KR101761092B1 - Composition for Transplanting of Islet Cells - Google Patents

Abstract

The present invention relates to a composition for pancreatic cell transplantation comprising a biocompatible polymer backbone bound with glycyrrhizin. When the composition for pancreatic islet transplantation or the method for producing pancreatic islet cells for transplantation of the present invention is used, the pancreatic islet cell composition of the present invention or the pancreatic islet cell of the present invention - Islet cell-deficiency When a pharmaceutical composition for improving, preventing or treating disease is used, it is possible to effectively transplant an islet cell without immunological rejection reaction, and effectively treat various diseases caused by an islet cell defect .

Description

[Technical Field] The present invention relates to a composition for transplantation of islet cells,

The present invention relates to a composition for pancreatic cell transplantation comprising a biocompatible polymer backbone bound with glycyrrhizin.

In pancreatic islet transplantation, the focus is on the immune response to transplanted pancreatic islet cells and the regulation of the inflammatory response that occurs in the transplantation site. More than 60% of the transplanted pancreatic islet cells are rejected due to immune and inflammatory responses at the early stage of transplantation. The major cause is the HMGB1 protein, a transcription factor protein in the cell. The HMGB1 protein is present in the cell as a gene transcriptional protein, but it is secreted out of the cell due to the surgical damage and immune reaction caused by the transplantation, and binds to the cell membrane receptor such as TLR2, TLR4 and RAGE of peripheral immune cells to induce immune reaction and inflammatory reaction In the end, through this process, the transplanted pancreatic islet cells are rejected. In particular, the development of techniques to regulate the release of HMGB1 protein in successful pancreatic islet transplantation is a very important issue because of the presence of HMGB1 protein in the pancreas compared to other organs.

In order to prevent the action of HMGB1 early in the transplantation of islet cells, an attempt was made to reduce the immune response by intravenously injecting an antibody of HMGB1 into the recipient animal (Matsuoka N, et al. High-mobility group box 1 is involved in the initial events of early transfusion of islets in mice. Journal of Clinical Investigation 120: 735-743 (2010).

In addition, DHMEQ, which blocks NF-κB, which is one of the transcription factors, and antithromin III, which is an anticoagulant when transplanted with islets in the liver, (Kuraya D, et al., Efficacy of DHMQ, a NF-kappaB inhibitor, in islet transplantation: I. HMGB1 suppression by DHMEQ and early islet graft damage. Transplantation 96: 445-453 (2013); Kojima D, et al ., Prevention of high-mobility group box 1-mediated early loss of transplanted mouse islets in the liver by antithrombin III Transplantation 93:. 983-988 (2012)).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a composition for pancreatic cell transplantation for reducing the immune rejection reaction during pancreatic cell transplantation. As a result, the present inventors have completed the present invention by confirming that when the biocompatible polymer backbone bound with glycyrrhizin is used, the immune rejection reaction upon islet cell transplantation can be significantly reduced.

Accordingly, an object of the present invention is to provide a composition for pancreatic cell transplantation.

It is another object of the present invention to provide a method for producing an islet cell for transplantation.

It is still another object of the present invention to provide an islet cell composition for transplantation.

It is another object of the present invention to provide a pharmaceutical composition for improving, preventing or treating an islet cell-deficiency disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for pancreatic cell transplantation comprising (a) a biocompatible polymer backbone and (b) glycyrrhizin bound to the biocompatible polymer backbone.

The present inventors have made extensive efforts to develop a composition for pancreatic cell transplantation for reducing the immune rejection reaction during pancreatic cell transplantation. As a result, it was found that the use of glycyrrhizin - conjugated biocompatible polymer backbone could significantly reduce the immune rejection response of pancreatic islet transplantation.

It has been known that "glycyrrhizin" of the present invention, as a licorice extract component, directly binds to the HMGB1 protein and inhibits its activity. However, there is no known technology for the surface modification of islet cells using glycyrrhizin. The present inventors have found that biocompatible polymer backbone binds glycyrrhizin and glycyrrhizin-bound biocompatible polymer backbone is used for islet cell surface modification , The transplantation success rate of islet cells can be significantly improved. As shown in the following examples, when the biocompatible polymer backbone with glycyrrhizin of the present invention was used, it was confirmed that the survival rate of the pancreatic islet cell increased up to 3-fold.

