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Title:  Crosslinked gelatin gel preparation containing basic fibroblast growth factor

United States Patent:  6,831,058

Issued:  December 14, 2004

Inventors:  Ikada; Yoshiro (Kyoto, JP); Tabata; Yasuhiko (Kyoto, JP)

Assignee:  Kaken Pharmaceutical Co., Ltd. (Tokyo, JP)

Appl. No.:  567355

Filed:  November 30, 1995

Abstract

The present invention relates to a crosslinked gelatin gel preparation containing a basic fibroblast growth factor hereinafter referred to as bFGF wherein a crosslinked gelatin gel is used as a sustained release carrier, and which have different water content, i.e. in vivo degradation and absorption by varying the condition for preparing the crosslinked gelatin gel. By the crosslinked gelatin gel preparation of the present invention, the sustained release rate of bFGF can be varied as required and the durability of in vivo activity of bFGF can be controlled.

Description of the Invention

FIELD OF INDUSTRIAL UTILIZATION

The present invention relates to a crosslinked gelatin gel preparation containing a basic fibroblast growth factor (to be abbreviated as "bFGF" hereinafter).

PRIOR ART

In 1976, bFGF was found in bovine pituitary gland by Gospodarowicz as a protein which highly stimulates the proliferation of fibroblast (Nature, Vol. 24, page 124, 1974). Then, bFGF-coding genes have been cloned so that bFGF can be produced in large quantities by genetic recombination technologies, and bFGF has been therefore energetically studied. As a result, it has been revealed that bFGF simulates the proliferation of a variety of cells such as capillary endotherial cells, blood vessel smooth muscle cells, cornea endothelial cells, osteoblast and chondrocyte as well as the proliferation of fibroblast.

Like other polypeptides and proteins, however, bFGF has a short in vivo half-life, and fails to provide an effect as expected when administered as an aqueous solution. It is therefore desirable to formulate bFGF as a sustained release preparation which can stabilize bFGF and can gradually release bFGF for a definite period of time. The present inventors have been therefore engaged in the development of sustained release carriers for formulating bFGF as a sustained release preparation.

In recent years, sustained release preparations of physiologically active peptides and proteins have been extensively studied, and as sustained release carriers therefor, there are synthetic polymers that can undergo degradation by a living body, such as polyglycolic acid.lactic acid and polyanhydride, and natural polymers that can undergo in vivo degradation and absorption, such as polysaccharides and proteins.

PROBLEMS TO BE SOLVED BY THE INVENTION

The natural polymers that can undergo in vivo degradation and absorption have excellent suitability to a living body and cause almost no stimulation to a living body so that they are preferred as sustained release carriers. Since, however, most of these natural polymers are water-soluble, they are not suitable as a sustained release insolubilizing carrier for bFGF which is a water-soluble physiologically active peptide.

The present inventors have therefore made studies for water-insolubilizing natural polymers that can undergo in vivo degradation and absorption, by some method in order to obtain some which can be used as a sustained release insoluble carrier for bFGF.

As a result, it has been found that a crosslinked gelatin gel insolubilized in water by crosslinking a gelatin which is a natural polymer that can undergo in vivo degradation and absorption, is suitable as a sustained release carrier for bFGF, and the present invention has been accordingly completed.

DISCLOSURE OF THE INVENTION

That is, the gist of the present invention consists in a crosslinked gelatin gel preparation containing a basic fibroblast growth factor.

The present invention characteristically provides a sustained release preparation of bFGF, which can achieve a desired sustained release rate as required, on the basis of a crosslinked gelatin gel which has excellent suitability to a living body, causes almost no stimulation on a living body and has excellent properties as a sustained release carrier. The sustained release rate can be varied depending upon the crosslinking degree of a gelatin, the water content of the crosslinked gelatin and the properties of a used gelatin (isoelectric point, etc.).

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will be explained in detail hereinafter.

