JP3914284B2 - Fibroblast growth factor complex - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fibroblast growth factor (hereinafter referred to as FGF) complex effective as a cell growth promoter such as a wound healing promoter, a method for producing the same, and a fibroblast growth promoter containing this complex as an active ingredient About.
[0002]
[Prior art]
FGF is a series of proteins that have been attracting attention for a long time as growth factors such as fibroblasts and vascular endothelial cells. Based on their isoelectric points, FGF is largely divided into basic FGF (bFGF) and acidic FGF (aFGF). Separated. Among these, bFGF is known to have a strong cell growth promoting activity and a strong angiogenic action. These actions are essential for the repair of tissue damage, and bFGF is thought to play an important role in promoting healing of the damaged site.
[0003]
bFGF has a heparin-binding site and is known to form a complex with a heparin-like polysaccharide. In addition, bFGF forms a complex with glycosaminoglycan, thereby binding to the membrane receptor of the target cell and expressing cell proliferation activity [Kragsburn, Cell: 67, 229. (1991)]. Furthermore, it has been reported that the stability of bFGF in a solution is improved by forming a complex with glycosaminoglycan (see JP-A-2-40399).
[0004]
[Problems to be solved by the invention]
Thus, the complex of bFGF and glycosaminoglycan has excellent properties, but glycosaminoglycan does not have an action of enhancing the cell proliferation activity of bFGF itself, Since glycosaminoglycan is expensive, it has a problem that it is difficult to supply a large amount of a complex with bFGF at low cost.
Accordingly, the present invention provides a complex of FGF activity enhancing substance and FGF that can be supplied in large quantities at low cost, a method for producing the same, and a fibroblast proliferation promoter effective as a wound healing promoter, etc., using this complex as an active ingredient Is to provide.
[0005]
[Means for Solving the Problems]
The present inventors have found that fucoidan, which is an extract of brown algae or red algae, particularly mozuku, has an excellent healing promoting effect on gastric ulcers (see JP-A-7-138166). Subsequent investigations have found that fucoidan binds to FGF in an aqueous medium to form a complex, and that the resulting complex has an excellent cell growth promoting action, thereby completing the present invention.
[0006]
That is, the present invention provides a fibroblast growth promoter comprising a complex of fibroblast growth factor and fucoidan as an active ingredient.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The FGF used in the present invention may be bFGF or aFGF, but bFGF is preferable from the viewpoint of the efficiency of binding to fucoidan.
[0008]
Specific examples of FGF include those derived from mammals, that is, those extracted from various organs such as the pituitary gland of humans, monkeys, pigs, cows, sheep and horses.
[0009]
As the fucoidan used in the present invention, a commercially available reagent or purified product may be used, and an extract or a purified product thereof extracted from hot algae or brown algae containing abundant red algae or brown algae with hot water or dilute hydrochloric acid aqueous solution. Also good. Examples of brown algae include mozuku, sea urchin, wakame, hibamata, Himansria, bifkaria, Fucus becyclosas and the like.
Fucoidan extracted from red algae and brown algae usually has a molecular weight of around 100,000, about 50-60% or more is fucose and contains some uronic acid. Some of the constituent sugars are sulfated. In the present invention, fucoidan extracted from mozuku is preferably used.
[0010]
In order to extract fucoidan from red algae and brown algae, the hot water extraction method or the acid extraction method described in JP-A-7-138166 may be performed. It can be a thing.
[0011]
A complex of FGF and fucoidan can be produced by simply mixing FGF and fucoidan in an aqueous medium. The amount of FGF and fucoidan used is preferably 2 to 20 mol of fucoidan, particularly preferably 5 to 10 mol, relative to 1 mol of FGF.
[0012]
The concentration of FGF in the aqueous medium is preferably 0.0001 to 0.1 W / V%, particularly preferably around 0.001 W / V%.
Further, the concentration of fucoidan in the aqueous medium is preferably 0.001 to 1 W / V%, particularly preferably around 0.01 W / V%.
[0013]
In order to mix FGF and fucoidan in an aqueous medium, the respective aqueous medium solutions can be mixed, or one of them can be dissolved in an aqueous medium, and then the other can be charged. The mixing of FGF and fucoidan is preferably carried out at pH 3-10, particularly preferably pH 5-9. When water (preferably distilled water for injection) is used as the aqueous medium, the pH is adjusted using Tris-hydrochloric acid or disodium phosphate-monopotassium phosphate. Moreover, you may use what adjusted pH beforehand, such as PBS (physiological phosphate buffer solution) (pH 7.5), as an aqueous medium.
[0014]
The temperature at the time of mixing is preferably 2 to 40 ° C, particularly preferably around 25 to 35 ° C. The time required for forming the complex is about 2 to 24 hours. For example, the complex is formed in about 24 hours at a mixing temperature of about 37 ° C. and about 24 hours at a mixing temperature of about 4 ° C.
[0015]
The complex thus obtained has an action of promoting the proliferation of fibroblasts and is highly safe. Therefore, the fibroblast proliferation promoter containing this as an active ingredient is a wound healing promoter, a burn-inducing agent. It can be used as a therapeutic agent. The dosage form and dosage in the case of these pharmaceuticals can be arbitrarily selected. In general, an acceptable liquid or solid carrier is blended in the composite, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant as necessary. It is appropriate to add a bulking agent and the like to prepare a tablet, granule, powder, powder, capsule or the like.
