KR20110080084A - 4-arm polyethylene glycol modified with anti-diabetic peptides and fatty acids - Google Patents

Abstract

PURPOSE: A composition containing a conjugate or derivative thereof for reducing blood glucose is provided to enhance effective molecular size and to treat physiological activity in vivo. CONSTITUTION: A conjugate is prepared by conjugating fatty acid and anti-diabetic peptide to polyethylene glycol(PEG). The PEG contains a derivative thereof. The each end of the PEG is succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, N-hydrosucciinimide, benzotriazole carbonate, maleimide, or OPSS. The anti-diabetic peptide is GLP-1, exendin-4, insulin or agonist, derivative, fragment, variant, or mixture thereof. A composition for reducing blood glucose contains an anti-diabetic peptide-PEG-fatty acid conjugate as an active ingredient.

Description

Anti-diabetic peptide and fatty acid conjugated to 4-arm polyethylene glycol {4-arm polyethylene glycol modified with anti-diabetic peptides and fatty acids}

The present invention relates to a compound obtained by conjugating antidiabetic peptide and fatty acid to polyethylene glycol (PEG) having four arms, or a derivative thereof and a composition for lowering blood sugar.

Glucagon-like peptide-1 (GLP-1) is a 30 (7-36) or 31 (7-37) amino acid produced from proglucagon secreted from intestinal L cells in response to oral ingestion. As a composed peptide, it is known that the glucose concentration dependent on the plasma stimulates insulin secretion and inhibits glucagon secretion, thereby stably lowering blood glucose. It also has a function of delaying gastric hunger and inducing satiety to reduce food intake and, in particular, to inhibit apoptosis and stimulate angiogenesis. Such excellent properties are known to effectively treat or prevent hyperglycemia or obesity (Diabetes 53: 2181-2189, 2004).

However, GLP-1 is rapidly cleaved and inactivated by two enzymes, N-terminal end of His ⁷ -Ala ⁸ , by enzymes Dipeptidyl peptidase IV and NEP 24.11 present in mammalian blood. It is known to have an extremely short biological half-life of about a minute.

Exendin-4 (Ex4) is a peptide substance isolated and purified from the saliva of a toxic lizard named Gila monster in the desert of the southwestern United States, consisting of 39 amino acid sequences and endogenous glucagon-like peptide-1 (GLP-1) 53% sequence similarity (Diabetes 53: 2181-2189, 2004). It has a physiological effect similar to GLP-1, which promotes insulin secretion dependent on blood glucose concentration, while inhibiting glucagon release, increasing insulin sensitivity, inducing proliferation of pancreatic beta cells, suppressing gastric emptying and appetite It is known to cause degradation. This physiological effect is known to be very specific for type 2 diabetes and obesity (Diabetes 48: 1026-1034, 1999).

In general, GLP-1 is rapidly cleaved and inactivated by Dipeptidyl peptidase IV to have a biological half-life of about 2 minutes, whereas Ex4 has Gly 2 as the amino acid. In addition to strong resistance, glomerular filtration rate in kidneys is much slower than GLP-1, resulting in prolonged half-life and physiological activity than GLP-1 (Diabetologia 46: 706-712, 2006; Diabetes 53: 2181). 2189, 2004). In vivo experiments have shown a half-life of 2-4 hours and can be reached at a certain concentration suitable for treatment by intraperitoneal administration 2-3 times a day (Drug Development Res., 260-267; 2001).

As described above, Ex4 is known to inhibit gastrointestinal motility, reduce food intake, and inhibit plasma glucagon secretion (US Pat. Nos. 6,885,576, 6,560, 6,687,003), and sulfonyl urea. In combination with oral antidiabetic drugs such as metformin, it has been reported that the levels of glycated hemoglobin (Hb _A1C ) were lowered within 1% after 28 days of administration.

