KR20130038426A - Glycol chitosan hydrogel modified hydrophobically with fatty acid-bonded antidiabetic peptide and a pharmaceutical composition for prevention or treatment of diabetes comprising the same - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing glycol chitosan hydrogel which is hydrophobically conjugated with a fatty acid-bonded antidiabetic peptide is provided to enhance gel formation in vivo and to enable long-term drug release. CONSTITUTION: Glycol chitosan hydrogel contains a fatty acid-bonded antidiabetic peptide and fatty acid-bonded glycol chitosan which are hydrophobically conjugated. The fatty acid has 12-18 carbon atoms. The fatty acid is substituted with N-hydroxysuccinimidyl ester or sulfo N-hydroxysuccinimidyl(sulfo-NHS) ester. The fatty acid is bonded by an acylate bond.

Description

Glycol chitosan hydrogel modified hydrophobically with fatty acid-bonded antidiabetic peptide and a pharmaceutical composition for prevention or treatment of diabetes configuring the same}

The present invention relates to a hydrophilic glycol chitosan hydrogel in which an anti-diabetic peptide to which a fatty acid is bound and a pharmaceutical composition for preventing or treating diabetes comprising the same, and more particularly, to an anti-diabetic peptide to which a fatty acid is bound, and a fatty acid to be bound. The present invention relates to a glycol chitosan hydrogel formed by self-assembling glycol chitosan through hydrophobic bonds and a pharmaceutical composition for preventing or treating diabetes.

Hydrogels have been of great interest as injectable drug carriers in the biomedical and pharmaceutical fields. These drug carriers provide a three-dimensional hydrophilic network structure, enabling physical and chemical encapsulation, retaining large amounts of water and drugs, and sustaining patterns of drug release. In addition, it is easily dosed through simple infusion through minimally invasive procedures and improves patient comfort. Recently, many hydrogels in the form of gels of self-cohesive and in situ have been studied, and these include a series of methods such as enzyme-mediated or complementary groups of chemical bonds, ionic bonds, lipophilic bonds, and complexes. do.

Various synthetic or natural polymers are used in the preparation of hydrogels. For example, there are PVA, poly (2-hydroxyethyl methacrylate), polyethylene glycol, alginate, chitosan, hyaluron and the like. In particular, natural polymers are mostly polysaccharides and do not present immunogenic weaknesses and the potential risk of pyrogenic substances of animal origin. Of these, chitosan is one of the most attractive natural polysaccharides, which are biosynthetic, low toxicity, susceptible to enzymatic degradation, and no immune response. Chitosan is prepared as a hydrogel through well established binding procedures using glutaraldehyde, glyoxal, diacetaldehyde, PEG and the like. In addition, chitosan exhibits hydrogelatinous properties by pH, because chitosan has a pKa of 6.0-6.5 and appears in a pH environment that exhibits physiological activity. Hydrogels using such chemically and physically bound chitosan are therefore of considerable interest as one of the most practical drug delivery systems.

Recently studied among such hydrogel systems are cross-linked polyphosphazene blend hydrogels ( Biomaterials 31: 8107-8120, 2010). This system attempted to maximize the gel formation ability and secure stable sustained-release drug characteristics by simultaneously using chemical cross-linking and physical cross-linking. Attempts have also been made to prepare injectable hydrogels that respond to pH changes using N-palmitoyl chitosan ( Biomaterials 30: 4877-4888, 2009). The study focused on the biocompatibility of glycolchitosan and its ability to gel at in vivo pH. In hydrogels, physical crosslinking methods include ionic complexes, polymer electrolyte complexes, thermoreversible hydrogels and hydrophobic bonding methods, and chemical crosslinking methods include a method of permanently bonding a polymer by using glutaraldehyde. Adv . Drug Deliv . Rev. 62: 83-99, 2010).

However, the method reported in the above literature has a clear limitation that the microcontrolling, biocompatibility and toxicity problems have not been completely solved in bioapplications, and thus a more specific and practical hydrogel method is required.

