JP2006347883A - Composition for medical treatment comprising sugar chain-containing chitosan derivative and glycosaminoglycan - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for medical treatments that performs a function of positively accelerating healing of an intractable wound and promotes vascularization and tissue regeneration without a risk of causing side effects such as cancerization. <P>SOLUTION: The composition for medical treatments comprises a hydrogel formed from a sugar chain-containing chitosan derivative and an acidic polysaccharide and, carried thereon, a drug for promoting healing of wounds. Preferably, the sugar chain-containing chitosan derivative is one obtained by incorporating a saccharide having a reducing end into at least a part of amino groups of a chitosan skeleton. Preferably, the acidic polysaccharide is a glycosaminoglycan such as periodic acid-oxidized heparin. Preferably, the drug for promoting healing of wounds is a cell growth factor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a medical composition comprising a hydrogel formed from a sugar chain-containing chitosan derivative and an acidic polysaccharide, and a wound healing promoter, particularly for intractable skin diseases such as diabetic skin ulcers and wounds. The present invention relates to a composition capable of promoting healing by removing a drug carried in close contact with a wound.

Wounds such as skin are mainly classified into releasable wounds in which normal skin / mucosal tissue is released, burns caused by heat or radiation, ulcers (circumscribed tissue defects), or complex wounds thereof. In particular, for a severe wound having a large area and a deep depth, a treatment that prevents exogenous inflammation due to infection and promotes the induction of granulation tissue is essential.
For the treatment of such wounds, the use of wound dressings such as gauze has been practiced for a long time, but since the dressing itself has no healing effect, it improves the healing effect in combination with drugs such as antibiotics. Ingenuity has been made. In recent years, advances in material chemistry have led to the use of transparent adhesive films and jelly-like hydrogels that have gas permeability and prevent moisture ingress, as well as artificial skin coverings that use animal biomembranes, etc. However, it has not yet been provided what has a protection function such as wound protection and infection protection and a healing function that promotes healing at the same time.

In particular, in intractable wounds such as wounds and skin ulcers derived from diabetes, the blood circulation of the defective tissue is inhibited, so that the regeneration of the defective tissue is significantly delayed. At present, there are no effective treatments or coatings to promote healing, so there is a risk of complications of serious external infections over the long period of time required for healing. In the case of merging, surgical treatment with a large burden on the patient such as uprooting and excising necrotic tissue is performed to treat them.
For the treatment of such intractable wounds, it has been studied to use a regenerated tissue obtained by culturing tissue cells outside the body, but problems such as simplicity, cost, time, and safety remain. Recently, research on therapeutic methods using genes that promote angiogenesis in order to induce tissue regeneration has also been promoted, but the methodology for introducing genes and the proteins produced by the transgenes have clinical effects? Many issues such as are unresolved.

  On the other hand, it has been known that cell growth factor (GF) contributes to tissue regeneration by controlling cell proliferation and differentiation. In particular, studies of fibroblast growth factors FGF-1 and FGF-2 are in progress, and these control cell growth, migration, differentiation, life span, etc., and fetal development, angiogenesis, bone formation, nerves It became clear that it contributed to formation and wound repair. That is, it is expected to promote angiogenesis and promote healing by administering a growth factor such as FGF to an ischemic disease site including diabetic skin ulcer. However, GF is difficult to maintain and preserve its activity, and normally interacts with heparin molecules in proteoglycans that constitute the extracellular matrix to protect against degradation and regulate functions. In addition, if a high concentration of GF acts at any one time, there is a risk of causing cancerous factors, so care must be taken in its usage. That is, while the tissue regeneration function of GF is expected to be used for wound treatment, problems such as the high diffusibility of GF itself and the speed of functional deterioration have hindered actual clinical application.

The present inventors have advanced the development of a wound treatment agent using chitosan, which is a polysaccharide (see Patent Document 1). Chitosan was known as a material that combines wound healing and antibacterial properties, but it was difficult to control water-solubility and gelation, so it should be commercialized as a general-purpose and functional adhesive or coating material. It was difficult. However, as described in Patent Document 1, the present inventors succeeded in improving the physical properties of chitosan solubilized only in the acidic region to be easily soluble by sugar chain modification, and thus obtained sugar chain-containing chitosan derivatives. Forms a viscous aqueous solution at physiological pH. Further, when a photoreactive group is further introduced into the sugar chain-containing chitosan derivative, the aqueous solution containing the derivative becomes a close contact insoluble gel body by light irradiation. Therefore, since the aqueous solution of the derivative can be applied to any affected part and immediately becomes an adhesive gel body by a photoreaction at the site, it can be effectively used as an adhesive for a wound site.
International Publication No. WO00 / 27889 Pamphlet

  However, the above-mentioned photoreactive chitosan derivative has an advantage that an insoluble gel can be easily formed by ultraviolet irradiation. For example, when it is administered subcutaneously where light does not reach, there is no means for irradiating the administration site with light. However, conversely, if light was irradiated before administration, gelation occurred in the syringe, which sometimes caused a problem that injection was impossible.

As a result of intensive investigations to solve the above problems, the present inventors have found that an injectable hydrogel can be obtained by mixing a sugar chain-containing chitosan derivative and an acidic polysaccharide such as glycosaminoglycan. The headline and the present invention were made.
That is, the present invention provides a medical composition in which a wound therapeutic agent such as FGF is supported on a hydrogel containing a sugar chain-containing chitosan derivative and at least one acidic polysaccharide.