In one embodiment of the invention, the glycyrrhizin of the invention is an oxidized form of glycyrrhizin. As shown in Fig. 1, the oxidized form of glycyrrhizin causes the bond between the carbon 2 and carbon 3 of the end glucuronic acid ring to be opened by oxidation to form an aldehyde substituent. By virtue of the aldehyde groups thus formed, glycine can form bonds with the biocompatible polymer backbone.

As used herein, the term "biocompatible polymer backbone" means a polymer having tissue compatibility and blood compatibility that does not cause tissue destruction or coagulation of blood in contact with living tissue or blood, Which is capable of forming a bond with the glycyrrhizin of the present invention.

In one embodiment of the present invention, the biocompatible polymer backbone of the present invention includes a functional group capable of forming a chemical bond with an aldehyde group. As described above, the oxidized form of glycyrrhizin includes an aldehyde group, and any biocompatible polymer containing a substituent capable of forming a chemical bond with an aldehyde group of glycyrrhin can be used without limitation.

In one embodiment of the present invention, the functional group contained in the biocompatible polymer backbone of the present invention is an amine group, a carboxyl group, a thiol group, or a hydroxide group. Preferably, a biocompatible polymer backbone containing an amine group can be used.

In one embodiment of the present invention, the biocompatible polymer backbone of the present invention comprises at least one of glycol chitosan, poly-L-lysine, poly (4-vinylpyridine / divinylbenzene), chitin, poly (butadiene / acrylonitrile) (2-vinylpyrrolidone), poly (vinylamine) hydrochloride, poly (2-vinylpyridine), poly (2-vinylpyrrolidone) (Allylamine), poly (allylamine hydrochloride), poly (N-methylvinylamine), poly (N-methylvinylamine) , Poly (diallyldimethylammonium chloride), poly (N-vinylpyrrolidone), chitosan, or poly (4-aminostyrene).

The transplantation of the pancreatic islets may be performed by selecting appropriate implantation sites known in the art (e.g., under the renal capsule and portal vein, etc.) and transplanting the transplantation site percutaneously through the portal vein in a known manner, such as ultrasound- And injecting the prepared islets into the liver. The condition of the suitable graft for transplantation is the ABO type, with an islet number of 5,000 / kg (object weight), an islet purity of 30% or more, a final volume of 10 ml or less, Endotoxin negative conditions result in transplantation conditions (Shapiro J et al., International trial of the edmonton protocol for islet transplantation, N Engl J Med 355: 1318-1330 (2006)).

The composition of the present invention is used in at least one of (a) pre-implantation cell pretreatment and (b) local administration of the transplantation site to the subject to which the cell has been implanted. In the present invention, pretreatment of cells for transplantation means preparing cells for transplantation into an object and treating the composition of the present invention to transplantable cells before transplanting to an object. And the composition of the present invention is brought into contact with the grafted cells under the condition that the composition of the present invention can be bound to the surface to form mutual bonds. As a specific example of a method capable of forming the above-described "binding condition ", there are a method of modifying a polymer backbone so as to have a substituent capable of forming a chemical bond with an amine group on the surface of an islet cell, And a linker capable of mediating the link. As a concrete example of the linker, PEG can be exemplified. In particular, pegyline islets can be pegylated using various bifunctional PEGs, and the polymer backbone bound with glycyrrhizin can be additionally bound to the pegylated surface. The local administration of the transplantation site to the cell-implanted object can be performed either at the same time as the cell transplantation or immediately after the cell transplantation, as described above, by modifying the islet cell surface to have a substituent capable of forming a chemical bond with the amine group By using a polymer backbone or by transplanting pegylated islet cells pegylated with, for example, a bifunctional PEG, which is specifically linked, for example, with the linker described above, and then locally injecting the composition of the present invention into the transplantation site, The biocompatible polymer backbone to which the glycyrrhizin is bound can be bound to the surface of the cell.

The composition of the present invention is preferably used for cell pretreatment before implantation. The pre-treatment of cells prior to transplantation can prevent rejection of transplantation. Therefore, it is more advantageous in terms of efficiency and stability than existing drugs that can only be administered systemically after transplantation or local administration of transplantation site. .

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

According to another aspect of the present invention, the present invention provides a method of treating a pancreatic islet cell isolated from a donor by treating the pancreatic cell transplantation composition described above with a glycyrrhizin-conjugated biocompatible polymer backbone The method comprising the steps of:

The method for producing an islet cell for transplantation according to the present invention relates to a method using an islet cell transplantation composition as an embodiment of the present invention described above, and redundant contents are omitted in order to avoid the excessive complexity described in the present specification.