The crosslinked gelatin gel preparation of the present invention is obtained by incorporating bFGF as an active ingredient into a sustained release crosslinked gelatin gel. The gelatin as a raw material for the crosslinked gelatin gel used in the present invention is not specially limited, and can be selected from generally available ones. Examples of the gelatin include alkali-treated gelatin having an isoelectric point of 4.9 (supplied by Nitta Gelatin Inc.) and acid-treated gelatin having an isoelectric point of 9.0 (supplied by Nitta Gelatin Inc.). As a gelatin, not only one kind of gelatin may be used, but a mixture of gelatins different in physical properties such as solubility, molecular weight, isoelectric point and material may be used.

The crosslinking agent for crosslinking the gelatin, used in the present invention, can be selected from those which are free of toxicity to a living body. For example, it is preferably selected from glutaraldehyde, water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate, a bisepoxy compound and formalin. Glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are particularly preferred.

Further, the gelatin can be crosslinked by heat treatment or irradiation which ultraviolet light.

Although not specially limited in form, the crosslinked gelatin gel as a sustained release carrier used in the present invention has the form, for example, of a cylinder, a prism, a sheet, a disk, spheres or particles. The crosslinked gelatin gel in the form of a cylinder, a prism, a sheet or a disk is generally used for implantation in many cases, and the crosslinked gelatin gel in the form of spheres or particles may be used for the administration by injection as well.

The crosslinked gelatin gel in the form of a cylinder, a prism, a sheet or a disk can be prepared by adding an aqueous solution of the crosslinking agent to a gelatin aqueous solution, or adding the gelatin to an aqueous solution of the crosslinking agent, casting the resultant mixture into a mold having a desired form, and allowing the mixture to react in a crosslinking reaction. Otherwise, and an aqueous solution of the crosslinking agent may be added to gelatin gel obtained by shaping a gelatin as it is or after it is dried. The crosslinking reaction is terminated by bringing the reaction product into contact with a low-molecular-weight substance having an amino group as ethanolamine or glycine, or by adding an aqueous solution having a pH of 2.5 or less. The so-obtained crosslinked gelatin gel is washed with distilled water, ethanol, 2-propanol (to be referred to as "IPA" hereinafter) or acetone, and used for preparing the preparation.

The above-obtained crosslinked gelatin gel has a water content of 50 to 99 w/w % (to be simply denoted by % hereinafter). This water content of a gel shows a ratio of the weight a water content in the gel based on the total gel weight when the gel is wet.

The crosslinking gelatin gel in the form of spheres or particles can be prepared, for example, by either placing an aqueous solution of the gelatin in an apparatus formed of a three-necked flask, a fixed stirrer motor (e.g., three one motor supplied by Shinto Scientific Co., Ltd., EYELA mini D.C. stirrer, or the like) and a stirring propeller of Teflon attached thereto, adding an oil such as olive oil and stirring the mixture at a rate of about 200 to 600 rpm to prepare a W/O emulsion, adding an aqueous solution of the crosslinking agent, or pre-homogenizing a gelatin aqueous solution in an olive oil (e.g., with a vortex mixer Advantec TME-21, a homogenizer polytron PT10-35, or the like), dropwise adding the resultant product in an olive oil to prepare, a finely milled W/O emulsion and adding an aqueous solution of the crosslinking agent, to cause a crosslinking reaction, centrifugally recovering a crosslinked gelatin gel, washing it with acetone or ethyl acetate or the like, and further, immersing the washed crosslinked gelatin gel in IPA ethanol, or the like to terminate the crosslinking reaction. The so-obtained crosslinked gelatin gel is consecutively washed with IPA, distilled water containing Tween 80 and distilled water, and used for preparing the preparation.

When the crosslinked gelatin gel particles from aggregates, for example, they may be ultasonically irradiated (preferably approximately for up to 1 minute with cooling).

In addition, when the pre-emulsification is carried out, there is obtained a crosslinked gelatin gel in the form of fine particles having a particle size of 20 .mu.m or less.