  Since the medical composition of the present invention is prepared at a physiological pH, it can be mixed well without modifying a wound healing promoter having physiological activity such as GF. The resulting composition can be applied to any wound site, but is particularly suitable for application by injection, resulting in an insoluble hydrogel at the injection site. The hydrogel not only firmly supports a wound treatment promoter such as a growth factor, but can also slowly release the supported GF and the like at an appropriate rate. With this moderate sustained release, angiogenesis-inducing effects can be obtained over a long period of time without causing canceration of tissues due to large doses of growth factors, resulting in the promotion of healing of refractory ulcers and wounds To do.

    The sugar chain-containing chitosan derivative used in the medical composition of the present invention has a structure in which a sugar chain structure is introduced into a polymer skeleton generally called chitin / chitosans. Is preferably obtained by introducing a saccharide having a reducing end into at least a part of the 2-position amino group of the glucosamine unit constituting the deacetylated chitin / chitosan.

Usually, chitin / chitosans are deacetylated acid-soluble fractions obtained by treating chitin derived from crab shell (poly-N-acetylglucosamine) with an alkali, and generally have the following formula (1), ( 2).

Of these chitins and chitosans, those with a low degree of deacetylation (usually less than 40%) are called “chitin”, and those with a high degree of deacetylation (usually 40% or more) are called “chitosan”. However, hereinafter, in this specification, chitin / chitosans at least partially deacetylated are collectively referred to as “chitosan”. The chitosan in the present invention is not limited to a naturally derived one, and may be a chemically modified sugar chain having a similar structure synthesized chemically or genetically.
Here, the “degree of deacetylation” is the ratio at which the acetylamino group at the 2-position of the sugar unit constituting chitosan (or poly-N-acetylglucosamine) is converted to a free amino group by deacetylation. is there. In the present specification, the degree of deacetylation is quantified by the “colloidal titration method” described in “Health Food Standards Collection (Part 4)”, Japan Health and Nutrition Food Association (1996), p. 55.

  The chitosan derivative of the present invention is functionalized by further chemically modifying this chitosan. The chitosan used as a raw material has a degree of deacetylation of at least 40%, particularly 60 to 100%, In particular, those in the range of 65 to 95% are suitable. Chitosan having a degree of deacetylation of 100% consists only of the structural unit of the above formula (1) and does not include the structural unit of the formula (2).

The molecular weight of the chitosan is not particularly limited and can be varied within a wide range depending on the purpose of use of the final chitosan derivative. In general, the number average molecular weight is 5,000 to 2,000,000, preferably Is in the range of 10,000 to 1,800,000, more preferably 40,000 to 1,500,000.
Preferred chitosan derivatives in the present invention are those in which a saccharide having a reducing end is introduced into at least a part of the amino group at the 2-position of the glucosamine unit of the formula (1) constituting the chitosan.

  Examples of the saccharide having a reducing end to be introduced into the chitosan derivative include aldoses and ketoses. Among them, those having 20 or less, particularly 1 to 7 constituent sugar units are preferably used. Specifically, for example, pentaose and hexaose such as glucose, fructose, galactose, fucose, mannose, arabinose, xylose, erythrose, heptulose, hexylose; aminosaccharides such as glucosamine, N-acetylglucosamine, galaxamine; uronic acids and deoxysaccharides Sugar derivatives such as: disaccharides composed of a combination of these monosaccharides, di- or trisaccharides such as maltose, isomaltose, lactose, melibiose, maltotriose; various oligosaccharides, among which maltose, lactose, Neutral disaccharides such as melibiose are preferred.

The introduction of the saccharide to the 2-position amino group of the glucosamine unit of the formula (1) of chitosan can be performed using a method known per se, for example, after carboxylating the reducing end of the saccharide, A method of binding to the 2-position amino group by an amide bond (see, for example, JP-A No. 10-120705), or aldehyde-forming or carbonylation of the reducing end of a saccharide, followed by a shift to the 2-position amino group of the glucosamine unit Coupling by a reductive alkylation method via a base (see, for example, chitin / chitosan study group, “Application of chitin / chitosan”, pages 53-56, February 20, 1990, published by Ryodo Publishing Co., Ltd.) Is included.
The saccharide introduced into chitosan in the present invention is not limited to one kind, and two or more kinds can be used in combination.

Specific examples of the sugar chain constituting the chitosan derivative of the present invention include the following, but are not limited thereto.
(i) Sugar chains derived from lactose:

(iii) メリビオースから誘導される糖鎖：

(ii) Sugar chains derived from maltose:

(iii) Sugar chains derived from melibiose:

(v) ラミナリビオースから誘導される糖鎖：

(iv) Sugar chains derived from cellobiose:

(v) Sugar chains derived from laminaribiose:

(vii) Ｎ-アセチルキトビオースから誘導される糖鎖：

(vi) Sugar chains derived from mannobiose:

(vii) Sugar chains derived from N-acetylchitobiose:

  Among the sugar chains of the above (i) to (vii), those described on the left side represent residues introduced by condensation of the sugar carboxyl group and the 2-position amino group of chitosan, and those described on the right side Represents a residue linked via a Schiff base.

