 27 of exendin, exhibits biological activity similar to that of natural exendin while increasing the half-life of the drug and minimizing side effects from the diversity of the location and number of bonds of polyethylene glycol. By formulating exendin, it may be useful for the treatment 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, and in particular diabetes. It can be useful for obesity and irritable bowel syndrome.

Figure 1 shows an HPLC chromatogram of the mixture and finally separated N-terminal polyethylene glycol (PEG) modified exendin-4 after completion of the N-terminal reaction;

Figure 2 shows the reaction of lysine residues with PEG and HPLC chromatograms of the final isolated materials;

Figure 3 shows the MALDI-TOF mass spectrometry of the reaction solution after the lysine-C enzymatic reaction;

4 shows MALDI-TOF analysis of single-modified exendin-4 at position lysine 27 prepared in Example 2;

5 is a result of insulin secretion promoting experiments of natural exendin-4 and PEG modified exendin-4;

Figure 6 shows the binding behavior of natural exendin-4 and PEG modified exendin-4 with GLP-1 receptor on the surface of insulin secreting cells;

7 shows the results of measuring oral glucose tolerance in an animal model of exendin-4 according to a single modified position of PEG;

FIG. 8 plots the area under the glucose concentration curve between 0 and 180 minutes based on the results of FIG. 7;

9 is the result of an insulin secretion promoting experiment according to the modified PEG molecular weight;

10 is the result of a GLP-1 receptor binding experiment according to modified PEG molecular weight;

FIG. 11 is a schematic of blood glucose changes in experimental animals following injection of drug according to modified PEG molecular weight; FIG.

12 shows changes in blood concentrations after subcutaneous injection of natural exendin-4, PEG2k-exendin-4 and PEG20k-exendin-4;

FIG. 13 shows the binding capacity of exendin-4 modified with two different molecular forms of PEG and insulin secreting cells to a receptor of about 20,000 Daltons in average molecular weight;

14 shows the results of antidiabetic efficacy in experimental animals according to the modified PEG molecular structure;

FIG. 15 shows the pharmacokinetic behavior and results of PEG-modified exendin-4 with three different molecular structures with molecular weights of about 20,000;

FIG. 16 shows the concentration-dependent insulin secretion promoting efficacy of exendin-4 modified with native exendin-4 and trimolecular weight 23000 PEG;

FIG. 17 shows the results of antidiabetic efficacy in animals according to the molecular weight of a single modified tri-branched PEG at position lysine 27;

FIG. 18 shows the results of antidiabetic efficacy in animals according to the concentration of a single modified tri-branched PEG conjugate at position lysine 27. FIG.

Claims (19)

Diabetes, obesity, or hypersensitivity, wherein the tri-branched polyethylene glycol derivative represented by the following formula (1) having a molecular weight greater than 20 kDa and less than or equal to 60 kDa contains a single modified exendin as an active ingredient at position 27 of exendin-4 Prevention and treatment of intestinal syndrome. <Formula 1> (In the above formula n has an integer value of 1 to 1,000, m has an integer value of 10 to 1,000, and the functional group capable of chemically reacting with proteins and peptides including exendin is N-hydroxysuccinimide, and Z is exyndine and polyethylene glycol. A (CH ₂ ) _S or (CH ₂ ) _S NHCO (CH ₂ ) _S group that acts as a linker between _S , where S represents an integer value from 1 to 6. delete delete delete delete delete delete delete The polyethylene glycol derivative according to claim 1, wherein the polyethylene glycol derivative has a molecular weight of more than 20 kDa and is less than or equal to 45 kDa, wherein the single modified exendin at position 27 of exendin-4 is used as an active ingredient. Preventive and treatment of diabetes, obesity or irritable bowel syndrome containing. delete delete delete delete delete delete delete delete delete delete