The duration of action of Ex4 is still assessed to be short in light of the specificity of diabetes disease, which may lead to decreased compliance of diabetic patients who have to be frequently administered by daily injection. Competitive long-acting insulin injections, Lantus ^® and Humulin U ^® , are also known to have an action time of ~ 24 hours and ~ 36 hours, respectively. Therefore, there is a need for extended therapeutic molecules that can have higher plasma stability in vivo and administered less frequently than Ex4 administered 2-3 injections per day, thereby improving the patient's potential adaptability.

Insulin is a polypeptide used for type 1 diabetes and is secreted from the pancreatic islets of Langerhans islet and maintains a constant level of plasma glucose. When the blood sugar level rises above a certain level, insulin is secreted and promotes the action of introducing glucose in the blood into cells and storing it again in the form of polysaccharides (glycogen). Insulin is a polypeptide in which the A chain of 21 amino acids and the B chain of 30 amino acids are connected by disulfide bonds, and one Lys amino acid is in the B chain.

However, due to the characteristics of diabetics, insulin has to be administered for a long time, and even the longest sustained type has a disadvantage of less than 2 days.

Recently, attempts have been made to solve the above problems by conjugating albumin to polypeptide antidiabetic agents such as GLP-1, Ex4 and insulin by physicochemical or genetic recombination methods. Human serum albumin (HSA) is a protein of about 67 kDa consisting of 585 amino acids and is present at a concentration of 35-50 g per liter of human serum and is produced mainly in the liver by 10-15 g per day, with a half-life of about 19 Is known to be nontoxic and immunogenic (J. Control. Release 132: 171-183, 2008). It is known to prolong blood half-life by regulating the osmotic pressure of blood and protein binding with long-chain fatty acids as well as partially hydrophobic drugs (Pharm. Res. 19: 569-577, 2002).

Albumin fusion technology was developed by Human Genome Sciences (PCT / US2006 / 029391), and is a method of fusion of human albumin genes into genes encoding polypeptides to create new fusion proteins. It has been applied to cytokines such as

Polyethylene glycol conjugation (PEGylation) is a technique applied to increase the biological half-life of drugs such as peptides and proteins. In general, the size of globular proteins that can be excreted through glomerular filtration is known to be about 70 kDa with a molecular weight of 5 to 6 nm. Have no choice but to. Since PEG molecules are linear in nature, they exhibit molecular sizes of about 2 to 9 times that of spherical proteins of the same molecular weight. In particular, PEG molecules tend to increase rapidly as molecular weight increases. Eventually, this PEG property allows PEG conjugated peptides and proteins to have strong resistance to glomerular filtration, resulting in extended biohalf life and increased bioavailability (Adv. Drug Deliv. Rev. 55: 1261-1277, 2003).

As described above, in order to overcome the problem of frequent long-term administration due to the short half-life and duration of the antidiabetic peptide, a conjugate in which an antidiabetic peptide and a fatty acid conjugated to an activated PEG molecule having four arms may increase the effective molecular size. In addition, the present invention was completed by confirming that the half-life and hypoglycemic effect were greatly improved by inducing reversible binding with albumin present in plasma.

Accordingly, an object of the present invention is to provide a conjugate or derivative thereof in which an anti-diabetic peptide and a fatty acid are conjugated to a PEG molecule.

Another object of the present invention is to provide a method for preparing a conjugate in which an anti-diabetic peptide and a fatty acid are conjugated to the PEG molecule.

In another aspect, the present invention to provide a composition for lowering blood sugar comprising the conjugate or a derivative thereof.

The present invention provides a conjugate in which an anti-diabetic peptide and a fatty acid are conjugated to a polyethylene glycol (PEG) molecule.

More specifically, the final derivative in which the 4-armed polyethylene glycol molecule reacts covalently with a 2-molecular anti-diabetic peptide primary amine group (-NH ₂ ) and 2-molecular fatty amine. To provide.

The PEG includes derivatives thereof.

In addition, the PEG or PEG derivative may be selected from linear or branched (branched) form of the branch may be two or more multi-branch.

For the preparation of the conjugate, polyethylene glycol (PEG), which is the center of the conjugate, is preferably in the form of four arms. That is, PEG is a form having four linear PEG groups different from the center, it is preferred that the form is connected to each other from the center (Fig. 1).