[0016]
A suitable daily dose of the complex for adults is about 1 to 500 mg, preferably 5 to 50 mg per kg body weight, and can be added to any food or drink and taken daily.
[0017]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.
[0018]
Test example 1
In examining the binding property between bFGF and fucoidan, fucoidan was first solid-phased. Since fucoidan is a compound that does not have a hydrophobic group, it cannot be immobilized on a plastic plate by the usual method for immobilizing proteins and the like. Thus, an ELISA-immobilized plate was prepared in the following manner in which the reducing end of fucoidan was aminated and bound to the activation plate.
[0019]
100 μl of 10 mg / ml NaBH ₄ solution was added to 5 ml of 50 mg / ml fucoidan aqueous solution and left at room temperature for 3 hours to open the reducing terminal fucose residue. After desalting by dialysis, 200 μl of 0.2 M periodic acid solution and 1 ml of 50 mM imidazole hydrochloric acid were added, and the mixture was left to stand for 1 hour on ice to make an aldehyde group. After dialysis, 1 ml of 20 mg / ml diaminopropanol was added and reacted at 60 ° C. for 1 hour to form a Schiff base. Thereafter, 20 μl of 10 mg / ml NaBH ₃ CN was added and reacted at 60 ° C. for 18 hours. 50 μl of the reaction solution was dispensed into CovaLink (manufactured by Nunk Inter Med; activated 96-well plate), and allowed to stand at room temperature for 18 hours to couple fucoidan to the plate. Thereafter, it was blocked with a 5% BSA solution, dried and stored.
[0020]
Using the fucoidan solid-phase plate prepared by the above method, fucoidan binding to bFGF was examined by ELISA (enzyme immunoassay). As a control, fucoidan binding to EGF without a heparin binding site was also examined.
[0021]
First, a solution (0 to 1 μg / ml in PBS) of bFGF (human-derived genetic recombination; manufactured by Sigma Immuno Chemicals) and EGF (human-derived genetic recombination; manufactured by Sigma Immuno Chemicals) is placed on a fucoidan-immobilized plate. Dispense 100 μl. 100 μl of primary antibody solution (anti-bovine bFGF rabbit IgG; manufactured by R & D systems and anti-human EGF mouse IgG; manufactured by Becton Dickinson) diluted 2,000 times after standing at 37 ° C. for 1 hour and washing 3 times Poured and left at 37 ° C. for 90 minutes. After washing, 100 μl of a 2,000-fold diluted secondary antibody solution (peroxidase-conjugated anti-rabbit IgG goat IgG; manufactured by Cappel and peroxidase-conjugated anti-mouse IgG goat IgG; manufactured by Cappel) was dispensed at 60 ° C. at 60 ° C. Left for a minute. After washing, a chromogenic substrate was added. After the reaction, 1% SDS solution was added to stop the reaction, and the absorbance at a wavelength of 405 nm was measured. From the results, the absorbance increased in a dose-dependent manner in the bFGF addition group, so that the binding of fucoidan to bFGF can be confirmed (FIG. 1).
[0022]
Production Example 1
In the above test, it was found that fucoidan has an affinity for bFGF. Therefore, Mozuku fucoidan-bFGF complex was prepared by the following method. When fucoidan in a molar ratio of 5 times was added to 10 mM phosphate buffer solution (pH 7.5) of bFGF and reacted at 37 ° C. for 2 hours, most of the bFGF in the solution formed a complex with fucoidan. did.
[0023]
Test example 2
With respect to the Mozuku fucoidan-bFGF complex obtained in Production Example 1, the cell proliferation activity was compared with that of bFGF. In this experiment, 3T3-SV-40 (3T3 Swiss albino SV40 transformed; mouse-derived fibroblast cell line) used for bFGF titer measurement was used. The cell proliferation activity was evaluated by the reduction activity derived from the cytochrome system (WST1 method). The specific method is as follows.
[0024]
3T3 cells pre-cultured in SFM 101 medium (manufactured by Nissui Pharmaceutical Co., Ltd .; containing 1% FCS) for 3 days were adjusted to 1 × 10 ⁴ cells / ml, and dispensed in 1-ml portions into 24-well plates. Then, bFGF or bFGF preincubated with 5 times equivalent amount of fucoidan was added at 0, 1, 5, 10 ng / well in bFGF equivalent amount, and cultured in a CO ₂ incubator. Three days later, the supernatant was aspirated, 1 ml of WST1 solution was added, and cultured in a CO ₂ incubator for 1 hour. The WST1 solution is 0.5 ml of 0.7 mg / ml 1-methoxymethylphene in 4.5 ml of Eagle MEM medium (manufactured by Nissui Pharmaceutical; 10% FCS) containing 16.7 mg of WST1 (manufactured by Dojindo). A mixture of dinium methylsulfuric acid (Dojindo) solution. After incubation, the absorbance was measured at a measurement wavelength of 450 nm and a control wavelength of 620 nm, and used as an index of the number of cells. As a result of the t-test, it can be seen that cell growth was significantly promoted in the fucoidan-bFGF complex added group as compared to the bFGF added group (FIG. 2).
[0025]
【The invention's effect】
The FGF complex of the present invention has a strong cell growth promoting action and is effective as a wound healing promoter and the like.
[Brief description of the drawings]
FIG. 1 is a graph showing bFGF binding to a fucoidan-immobilized plate.
FIG. 2 is a graph showing the influence of fucoidan on bFGF cell proliferation activity.

Claims (1)

  A fibroblast growth promoter comprising a complex of fibroblast growth factor and fucoidan as an active ingredient.

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