CA2048638C

CA2048638C - Fibroblast growth factor (fgf)-based stabilized compositions and the use thereof

Info

Publication number: CA2048638C
Application number: CA002048638A
Authority: CA
Inventors: Denis Barritault; Jacqueline Jozefonvicz; Faouzi Slaoui; Michele Tardieu; Jean-Pierre Caruelle; Jose Courty
Original assignee: Universite Sorbonne Paris Nord
Current assignee: Universite Sorbonne Paris Nord
Priority date: 1989-03-09
Filing date: 1990-03-09
Publication date: 2002-06-11
Anticipated expiration: 2010-03-09
Also published as: FR2644066A1; DE69009748T2; FR2644066B1; JPH04505756A; ATE106743T1; ES2057544T3; DK0462194T3; EP0462194B1; JP3236916B2; DE69009748D1; CA2048638A1; WO1990010456A1; AU5283890A; EP0462194A1

Abstract

A novel stabilized composition is provided herein. The stabilized composition includes an agent having a cell and tissue regenerating activity. The agent comprises at least one functionalized, substituted dextran containing the following functions, namely carboxymethyl, benzylamide or benzylamide sulphonate, in association with at least one of the following: an acid FGF; a basic FGF; a derivative thereof; an analogue thereof;
a fragment thereof, the fragment thereof having a biological activity. The agent is capable of at least partially restoring the biological activity of the acid and/or basic FGF/FGFs which have been inactivated by prolonged storage or temperature.
Novel uses for this stabilized composition are also provided.

Description

44~63~

(a) TITLE OF THE INVENTION
Fibroblast Growth Factor (FGF)-Based Stabilized Compositions and the Use Thereof.
(b) TECHNICAL FIELD TO WHICH THE INVENTION RELATES
The present invention relates to agents having a cell and tissue regenerating activity, consisting of specified dextrans, to stabilized compositions containing the agents in association with fibroblast growth factors (FGFs), and to their in vitro applications, e.g., the storage of FGFs and cell cultures, and in vivo applications, e.g., as therapeutic agents, especially for healing and tissue regeneration, or as cosmetic agents.
(c) BACKGROUND ART
The existence of fibroblast growth factors (FGFs) has been demonstrated by numerous teams as a result of studying the biological activities of growth factors obtained from extracts of a very large number of tissues or organs (brain, pituitary gland, retina, vitreous humour, choroid, iris, cartilage, kidney, liver, placenta, corpus luteum, prostate gland, bone, muscle etc.).
The very diversity of the tissues studied and of the cells stimulated by these factors in vitro and in vivo, together with the large number of teams which have independently contributed to the characterization, isolation and complete identification of these factors, explains the multitude of names and initials used by these various authors to denote the factors.
It appears that all these extracts contain growth factors from the family of the FGFs and that this family can be divided into two main branches.
The first branch has been described under the names basic FGF, basic fibroblast growth factor or heparin binding growth factor II (FBGF II), brain derived growth factor (BDGF), eye-derived growth factor (EDGF II), astrocyte growth factor (AGF II), cartilage-derived growth factor (CDGF) etc. , while the second branch of the FGF family has been described ~0 _4~~ fi 3 under the names acid FGF or heparin binding growth factor I (HBGF I), brain-derived growth factor I (BDGF
I) etc.
These factors have been named either according 05 to the type of target cells used (fibroblast, astrocyte or endothelial cell growth factors with the initials FGF, AGF, ECGF), or according to the source from which this factor is extracted (for example growth factors derived from brain, retina or eyes, cartilage or hepatocytes in culture, with the respective initials BDGF, RDGF, EDGF, CDGF, HDGF) , or else according to a biochemical or biological property (heparin binding growth factors (HBGF) or tumour angiogenic factor (TAF)); the two main branches of the family are named according to these initials, preceded or followed by acid or basic or by type I or type TI.
It is by following the biological activity on cells in culture that these factors could be purified.
The first physicochemical characteristics (molecular weight and isoelectric paint) were published as early as 1975 (GOSPODAROWICZ, J. Biol. Chem., 250, 2515) for the basic form and in 1982 (BARRITAUZaT et al., J.
Neurosci., 8, 477-490) for the acid form.
Purification of the two forms of FGF to homo geneity made it possible to establish their primary structures (ESCH et al., 1985, Proc. Natl. Acad. Sci.
US, 82, 6507, for the basic form, and GIMENEZ G. et al., 1985, Science, 230, 1385-1388, for the acid form).
Isolation of the two forms was greatly favoured by the demonstration of a strong affinity of these factors for heparin and the subsequent use of affinity chromatography on immobilized heparin (SHING et al., 1984, Science, 223, 1296-1299).
It is known that, in vitro, FGFs are capable of stimulating the proliferation and differentiation of a 204os.~' large number of cells originating from different tis-sues and species.
There may be mentioned especially fibroblasts;
endothelial cells, epithelial cells, keratinocytes, 05 chondrocytes, myoblasts, astrocytes etc., and also neuronal survival.
It is also known that, in vivo, FGFs have neurotrophic, angiogenic and healing properties.
French patent 7918282 , which teaches a method of stimulating the growth of epidermal cells, may be cited especially; this method shows in particular the role of a partially purified aqueous retina extract, containing FGF, on the stimulation of said epidermal cells.
US patent 4 477 435, which teaches a method of healing the corneal epithelium with the aid of a com position containing an aqueous retina extract, is also - known.
Numerous studies are also known which involve demonstrating the exister3ce of and characterizing FGFs and their role in the regeneration and healing of the skin, vessels, nerves, bones, muscles etc., both in vitro and in vivo.
There may be cited especially US patent 4 444 760, which describes a brain-derived acid growth factor, its method of extraction and its application to the healing of wounds, and European patent application 186 084, which describes a method of stimulating the growth of vascular endothelial cells with the aid of a composition containing the brain-derived acid growth factor described above.
The FGFs described above are obtained by puri fication; FGFs obtained by genetic recombination are also known from international patent application PCT
US86/01879.