Each terminal portion of the PEG of the present invention is either N-hydroxysuccinimidyl (NHS) esters or sulfo N-hydroxysuccinimidyl (Sulfo N-hydroxysuccinimidyl) which can react with amines. ) Esters and the like.

More specifically,

Terminals of the PEG are succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, N-hydroxy succinimide And benzotriazole carbonate (Benzotriazole carbonate) is preferably modified with any one or more groups selected from the group consisting of.

In some cases, it may be modified with maleimide (MAL) or orthopyridyl disulfide (OPSS) to react with thiol (-SH) groups.

PEG of the present invention preferably has a total molecular weight of 10,000 ~ 40,000 Da. In other words, the molecular weight range of each PEG corresponding to each arm is preferably about 2,500 to 10,000 Da.

If the molecular weight of each arm is too small to 2,500 or less, there is a risk of Steric hindrance when the anti-diabetic peptide molecules are conjugated, and the effective molecular size is too small to expect satisfactory in vivo in vivo. Not only is it difficult, but there are concerns that albumin conjugation can be difficult.

The antidiabetic peptides of the present invention may be synthetic or recombinant.

The antidiabetic peptide may be selected from the group consisting of GLP-1, exendin-4, insulin or agonists, derivatives, fragments, variants, and combinations thereof. And preferably GLP-1, exendin-4 or insulin.

GLP-1 of the present invention may have the following amino acid sequence, GLP-1 (Glucagon-like peptide-1) is 30 (7-36) or 31 (7-37) amino acids produced from proglucagon It is composed of a peptide consisting of stimulating insulin secretion and inhibiting glucagon secretion depending on the glucose concentration present in the plasma to lower the blood sugar stably.

GLP -1 (7-36) amino acid sequence

His ⁷ -Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys ²⁶ -Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys ³⁴ -Gly-Arg ³⁶

Exendin-4 (Exendin-4) is a peptide substance isolated and purified from the saliva of toxic lizards. It consists of 39 amino acid sequences as follows and has a physiological effect similar to that of GLP-1. On the other hand, it is known to inhibit glucagon release, increase insulin sensitivity, induce proliferation of pancreatic beta cells, inhibit gastric emptying, and cause appetite loss.

Exendin -4 amino acid sequence

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

Insulin is a polypeptide used for type 1 diabetes and is secreted from the pancreatic islets of Langerhans islet and maintains a constant level of plasma glucose. When the blood sugar level rises above a certain level, insulin is secreted and promotes the action of introducing glucose in the blood into cells and storing it again in the form of polysaccharides (glycogen). As shown in the figure below, insulin is a polypeptide in which A chain consisting of 21 amino acids and B chain consisting of 30 amino acids are connected by disulfide bond. Lys amino acid has one B chain.

Insulin  Amino acid sequence

In PEG binding, Lys amino acids of antidiabetic peptides can be used for conjugation.

For example with Ex4 one of the Lys ¹² and Lys ²⁷ amines can be used for conjugation. Literature suggests that Ex4 avoids conjugation to His ¹ alphaamine because of its bioactive site at the amino terminus (Diabetes 53: 2181-2189, 2004).

Similarly, in the case of GLP-1, one of Lys ²⁶ and Lys ³⁴ amines can be used for conjugation because there is a bioactive site at the amino terminus.

In the case of insulin, conjugated to Lys ²⁹ amino acid of B chain is used.

In the case of fatty acid conjugation that binds albumin in the body as a component of the final derivative, the carbon number is preferably about 6 to 20. If the carbon number is too small, the hydrophobicity is very low enough to be dissolved in water. If the carbon number is too large, it may not be easily dissolved in an organic solvent, but the albumin bond may be reduced in the body. It is also activated by N-hydroxysuccinimide (NHS) esters at each end of the PEG with four arms, so that each fatty acid itself can react with the fatty acid (-COOH) itself. It should be a non-fatty acid amine.

In the examples of the present invention, palmitic acid was used as the fatty acid amine.

