JP2002128694A - bFGF SUSTAINED RELEASE PREPARATION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preparation for sustainedly releasing bFGF, namely bFGF sustained release preparation and a method for producing the preparation. SOLUTION: This sustained release preparation comprises an insoluble combination produced by dissolving a small amount of bFGF in a human serum protein solution having no pharmaco dynamic action and then mixing the solution with an acidic mucopolysaccharide solution to make the solution acidic.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a bFGF comprising bFGF, an insoluble conjugate formed by acidifying a mixture of a protein such as human γ-globulin and albumin and an acidic mucopolysaccharide solution such as sodium chondroitin sulfate.
The present invention relates to a GF sustained release preparation and a method for producing the sustained release preparation.

[0002]

2. Description of the Related Art Japanese Patent Application No. 2000-203850 filed on Jul. 5, 2000 by the present applicant as an invention relating to an excellent sustained-release preparation for injection applicable to many protein drugs and the like.
There is.

[0003] Japanese Patent Application No. 2000-203850 discloses a method for preparing a pharmaceutical preparation which is extremely simple and provides a sustained-release preparation comprising a protein capable of being applied to almost all protein drugs and having an excellent sustained-release effect, and a polysaccharide inactive conjugate. A method for producing the sustained release formulation is described.

[0004] Further, the above-mentioned literature discloses F as a protein preparation.
It is described that there is GF.

[0005]

However, FGF has a
Although there are FGF and bFGF, Japanese Patent Application No. 2000-2038
In 50 documents, there is only a description of FGF,
There is no description about F and bFGF. That is, Japanese Patent Application 200
At the time of filing of 0-203850, aFGF and bFGF
Has not been studied yet.

Accordingly, the present inventors have conducted intensive studies on bFGF, and found that a sustained-release preparation of bFGF is bFGF.
F and acid mucopolysaccharides such as sodium chondroitin sulfate alone do not precipitate and become muddy and do not form a suspension of an insoluble conjugate suitable as an injectable preparation, but human gamma globulin, human serum albumin, human fibrinogen It has been found that the addition of a human serum protein having no medicinal effect such as that described above can form a suspension of an insoluble conjugate suitable as an injectable preparation.

Accordingly, an object of the present invention is to provide a preparation for sustained release of bFGF, that is, a sustained release preparation of bFGF and a method for producing the same.

[0008]