Another healing composition based on at least one FGF is described in European patent application 243 179 and additionally comprises collagen and heparin and/or a glycoaminoglycan.
05 In these various documents, the topical appli-cation of FGF, by itself or in association, is effected with the aid of customary formulations such as creams, pastes, solutions and gels, or formulations associated with polymers, sponges and pumps permitting a slow release of the FGFs, as described in particular in international patent application PCT US86/01879, where it is specified that~formulations comprising recombi-nant FGFs and appropriate excipients or carrier mole-cules can be prepared, especially lotions, gels, delayed-release forms or creams, said formulations being associated, if appropriate, with other active principles such as antibiotics. The delayed-release forms described in said patent application comprise polymers in particular.
The compositions'obtained can be used espe-cially.as heeling agents in the control of clotting, in the improvement, of neurological damage and in the regeneration of hard tissues.
It is apparent, however, that FGF does not systematically stimulate healing: in fact, the absence of stimulation has been reported especially in J.
Dermatol. Sing. Oncol.; the topical application of acid or basic FGF must therefore often be repeated in order to achieve the maximum effects, although some composi tions of the prior art, such as FGF-impregnated poly-vinyl alcohol sponges applied under the skin, induce the proliferation of fibroblasts and myoblasts.
This is due to thermal instability of the mole cule, pH-related inactivation of the molecule, proteo lysis by enzymes and interaction between the FGFs and 20~~s3 the glycoaminoglycans, such as heparan sulphate or proteoheparan sulphate, of the cell membranes or basal membranes, leading to immobilization of the FGFs which can deny them access to the cell receptors.
05 Such disadvantages limit the possibilities of storing and using FGFs.
To mitigate this disadvantage, European patent application 267 015 has proposed a composition con-taining a polypeptide growth factor, more particularly EGF, and a sufficient amount of water-soluble poly-saccharide to stabilize said factor against the loss of biological activity, especially in the presence of water. It is specified in said patent application that the water-soluble polysaccharides which can be used include cellulose derivatives, starch, agar, alginic acid, gum arabic, dextrans, fructans, inulin, mannans, xylans, arabinans, chitosans, glycogen and glucans.
Pursuing their studies on dextrans, the inven tors have demonstrated novel properties of functiona lized substituted dextraris: said dextrans are found to have an inherent~cell and. tissue regenerating activity and,,. in addition, they not only have a stabilizing action on an FGF composition-but also cooperate with FGF in the biological activity of the latter.
The Applicant consequently set out to provide an agent having a cell and tissue regenerating activity and compositions containing said agent in association with FGFs, said compositions meeting the practical needs better than the compositions proposed in the prior art for serving the same purpose, especially in that the compositions according to the invention have a markedly improved stability, permitting easier storage and hence a superior therapeutic effect to that of the compositions of the prior art, and in that their fre-quency of application is thereby markedly reduced.

