KR20110080071A - Albumin-conjugated anti-diabetic peptide derivatives via polyethylene glycol linkages - Google Patents

Abstract

PURPOSE: A composition containing an anti-diabetic peptide-PEG-albumin conjugate is provided to reduce renal clearance rate and to improve blood glucose reduction. CONSTITUTION: An anti-diabetic peptide-PEG-albumin conjugate is prepared using polyethylene glycol(PEG) as a leakage compound. PEG is modified to succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, N-hydroxy succinimide, or benzotriazole carbonate at one end and is modified to maleimide or OPSS at the other end. The anti-diabetic peptide is synthetic type or gene recombination type. 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 the diabetic peptide-PEG-albumin conjugate as an active ingredient.

Description

Albumin-conjugated anti-diabetic peptide derivatives via polyethylene glycol linkages

The present invention relates to a conjugate or albumin conjugated with albumin by using polyethyleneglycol as an antidiabetic peptide and a composition for lowering blood glucose.

Glucagon-like peptide-1 (GLP-1) is a 30 (7-36) or 31 (7-37) amino acid produced from proglucagon, which is secreted from intestinal L cells in response to oral ingestion. As a peptide consisting of, it is known to stimulate insulin secretion and inhibit glucagon secretion in a concentration-dependent manner of glucose present in plasma to stably lower blood sugar. 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

Albumin has 35 Cys amino acids, but 34 of them form disulfide bonds with each other, and only Cys ³⁴ amino acids are freely exposed. It is known.

In order to overcome the problem of long-term, frequent administration due to the short half-life and duration of the antidiabetic peptide as described above, an albumin-conjugated conjugate or derivative thereof is prepared by using polyethylene glycol (PEG) as a linking compound to the antidiabetic peptide. By confirming that the half-life and hypoglycemic effect are greatly improved when it is injected in vivo, the present invention has been completed.

Accordingly, an object of the present invention is to provide a conjugate or derivative thereof in which albumin is conjugated by using polyethylene glycol as a linking compound to an antidiabetic peptide.

Still another object of the present invention is to provide a composition for lowering blood sugar, including the conjugate or a derivative thereof.

In the present invention, a polyethylene glycol (PEG) molecule covalently reacting with the primary amino group (-NH ₂ ) of the antidiabetic peptide reacts secondly with the thiol group (-SH) of albumin and is covalently bound to antidiabetic Peptide-PEG-albumin conjugates and derivatives thereof are provided.

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) located in the middle is preferably bi-functional at both ends.

One side of the PEG may be N-hydroxysuccinimidyl (NHS) esters or sulfo N-hydroxysuccinimidyl (Sulfo-NHS) esters to react with amines. It is preferable that it is done.

More specifically,

One end of the PEG is Succinimidyl succinate, Succinimidyl propionate, Succinimidyl butanoate, N-hydroxy succinimide And at least one group selected from the group consisting of Benzotriazole carbonate.

It is also preferred that the other end of PEG is modified with maleimide (MAL) or orthopyridyl disulfide (OPSS) to react with thiols.

It is preferable that PEG of this invention is 1000-20,000 Da of the molecular weight range. If the molecular weight is too small and less than 1000, the antidiabetic peptide molecule is too close to albumin, which may reduce the degree of receptor binding. If the molecular weight is more than 20,000, the effective molecular size of its own increases, and albumin conjugation shows a great effect. This is because it may not be, and there is a fear of a significant decrease in physiological activity in vitro.

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 As a peptide consisting of, it stimulates insulin secretion depending on the glucose concentration present in plasma, and suppresses glucagon secretion, thereby stably lowering blood glucose.

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 has been 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 by 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.

When albumin and PEG (maleimide (MAL) or OPSS (orthopyridyl disulfide) are combined, the albumin's thiol (-SH) group is conjugated. As described above, only one of the 35 Cys amino acids can react with albumin, but the Cys ³⁴ amino acid is also known to be very poor. Therefore, in the present invention, a method of introducing a thiol group into the amine of albumin and reacting it with PEG is preferable. .