In addition, the present invention,

(1) dissolving PEG-NHS with four arms in dimethylformamide and then adding fatty acid amine;

(2) adding antidiabetic peptide dissolved in dimethyl sulfoxide to the solution prepared in step (1) and reacting for 6 hours or more; And

(3) dialysis with dimethylformimide and dimethyl sulfoxide in the solution prepared in step (2);

It provides a method for producing a conjugate conjugated to an anti-diabetic peptide and a fatty acid in polyethylene glycol (PEG) comprising a.

In one embodiment of the present invention, when preparing the primary conjugate of fatty acid amine and PEG, the reaction was induced in dimethylformamide (DMF, dimethylformamide), the fatty acid should be 0.5 equivalent to the number of PEG ends and the reaction for up to 2 hours. Was taken. An appropriate amount of triethylamine (TEA, triethylamine) or dimethylaminopyridine (DMAP) may be added thereto to shorten the reaction rate. In addition, since anti-diabetic peptides are not soluble in dimethylformamide, when anti-diabetic peptides are conjugated to intermediate derivatives, anti-diabetic peptides dissolved in the same amount of dimethyl sulfoxide are added to the mixed solution to induce a reaction, and the reaction time is at least 6 hours. This increased the reactivity to the maximum. In addition, about 1.5 equivalents of DCC (dicyclocarbodiimide) and N-hydroxysuccinimidyl esters (N-hydroxysuccinimidyl (NHS) esters) were added to each arm to enhance the completion of the reaction (Example 1). .

In another aspect, the present invention provides an anti-diabetic peptide-PEG-fatty acid conjugate composition as an active ingredient.

The antidiabetic peptide-PEG-fatty acid conjugate prepared as described above can reduce glomerular filtration in the kidney and resist various enzymatic attacks in vivo as well as increase the remaining period in the body by combining with albumin in the body (hypoglycemia) It has a (Example 5).

Therefore, the composition of the present invention lowers blood sugar in the body, and is used for the treatment of diabetes, obesity, irritable bowel syndrome and the like.

The composition may consist of an effective amount of the antidiabetic peptide-PEG-fatty acid conjugate of the present invention and a pharmaceutically acceptable diluent, preservative, solubilizer, other adjuvants and / or carriers.

The composition of the present invention may be used in the form of an aqueous injection solution, a non-aqueous injection solution, an emulsion injection solution, a suspension injection solution or an injectable (freeze-dried) powder to be dissolved in sterile injection water when used. Various formulations, such as for other injections and parenteral administration, can be prepared according to techniques described or commonly used in books well known in the art.

Additives that may be included in the composition may be any base commonly used in injections. For example, the base may be distilled water, sodium chloride solution, mixtures of sodium chloride and mineral salts or the like, solutions of mannitol, lactose, dextran, glucose, etc., amino acid solutions such as glycine, arginine, organic acid or salt solutions, and glucose solutions. Mixing and similar solutions. Injectables may also be prepared by conventional methods such as osmotic pressure regulators, pH regulators, antiseptics such as methylhydroxybenzoate or propylhydroxybenzoate, vegetable oils such as sesame oil or soybean oil, or surfactants such as lecithin or nonionic surfactants. It can be prepared into a solution, a suspension or a colloidal solution, and the like in addition to.

In addition, the effective dosage of the composition according to the present invention can be varied depending on the age, physical condition, weight, etc. of the patient, but generally can be administered once every three days to one week. And in a daily effective dosage range (10-250 nmol / kg as antidiabetic peptide) can be added once a day or divided several times a day.

The antidiabetic peptide conjugate conjugated with the polyethylene glycol and fatty acid of the present invention can provide an antidiabetic peptide compound, its derivatives and pharmaceutical compositions, which have a reduced half-life due to reduced kidney clearance and ultimately extended physiological activity and therapeutic effect. They can be used to treat diabetes, obesity and irritable bowel syndrome by greatly improving the sustained efficacy of each peptide, such as lowering plasma glucose, inhibiting stomach and intestinal motility, suppressing stomach and intestinal fasting, or inhibiting food intake. .