By a first broad aspect of this invention, a stabilized composition is provided comprising an agent having a cell and tissue regenerating activity, the agent comprising at least one functionalized, substituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, in association with at least one of the following: an acid FGF; a basic FGF; a derivative thereof; an analogue thereof; and a fragment thereof, the fragment thereof having a biological activity; the agent being capable of at least partially restoring the biological activity of the acid and/or basic FGF/FGFs which have been inactivated by prolonged storage or temperature.
By a second broad aspect of this invention, a stabilized composition is provided comprising an agent having a cell and tissue regenerating activity, the agent comprising at least one functionalized, substituted dextran, the dextran being selected from the group consisting of soluble dextrans and insoluble dextrans, the selected dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate in association with at least one of the following:
an acid FGF; a basic FGF; a derivative thereof; an analogue thereof; and a fragment thereof, the fragment thereof having a biological activity; the agent being capable of at least partially restoring the biological activity of the acid and/or basic FGF/FGFs which have been inactivated by prolonged storage or temperature.
By a first variant of the first and second aspects of this invention, the composition comprises from 0.1 to 1000~g/ml of at least one the agent having a cell and tissue regenerating activity, and from 0.41 ng to 300 ~cg of at least one FGF which is selected from the group consisting of acid FGFs, basic FGFs and their derivatives, their analogues and their fragments, the fragments having a biological activity.
By a second variant of the first and second aspects of this invention, and/or the above variant thereof, the composition also includes other associated active principles.
By one variation thereof, the associated active principles are selected from the group consisting of local anaesthetics, anti-infectious agents, serum proteins and collagen.

By a third variant of the first and second aspects of this invention, and/or the above variants thereof, the composition also contains at least one appropriate pharmaceutically-acceptable vehicle and/or physiologically-acceptable support.
By one variation thereof, the vehicle is water, and the composition also includes buffers and/or salts so as to keep the mixture at a pH of between 6.8 and 7.4 and at an ionic strength of between 0.1 and 0.2 in NaCI equivalents.
By a fourth variant of the first and second aspects of this invention, andlor the above variants thereof, the composition is incorporated into appropriate liposomes. By one variation thereof, the support is selected from the group consisting of dressings and biomaterials. By a second variation thereof, the composition is in the form of an aerosol, and the vehicle is an appropriate gas.
By a fifth variant of the first and second aspects of this invention, and/or the above variants thereof, the composition is in the form of an ointment, cream, paste or lotion.
By a sixth variant of the first and second aspects of this invention, and/or the above variants thereof, the composition is impregnated in a gel. By one variation thereof, the gel is a collagen gel.
By a seventh variant of the first and second aspects of this invention, and/or the above variants thereof, the composition is included in a dressing or biomaterial, which directly or indirectly favours cell repair.
By an eighth variant of the first and second aspects of this invention, andlor the above variants thereof, the composition is impregnated in a dressing or biomaterial, which directly or indirectly favours cell repair.
By a third aspect of this invention, the use is provided of at least one functionalized substituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for obtaining a composition intended for a therapeutic activity of cell and tissue regeneration.
By a fourth aspect of this invention, the use is provided of at least one functionalized substituted dextran which is selected from the group consisting of soluble dextrans and insoluble dextrans, the selected dextrans containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for obtaining a drug intended for a therapeutic activity of cell and tissue regeneration.
By a fourth aspect of this invention, the use is provided of at least one functionalized substituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate for a therapeutic activity of cell and tissue regeneration.
By a sixth aspect of this invention, the use is provided of at least one functionalized substituted dextran which is selected from the group consisting of soluble dextrans and insoluble dextrans, the selected dextrans containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for a therapeutic activity of cell and tissue regeneration.
By a seventh aspect of this invention, the use is provided of a composition, as described hereinabove, for a therapeutic activity of cell and tissue regeneration.
As used in the present specification, the term "soluble functionalized substituted dextrans" is understood as meaning as those described especially in French Patent Number 2,5559 or in French Patent Number 2,461,724.
As used in the present specification, the term "insoluble functionalized substituted dextrans" is understood as meaning those described especially in Published French Patent Application Number 82,01641 or in French Patent Number 2,461,624.
The dextrans as above-described are stable and do not lose their properties with time. Furthermore, they possess the unexpected property of having an inherent cell and tissue regeneration activity at low doses and, more particularly, a healing activity.
The compositions of aspects of this invention have a cell and tissue regenerating activity, and especially a healing action, which is superior to that of the compositions of the prior art.
Lidocaine may be mentioned in particular as a local anaesthetic and sodium salts, silver salts, derivatives thereof or sulphadiazines may be mentioned in particular as bacteriostatic substances. Streptomycin may be mentioned as an antibiotic.
Serum albumin or fibronectin may be mentioned as serum proteins. Soluble collagens and elastin may also be mentioned.
Such associations according to aspects of the invention are hereafter referred to as "matrix compositions" .
The "matrix" composition is advantageously applied directly in solution or as an aerosol. According to aspects of this invention, the compositions, especially the "matrix"
compositions, may be included and/or impregnated in an appropriate support, e.g., a dressing or biomaterial, which directly or indirectly favours cell repair (for example, a surgical suture thread or coral for a bone graft) .
The "matrix" compositions of aspects of this invention can be included in traditionally-used creams or lotions, in particular, lanolin-based creams, e.g., those known by the trade-marks SILVEADENF.~.M , MARIOTM, AQUAPHORTM and EQUALIATM, for application to the skin. They can also be included or impregnated in dressings, e.g., those made of textiles, synthetic fabrics or sponges, or natural products which are used for covering wounds, for example, collagen gels or dermis of animal origin.
The "matrix" compositions according to aspects of this invention impregnate these various forms of dressings so that the FGF and/or the substituted functionalized dextran can be in contact with or diffuse as far as the target tissues.
Compositions according to aspects of the invention are hereafter referred to as FGF/functionalized dextranlliposome compositions and functionalized dextran/liposome compositions.
The compositions according to aspects of this invention are kept especially on the site of the injury and on open injuries so as to maintain hydration in accordance with the techniques of those skilled in the art, which are particularly developed in the field of skin grafts.
Occlusive dressings can be impregnated in the same manner, or absorbed, or they can cover natural or synthetic supports.