In the method of the present invention, the "treatment" of the composition for pancreatic cell transplantation to the pancreatic islet cell is the same as that of the cell pretreatment before transplantation described above. That is, under the condition that the composition of the present invention can bind to the surface of the isolated transplantable cell The cells for transplantation are brought into contact with the composition of the present invention to form mutual bonds. The above "condition that can be combined" is the same as described above.

In one embodiment of the invention, the pancreatic islets of the invention are pegylated. Although a polymer backbone incorporating a substituent capable of forming a bond with an amine group on the surface of an islet cell can be used, the efficiency of binding between the islet cell and the polymer backbone can be enhanced by pegylation.

In one embodiment of the invention, the pegylation of the present invention is carried out using heterobifunctional PEG. The first step of the pegylation is to substitute one end or both ends of the PEG polymer with an appropriate functional group. In the case of the present invention, since pegylation is used to mediate the chemical bonding between the islet cell and the polymer backbone, Functional groups can be used. As used herein, the term " heterobifunctional PEG "means a PEG in which both ends of the PEG are each substituted with a suitable functional group, as described above.

In one embodiment of the invention, the heterobifunctional PEG of the present invention comprises a maleimide substituent at one end and an NHS ester or carboxylic acid substituent at the other end. The "maleimide" substituent of the present invention is a substituent capable of forming a chemical bond by reaction with a sulfhydryl (-SH) group, and the "NHS ester" or "carboxylic acid" substituent reacts with an amine group to form a chemical bond Lt; / RTI >

Thus, by using the heterobifunctional PEG of the present invention, a bond between the NHS ester or carboxylic acid substituent at one end of the PEG and the amine group of the pancreatic cell surface can be formed. On the other hand, it is necessary to form a bond between the secondary pegylated islet cell and the biocompatible polymer backbone linked with glycyrrhizine. When the other untreated end of the PEG contains a maleimide substituent, the residual amine group of the polymer backbone It is necessary to replace it with a drill group. As a specific example, in the examples of the present invention, Traut's reagent was used to replace the remaining amine groups with sulfhydryl groups.

According to another aspect of the present invention, the present invention provides an islet cell composition for transplantation comprising:

(a) an islet cell isolated from a donor;

(b) a bifunctional PEG as a linker; And

(c) (i) a biocompatible polymer backbone and (ii) glycyrrhizin bound to the biocompatible polymer backbone; The biocompatible polymer backbone is bound to the pancreatic islet cell via the linker.

In order to transplant an islet cell isolated from a donor into a subject, the islet cell composition of the present invention is prepared by treating the islet cell with a composition for pancreatic cell transplantation, which is another aspect of the present invention described above, And the overlapping content is omitted in order to avoid the excessive complexity described in this specification.

In one embodiment of the invention, the glycyrrhizin of the invention is an oxidized form of glycyrrhizin. The oxidized form of glycyrrhizin has already been described with reference to another aspect of the present invention.

In one embodiment of the present invention, the biocompatible polymer backbone of the present invention is prepared by reacting a mixture of glycol chitosan, poly-L-lysine, poly (4-vinylpyridine / divinylbenzene), chitin, poly (butadiene / Poly (vinylamine) hydrochloride, poly (2-vinylpyridine), poly (vinylpyrrolidone), poly (Allylamine), poly (allylamine hydrochloride), poly (N-vinylpyridine N-oxide), poly-epsilon Cbz-L-lysine, poly (2- dimethylaminoethyl methacrylate) Methyl vinyl amine), poly (diallyldimethylammonium chloride), poly (N-vinylpyrrolidone), chitosan, or poly (4-aminostyrene).

In one embodiment of the present invention, the dual functional PEG of the present invention is a heterobifunctional PEG.

In one embodiment of the invention, the heterobifunctional PEG of the present invention comprises a maleimide substituent at one end and an NHS ester or carboxylic acid substituent at the other end. The heterobifunctional PEG has been described above with reference to another aspect of the present invention.

The pancreatic cell composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient, like the above-described composition for pancreatic cell transplantation.

The pancreatic islet cell composition for transplantation of the present invention is not particularly limited, but it may contain glycyrrhizin at a concentration of about 1-300 μg / ml per 300 pancreatic cells. More specifically about 5-200 占 퐂 / ml, more specifically about 10-150 占 퐂 / ml, more specifically about 20-120 占 퐂 / ml, of glycyrrhizin per 300 pancreatic cells.

According to another aspect of the present invention, there is provided a pharmaceutical composition for improving, preventing or treating an islet cell-deficiency disease of an islet comprising the above-described islet cell composition for transplantation.