Thus, by substituting the 2-position amino group of the glucosamine unit of chitosan with a saccharide, the acid-dependent solubility of chitosan is relaxed, and solubilization in the neutral region is achieved.
The degree of substitution of the 2-position amino group of the glucosamine unit of chitosan with a sugar chain can be changed according to the physical properties desired for the final chitosan derivative, but the degree of substitution is generally 0.1 to 80%, It is particularly preferable that it is in the range of 0.5 to 60%, more particularly 1 to 40%. Here, the “degree of substitution” with a sugar chain is the degree to which the amino group at the 2-position of the sugar unit constituting chitosan is substituted with the sugar chain, and the free amino group at the 2-position of the sugar unit constituting chitosan. And the ratio of the substituted amino group to the total of the substituted amino group. In the present specification, the degree of substitution with a sugar chain is measured by the “phenol-sulfuric acid method” in which a characteristic color development based on the reaction of a sugar chain with phenol in sulfuric acid is detected at an absorbance of 490 nm (JEHodge, BTHofreiter , "Methods in Carbohydrate Chemistry", ed. By RLWhistler, MLWolfrom, vol.1, p388, Academic Press, New York (1962)).

  In the chitosan derivative of the present invention, at least the amino group at the 2-position of the saccharide unit of the formula (1) constituting the chitosan and the hydroxyl group at the 3- or 6-position of the saccharide unit of the formula (1) or (2) In part, an amphiphilic group may be introduced, which can add dramatically improved water content to the crosslinked hydrogel. This amphiphilic group is a group having a hydrophobic block having a hydrophobic group and a hydrophilic block having a hydrophilic group, and generally has a surfactant function in many cases. Among them, the ratio of the molecular weight of the hydrophobic block (X) and the hydrophilic block (Y) is preferably X: Y = 1: 5 to 5: 1, and is nonionic having no dissociable ionic group. The group can be used more suitably. In particular, a polyoxyalkylene alkyl ether having a molecular weight composed of a hydrophobic alkyl block and a hydrophilic polyoxyalkylene block of at least 90, more preferably 500 to 10,000 is preferred. Polyethers having no hydrophobic block can be used, but polyoxyalkylene alkyl ethers having both a hydrophobic block and a hydrophilic block are preferred from the viewpoint of improving water content.

  Such an amphiphilic group can be introduced into chitosan, for example, at the end of either the hydrophilic block or the hydrophobic block of the amphiphilic group, a group that can react with an amino group to form a covalent bond, such as an aldehyde group. Or a compound having an epoxy group or the like and then reacting with the 2-position amino group of glucosamine of chitosan, a method of reacting a polyoxyalkylene alkyl ether derivative having a carboxyl group and chitosan in the presence of a condensing agent, Can be performed using a method of reacting a polyoxyalkylene alkyl ether derivative having an acid chloride group with a hydroxyl group or an amino group of chitosan.

  For example, when a polyoxyalkylene alkyl ether group derived from an epoxy group at the terminal or a polyoxyalkylene alkyl ether group derived from an aldehyde group at the terminal is introduced into the amino group of chitosan, the side chain bonded to the chitosan skeleton has the following formula ( It is represented by a) or (b). In addition, when a polyoxyalkylene alkyl ether group derived from an acid chloride group at the end is bonded to the hydroxyl group at the 3-position or 6-position of chitosan, the side chain bonded to the chitosan skeleton is represented by the following formula (c): . However, n and m in the following formulas (a) to (c) are one or more repeating units.

  The degree of introduction of the amphiphilic group in the chitosan derivative of the present invention is not particularly limited, but is usually 5 to 70%, preferably 15 to 55%, based on the weight change of the chitosan derivative after introduction. Can be within range.

The composition of the present invention includes a hydrogel formed from the sugar chain-containing chitosan derivative as described above and at least one acidic polysaccharide. The “acidic polysaccharide” used in the present invention includes a polysaccharide having an acidic group such as a sulfate group or a carboxylic acid group or a natural or synthetic organic molecule having a part of its structure, preferably heparin or heparan. It is selected from sulfated mucopolysaccharides including glycosaminoglycans such as sulfuric acid, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and derivatives thereof. Among these acidic polysaccharides, low molecular weight heparin obtained by enzymatic or chemical degradation of heparin is preferred, and low molecular weight heparin in which the anticoagulant activity of unmodified (natural) heparin is reduced by degradation is preferred. . Particularly preferred is periodate-decomposed heparin (hereinafter referred to as “IO ₄ heparin”). The preparation method of IO ₄ heparin and the like are described in, for example, K. Ono et al., Br. J. Cancer; 86 , 1803-1812 (2002). Periodic acid oxidized heparin (IO ₄ heparin) is known not to have a unique pentasaccharide structure that interacts with antithrombin III, and thus its anticoagulant activity is better than unmodified heparin. Very low.

In the present invention, a basic chitosan molecule is complexed with an acidic polysaccharide (such as IO ₄ heparin) to form a hydrogel (polyion complex) through ionic interaction.
In the sugar chain-containing chitosan derivative used in the medical composition of the present invention, a saccharide having a reducing end is introduced into the chitosan skeleton, and an amphiphilic group may optionally be introduced. By introducing a sugar chain, the chitosan derivative is excellent in solubility in the neutral region, and can be dissolved in a physiological buffer or medium, and may be denatured with acids or alkalis such as proteins. It can be carried without losing the activity of the drug.