to For applications to the cornea, the vehicle must be compatible with the tolerance of the eye [for example, the product marketed under the trade-mark LACRIBULETM, saline solutions or isotonic solutions, for example, that known by the trade-mark NEOCADRONTM (Merck-Sharp-Dohme)] .
These vehicles can also contain preservatives, e.g., benzyldimethylalkyl-ammonium chlorides or sodium ethylenediaminetetraacetate (EDTA).
According to aspects of this invention, the FGFlfunctionalized dextranlliposome composition or the functionalized dextran/liposome composition is included in a medicinal form, e.g., an ointment, cream, paste or lotion, or impregnated in a gel, especially a collagen gel.
The insoluble functionalized dextrans can also be included, by themselves or in association with FGF, in carriers, e.g., creams, gelatins or collagen gels, or on synthetic or natural fibres, which are the usual supports for cover dressings. The insoluble functionalized polymers can be included by the addition of collagen solution and gelling.
The procedures described in a series of patents in the name of YANNAS can be used.
In one of these patents (namely U.S. Patent Number 4,060,081), a composite laminar composition gives an equivalent skin in which the part in contact with the injury is covered with collagen cross-linked with a glycosaminoglycan, the mixture being obtained by adding glycosaminolgycans to the solubilized collagen and the whole being precipitated or cross-linked with glutaraldehyde (see U.S. Patent Number 4,418,691).
The compositions according to aspects of this invention may be prepared by mixing at least one appropriate FGF with at least one agent having a regenerating and stabilizing activity.
The FGFs may be obtained by extraction and purification from natural sources, by chemical synthesis or else by appropriate genetic recombination techniques.
The FGFs may be of human origin or else originate from other animals, especially other mammals.
Numerous purification methods for extracting and isolating the two forms of FGF
from these natural sources (retina, brain, pituitary gland, placenta, kidney etc.) have been described in the prior art.