Since the composition of the present invention relates to an islet cell composition that has been surface-modified for transplantation of an islet cell, it can be used for all diseases that may be caused by an islet cell-defect.

In one embodiment of the present invention, the pancreatic cell-defective disease of the present invention is a disease selected from the group consisting of type 1 diabetes, type 2 diabetes, and diabetic chronic renal disease. The type 1 diabetes, type 2 diabetes and diabetic chronic renal disease are all treatable by islet cell transplantation.

In one embodiment of the present invention, the pancreatic cell-defective disease of the present invention is type 1 diabetes. In the case of type 1 diabetes, the insulin secretory function is lost due to the destruction of the beta cells of the insulin-secreting insulin, and islet cell transplantation may be a good treatment for type 1 diabetes.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered parenterally and topical administration to the implant site is preferred.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for pancreatic cell transplantation.

(b) The present invention provides a method for producing an islet cell for transplantation.

(c) The present invention provides an islet cell composition for transplantation.

(d) The present invention provides a pharmaceutical composition for improving, preventing or treating an islet cell-deficiency disease.

(e) When the composition for pancreatic islet transplantation or the method for producing pancreatic islet cells for transplantation of the present invention is used, the pancreatic islet cell to be transplanted may be surface-modified to effectively reduce the immune rejection reaction.

(f) When a pharmaceutical composition for improving or preventing an islet cell-deficiency disease or an islet cell-deficiency disease of the present invention is used, the islet cell can be effectively transplanted without an immunological rejection reaction, It can effectively treat various diseases caused by defects in cells.

1 shows a schematic diagram of the conjugation process of pegylated islet cells with glycol chitosan and glycyrrhizin.
Fig. 2 shows the results of FT-IR determination of the binding of glycyrrhizin and glycol chitosan. FT-IR was confirmed for four groups of glycol chitosan, glycol chitosan, oxidized glycyrrhizin, and glycyrrhizin-conjugated glycol chitosan.
FIGS. 3A to 3D show NMR results of PEG bonds confirmed in the binding state of glycyrrhizin and glycol chitosan. FIG. The results were confirmed by NMR for glycol chitosan (FIG. 3A), glycyrrhizin (FIG. 3B), PEG (MAL-PEG-NHS) (FIG. 3C) and PEG-glycol chitosan-glycyrrhizin (FIG. 3D)
Fig. 4 is a schematic diagram of a binding reaction process of glycyrrhizin to poly-L-lysine polymer.
FIG. 5 shows the result of confirming the generation of the bond through FT-IR with respect to glycyrrhizin bound to poly-L-lysine. Fig. 5 (A) shows the FT-IR result, and Fig. 5 (B) shows the combined structure.
6 shows cell viability according to the treatment concentration of glycol chitosan-oGL.
Fig. 7 shows the result of cell surface observation according to the concentration of glycol chitosan-oGL treatment.
FIG. 8 shows the result of observation of confocal imaging of whether glycol chitosan -oGL binds to the surface of pancreatic islet cells via PEG.
FIG. 9 shows the results of comparing SI values according to insulin secretion amount (left panel) and insulin protein secretion amount (right panel) of pancreatic islets via GSIS.
FIG. 10 shows binding affinity for HMGB1 through surface plasmon resonance (SPR). The binding affinity for HMGB1 was measured by dividing into glycyrrhizin, glycol chitosan, HMGB1 Ab, and cholol chitosan-oGL groups.
Fig. 11 shows the results of blood glucose change test according to pancreatic islet transplantation in a mouse.
FIG. 12 shows the results of observing blood glucose changes after transplantation of glycyrrhizin-treated islet cells into an animal model of diabetes mellitus.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1: glycyrrhizin ( glycyrrhizin ) And glycol chitosan (glycol chitosan )

(1) Glycyrrhizine 0.41 mg (0.5 mM) (Sigma Aldrich, St. Louis, Mo., USA) was dissolved in 1 ml of a carbonate buffer solution (pH 9.5) at 4 ° C. (2) The same volume of sodium periodate solution (50 mM) was added and reacted for 90 minutes at 4 ° C in the absence of light to obtain oxidized glycyrrhizin. (3) Glycol chitosan (80,000-120,000 Da; Wako Pure Chemical Industries, Tokyo, Japan) was dissolved in a carbonate buffer solution (pH 9.5) at 1 mg / ml < / RTI > (4) The reaction was carried out at 4 캜 for 24 hours while blocking the light. (5) Cyanoborohydride (5 M) was added to the solution of (4) in an amount of 0.5 μl per ml. (6) The reaction was carried out at 4 캜 for 24 hours while blocking the light. (7) dialysis (MWCO 3500-5000) for 48 hours in a carbonate buffer solution (pH 9.5). (8) Secondary distilled water was dialyzed (MWCO 3500-5000) for 72 hours. (9) were stored in powder form through lyophilization. (10) To the phosphate buffer (pH 8.0, 5 mM EDTA), the powder of glycyrrhizin and glycol chitosan obtained in (9) was dissolved at 1 mg / ml. (11) 69 μl of Traut's reagent (14 mM) dissolved in phosphate buffer (2 mg / ml) was added per 10 ml of solution (10). (12) Reaction was carried out at room temperature for 4 hours.