The medical composition of the present invention includes at least one wound healing promoter in a hydrogel formed from a sugar chain-containing chitosan derivative and an acidic polysaccharide. The wound healing promoter in the present invention may be any substance that promotes the wound healing effect at the site to which the composition is applied in any sense. This includes drugs with wound healing effects described in "Pharmaceuticals Manual 5th Edition" (written by Osaka Prefectural Hospital Pharmacists Association, Yakuho Jihosha, 1992) and its supplement (1992), For example, antibacterial agents that promote healing by preventing infection and inflammation at the wound site, antibiotics, analgesics and anesthetics that relieve pain caused by wounds, and the like are also included. In particular, when applied to refractory skin wounds with impaired blood circulation such as wounds and diabetic skin ulcers, substances that induce angiogenesis, such as growth factors and angiogenic factors, are preferred. The growth factor used here is not particularly limited as long as it is a cell growth factor having a function of inducing angiogenesis and promoting granulation formation. For example, FGF-1, FGF-2, HB-EGF , HGF and VEGF _165, and HGF. In addition, since the sustained-release drug of the present invention has extremely high tissue adhesion, it is also possible to include a plasmid incorporating a gene that expresses the above-mentioned growth factor to supply the growth factor spontaneously at the wound site. .

  Furthermore, the composition of the present invention may optionally contain other additives acceptable for medical use. Examples include interleukin, leukemia inhibitory factor, interferon, TGF-β, erythropoietin, thrombopoietin and the like. Other agents known to induce apoptosis in mammals can also be used and include TNF-α, TNF-β, CD30 ligand and 4-1BB ligand. Chemotherapeutic agents useful for the treatment of cancer may also be used, examples of which include alkylating agents, folic acid antagonists, metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purines Also included are nucleoside, amines, amino acids, triazole nucleosides, or corticosteroids, or hormonal drugs that function to modulate or inhibit hormonal effects on tumors such as tamoxifen and onapristone.

  The medical composition of the present invention is prepared by dissolving a sugar chain-containing chitosan derivative, an acidic polysaccharide and a wound healing promoter and other optional components in a solvent, preferably an aqueous medium, preferably at a neutral pH. can do.

  In consideration of administration by injection, the composition thus prepared is measured by, for example, a commercially available rotational viscometer (for example, a B-type viscometer, TOKIMEC INC. (Tokyo Japan)). Preferably, the viscosity is less than about 300 cps (centipoise (mPa · s)), further less than about 200 cps, and even less than about 100 cps. In addition, a composition having a low viscosity comparable to that of pure water can be applied using a spray device or the like. In endoscopic treatment via a catheter or the like, fluidity in the tube or endoscopic surgery Appropriate viscosity can be adopted in consideration of retention at the resection wound. Further, when applied to a non-horizontal wound surface, the viscosity is preferably about 100 to 10,000 cps, preferably about 150 to 8,000, more preferably about 200 to 6,500 cps. Such viscosity adjustment can be achieved by appropriately selecting the concentration of the sugar chain-containing chitosan derivative or the like.

That is, the content of the sugar chain-containing chitosan derivative in the medical composition of the present invention is at least 3 mg / mg in order to ensure retention at the application site and reliably carry the encapsulated drug by the hydrogel after gelation. ml, more preferably at least 5 mg / ml, at least 7.5 mg / ml, more preferably at least 10 mg / ml, still more preferably at least about 15 mg / ml.
The acidic polysaccharide such as IO ₄ heparin preferably has a content of about 10 to 5000 μg / ml, more preferably about 50 to 1000 μg / ml, and still more preferably about 100 to 500 μg / ml.
The content of the wound healing promoter is appropriately determined so that the amount required at the application site is gradually released. For example, the growth factor when applied to the skin wound site is about 1-1000 μg / ml, more preferably about 5-500 μg / ml, and still more preferably about 10-200 μg / ml.
The content of other optional components is a content that is usually used in the medical field, and the optimal amount can be easily determined by those skilled in the art.

  The sugar chain-containing chitosan derivative forms an insoluble hydrogel when mixed with an acidic polysaccharide. The formed chitosan hydrogel contains wound healing promoters such as growth factors and other additives. Although these drugs may be released to some extent due to the diffusibility of the drugs themselves, most of them are firmly supported in the chitosan hydrogel. Thereafter, when the chitosan hydrogel is biodegraded, the supported drug is released slowly at an appropriate rate.

More specifically, the medical composition of the present invention is injected into, for example, a subcutaneous tissue having a skin ulcer to form an insoluble gel body (hydrogel). This chitosan hydrogel promotes wound protection and healing by gradually releasing the encapsulated drug into the surrounding tissues.
In addition, when an amphiphilic group such as polyoxyalkylene alkyl ether is introduced into the sugar chain-containing chitosan derivative, the chitosan hydrogel has a high water content such that it can quickly absorb moisture close to 100 times its own weight, for example. It is also possible to improve the hygiene of the affected area by absorbing bleeding and exudate from the wound site or surgical site.