Figure 1 is a simplified diagram showing the molecular structure of the ratio of Exendin-4 and fatty acids 1: 1 (two molecules each) in the final derivative conjugated with Exendin-4 and fatty acid in polyethylene glycol having four arms;
2 is a schematic of a chemical reaction for conjugating Exendin-4 and a fatty acid to polyethylene glycol having four arms;
FIG. 3 shows a chromatogram of molecular exclusion high-performance liquid chromatography (SE-HPLC) of a molecular glycol derivative having four arms of Exendin-4 and fatty acid conjugated 4 prepared in Example 2;
Figure 4 shows the results of the albumin binding degree of the prepared polyethylene glycol derivative having four arms of Exendin-4 and fatty acid conjugates;
Figure 5 shows the results of the persistence of the hypoglycemic effect of the prepared type 2 diabetes-induced db / db mice of the polyethylene glycol derivative having four arms of Exendin-4 and fatty acid conjugate.

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

< Example  1> with 4 arms Polyethylene glycol  Palmitic acid ( Palmitic acid )and Ex4 2 each Bonded  Derivatives (4 PEG _5k -2 C ₁₆ -2 Ex4 )Produce

50 mg of 4-arm PEG-NHS (Mw: 20,000 Da) was dissolved in 5 ml of dimethylformamide (DMF) at room temperature, followed by 500 μl of palmityl amine (Sigma) dissolved in dimethylformamide. (2.5 mg / ml) was added and the reaction was induced at room temperature for about 1 hour. Thereafter, 15 mg of Ex-4 (American Peptide Company) dissolved in 3 ml of dimethyl sulfoxide was added to the above solution to induce a reaction for a minimum of 6 hours. The reaction solution was placed in a semi-permeable membrane (Mw CO: 8,000 Da) tube, and dialyzed five times with 500 ml of dimethylformimide for 4 hours. It was then dialyzed three times for 6 hours with 500 ml of dimethyl sulfoxide (DMSO) again in the same manner. Thereafter, the final solution was dialyzed twice with 2,000 ml of deionized water, and the precipitate was filtered off with a filter of 0.45 μm in pore size, and then the filtrate was concentrated using Centricon (MW cut off 10,000). After freezing and drying with a lyophilizer to obtain the final dried derivative sample. The chemical reaction is shown in FIG.

< Example  2> with 4 arms Polyethylene glycol  Palmitic acid ( Palmitic acid )and Ex4 2 each Bonded  Derivatives (4 PEG _5k -2 C ₁₆ -2 Ex4 Separation and purification of

About 0.2 ml of the final filtrate prepared by the above method was loaded on a Superdex 200 (GE Healthcare, 1 ⅹ 30 cm) column filled with phosphate buffer solution (10 mM, pH 7.0) added with 0.9% NaCl to fractionate the monomer peak portion. And stored until later albumin conjugation.

< Example  3> 4 PEG _5k -2 C ₁₆ -2 Ex4 Ok

Size-Exclusion HPLC and SDS-PAGE were used to confirm the molecular weight and purity of the prepared 4PEG _5k -2C ₁₆ -2Ex4. A Superdex 200 (GE Healthcare, 1 × 30 cm) column was used, with the mobile phase being 0.9% NaCl added phosphate buffer solution (10 mM, pH 7.0). As it is shown in FIG. 3 4PEG-2C ₁₆ -2Ex4 four PEG5k, similarly molecular Ex4 2 molecule of about 4200 Da and palmitic acid (palmitic acid) calculating a second molecule binding the molecular weight of the conjugates of 28,400 Da with a linear molecular structure However, in size-exclusion chromatography, molecular weight markers were found to be macromolecules with approximately ˜150 kDa.

At higher concentrations, one more peak was formed before the main molecule, which is believed to be a micelle structure. In the case of SDS-PAGE, a 0.5 mm thick 12% acrylamide gel was prepared and subjected to electrophoresis under denaturing conditions. Each sample was loaded with a molecular weight marker (BioRad, Precision Plus Protein Standard ^TM ). Electrophoresis was performed by flowing a current of 35 mA for 40-50 minutes, and 4PEG _5k -2C ₁₆ -2Ex4 was identified at higher positions than 4-arm PEG20k and 4-arm PEG10k as shown in the photograph of FIG. 3. In this case, after fixing with glutaraldehyde solution, bands of PEG moieties, not protein moieties, were stained using BaCl ₂ and iodine solution.