Exendin-4 (Ex4) is a peptide substance isolated and purified from the saliva of a lizard called Gila monster (Heloderma suspectum), consisting of the following 39 amino acid sequences and 53% sequence similarity to endogenous glucagon-like peptide-1 (GLP-1) ( Diabetes 53: 2181-2189, 2004). It has a similar physiological effect as GLP-1, but the amino acid 2 is Gly, which not only has a strong resistance to DPP IV but also has a slower glomerular filtration rate than GLP-1, resulting in prolongation of GLP-1. It is known to have half-life and bioactivity ( Diabetologia 46: 706-712, 2006; Diabetes 53: 2181-2189, 2004). It was first developed by John Eng and it is known that the material patent applies only to the United States (U.S. Pat.

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

As described above, Ex4 is known to inhibit gastrointestinal motility, reduce food intake, and inhibit plasma glucagon secretion (US Pat. Nos. 6858576, 6956026, and 6872700), oral sulfonyl urea and metformin. Both antidiabetic and combination therapies have been reported to lower the levels of glycated hemoglobin (Hb _A1C ) within 1% after 28 days of administration ( Diabetes 53 suppl. 3: S197-S204, 2004). Recently, Byetta ^? Synthetic Ex4 (Exenatide; Amylin & Eli Lily) has been marketed by the US Food and Drug Administration (FDA) since April 2005 and has been used as a drug.

Recently, attempts have been made to solve the problem of short biological half-life of polypeptides by physicochemically binding albumin to polypeptide antidiabetics such as Ex4, glucagon-like peptide-1 and insulin. 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 has a half-life of about 19 days ( 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).

The albumin protein binding induction technique using a long fatty acid chain was developed by Novo Nordisk and introduced a C ₁₄ ~ C ₁₆ fatty acid to the lysine amine of the polypeptide. In general, one molecule of albumin has a binding constant of 10 ⁷ to 10 ⁸ , and about five fatty acids can be hydrophobicly bound. Novo Nordisk sold Insulin detemer (Levemir ^? ), Produced by binding myristic acid (C ₁₄ ) to human insulin, in the United States in June 2005 (http://www.levemir-us.com). It is known that it will be marketed jointly with Eli Lily after completing the clinical study of NN2211 (Liraglutide ^? ), Which produced acid (C ₁₆ ) by binding amino acids to Lys ²⁶ of the GLP-1 analogue ( Diabetes 53: 2181-2189). , 2004).

In view of the above, the inventors of the present invention, while preparing a glycol chitosan hydrogel by self-assembly of a fatty acid-bound anti-diabetic peptide and a glycol chitosan to which a fatty acid is bound through a hydrophobic bond, lowering its blood sugar The present invention was completed by confirming the effect.

It is an object of the present invention to provide an antidiabetic peptide to which a fatty acid is bound, and a glycol chitosan hydrogel to which a glycol chitosan to which a fatty acid is bound is hydrophobicly bound.

Another object of the present invention to provide a pharmaceutical composition for preventing or treating diabetes comprising the glycol chitosan hydrogel.

In order to solve the above problems, the present invention provides an anti-diabetic peptide combined with a fatty acid, and a glycol chitosan hydrogel in which a hydrophilic glycol chitosan is bonded to a fatty acid.

In the present invention, the fatty acid-bound antidiabetic peptide is surface-modified to the glycol chitosan to which the fatty acid is bound through hydrophobic interaction to form a glycol chitosan hydrogel.

Specifically, the chemical structure and manufacturing method of glycol chitosan hydrophobicity enhanced through palmitic acid binding in Figure 1 (a), Exendin-4, and palmitic acid-bound glycol chitosan combined with palmitic acid through a hydrophobic bond with each other The process (b) of self-assembly to form a glycol chitosan hydrogel is shown briefly.

The fatty acid is preferably a fatty acid having a carbon number of about 12 to about 18 so as to induce protein binding with albumin in the body. In addition, since the fatty acid has a higher degree of albumin binding than the nonlinear cyclic type, the fatty acid is preferably linear.