  Furthermore, since the chitosan gel of the present invention has the property of containing a cell growth factor therein and gradually releasing it, it can also be used as a cell culture substrate for tissue regeneration. For example, when the medical composition of the present invention is applied to the surface of a cell culture plate or the like to form a chitosan hydrogel, and the target cell culture medium is placed on the coated portion of the cell culture plate and incubated, Proliferation is stimulated. Therefore, in the present invention, the above-described chitosan hydrogel, especially a hydrogel containing a growth factor can be effectively used as a cell culture substrate for tissue regeneration.

  Furthermore, it has been found that the hydrogel of the present invention is enhanced by the addition of serum proteins. Specifically, the strength of the formed hydrogel is improved by adding serum proteins such as albumin and globulin during the formation of the hydrogel. This indicates that when the hydrogel of the present invention is injected into a living body, it is stably present without being deteriorated by the influence of various proteins present in the living body, and is suitable for a sustained drug release carrier in vivo. Suggests.

  Hereinafter, the present invention will be described in more detail with reference to specific examples, but these specific examples do not limit the scope of the present invention.

Preparation of medical composition of the present invention (1) Synthesis of sugar chain-containing chitosan derivative UV-RC having an ultraviolet-reactive functional group and a sugar chain introduced into a chitosan skeleton was synthesized according to the method described in WO00 / 27889. did. Specifically, lactose (lactobionic acid) was introduced into the amino group of chitosan (manufactured by Yaizu Suisan Kogyo Co., Ltd.) having a molecular weight of 800 to 1000 kDa derived from crab and a deacetylation degree of 0.8 by a condensation reaction. . It was confirmed that it was soluble at neutral pH by introduction of lactose and the degree of substitution was about 2% (the obtained sugar chain-containing chitosan derivative was referred to as “CH-LA”).

(2) Preparation of non-anticoagulant (IO ₄ ) heparin IO ₄ heparin was prepared as described in K. Ono et al., Br. J. Cancer; 86 , 1803-1812 (2002). Specifically, heparin 25 g (185.8 USP units / mg) from porcine small intestine was dissolved in 400 ml of 0.1 M NaIO ₄ solution in 0.05 M sodium acetate buffer (pH 5) and stirred at 4 ° C. for 3 days. Unreacted NaIO ₄ was then neutralized by the addition of glycerol (25 ml), followed by dialysis and lyophilization of the reaction mixture. The product (periodate oxidized heparin) was then chemically reduced by mixing with 0.2 M sodium borohydride solution in 0.25 M sodium bicarbonate at 4 ° C. for 3 hours. Excess borohydride in the reaction solution was neutralized by adding acetic acid (pH 5). Reduced periodate oxidized heparin (IO ₄ heparin) was recovered after neutralization with NaOH, dialysis, and lyophilization.

(3) Preparation of FGF-2-supported chitosan / IO ₄ heparin hydrogel
1 ml phosphate buffer (PBS) containing 50 μg / ml FGF-2 (Fiblast: Kaken Pharmaceutical) and 1 mg / ml IO ₄ heparin (above (2)) was added to 40 mg / ml CH-LA ( The composition (1)) was mixed with 1 ml of an aqueous solution to obtain a composition (hydrogel) of the present invention.

Release of FGF-2 and IO ₄ heparin from hydrogel The hydrogel (50 μl) obtained in Example 1 (3) was dropped into each well of a 24-multiwell tissue culture plate. The hydrogel in each well was washed with 199 medium (Life Technologies Oriental) supplemented with 10% by weight heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U / ml penicillin G and 100 μg / ml streptomycin), 1 ml of medium was added and shaking was continued at room temperature on a rotary shaker. The medium was changed every day. On each measurement day, the concentration of FGF-2 released into the medium was measured by ELISA assay using heparin beads (Sigma-Aldrich) (for details of the measurement method, see Ono K. et al., Glycobiology, 9 , 705). -711 (1999)), IO ₄ heparin concentration was measured using the dimethylmethylene blue (DMB) method described in Farndale, RW et al., Biochem. Biophys. Acta, 883 , 173-177 (1986) (glyco Saminoglycan assay kit: manufactured by Funakoshi Co., Ltd. was used).

The release profile of FGF-2 and IO ₄ heparin from the hydrogel to the medium at room temperature is shown in FIG. Only a small amount of IO ₄ heparin was released from the hydrogel, and 90% or more was retained in the gel even after 7 days. About 20% of FGF-2 was released on the first day, but no significant release was observed thereafter.

Effect of FGF-2-supported chitosan / IO ₄ heparin hydrogel on HUVEC growth First, 50 μl of CH-LA aqueous solution (hydrogel) containing FGF-2 (1.25 μg) and IO ₄ heparin (25 μg) was added to a 24-multiwell plate. It was dripped at the center of each well. The FGF-2-supported chitosan / IO ₄ heparin hydrogel was washed in 1 ml of 199 medium (Life Technologies Oriental) with shaking at room temperature. The medium was changed every day. Then, human umbilical vein endothelial cells (HUVEC; Takara Biochemistry) in 199 medium supplemented with 10 wt% heat-inactivated FBS and antibiotics (100 U / ml penicillin G and 100 μg / ml streptomycin) without FGF-2 Co., Ltd.) was seeded at an initial density of 15,000 cells / well and incubated for 3 days. After incubation, the medium used was replaced with 100 μl fresh medium containing 10 μl WST-1 reagent (Cell Counting Kit, Dojindo Co. Ltd.), and after 1 hour incubation at 37 ° C., immunomini The optical density at 450 nm was read using a plate reader 89 (Nunc InterMed). FIG. 2 shows the growth rate (relative value) of HUVEC calculated from the obtained values and plotted against the washing time. The vertical axis in FIG. 2 shows relative values when the growth rate of HUVEC in a medium without hydrogel (FGF-2) is 1.0.