Means for Solving the Problems Therefore, B of the present invention
FGF sustained-release preparations are prepared by (1) dissolving a small amount of bFGF in a non-medicated human serum protein solution, then mixing an acidic mucopolysaccharide solution, and acidifying the solution to form an insoluble bond. BFGF sustained-release preparation comprising body, (2) bF
When a small amount of GF is dissolved in a non-medicated human serum protein solution, if the binding is weak, lower the ionic strength and / or temperature, and / or add a binding enhancer, and then add the acidic mucopolysaccharide. BFGF sustained-release preparation comprising an insoluble conjugate produced by mixing a body solution and making the solution acidic,
(3) When the two solutions are mixed to bind a small amount of bFGF to human serum protein, if the binding is weak, lower the ionic strength and / or temperature, and / or add a binding enhancer, and then BFG comprising an insoluble conjugate formed by mixing an acidic mucopolysaccharide solution and acidifying the solution
F sustained-release preparation, (4) bFG consisting of an insoluble conjugate formed by covalently binding when bFGF is dissolved in a human serum protein solution to measure binding and when binding is weak
F sustained release preparation, (5) bFGF, a chondroitin sulfate solution and a human γ globulin solution are mixed, an insoluble conjugate produced by acidification is suspended, and a bFGF sustained release preparation comprising the suspension, 6) The final concentration of the sodium chondroitin sulfate solution is 0.1% to 2%, and the final concentration of the human gamma globulin solution is 0.1% to 2%.
(7) bFGF sustained release preparation using sodium hyaluronate or all acidic mucopolysaccharides present in the human body instead of sodium chondroitin sulfate, (8) human serum albumin instead of human γ globulin 1. a sustained-release bFGF preparation using human fibrinogen; The method for producing the bFGF sustained-release preparation of the present invention comprises:
(1) mixing bFGF, a chondroitin sodium solution and a globulin solution containing a human γ globulin solution,
Produce insoluble conjugate by acidification, spin down,
Removing the supernatant, resuspending the insoluble conjugate into small particles in a buffer solution at pH 6 to pH 8, and freeze-drying the same to produce a sustained-release bFGF preparation;
(2) bFGF, sodium chondroitin sulfate solution and human gamma globulin solution are mixed and acidified to form an insoluble conjugate, spun down, and the supernatant is removed.
A process for producing a bFGF sustained-release preparation produced by resuspending the insoluble conjugate in a buffer solution of pH 8 to a small particle and centrifuging again, (3) the insoluble conjugate according to (1) above. For producing a sustained-release bFGF formulation by resuspending the insoluble conjugate described in the above (2) into small particles in a weakly acidic buffer. (5) A final concentration of the sodium chondroitin sulfate solution is 0.1% to 2%.
Wherein the final concentration of the human gamma globulin solution is 0.1
(6) a method for producing a sustained release bFGF preparation using sodium hyaluronate or all acidic mucopolysaccharides present in the human body in place of the sodium chondroitin sulfate, 7) The human γ
A method for producing a sustained-release bFGF preparation using human serum albumin and human fibrinogen instead of globulin,
Further, 3. The bFGF sustained-release preparation of the present invention comprises: (1) mixing the sodium chondroitin sulfate solution and the globulin solution containing the human γ globulin solution and making the mixture acidic to produce an insoluble conjugate; BFGF sustained-release preparation produced by removing the insoluble conjugate into small particles in a buffer solution of pH 6 to pH 8 and lyophilizing the same; (2) the sodium chondroitin sulfate solution and the human γ The globulin solution is mixed and acidified to form an insoluble conjugate, spun down, the supernatant is removed, and the insoluble conjugate is resuspended in a buffer of pH 6 to pH 8 into small particles, BFGF sustained-release preparation produced by centrifuging again, (3)
BF produced by resuspending the insoluble conjugate according to (1) above in a weakly acidic buffer so as to be small particles.
A GF sustained-release preparation, (4) a bFGF sustained-release preparation produced by resuspending the insoluble conjugate according to the above (2) into small particles in a weakly acidic buffer, (5) the sodium chondroitin sulfate solution Final concentration of 0.1% to 2
% And the final concentration of the human gamma globulin solution is 0.1%.
BFGF sustained-release preparation of 1% to 2%, (6) bFGF sustained-release preparation using sodium hyaluronate or all acidic mucopolysaccharides present in the human body in place of sodium chondroitin sulfate, (7) human γ This is a bFGF sustained-release preparation using human serum albumin and human fibrinogen instead of globulin.

As described above, by using bFGF as a sustained release preparation, the stability of bFGF can be increased, and bFGF can be administered locally (ie, local injection, implanted pellet, ointment). The present invention can be applied to treatment of pressure ulcers and early healing at the time of fracture or intestinal surgery due to angiogenesis and cell proliferation.

[0010] The bFGF sustained-release preparation is also called a bFGF muco preparation since it uses an acidic mucopolysaccharide.

[0011]

EXAMPLE The present invention was prepared by using a suspension under the following formula: bFGF 1 μg / 10 μl CS-A 3 mg (1% CS-A / PBS 300 μl) γ-globulin 9 mg (2% γ-globulin / PBS 450 μl) The results of the study of sustained release in vitro were shown in Example 1 below. The suspension was injected subcutaneously into the back of rats, and after 7 days, the results of angiogenesis were shown in Example 2 below. Was implanted into the back of a rat, and the results of angiogenic action 7 days later are shown in Example 3 below.

Example 1 1 μg / 10 μl of bFGF and 9 mg of γ-globulin (2% γ-g
lobulin / PBS 450 μl), mix well, and then add CS-A
3 mg (300 µl of 1% CS-A / PBS) was added and mixed well. 0.2N HCl was added to pH 3 (p
H3). After the precipitate has fallen, the mixture is immediately centrifuged (1000 rpm, 10 min), and the supernatant is separated with 1% HSA.
The solution was replaced with PBS (buffer). Thereafter, the supernatant was replaced daily with 1 ml of 1% HSA-containing PBS, and the room temperature (25-3
0 ° C.). As a result, as shown in FIG. 1, gradual sustained release for 7 days was shown. In addition, it was shown that 100% of the bFGF was recovered and was stable in the preparation. On the other hand, the stability of bFGF alone in physiological saline was as simple as 7 days and 50% of the initial concentration as shown in FIG.
, Suggesting that the bFGF muco preparation (also referred to simply as muco agent) is excellent in stability.