Example 2: Pancreatic islet surface modification

(1) Glycyrrhizin was oxidized to conjugate glycol chitosan and glycyrrhizin to form an aldehyde group in the glucuronic acid moiety (see 1 in Fig. 1). (2) After the oxidized glycyrrhizin was bound to the amine group of the glycol chitosan, the amine group remaining through the Traut's reagent was replaced with the SH group (see FIG. 1, 2). (3) Amine groups on the surface of pancreatic islet cells were pegylated by binding of NHS of PEG (MAL-PEG-NHS) (see 3 in FIG. 1). (4) The maleimide of pegylated pancreatic islets and the SH group of glycol chitosan bound to glycyrrhizin were combined to modify the surface (see Fig. 1, 4).

Example 3: Confirmation of binding of glycyrrhizin and glycol chitosan

3-1. FT-IR measurement

(1) glycyrrhizin, glycol chitosan, oxidized glycyrrhizin, and glycyrrhizin bound to glycol chitosan were prepared, respectively. (2) Oxidized glycyrrhizin and glycol Chitosan bound to chitosan was dried in powder form through lyophilization. (3) Each of the four groups (1 mg) was added to NaCl (100 mg) in a mortar and then measured by FT-IR. The results are shown in Fig.

As shown in FIG. 2, the saccarin structure of glycol chitosan was confirmed at 1065 cm ^-1 , and the peak of the amine group was confirmed at 1651 cm ^-1 and 3425 cm ^-1 . Of a carboxyl group was confirmed geulrisirijin at 1442 cm ^-1, for the coarse oxide process geulrisirijin a new peak was observed at 1632 cm ^-1, a peak at 1632 cm ^-1 was confirmed that the peak of the bar represents an aldehyde, the oxidation .

The synthesized data of glycyrrhizin and glycol chitosan showed that the aldehyde groups bonded at 1628 cm ^-1 and the remaining aldehyde groups and oxidized glycyrrhizin at 1320 cm ^-1 bound to amine groups of glycol chitosan to form amide bonds, and 940 cm ^-1 , It was confirmed that the peak shifts as the saccharin structure of glycol chitosan undergoes synthesis.

FT-IR data of the combined groups confirmed that the glycyrrhizin was oxidized to form an aldehyde group to be linked to the glycol chitosan. Also, it was confirmed that the amide bond was formed by bonding the aldehyde group of glycyrrhizin formed and the amine group of glycol chitosan.

3-2. NMR measurement

(1) Glycyrrhizin, glycol chitosan, PEG (MAL-PEG-NHS) and PEG-glycol chitosan-glycyrrhizin (1 mg) were dissolved in D ₂ O (600 ml). (2) The four groups were measured by NMR, respectively, and the results are shown in FIGS. 3A to 3D.

6.89 ppm of maleimide, 3.72 ppm of PEG, and 2.51 ppm and 2.73 ppm of NHS, respectively, of PEG (MAL-PEG-NHS). After binding of glycol chitosan to glycine and binding of PEG, it was confirmed that the peak showing glycine at 0.81 ppm, 1.42 ppm and 5.70 ppm was confirmed at 2.51 ppm and 2.73 ppm, respectively, indicating NHS attached to one side of PEG .

It was confirmed that the peak appeared very strongly at 3.4 ppm at 3.4 ppm, which indicates the -CH- of the glycol chitosan and the hydroxyl radical of glycyrrhizin, and the ethylene oxide backbone of PEG. As the points where the peaks appear overlap each other, , But it was confirmed that both the peak of NHS and glycyrrhizine of PEG were confirmed at the same time and that the peak was strongly observed in the overlapping region of the peaks and that PEG was bound to the peptide.