Next, the FGF-2-supported chitosan / IO ₄ heparin hydrogel washed for 7 days was treated with 1 mg / ml chitinase (chitinase-RS; Biochemical Co., Ltd.) and 2 mg / ml chitosanase (chitosanase-RD; biochemical ( And a solution containing the same)). Subsequently, HUVECs were seeded in wells containing enzyme-treated FGF-2-supported chitosan / IO ₄ heparin hydrogel and incubated as described above. The growth rate at that time is also shown in FIG.

  It is well known that FGF-2 is a stimulator of endothelial cell growth. Therefore, if FGF-2 was released from the hydrogel, it was predicted that the growth rate of HUVEC would be improved by the stimulation. Actually, the growth rate (relative value) at the washing time of 1 day was about 2.0, but after that, the growth rate decreased, and it was equivalent to the case where FGF-2 was not added after the 5th day. Proliferation rate. These results are consistent with the results of Example 2 (FIG. 1) where about 20% of FGF-2 is released on the first day and no significant release occurs thereafter.

However, when HUVEC was cultured in the presence of chitinase and chitosanase together with FGF-2-supported chitosan / IO ₄ heparin hydrogel washed for 7 days, the stimulating activity on HUVEC proliferation was remarkably recovered (FIG. 2: ●). It has been confirmed that HUVEC grows normally in a medium containing chitinase and chitosanase, and that chitinase and chitosanase do not affect the growth of HUVEC (data not shown). In addition, after 3 days incubation with chitinase and chitosanase and HUVEC, many cracks were seen in the hydrogel and small pieces of hydrogel were seen in the medium (data not shown). That is, it is clear that chitosan hydrogel was decomposed by the action of chitinase and chitosanase, and FGF-2 contained therein was released to stimulate HUVEC proliferation. This also indicates that FGF-2 retained a biologically active form in the hydrogel of the present invention.

Evaluation of in vivo degradation of hydrogels
Chitosan / IO ₄ -heparin hydrogel (100 μl) containing 100 μg / ml trypan blue was applied to the left and right of the spine of a mouse (C57BL / 6, female, 10 weeks old, Crea Japan Inc.) weighing approximately 30 g. A careful injection was made 2 cm from the base of the tail. Mice were sacrificed at 2, 5, 9, 14, and 21 days after hydrogel injection and the tissue containing the injected hydrogel was removed. The blood around the removed tissue was washed with PBS, and the tissue adhered to the hydrogel was completely removed. The remaining hydrogel was treated with 0.5 ml of 100 mM NaNO ₂ at pH 3.0 and centrifuged at 2000 rpm. The trypan blue in the supernatant was quantified by measuring OD ₆₄₀ , and the results are shown in FIG. Each experimental group consists of 3 mice.

In order to facilitate observation, trypan blue (acidic dye; manufactured by sigma-Aldrich), which is an acidic molecule, was used as in FGF-2. Prior in vitro experiments have confirmed that trypan blue supported on chitosan / IO ₄ heparin hydrogel is not released without degradation of the hydrogel itself. That is, it is inferred that the reduction of trypan blue in the hydrogel in vivo is due to biodegradation of the hydrogel.

The amount of the dye trypan blue in the chitosan hydrogel injected into the back of the mice decreased with the time of implantation (FIG. 3). The chitosan / IO ₄ heparin hydrogel of the present invention retained about 80%, 60% and 30% dye molecules on days 2, 5, and 14 respectively. On days 9, 14, and 21, a blue gel fragment was also observed in the vicinity of the embedded site. In combination with the results of the above in vitro experiments, it was found that the chitosan / IO ₄ heparin hydrogel of the present invention was biodegraded in vivo and could gradually release the encapsulated trypan blue to about 90% over about 21 days. On the other hand, when the same amount of trypan blue was added to the sugar chain-containing chitosan derivative aqueous solution not containing IO ₄ heparin, the stored trypan blue was about 40% on the second day and the fifth day. It is only about 20% and about 90% is released on the 9th day (FIG. 3: ●). That is, the hydrogel of the present invention stably includes acidic molecules such as FGF-2, and is suitable for use as a matrix material that releases slowly by biodegradation of the hydrogel.

Considering the results of Examples 2, 3 and 4, the chitosan / IO ₄ heparin hydrogel of the present invention interacts (electrostatically) with acidic molecules such as FGF-2 contained therein, It is considered that the activity of the encapsulated molecule is maintained relatively firmly while maintaining the activity. However, in vivo, the chitosan / IO ₄ heparin hydrogel can be biodegraded and gradually release the encapsulated molecules. Therefore, introduction of FGF-2 or the like into the chitosan / IO ₄ heparin hydrogel of the present invention provides an excellent drug sustained release system.