These reactions include Traut 'reagent (2-iminothiolane), SATA (N-succinimidyl S-acetylthioacetate), SATP (succinimidyl acetylthiopropionate), SPDP (N-succinimidyl 3- (2-pyridyldithio) propionate), SMPT (succinimidyloxycarbonyl-α-methyl -α- (2-pyridyldithio) toluene) and the like can be used.

The albumin may be wild-type or recombinant, and the anti-diabetic peptide may be synthetic or recombinant.

In the embodiment of the present invention, when preparing the primary conjugate of the anti-diabetic peptide and PEG induce a reaction in dimethyl sulfoxide (DMSO, dimethyl sulfoxide), 1.5 equivalents of PEG than the anti-diabetic peptide and takes up to 1 hour It became.

An appropriate amount of triethylamine (TEA, triethylamine) or dimethylaminopyridine (DMAP) may be added thereto to shorten the reaction rate.

When preparing a final conjugate in which the anti-diabetic peptide-PEG conjugate was combined with albumin, a pre-calculated equivalent number of albumin was added to the albumin and reacted at pH 6.5-7.5 for up to 3 hours.

The present invention also provides a composition for lowering blood glucose, which contains an anti-diabetic peptide-PEG-albumin conjugate as an active ingredient.

The anti-diabetic peptide-PEG-albumin conjugate prepared as described above has reduced glomerular filtration in the kidney and resists various enzyme attacks in vivo, thereby increasing the remaining period in the body to have an improved diabetes treatment effect (hypoglycemia) (Example 5 ).

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

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 according to 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 (10-500 nmol / kg as antidiabetic peptide) can be added once a day or divided several times a day.

The antidiabetic peptide conjugate conjugated with polyethylene glycol and albumin of the present invention can provide antidiabetic peptide compounds, derivatives and pharmaceutical compositions having reduced kidney clearance and thus prolonging half-life and ultimately extending 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 shows the molecular structure of the 3: 3: 1 ratio of the final derivative in which albumin is conjugated using polyethylene glycol as a linking compound to the antidiabetic peptide.
FIG. 2 is a schematic of a conjugation scheme of Ex4 and polyethylene glycol, an example of a scheme when converting a primary amine of Lys amino acid of albumin to thiol, and an overall scheme of conjugation of these two molecules to prepare a final derivative. It is shown.
Figure 3 shows the chromatogram and SDS-PAGE of the molecular exclusion liquid chromatography (SE-HPLC) of the Ex4-PEG-albumin derivatives prepared in Example 2 (size exclusion high-performance liquid chromatography).
Figure 4 shows the results of the persistence of the hypoglycemic effect of the prepared Ex4-PEG-albumin derivatives in type 2 diabetes-induced db / db mice.
Figure 5 shows the results of comparing the persistence of the hypoglycemic effect of the prepared Ex4-PEG-albumin derivatives at the high dose with Ex4 itself.

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 Lys ¹² Or Lys ²⁷ Preparation of Ex4 Derivative Conjugates One Molecular Polyethyleneglycol to an Amine at the Amino Acid Position

3 mg of Ex-4 (American Peptide Company) was dissolved in 1 ml of dimethylsulfoxide, and to this was added a solution of MAL-PEG-NHS (5.4 mg; Mw: 5000 Da; 1.5 equiv) dissolved in 0.5 ml of dimethylsulfoxide. It was left at room temperature for about 1 hour. Triethylamine can be added to dimethylsulfoxide in concentration ranges up to 0.3% (v / v) for rapid reaction progress. After 1 h of reaction, dimethyl sulfoxide was removed using a column filled with Sephadex G-25 (GE Healthcare) resin (1 ⅹ 30 cm, Bio-rad) and the final reaction solution was phosphate buffered solution (10 mM, pH 7.4). To be preserved. This was quickly concentrated using Centricon (MW cut off 3000), and then the whole amount was loaded on a Superdex 200 (GE Healthcare, 1 ⅹ 30 cm) column to fractionate the monomer peaks and stored until later albumin conjugation. In this case, a phosphate buffer solution (10 mM, pH 7.0) was used as the mobile phase.