Example 2 1 μg / 10 μl of bFGF and 9 mg of γ-globulin (2% γ-g
lobulin / PBS 450 μl), mix well, and then add CS-A
3 mg (300 µl of 1% CS-A / PBS) was added and mixed well. 0.2N HCl was added to pH 3 (p
H3). After the precipitate has fallen, the mixture is immediately centrifuged (1000 rpm, 10 min), and the supernatant is separated with 1% HSA.
It was replaced with PBS containing. This suspension was administered subcutaneously to the back of the rat. After 7 days, new blood vessels were observed under the back (FIG. 3).

FIG. 3A shows the state of blood vessels when only bFGF is administered subcutaneously (SC), and shows a state in which no new blood vessels have appeared. FIG. 3b shows bFG
It shows the condition of blood vessels when a sustained-release F preparation (bFGF muco preparation) is administered subcutaneously, and shows the state where new blood vessels are emerging at the locations indicated by arrows. FIG. 3c shows the state of the blood vessel when only chondroitin sulfate sodium containing no bFGF and human gamma globulin (an empty pellet) are implanted, and shows a state in which no new blood vessel has emerged. FIG. 3d shows the state of the blood vessel when the pellet of the bFGF sustained-release preparation (bFGF muco preparation) is embedded, and shows a state in which a new blood vessel appears at the location of the arrow.

Example 3 1 μg / 10 μl of bFGF and 9 mg of γ-globulin (2% γ-g
lobulin / PBS 450 μl), mix well, and then add CS-A
3 mg (300 µl of 1% CS-A / PBS) was added and mixed well. 0.2N HCl was added to pH 3 (p
H3). After the precipitate has fallen, the mixture is immediately centrifuged (1000 rpm, 10 min), and the supernatant is separated with 1% HSA.
It was replaced with PBS containing. This suspension is further centrifuged,
(1000 rpm, 10 min), the supernatant was removed, and the residue pellet was subcutaneously implanted in the back of the rat. After 7 days, many new blood vessels were observed under the back (FIG. 4).

Here, FIG. 4A shows a state in which the state of the blood vessel in FIG. 3A is raised by injecting intrapolis, and shows a state in which a state in which no new blood vessel is seen can be clearly understood. FIG. 4B shows a state in which the state of the blood vessel in FIG. 3B is raised by injecting intrapolis, and shows a state in which a new blood vessel appears at the position of the arrow. FIG. 4C shows a state in which the state of the blood vessel in FIG. 3C is made to stand out by injecting intrapolis, and shows a state in which no new blood vessel is seen. FIG. 4D shows a state in which the state of the blood vessel in FIG. 3D is raised by injecting intrapolis, and shows a state in which a new blood vessel is clearly visible at the position of the arrow.

[Brief description of the drawings]

FIG. 1 is a diagram showing sustained release of bFGF in PBS containing 1% HSA.

FIG. 2 is a graph showing the time course of bFGF activity at room temperature.

FIG. 3A is a view showing a state of a blood vessel when only bFGF is subcutaneously administered (SC), and is a view showing a state in which a new blood vessel has not appeared. b is a bFGF sustained release preparation (b
It is a figure which shows the state of the blood vessel at the time of subcutaneously administering (FGF muco preparation), and is a figure which shows the state in which the new blood vessel has appeared in the place of the arrow. c is a view showing the state of the blood vessel when only chondroitin sulfate sodium containing no bFGF and human gamma globulin (an empty pellet) are embedded, and is a view showing a state in which no new blood vessel has appeared. d is bFG
It is a figure which shows the state of the blood vessel when the pellet of the F sustained-release preparation (bFGF muco preparation) is embedded, and shows the state where the new blood vessel appears at the location of the arrow.