The FGF-2 carrying chitosan / IO ₄ heparin evaluation of angiogenesis induced by hydrogel FGF-2 carrying chitosan / IO ₄ heparin hydrogel (100 [mu] l) into the left and right of the spine of the mouse, carefully from the base of the tail on the position of 2cm Injected. To assess angiogenesis, mice were sacrificed a predetermined day after hydrogel injection and the tissue hemoglobin weight was measured. Specifically, the skin (1 cm × 1 cm) around the fixed region of the subcutaneous tissue and the hydrogel injection site was cut out with a scalpel, and excess blood was washed with PBS. The removed tissue and skin were soaked in 2 ml of erythrocyte lysis reagent (Sigma and Aldrich) and then minced with a scalpel. Hemoglobin from tissue and skin was extracted for 24 hours at 4 ° C. with several vortex mixing and finally centrifuged at 2000 rpm to remove solid tissue. The amount of extracted hemoglobin in the supernatant was quantified using a total hemoglobin assay kit (Sigma and Aldrich) based on a calibration curve generated using a hemoglobin standard solution (Sigma and Aldrich).

FIG. 4 shows changes in the amount of tissue hemoglobin when the FGF-2 concentration introduced into the chitosan / IO ₄ heparin hydrogel is changed. It can be seen that inclusion of 1 μg or more of FGF-2 in the hydrogel (100 μl) is effective in inducing angiogenesis in vivo in a concentration-dependent manner.
FIG. 5 shows the time course of angiogenesis induced by FGF-2-supported chitosan hydrogel (200 μl). The amount of tissue hemoglobin increased remarkably until day 8 after the injection of FGF-2 loaded chitosan hydrogel, and decreased slightly thereafter, but remained at a high level even on day 21 (FIG. 5: ●). On the other hand, injection of chitosan / IO ₄ heparin hydrogel without FGF-2 (FIG. 5: □) and chitosan with or without FGF-2 (FIG. 5: ■, ○) did not show significant angiogenesis. In addition, when an FGF-2 aqueous solution, IO ₄ heparin solution, or FGF-2 / IO ₄ heparin solution was injected, angiogenesis was not observed (data not shown). That is, only when FGF-2 and chitosan / IO ₄ heparin were administered at the same time, the angiogenic effect was significantly improved.

Histological observation Rats (male, Sprague Dawley rats; Crea Japan Inc.) were injected subcutaneously with FGF-2-supported chitosan / IO ₄ heparin hydrogel, and the tissue containing the hydrogel was taken out after a predetermined date to obtain a 10% formaldehyde solution. It was fixed in, embedded in paraffin, and cut into 4 μm thicknesses (Yamato Kohki). Sections were perpendicular to the anteroposterior axis and perpendicular to the wound surface. Sections were placed on glass slides and stained with hematoxylin-eosin (HE) reagent. In each section, a random area showing the highest capillary density (microscopic field, 100X) was photographed and the number of capillaries per micrograph was counted. Only mature blood vessels containing red blood cells were counted.
For comparison, in addition to the FGF-2-supporting chitosan / IO ₄ heparin hydrogel of the present invention, the same experiment was performed when chitosan, chitosan / IO ₄ heparin hydrogel, and FGF-2 support chitosan were injected.

FIG. 6 is a photomicrograph of the vicinity of the injection site on days 4, 8, and 14 after injection. Regardless of the presence or absence of FGF-2, it is shown that a large number of neutrophils that migrated into the gel after 14 days are retained at the site injected with chitosan / IO ₄ heparin hydrogel. The area holding the FGF-2-supported chitosan hydrogel in the tissue removed 4, 8, and 14 days after injection is covered with a glossy smooth membrane. The hydrogel is surrounded by a fibrous structure and covered with a regenerated thick film (FIG. 6). These fibrous tissues were not observed at the injection site of chitosan and chitosan / IO ₄ heparin hydrogel without FGF-2, but a slight fibrous tissue was observed around the injection site of FGF-2-loaded chitosan.

FIG. 7 shows representative micrographs of surrounding tissues on the 8th day after injection at each injection site. These correspond to the portions enclosed by squares in FIG. In FIG. 7, black triangles indicate portions where mature blood vessels including erythrocytes are formed. In the peripheral part of the FGF-2 loaded chitosan / IO ₄ heparin hydrogel, significantly more mature blood vessels were already observed than other conditions.

FIG. 8 shows the number of newly formed blood vessels plotted against the number of days. Even on the 4th day, compared with other conditions (chitosan only, chitosan / IO ₄ heparin hydrogel, and FGF-2 loaded chitosan), the site where FGF-2 loaded chitosan / IO ₄ heparin hydrogel was injected About twice or more of the blood vessels have been generated, and the difference does not change even on the 14th day. These results indicate that, as shown in Example 4 (FIG. 3), the FGF-2-supporting chitosan / IO ₄ heparin hydrogel of the present invention was gradually degraded in vivo and encapsulated with FGF- 2 is considered to reflect the sustained release.
Similarly, in animal experiments in the ischemic limb model (skin ulcer model of diabetic mice), FGF-2-loaded chitosan / IO ₄ heparin hydrogel promotes angiogenesis and granulation tissue formation, and promotes wound occlusion and healing. Results were obtained.