Example 4: Confirmation of binding with glycyrrhizin using PLL (Poly-L-Lysine) in addition to chitosan

In order to confirm the binding of glycyrrhizin to the polymer other than chitosan, PLL (Poly-L-Lysine) (30,000 ~ 70,000 Da; Sigma Aldrich, St. Louis, MO, USA) having the most amine group To bind glycyrrhizin (cf. FIG. 4). The binding conditions were carried out in the same manner as for chitosan, and the results were confirmed using FT-IR (see FIG. 5).

FT-IR results confirmed bound peaks at 3055 cm ^-1 and 1320 cm ^-1 . From the above results, it was confirmed that the polymer capable of binding glycyrrhizin was not limited to chitosan but could be used as various polymers having PLL and other amine groups.

Example 5: Isolation of pancreatic islet cells

(1) Seven-week-old male SD-rats were anesthetized with anesthetics and then abdominal incisions were made. (2) Pancreatic organs were exposed and pancreas organs were removed after pancreatic organs were inflated using a collagenase P solution through a common bile duct. (3) After 15 minutes of enzyme reaction, cold M199 medium was added to stop the enzyme reaction and then washed twice. (4) Density gradient-centrifugation using Ficoll-histopaque and M199 medium in a double-layer was performed for 24 minutes. (5) The pancreatic islets between the separated layers were collected and hand-picked using a stereoscope. (6) Cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotic for 1 day. (7) The next day, the culture medium was changed once.

Example 6: Establishment of the concentration of glycol chitosan-oGL (oxidazed glycyrrhizin) in pancreatic islets

CCK-8 was used as an experiment to set the optimal concentration of glycyrrhizin that does not affect the cell viability when progressing pancreatic islet cell surface modification. The control group did not undergo PEGylation with glycol chitosan-OGL, and all of the comparative groups reacted with 0.25% of PEG. The glycol chitosan-oGL was treated with 0%, 0.05%, 0.1%, 0.25% , And 0.5%, respectively. The number of n was 5 and the value was corrected by DNA assay. When the control (control) was set to 100%, the cell viability was about 93% from 0% to 0.25% of the glycol chitosan-oGL concentration and no statistical difference was observed. (See Fig. 6).

In order to measure the viability of pancreatic islet cells and to observe the state and surface of cells, glycol chitosan-oGL was treated by concentration, and then pancreatic islet cells of each group were microscopically examined by a microscope just before CCK- Lt; / RTI > 0%, 0.05%, and 0.1% of the control group and the number of cells were not different from each other. In 0.25% group, pancreatic islet cells were slightly broken compared to other groups, but the number of whole cells and the surface appearance were not different from the control group. It is confirmed that there is no problem in the ability of the pancreatic islet cells to survive, though there is a slight difference in the surface appearance when compared with the result of CCK-8. The 0.5% group was observed to have fewer apical cells and the cell surface was rougher than the other groups, and the cells were considerably heavily fragmented, confirming the same results as the CCK-8 results (cf. FIG. 7 ).

Example 7: Pancreatic islet cell surface binding of glycol chitosan -oGL

Glycol chitosan-oGL was conjugated to PEGylated pancreatic islet cells after attaching the fluorescent material FITC to the cell surface and confocal imaging experiments were performed to determine whether glycol chitosan -oGL binds to the cell surface via PEG Respectively.

The pegylated goat cell group without pegylation was compared with the group treated with glycol chitosan-OGL, and it was confirmed whether or not the PEG-mediated binding of glycyrrhizin was able to increase the amount of glycyrrhizin bound to the surface according to the number of treatments.

The groups treated with glycol chitosan-oGL in pegylated islet cells not subjected to pegylation were divided into groups treated with 1, 2, and 3 times of 0.25% of glycol chitosan-oGL determined by the experiment. The non-PEG group showed that the fluorescence was completely expressed from the inside of the cell, indicating that the glycol chitosan-oGL did not attach to the surface and was uptaked. It was confirmed that glycol chitosan-oGL was randomly uptake into cells because it was not expressed uniformly in all cells but expressed only in some cells. The group treated with 0.25% PEG showed fluorescence on the cell surface except that the fluorescence was expressed inward from some damaged cells. It was confirmed that glycol chitosan-oGL binds to the surface via PEG. The groups treated with No.1, No.2 and No.3 were similar in fluorescence intensity and distribution, respectively, and the condition for treating pancreatic islet cell surface was established as No.1 (see FIG. 8).