For the preparation of the antidiabetic peptide to which the fatty acid is bound, the fatty acid is preferably activated to react with the amine of the antidiabetic peptide. Specifically, the fatty acid is preferably substituted with N-hydroxysuccinimidyl (NHS) ester or sulfo N-hydroxysuccinimidyl (Sulfo-NHS) ester.

In the present invention, the fatty acid bond is preferably an acylate bond.

In the present invention, the antidiabetic peptide is preferably Exendin-4 (Exendin-4). Wherein the antidiabetic peptide exendin-4 may be synthetic and recombinant.

In the present invention, the average molecular weight of the glycol chitosan is preferably 30 to 90 kDa.

According to the present invention, when preparing a conjugate of a fatty acid and an antidiabetic peptide, both of them induce a reaction in a soluble solvent of dimethyl sulfoxide (DMSO) and induce a fatty acid to be reacted with the antidiabetic peptide in a 1: 1 equivalent. The reaction takes about 1 hour. An appropriate amount of triethylamine (TEA, triethylamine) or dimethylaminopyridine (DMAP) may be added thereto to shorten the reaction rate.

Since the hydrogel is applied to a living body, the polymer used in the preparation of the polymer should be biocompatible and biodegradable and therefore safe for living cells, and can be decomposed and lost at the end of the release of the fatty acid conjugated exendin-4. desirable. Therefore, chitosan (chitosan) is so large that the molecular weight of biodegradability should not be limited, so their molecular weight is preferably about ~ 40 kDa and fractions over 80 kDa should be 5% or less. In addition, it should be soluble in both organic solvents such as water and dimethyl sulfoxide, ethylene oxide (ethylene oxide) is preferably bonded to secure the amphiphilic.

Fatty acid binding to chitosan is excessive when the fatty acid is excessively bound to chitosan, so the hydrophobicity is too large to induce gel formation and tends to harden. If the fatty acid bond is too small, the formation of hydrogel by chitosan itself may be very weak. The adequacy of the ratio should be ensured.

Specifically, the ratio of fatty acid bound to chitosan is preferably 10 to 30 parts by weight based on 100 parts by weight of chitosan.

The fatty acid conjugated exendin-4-containing glycolchitosan prepared as described above continuously releases the drug and binds to albumin in the blood in the systemic circulation, thereby increasing the remaining period of the body and thus improving diabetes treatment (hypoglycemia).

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, including the glycol chitosan hydrogel.

These pharmaceutical compositions increase gel formation and induce hydrophobic bonds with fatty acid conjugated antidiabetic peptides, which are very useful for prolonged sustained release of the drug and are absorbed to induce reversible bonds with albumin in the blood to sustain bio-half and hypoglycemic effects. Has superior characteristics that can greatly improve.

In the experimental example of the present invention, it was confirmed that the glycol chitosan hydrogel has an excellent hypoglycemic effect.

The pharmaceutical compositions of the invention can be formulated in oral or injection dosage forms. Formulations for oral administration include, for example, tablets, capsules, etc. These formulations may contain, in addition to the active ingredient, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g. : Silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpicolidine, optionally starch, agar, alginic acid or its Disintegrating or boiling mixtures such as sodium salts and / or absorbents, colorant anti-flavoring agents, and sweetening agents. Injectable formulations are preferably aqueous isotonic solutions or suspensions.

The composition may be sterilized and / or contain adjuvants such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure and / or buffers, and other therapeutically useful substances.

The formulations may be prepared by conventional mixing, granulating or coating methods and may contain the active ingredient in the range of about 0.1 to 75% by weight, preferably about 1 to 50% by weight. The unit dosage form for about 50-70 kg mammals contains about 10-200 mg of active ingredient.

Preferred dosages of the glycol chitosan hydrogels of the invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer the glycol chitosan hydrogel of the present invention at 0.0001 to 100 mg / kg body weight, preferably 0.001 to 100 mg / kg body weight. Administration can be administered via oral or parenteral routes once or divided daily.