In vivo stability of hydrogel (strength measurement of hydrogel)
Since the chitosan / IO ₄ heparin hydrogel (with FGF-2) of the present invention is intended to be injected into a living body, it comes into contact with various protein components present in the living body. Therefore, using fetal bovine serum (FCS), which is a serum protein, as a protein component model, the change in strength of the hydrogel in the presence or absence of the protein was measured.

Measuring method:
The measurement system shown in FIG. 9 was configured. That is, a container (20) having an open upper surface was disposed on the upper dish of the upper balance (10), and a plunger (30) that can be vertically moved is disposed above the container (20).
Place 100 μl of 4% CH-LA aqueous solution (1) in the container 20 and overlay it with 100 μl of IO ₄ heparin-containing upper layer solution (2) with or without serum protein so as not to disturb the interface, and leave it for 1 hour or 24 hours. When the gel layer (3) is formed, the plunger (30) (diameter 2 mm) is lowered at a rate of 0.03 mm per second at room temperature to push the gel, and the gel layer (3) is broken. The load was measured with an upper pan balance.

As the upper layer liquid (3), PBS solutions of 1, 4 and 10 mg / ml of IO4 heparin were used, and measurements were made with and without the addition of 10 wt% FCS. For comparison, the same measurement was performed when sodium alginate (manufactured by Wako Pure Chemical Industries) was used instead of IO ₄ heparin. The results are shown in Table 1 below (data in Table 1 is an average value of four measurements).

A sugar chain-containing chitosan derivative (basic) such as CH-LA is considered to gel by forming an ion complex with an acidic (anionic) molecule. However, the above results show that a high-strength hydrogel is formed when an acidic polysaccharide such as IO ₄ heparin is used as the acidic molecule. It was also found that the strength of the hydrogel was improved when the concentration of acidic polysaccharide was higher and sufficient strength was obtained after about 24 hours. Moreover, the gel strength does not decrease even when a protein component is present, and when a serum protein such as FCS is added, the gel strength after 24 hours is rather improved. This is a tendency opposite to that when sodium alginate is used as an acidic (anionic) molecule.

  When the medical composition of the present invention is used, a wound healing promoter such as FGF-2 introduced into a hydrogel of a sugar chain-containing chitosan derivative / acidic polysaccharide is gradually released by biodegradation of the hydrogel, and angiogenesis is performed. And a significant effect on the induction of fibrous tissue formation. Furthermore, the composition of the present invention was injectable, and there was a tendency for strength to be improved by protein components present in the living body. Therefore, the medical composition of the present invention can be used as a novel medical material that induces angiogenesis and fibrous tissue formation in an ischemic limb.

It is a graph showing the release profiles of the IO ₄ heparin from the hydrogel of the present invention (●) and FGF-2 (○). It is a graph which shows the HUVEC proliferation stimulating effect of the hydrogel of this invention. It is a graph which shows release | release of the inclusion molecule (trypan blue) by the in vivo decomposition | disassembly of the hydrogel of this invention. ○: chitosan / IO ₄ heparin hydrogel; ●: chitosan only. It is a graph which shows the relationship between the FGF-2 density | concentration carry | supported by the hydrogel of this invention, and an angiogenesis effect. It is a graph which shows the angiogenesis effect in the structure | tissue which embedded the hydrogel of this invention. ●: FGF-2 carrying chitosan / IO ₄ heparin hydrogel; ■: FGF-2 carrying chitosan; ○: chitosan / IO ₄ heparin hydrogel; □: chitosan only. It is a microscope picture which shows the angiogenesis and the fiber tissue reproduction | regeneration in the structure | tissue which embedded the hydrogel of this invention. It is the microscope picture which expanded a part of structure | tissue shown in FIG. It is a graph which shows the number of blood vessels formed in the structure | tissue which embedded the hydrogel of this invention. ●: FGF-2 carrying chitosan / IO ₄ heparin hydrogel; ■: FGF-2 carrying chitosan; ○: chitosan / IO ₄ heparin hydrogel; □: chitosan only. It is a schematic diagram which shows the apparatus comprised in order to measure the intensity | strength of the hydrogel of this invention.

Explanation of symbols

1: CH-LA aqueous solution; 2: IO ₄ heparin-containing upper layer solution; 3: gel layer; 10: upper pan balance; 20: container; 30: plunger.

Claims (10)

A medical composition comprising a hydrogel formed from a sugar chain-containing chitosan derivative and an acidic polysaccharide, and a wound healing promoter supported thereon. The sugar chain-containing chitosan derivative is represented by the following formula (I):

A polymer obtained by introducing a sugar chain having a reducing end into at least a part of the 2-position amino group of a glucosamine unit of chitin / chitosans comprising a unit represented by 2. The composition according to 1. The composition according to claim 1 or 2, wherein the wound healing promoter is a growth factor. 4. Composition according to claim 3, characterized in that the growth factor is FGF-1, FGF-2, HB-EGF, VEGF ₁₆₅ or HGF. The composition according to claim 1, wherein the acidic polysaccharide is a glycosaminoglycan. 6. The composition of claim 5, wherein the glycosaminoglycan is periodate oxidized heparin. The composition according to any one of claims 1 to 6, further comprising a serum protein. A therapeutic agent for intractable skin ulcer, comprising the composition according to any one of claims 1 to 7. A sustained-release drug comprising the composition according to any one of claims 1 to 7. A tissue regeneration or cell culture substrate comprising the composition according to any one of claims 1 to 7.

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