Example 8: Measurement of glucose-stimulated insulin secretion (GSIS) by glucose stimulation

GSIS experiments were conducted to confirm normal insulin secretion in pancreatic islet cells at 0.25% of the treatment concentration of glycol chitosan-oGL. The experimental group was divided into two groups: a group not treated with glycol chitosan-OGL and a group treated with 0.25%. For comparative experiments, a low glucose concentration (2.8 mM) and a high concentration (Glucose solution: 20.2 mM) for one hour, and the amount of insulin was measured using an ELISA kit. The results are shown in FIG.

The control group had an insulin content of about 0.3 ng at the low glucose condition and about 1.2 ng at the high glucosone condition, about 0.4 ng at the low glucose condition for the glycine-conjugated group, about 0.4 ng at the low glucose condition, 1.3 ng, and the differences were similar for the control group and the condition. It was confirmed that when the glycyrrhizin was reacted with 0.25% of pancreatic islet cells, not only viability but also insulin secretion was regulated according to the condition, and the SI index was also observed at 2 or more, so that the insulin secretion function was normal Respectively.

Example 9: Confirmation of binding affinity for HMGB1 through surface plasmon resonance (SPR)

Reichert SR7500DC system, scrubber2 software, and CMDH was used as a gold chip. HMGB1 protein was used as a ligand, and the chip was coated with a 10 μl / ml flow, and the experiment was conducted using various concentrations of each analyte.

The binding affinity for HMGB1 was measured by dividing into glycyrrhizin, glycol chitosan, HMGB1 Ab, and glycol chitosan-oGL groups (see FIG. 10).

As a result of SPR analysis, it was confirmed that HMGB1 protein binds to glycyrrhizin and glycol chitosan -oGL, respectively. In the case of glycol chitosan, the binding affinity shows greater binding force than other groups except HMGB1 Ab, 120 kDa. In the SPR experiment, it was confirmed that the KD value was not significant and actually did not bind with HMGB1. In the case of glycol chitosan-oGL, the binding affinity of glycyrrhizin was about 260 times that of glycyrrhizin because of binding of glycyrrhizin to glycol chitosan to increase the binding site of HMGB1 to be targeted, This is very small, but it has meaning in itself. It was confirmed that glycyrrhizin synthesized with glycol chitosan has activity against HMGB1 inhibition and glycol chitosan acts as a polymeric backbone for introducing glycyrrhizin as much as possible.

Example 10: Islet transplantation in Balb / c mice induced type 1 diabetes

10-1. Diabetic model

(1) To induce diabetes mellitus, streptozotocin was dissolved in 200 μl of citrate buffer at a rate of 200 mg per kg body weight of Balb / C mouse. (2) It was rapidly injected into the peritoneal cavity and blood glucose was measured one week later. The blood glucose level was> 350 mg / dl for two consecutive days.

10-2. Renal xenotransplantation of pancreatic islets treated with glycol chitosan-oG1

(1) Diabetic-induced experimental animals were anesthetized with anesthetic. (2) The vicinity of the left kidney position of the mouse was thoroughly wiped with alcohol cotton, and then incised. (3) After carefully removing the kidney, 300 pancreatic islets treated with normal pancreatic islets and glycol chitosan -oGL treated with no treatment were transplanted into the left renal endothelial membrane using a Hamilton syringe. (4) The skin of the experimental animals was implanted and then sutured. (5) The animals were observed at appropriate temperature until they were awakened from the anesthesia. (6) Blood was obtained from the tail vein of mice transplanted with pancreatic islets between 1:00 and 3:00 pm on a daily basis, and blood glucose was measured using a one-touch ultra-high glucose test strip (see FIG. 11).

As a result of the blood glucose measurement, the survival rate of the group transplanted with the untreated islets was about 6.8 days, while the group transplanted with the glycol chitosan -oGL transplanted group had a survival rate of about 12 days. This is because the survival rate of islets transplanted with glycol chitosan -oGL increased up to about 3 times as much as the result of capturing HMGB1 secreted by post-transplantation injury and inhibiting the immune / inflammatory response.

Example 11: Confirmation of the survival rate of islets transplanted by binding of glycol chitosan -oGL

(1) D-glucosamine assay method for treating an equivalent amount of glycyrrhizin bound to the surface of a pancreatic cell (this is to confirm the degree of decomposition of glycol chitosan using alpha amylase and glucoamylase enzyme, The amount of glycyrrhizin bound to glycol chitosan-oGL was measured using a method of calculating the amount of chemically bound glycyrrhizin by quantitatively analyzing the degree of decomposition when it is chemically bonded to glycyrrhizin. As a result, the presence of glycyrrhizin at a concentration of about 56 μg / ml per 300 islets was confirmed. (2) Thereafter, pancreatic cells were newly isolated and treated with glycyrrhizin at a concentration of 56 μg / ml, and then blood glucose was measured by transplantation into an animal model of diabetes mellitus. The results are shown in FIG.