The obtained crosslinked gelatin gel particles have an average particle diameter of 1 to 1,000 .mu.m, and are sieved out to necessary sizes as required depending upon purposes. For example, for the topical administration for the therapy of the fracture of a human bone and osteoporosis, it is preferred to use crosslinked gelatin gel particles having a size of 10 to 150 .mu.m. Further, the obtained crosslinked gelatin gel has a water content of approximately 50 to 93%, and can be prepared as products having water contents as required.

The crosslinked gelatin gel in the form of spheres or particles is also produced by other method as below. An olive oil is placed in an apparatus similar to that in the above method and stirred at a rate of approximately 200 to 600 rpm, and a gelatin aqueous solution is dropwise added thereto to prepare a W/O emulsion. This W/O emulsion is cooled, then acetone, ethyl acetate or the like is added, and the mixture is stirred and centrifugally separated to recover gelatin particles. The recovered gelatin particles are further washed with acetone or ethyl acetate and then with IPA, ethanol or the like, and then dried. The dry gelatin particles are suspended in a crosslinking agent aqueous solution containing 0.1% Tween 80, and the mixture is moderately stirred to cause a crosslinking reaction. The crosslinking reaction is terminated by washing the reaction mixture with a 100 mM glycine aqueous solution containing 0.1% Tween 80 or with 0.004N HCl containing 0.1% Tween 80 depending upon the crosslinking agent used, thereby to obtain crosslinked gelatin gel particles.

The average particle diameter and the water content of the crosslinked gelatin gel particles obtained by this "other" method are similar to those of the crosslinked gelatin gel particles obtained by the above method.

The conditions of the crosslinking reaction should be properly selected as required, while, preferably, the reaction temperature is 0 to 40<sup>o</sup>C., and the reaction time is 1 to 48 hours.

The above-obtained crosslinked gelatin gel may be further dried under reduced pressure or lyophirized.

The freeze-drying is carried out, for example, by placing the crosslinked gelatin gel in distilled water, freezing it in liquid nitrogen for at least 30 minutes or at -80<sup>o</sup>C. for at least 1 hour, and then drying it with a freeze-dryer for 1 to 3 days.

The concentrations of the gelatin and the crosslinking agent for the preparation of the crosslinked gelatin gel should be properly selected depending upon a desired water content, while, preferably, the gelatin concentration is 1 to 100 w/v % (to be simply denoted by % hereinafter), and the crosslinking agent concentration is 0.01 to 100 w/v % (to by simply denoted by % hereinafter) equivalent to 1 to 5,400 mM).

The crosslinked gelatin gel can be arranged to have a desired water content by varying the concentrations of the gelatin as a raw material and the crosslinking agent. For increasing the water content, both the gelatin concentration and the crosslinking agent concentration are decreased. For decreasing the water content, conversely, both the gelatin concentration and the crosslinking agent concentration are increased.

For incorporating bFGF into the above-prepared crosslinked gelatin gel, the crosslinked gelatin gel is impregnated with bFGF by dropwise adding a bFGF aqueous solution to the crosslinked gelatin gel, or by suspending the crosslinked gelatin gel in a bFGF aqueous solution to re-shell the crosslinked gelatin gel.

The amount of bFGF which can be incorporated into the crosslinked gelatin gel differs depending upon the water content of the crosslinked gelatin gel, and the like, while bFGF can be incorporated in an amount of 0.1 to 500 .mu.g per mg of the crosslinked gelatin gel.

The sustained release period and the release amount of bFGF differ depending upon various conditions such as the amount of bFGF contained in the preparation, the water content of the crosslinked gelatin gel, physical properties of the gelatin used such as isoelectric point, an administration site, etc.

The bFGF-containing crosslinked gelatin gel obtained in the above manner (to be referred to as "crosslinked gelatin gel preparation" hereinafter) may be lyophilized. When lyophirized, for example, it is frozen in liquid nitrogen for at least 30 minutes or at -80<sup>o</sup>C. for at least 1 hour, and then dried with a freeze-dryer for 1 to 3 days.