Example 2 Introduction of Cheol Group to an Amine Group of Albumin

10 mg of albumin was dissolved in phosphate buffer (50 mM, pH 8.0) at a concentration of 1 ml, then 2-iminothiolane (about 62) dissolved in the same buffer so that about 3 of 59 Lys amino acid amines in one molecule of albumin could react. μg) was added to 0.5 ml. This was left at room temperature for about 1 hour to induce a reaction. The chemical reaction is shown in FIG.

Example 3 Binding of Polyethylene Glycol to Ex4 and Albumin Introduced with Thiol and Its Derivative

Each solution was mixed so that the equivalents of the polyethylene glycol conjugated Ex4 derivative prepared as in Example 1 and the thiol group-introduced albumin prepared as in Example 2 were 5: 1 in order, respectively, and the total amount of the solution was 5 ml. The pH was adjusted to 7.0 and the reaction was performed at room temperature for about 3 hours to complete the reaction. After the reaction, the reaction was loaded on a Superdex 200 (GE Healthcare, 1 × 30 cm) column to collect the first fractions which preceded albumin on the chromatogram, which was used as a sample for the in vivo animal experiment conducted in Example 5.

Example 4 Identification of Ex4-PEG-Albumin

Size-Exclusion HPLC and SDS-PAGE were used to confirm the molecular weight and purity of the prepared Ex4-PEG-albumin. A Superdex 200 (GE Healthcare, 1 × 30 cm) column was used where the mobile phase was phosphate buffered solution (10 mM, pH 7.0) added with 150 mM NaCl. As shown in FIG. 3, Ex4-PEG-albumin showed a much higher molecular weight (˜150 kDa) than albumin itself since Ex and PEG were each bonded by 3 molecules. In the case of SDS-PAGE, a 0.5 mm thick 12% acrylamide gel was prepared and subjected to electrophoresis under denaturing conditions. With each sample, a molecular weight marker (BioRad, Precision Plus Protein Standard) was used. ^TM ) was loaded. Electrophoresis was performed by flowing a current of 35 mA for 40-50 minutes, and as shown in the photograph of FIG. 3, Ex4-PEG-albumin showed a band showing a higher molecular weight than albumin and Ex4 itself.

<Example 5> Checking the hypoglycemic effect of Ex4-PEG-albumin

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 is collected from the tail vein at 30, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 hours immediately before and after injection of the sample using a glucometer. Blood glucose concentrations were measured. As shown in Figure 4, Ex4-PEG-albumin at a dose of 250 nmo / kg showed a sustained prolonged hypoglycemic effect than the Ex4-albumin group without Ex4 itself and PEG.

 The hypoglycemic efficacy of Ex4-PEG-albumin and Ex4 itself was compared at doses of 25 and 250 nmo / kg and showed about four times longer than that of Ex4 itself (FIG. 5). In addition, the dose of 500 nmo / kg showed a blood concentration below 150 mg / dl for about 61 hours after administration.

Claims (10)

An antidiabetic peptide-PEG-albumin conjugate comprising polyethylene glycol (PEG) as a linking compound.
The conjugate of claim 1 wherein said PEG comprises a derivative thereof.
The compound of claim 1, wherein one end of the PEG is succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, N-hydroxy succinimide and benzotriazole carbonate capable of reacting with an amine. The conjugate is modified with any one or more groups selected from the group consisting of, the other end is modified with maleimide or OPSS that can react with the thiol.
The conjugate according to claim 1, wherein the PEG has a molecular weight selected from the range of 1,000 to 20,000 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 combination of claim 1, wherein the albumin is a part of the amine is chemically modified with thiol.
A blood sugar lowering composition comprising the diabetic peptide-PEG-albumin conjugate of any one of claims 1 to 7 as an active ingredient.
The composition of claim 8, wherein the composition lowers blood sugar in the body and is used to treat diabetes, obesity, and irritable bowel syndrome.
The composition of claim 8, wherein the composition is administered in an effective amount of 10-500 nmol / kg.

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