 From these results, it was confirmed that the survival rate of transplanted pancreatic islets was not enhanced when the glycyrrhizin was simply transplanted into pancreatic cells (survival rate: about 4 days).

This is because the physically treated glycyrrhizin simply can not wash-out after transplantation and exert the ability to capture HMGB1 released from transplanted pancreatic cells. As a result, it was confirmed that the survival rate of the transplanted pancreatic cells was similar to that of the untreated pancreatic transplant group because there was no effect of inhibiting the immune / inflammatory reaction.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

A composition for pancreatic cell transplantation comprising (a) a biocompatible polymer backbone and (b) glycyrrhizin bound to the biocompatible polymer backbone.
The composition of claim 1, wherein the glycyrrhizin is an oxidized form of glycyrrhizin.
The composition of claim 1, wherein the biocompatible polymer backbone comprises a functional group capable of forming a chemical bond with an aldehyde group.
4. The composition of claim 3, wherein the functional group is an amine group, a carboxyl group, a thiol group, or a hydroxide group.
5. The composition of claim 4, wherein the functional group is an amine group.
The biocompatible polymeric backbone of claim 1 wherein said biocompatible polymer backbone is selected from the group consisting of glycol chitosan, poly-L-lysine, poly (4-vinylpyridine / divinylbenzene), chitin, poly (butadiene / acrylonitrile) amine end, polyethyleneimine, , Poly (ethylene glycol) bis (2-aminoethyl), poly (N-vinyl pyrrolidone), poly (vinylamine) hydrochloride, poly (2-vinylpyridine) , Poly (ε-Cbz-L-lysine, poly (2-dimethylaminoethyl methacrylate), poly (allylamine), poly (allylamine hydrochloride) Dimethylammonium chloride), poly (N-vinylpyrrolidone), chitosan, or poly (4-aminostyrene).
A pancreatic cell transplantation composition according to any one of claims 1 to 6, which is isolated from a donor, is treated to bind a biocompatible polymer backbone bound with glycyrrhizin to the surface of the pancreatic islet cell Gt; islet cell for transplantation.
8. The method of claim 7, wherein the pancreatic islets are PEGylated.
9. The method of claim 8, wherein the pegylation is performed using heterobifunctional PEG.
10. The method of claim 9, wherein the heterobifunctional PEG comprises a maleimide substituent at one end and an NHS ester or carboxylic acid substituent at the other end.
An islet cell composition for transplantation comprising:
(a) an islet cell isolated from a donor;
(b) a bifunctional PEG as a linker; And
(c) (i) a biocompatible polymer backbone and (ii) glycyrrhizin bound to the biocompatible polymer backbone; The biocompatible polymer backbone is bound to the pancreatic islet cell via the linker.
12. The composition of claim 11, wherein the glycyrrhizin is an oxidized form of glycyrrhizin.
The biocompatible polymeric backbone of claim 11, wherein the biocompatible polymer backbone is selected from the group consisting of glycol chitosan, poly-L-lysine, poly (4-vinylpyridine / divinylbenzene), chitin, poly (butadiene / acrylonitrile) amine end, polyethyleneimine, , Poly (ethylene glycol) bis (2-aminoethyl), poly (N-vinyl pyrrolidone), poly (vinylamine) hydrochloride, poly (2-vinylpyridine) , Poly (ε-Cbz-L-lysine, poly (2-dimethylaminoethyl methacrylate), poly (allylamine), poly (allylamine hydrochloride) Dimethylammonium chloride), poly (N-vinylpyrrolidone), chitosan, or poly (4-aminostyrene).
12. The composition of claim 11, wherein the bifunctional PEG is a heterobifunctional PEG.
15. The composition of claim 14, wherein the heterobifunctional PEG comprises a maleimide substituent at one end and an NHS ester or carboxylic acid substituent at the other end.
15. A pharmaceutical composition for the improvement, prevention or treatment of an islet cell-deficiency disease, comprising the composition according to any one of claims 11 to 15.
17. The composition of claim 16, wherein the islet cell-deficient disease is a disease selected from the group consisting of type 1 diabetes, type 2 diabetes, and diabetic chronic renal disease.
18. The composition of claim 17, wherein the islet cell-defective disease is type 1 diabetes.

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