The bFGF used as an active ingredient in the crosslinked gelatin gel preparation of the present invention includes those extracted from organs such as pituitary gland, brain, retina, corpus lutenum, adrenal, kidney, placenta, prostate and thymus, those produced by genetic engineering methods such as recombination DNA technique, and those which are modified products of these and work as fibroblast growth factor. The modified product of bFGF includes those obtained by adding amino acid(s) to the amino acid sequence of bFGF obtained by the above extraction or genetic engineering method, bFGF in which amino acid(s) is partly replaced with other amino acid(s), and bFGF in which amino acid(s) is lacking. In the present invention, these bFGFs or modified products may be used alone or in combination.

Examples of the above bFGF preferably include those disclosed in WO87/01728 (Japanese PCT Publication No. 63-500843), WO89/04832 (Japanese PCT Publication No. 2-504468), WO86/07595 (Japanese PCT Publication No. 63-500036), WO87/03885 (Japanese PCT Publication No. 63-501953), European Laid-open Patent Publication No. 237966 (JP-A-63-226287), European Laid-open Patent Publication No. 281822 (JP-A-193), European Laid-open Patent Publication No. 326907 (JP-A-2-209894), European Laid-open Patent Publication No. 394851 (JP-A-3-61494) and European Laid-open Patent Publication No. 493737 (JP-A-5-124975).

Of the above bFGFs, a polypeptide having a sequence of 154 amino acids in the following SEQ ID No. 1 and a polypeptide having a sequence of 153 amino acids in the following SEQ ID No. 2, produced by a genetic engineering method and disclosed in WO87/01728, are particularly preferred in view of stability and availability of a necessary amount on a constant basis.

bFGF having the amino acid sequence shown in the SEQ ID No. 1 is obtained by preparing a cDNA clone of human bFGF from a .lambda.<sub>gt</sub> 10cDNA library prepared from mRNA of human kidney using bovine 1.4 kb basic sub-fragment, and constituting an expression vector to express the above clone, as is described in Japanese PCT Publication No. 63-500843.

    SEQ ID No.:1
    Sequence characteristics:
    Sequence length: 154 amino acids
    Sequence type: amino acid
    Original source
    Organism: Homo sapiens
    Sequence
    Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly
     1               5                  10                   15
    Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr
                20                   25                  30
    Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val
             35                  40                  45
    Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln
         50                  55                  60
    Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg
    65                  70                   75                 80
    Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val
                    85                  90                  95
    Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn
                100                 105                 110
    Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg
            115                 120                 125
    Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala
        130                 135                 140
    Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
    145                 150
    SEQ ID No.:2
    Sequence characteristics:
    Sequence length: 153 amino acids
    Sequence type: amino acid
    Original source
    Organism: Homo sapiens
    Sequence
    Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser
     1               5                   10                  15
    Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys
                20                  25                  30
    Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp
            35                  40                  45
    Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala
        50                  55                  60
    Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr
    65                  70                  75                   80
    Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr
                    85                  90                  95
    Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr
                100                 105                 110
    Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr
            115                 120                 125
    Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile
        130                 135                 140
    Leu Phe Leu Pro Met Ser Ala Lys Ser
    145                 150

Claim 1 of 12 Claims

What is claimed is:

1. A method of treating a fracture comprising administering to an animal in need of same a therapeutically effective, fracture treating amount of basic fibroblast growth factor impregnated in a water insoluble, in vivo degradable cross-linked gelatin gel for sustained release of the basic fibroblast growth factor, the cross-linked gelatin gel having a water content of 63.1 to 99% w/w and produced by process comprising the steps of:

(a) cross-linking an alkali-treated gelatin having an isoelectric point of 4.9 to provide a cross-linked, water-insoluble gelatin gel;

(b) lyophilizing or drying the cross-linked, water-insoluble gelatin gel of step (a); and

(c) incorporating basic fibroblast growth factor into the cross-linked, water-insoluble gelatin gel of step (b).


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