The pharmaceutical composition of the present invention can be administered to mammals including rats, mice, livestock and humans by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerbroventricular injections.

The present invention can provide an antidiabetic peptide to which a fatty acid is bound, a glycol chitosan hydrogel to which a hydrophilic glycol chitosan is bonded to a fatty acid, and a pharmaceutical composition for preventing or treating diabetes, including the same. The hydrogel with enhanced hydrophobicity increases gel formation in vivo and is very useful for prolonged sustained release of the drug by inducing hydrophobic binding with antidiabetic peptides bound with fatty acids, as well as lowering plasma glucose due to exendin-4, It can be used therapeutically for insulin dependent and non-dependent diabetes mellitus, obesity, irritable bowel syndrome by exerting such effects as suppressing stomach and intestinal motility, suppressing stomach and intestinal fasting or inhibiting food intake.

1 is a chemical structure and manufacturing method of glycol chitosan hydrophobicity enhanced through palmitic acid binding (a), as well as exendin-4 and palmitic acid-bound glycol chitosan self-assembled through hydrophobic bonding (b) is a simplified illustration of the process of self-assembly to form a glycol chitosan hydrogel.
2 shows the synthesized Pal-GC shows H NMR. In this case, a represents an H NMR chart of glycol chitosan, and b represents an H NMR chart of parylated glycol chitosan.
Figure 3 shows the appearance of a glycol chitosan hydrogel of the present invention. In this case, a represents glycol chitosan and palytylated glycol chitosan before gel formation, and b represents glycol chitosan and palytylated glycol chitosan after gel formation.
Figure 4 shows the results of morphology confirmation of the present invention glycol chitosan hydrogel using a scanning electron microscope. Where a is a reaction molar ratio of glycol chitosan and N-hydroxysuccinimidyl (NHS) ester-bonded palmitic acid is 1: 0, b is 1: 45, c is 1: 75, d is 1: 100, e is 1: 150, and f is 1: 200.
5 is a graph showing the blood glucose lowering effect investigation result of the present invention glycol chitosan hydrogel.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only examples showing some experimental methods and compositions of the present invention, but the scope of the present invention is not limited thereto.

Example  One: Palmitic acid  ( palmitic acid ) Conjugated Glycol Chitosan Hydrogel  Produce

33-124.5 mg of N-hydroxysuccinimidyl-activated palmitic acid dissolved in 10 mL of DMSO containing 0.3% DMAP was obtained with 200 mg of glycol chitosan (GC) (average molecular weight: 62 kDa, manufactured by Sigma-Aldrich, country of origin: USA ). Then acylation was induced for 6 hours at room temperature. As a result, the mixture was dialyzed in DMSO, followed by dialysis in DW for 48 hours using a cut off 10 kDa membrane. Finally the obtained GC derivative solution was lyophilized and stored frozen until used at -70 ° C.

Synthesized Pal-GC was analyzed by H NMR and compared by adjusting the GC (Fig. 2).

In Figure 2 a is a H NMR Chart of glycol chitosan, b is a H NMR Chart of the parylated glycol chitosan.

Example  2: Palmitic acid  ( Palmitic acid )this Bonded Ex4 Preparation and Separation

N-hydroxysuccinimidyl Palmitic acid was dissolved in dimethylsulfoxide at a concentration of 2.5 mg / ml at room temperature, and 0.5 ml of this solution was taken. 0.3% triethylamine-containing dimethyl sulfoxide 3 dissolved in 15 mg of Ex-4 (American Peptide Company) 3 The reaction was induced by addition to ml. After about 1 hour, 1.5 ml of 50% distilled water / acetonitrile solution containing 1% trifluoroacetic acid was added to terminate the reaction. Thereafter, the reaction solution was injected into reversed-phase HPLC to separate Ex4 conjugated with palmitic acid, and the fractions were collected. The mobile phase used was distilled water and acetonitrile containing 0.1% trifluoroacetic acid, and the ratio of acetonitrile containing 0.1% trifluoroacetic acid was adjusted from 20% to 30%. The chromatogram was obtained at a wavelength of 215 nm. The collected fractions were acetonitrile removed under a nitrogen stream, the filtrate was concentrated using Centricon (MW cut off 3,000), and then replaced with a buffer solution of the desired pH to secure and store the final sample.