< Example  4> 4 PEG _5k -2 C ₁₆ -2 Ex4 Of albumin

Add 100 mg of CNBr-activated Sepharose 4B resin to 10 ml of phosphate buffer solution (10 mM, pH 8.0), add excess albumin (HSA 100 mg) to induce the reaction for 24 hours, and then about 500 rpm. After centrifugation for 30 seconds below the supernatant was removed. To this was added phosphate buffer solution and vortexed for 30 seconds and then washed five times by removing the supernatant in the same manner. Resin was identified by confocal laser scanning microscope (CLSM) by reacting rhodamine B with Sepharose 4B corresponding to 3 mg as albumin. In addition, 4PEG _5k -2C ₁₆ -2Ex4 conjugated with fluoresceine conjugated to the same amount of new Sepharose 4B at room temperature for 3 hours, and then the resin was similarly subjected to confocal laser scanning microscope (CLSM). Confirmed. The above result is shown in FIG.

< Example  5> 4 PEG _5k -2 C ₁₆ -2 Ex4 Blood sugar lowering effect

The hypoglycemic effect of the Ex4 derivative prepared by the present invention was confirmed and evaluated using 6-9 weeks old male C57BL / 6 db / db mice (Korea Research Institute of Bioscience and Biotechnology, Daejeon). db / db mice were acclimated for at least 1 week under constant conditions such as temperature (22 ± 3 ° C), humidity (55 ± 5%), and light (light and dark repeated 12 hours). Four to six mice were randomly divided per group before the experiment, and water and feed were freely taken in the middle of the experiment, and the prepared samples were intraperitoneally injected with 0.1 ml. Blood glucose levels were measured using a glucometer at 30 minutes, 1, 2, 3, 4, 6, 8, 12, 24, 36 and 48 hours immediately before and after injection. . As shown in FIG. 5 at a dose of 25 and _{250 nmo / kg 4PEG 5k -2C 16} -2Ex4 showed the hypoglycemic effect continuously extend more than 2.5 times its own Ex4.

Claims (11)

A conjugate of a polyethylene glycol (PEG) conjugated with an antidiabetic peptide and a fatty acid. The conjugate of claim 1 wherein said PEG comprises a derivative thereof.
The conjugate of claim 1, wherein the PEG is in a form having four linear PEG groups different from the center.
4. The PEG of claim 3 wherein each end of the PEG is succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, N-hydroxy succinimide, benzotriazole carbonate, maleimide and OPSS A conjugate, characterized in that it is modified in any one or more groups selected from the group consisting of. The conjugate according to claim 1, wherein the total molecular weight of PEG is selected from the range of 10,000-40,000 Da.
The conjugate of claim 1, wherein the antidiabetic peptide is synthetic or genetically modified.
The group of claim 1, wherein the antidiabetic peptide comprises GLP-1, exendin-4, insulin or agonist, derivative, fragment, variant, and combinations thereof. Combination characterized in that the selected from.
The conjugate according to claim 1, wherein the conjugated fatty acid has 6 to 20 carbon atoms.
(1) dissolving PEG-NHS with four arms in dimethylformamide and adding fatty acid amine;
(2) adding antidiabetic peptide dissolved in dimethyl sulfoxide to the solution prepared in step (1) and reacting for 6 hours or more; And
(3) dialysis with dimethylformimide and dimethyl sulfoxide in the solution prepared in step (2);
Polyethylene glycol (PEG) characterized in that it comprises an anti-diabetic peptide and a fatty acid conjugate method.
A blood sugar lowering composition comprising the anti-diabetic peptide-PEG-fatty acid conjugate of any one of claims 1 to 8 as an active ingredient. The composition of claim 10, wherein the composition lowers blood sugar in the body and is used to treat diabetes, obesity, and irritable bowel syndrome.

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