FIG. 4A is a view in which the state of the blood vessel in FIG. 3A is raised by injecting intrapolis. FIG. 3B is a diagram in which the state of the blood vessel in FIG. 3B is made to stand out by injecting intrapolis, and is a diagram showing a state in which a new blood vessel appears at the position of the arrow. FIG. 3C is a view in which the state of the blood vessel in FIG. FIG. 3D is a diagram in which the state of the blood vessel in FIG. 3D is made to stand out by injecting intrapolis, and is a diagram showing a state in which a new blood vessel appears at the position of the arrow.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61K 47/42 A61K 47/48 47/48 A61P 9/00 A61P 9/00 17/02 17/02 19/08 19 / 08 A61K 37/24 F term (reference) 4C076 AA22 AA31 AA94 CC11 CC19 EE37 EE41 EE56 FF16 FF31 GG08 4C084 AA02 AA03 BA41 CA62 DB54 MA05 MA23 MA44 NA12 ZA362 ZA892 ZB212

Claims (20)

[Claims] 1. A bFGF comprising an insoluble conjugate formed by dissolving a small amount of bFGF in a non-medicated human serum protein solution, mixing an acidic mucopolysaccharide solution, and acidifying the solution. Sustained release formulation. 2. When a small amount of bFGF is dissolved in a non-pharmacologically effective human serum protein solution, if the binding is weak, the ionic strength and / or the temperature may be lowered, and / or a binding enhancer may be added. Next, a bFGF sustained-release preparation comprising an insoluble conjugate produced by mixing an acidic mucopolysaccharide solution and acidifying the solution. 3. When the two solutions are mixed to bind a small amount of bFGF to human serum protein, if the binding is weak,
A bFGF sustained-release preparation comprising an insoluble conjugate produced by lowering the ionic strength and / or temperature and / or adding a binding enhancer, and then mixing the acidic mucopolysaccharide solution to make the solution acidic. 4. A sustained-release bFGF preparation comprising an insoluble conjugate produced by covalently binding when bFGF is dissolved in a human serum protein solution and binding is to be measured when binding is weak. 5. A sustained-release bFGF preparation comprising a suspension of an insoluble conjugate produced by mixing bFGF, a solution of sodium chondroitin sulfate, and a solution of human γ globulin and making the mixture acidic, and then suspending the mixture. 6. The method according to claim 5, wherein the final concentration of the sodium chondroitin sulfate solution is 0.1% to 2%, and the final concentration of the human gamma globulin solution is 0.1% to 2%. bFGF sustained release formulation. 7. The sustained-release bFGF preparation according to claim 5, wherein sodium hyaluronate or all acidic mucopolysaccharides present in the human body are used in place of sodium chondroitin sulfate. 8. The sustained-release bFGF preparation according to claim 5, wherein human serum albumin and human fibrinogen are used in place of human gamma globulin. 9. An insoluble conjugate is produced by mixing bFGF, a sodium chondroitin sulfate solution and a human γ globulin solution and acidifying the mixture, centrifuging the supernatant, removing the supernatant, and removing a buffer solution of pH 6 to pH 8. And resuspending the insoluble conjugate into small particles, followed by freeze-drying to produce a sustained-release bFGF preparation. 10. An insoluble conjugate is produced by mixing bFGF, a sodium chondroitin sulfate solution and a human γ globulin solution, and making the mixture acidic, spun down, removing the supernatant, and removing a buffer solution of pH 6 to pH 8. And resuspending the insoluble conjugate into small particles and centrifuging again to produce a sustained-release bFGF preparation. 11. A method for producing a sustained-release bFGF preparation, which is produced by resuspending the insoluble conjugate according to claim 9 or 10 in a weakly acidic buffer so as to become small particles. 12. The method according to claim 9, wherein the final concentration of the sodium chondroitin sulfate solution is 0.1% to 2% and the final concentration of the human gamma globulin solution is 0.1% to 2%. A method for producing the sustained-release bFGF preparation according to the above. 13. The method for producing a sustained release bFGF preparation according to claim 9, wherein sodium hyaluronate or any acidic mucopolysaccharide present in the human body is used in place of sodium chondroitin sulfate. 14. The method for producing a sustained-release preparation according to claim 9, wherein human serum albumin and human fibrinogen are used in place of human γ-globulin. 15. A sustained-release bFGF preparation produced by the production method according to claim 9. 16. A sustained-release bFGF preparation produced by the production method according to claim 10. 17. A sustained-release bFGF preparation produced by the production method according to claim 11. 18. A sustained-release bFGF preparation produced by the production method according to claim 12. 19. A sustained-release bFGF preparation produced by the production method according to claim 13. 20. A sustained-release bFGF preparation produced by the production method according to claim 14.