Example  3: Palmitic acid  join Ex4 , And Palmitic acid  Conjugated Glycol Chitosan Hydrophobic Combined  Glycol Chitosan Hydrogel  Produce

A portion (10 mg) of Pal-GC (45 eq.) Prepared in Example 1 was dissolved in 5 ml of DMSO, and this solution was conjugated to Ex4 (Ex4-C16) in which palmitic acid (Palmitic acid) prepared in Example 2 was conjugated. (250 nmol / kg) was added. The solution was mixed for 12 hours using a magnetic bar and dialyzed for 12 hours in DW.

Figure 3 shows the appearance of the prepared glycol chitosan hydrogel.

In Fig. 3, a denotes glycol chitosan and palytylated glycol chitosan before gel formation, and b denotes glycol chitosan and palytylated glycol chitosan after gel formation.

3, it can be seen that the gel-forming ability of the glycol chitosan hydrogel prepared in the present invention is excellent.

Experimental Example  1: Glycol Chitosan of the Invention Using a Scanning Electron Microscope Hydrogel  Check shape

The form of the glycol chitosan hydrogel prepared in Example 3 was confirmed by SEM.

First, the dried powder of each sample was attached to the sample stub using double-sided tape. And sputtered in the argon atmosphere. The morphology of the glycol chitosan hydrogel was then observed using SEM.

The observation results are shown in FIG. 4.

In Figure 4, the reaction molar ratio of palmitic acid is a is 1: 0, b is 1: 45, c is 1: 75, d is 1: 100, e is 1: 150, f Represents the form of 1: 200.

4, it can be seen that as palmitylation progresses, the internal structure of the glycolchitosan hydrogel becomes more dense.

Experimental Example  2: present invention glycol chitosan Hydrogel  Checking hypoglycemic effect

The hypoglycemic effect of the glycol chitosan hydrogel of the present invention prepared by Example 3 was confirmed and evaluated using 6-9 weeks old male C57BL / 6 db / db mice (Korea Research Institute of Bioscience and Biotechnology, Daejeon).

First, db / db mice were adapted 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. Male db / db mice (6-7 weeks old) were used for hypoglycemic effect testing. In non-fasting situations, animals were injected once with saline, Ex4, Ex4-C16-loaded Pal-GC hydrogels. Blood glucose was measured using a one-touch blood glucose meter and administered as mentioned above and monitored for 96 hours. The hypoglycemic effect was expressed as the total hypoglycemia and used the following formula.

[(AUC _{saline , 0- last time} -AUC _{test , 0- last time} ) / AUC _{saline , 0- last time} ] x 100

The hypoglycemic effect investigation result of the glycol chitosan hydrogel of this invention is shown in FIG.

5, it can be seen that the present invention glycol chitosan hydrogel has an excellent hypoglycemic effect.

Claims (7)

Antidiabetic peptides to which fatty acids are bound, and glycol chitosan hydrogels to which hydrolysis of glycol chitosan to which fatty acids are bound.
The glycol chitosan hydrogel of claim 1, wherein the fatty acid is a fatty acid having 12 to 18 carbon atoms.
The glycol chitosan hydrogel of claim 1, wherein the fatty acid is substituted with an N-hydroxysuccinimidyl (NHS) ester or a sulfo N-hydroxysuccinimidyl (Sulfo-NHS) ester.
The glycol chitosan hydrogel of claim 1, wherein the fatty acid linkage is an acylate linkage.
The glycol chitosan hydrogel of claim 1, wherein the antidiabetic peptide is Exendin-4.
The glycol chitosan hydrogel of claim 1, wherein the average molecular weight of the glycol chitosan is 30 to 90 kDa.
A pharmaceutical composition for preventing or treating diabetes, comprising the glycol chitosan hydrogel of any one of claims 1 to 6.

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