The preferred extraction methods used in the present invention are those which are described in Biochimie, 1986 (COURTY et al.), or that described in Published French Patent Application Number 2,613,936, which utilizes affinity chromatography on bio-specific substituted polystyrenes.
These preferred methods include a step for treating the tissue extract at very acid pH, thereby excluding any risk of viral contamination, and the use of chromatography on immobilized heparin or substituted polystyrene.
The two forms of FGF can thus be isolated and separated, with the other proteins or individually, with a sufficient degree of purity to be devoid of significant amounts of other contaminating materials.
Apart from the foregoing provisions, the invention in its various aspects also includes other provisions, which will become apparent from the following description referring to Examples of how to carry out the process for obtaining aspects of the present invention and to Examples showing the effect of functionalized substituted dextrans on the protection of the biological activity of FGFs.
It must be clearly understood, however, that these Examples are given solely to illustrate the subject of the invention.
(e) AT LEAST ONE MODE FOR CARRYING OUT THE INVENTION
Examples Example 1: Process of Stabilizing FGFs 1) Preparation of a functionalized substituted dextran (cell and tissue regenerating agent).
grams of dextran T40 (0.185 mol) are dissolved in 146 ml of distilled water 25 and cooled to 4°C in a bath of melting ice. 59.2 g of NaOH (1.48 mol) are dissolved in 100 ml of distilled water and then cooled to 4°C. The sodium hydroxide solution is poured slowly into the dextran solution, with stirring, and the whole is kept at 4°C for 20 minutes. 61 g of C1CHZCOOH (0.647 mol) are then added very gradually so that the temperature reaches 20°C after 5 minutes. The reaction medium is then heated to 40°C
30 over 10 minutes, kept at this temperature for 90 minutes and then cooled to 20°C. The pH is lowered to 7 with concentrated acetic acid. The whole is precipitated in 2 litres 11a of methanol, filtered off, washed twice with 1 litre of ethanol and then dried under vacuum at 40 ° C .
g of the above modified polymer are dis-solved in 55 ml of distilled water acidified to pH 3.
60 ml of dimethylformamide are added very gradually, with stirring, the pH being kept at a value of 3. The temperature is lowered to -15'C and 12.3 ml of N-05 methylmorpholine are added with 14.5 ml of isobutyl chloroformate. This is followed by the addition of 12.2 ml of benzylamine. After 30 minutes, the polymer is precipitated in 800 ml of methanol, filtered off and dried.
~ 9 g of the above modified polymer are dis-persed in 25 ml of anhydrous methylene chloride. A .
mixture of 0.26 ml of HS03C1 and 2.5 ml of methylene chloride is introduced into the reactor and the whole is kept at room temperature for 4 hours. After filtra-tion and washing with methylene chloride, the product is dried and dissolved in 30 ml of water and the pH is adjusted to a value of 7Ø The solution is ultra-- filtered against a buffer solution and then against distilled water. The solution is then lyophilized until the dry polymer is abtained.
Another method of preparing a functionalized substituted dextran can be used, such as that described in European patent n' 0 023 854.
2) Preparation of the FGF/FGFs - The cell extract/extracts are treated over-night in the presence of acetic acid at pH 3 and the FGFs are then separated out by chromatography on immobilized heparin or substituted polystyrene.
3) Preparation of a stable FGF composition according to the invention - A solution of dextran is prepared from the dry polymer obtained in 1) by dissolving it in an iso-tonic phosphate buffer (PBS) to give a concentration of 400 ~g/ml.
- The FGFs extracted in 2) are dissolved in 2o~~s3 this buffer, containing the appropriate substituted dextrans, so as to give an FGF concentration of 100 ~cg/ml .
Example 2: Stabilized ointment according to the 05 invention FGF 10 ~g FD 5 mg Carboxymethyl cellulose 2.5 g Apyrogenic sterile purified water 100 ml FD - type E functionalized dextran as defined in Table III below.
The cream obtained can be applied for three days to a scarification-type wound on a rat.
Example 3: Stabilized dressing according to the invention The support for the dressing consists of a "Pangil" collagen film from Laboratoires FOURNIER, impregnated by passive adsorption with a mixture of FGF
and functionalized dextran in the following propor tions: ' FGF lu ~g FD 500 ~cg Isotonic solution 10 ml After incubation of the collagen film for 30 minutes at 4°C in the solution described above, a dres-sing is obtained which can be used in cases of ulcera-tions of various kinds and superficial or deep wounds.
This dressing can be stored under vacuum and packed.
* STUDY OF THE EFFECT OF FUNCTIONALIZED BIO-SPECIFIC POLYMERS ON THE PROTECTION OF THE BIOLOGICAL
ACTIVITY OF FGFs in vitro Methodology used for measurement of the bio-logical activity of FGFs in vitro The methods of evaluating the biological x~~. 204~fi3 activity of FGFs in vitro are described in numerous publications and are all based either on measurement of the increase in the number of cells induced by increasing doses of factors added to the cell culture 05 medium, or on an increase in the incorporation of tri-tiated thymidine into the DNA of cells stimulated by the growth factor. In the two methods referred to, these increases are dependent on the dose of factor added and it is therefore possible to establish dose effects and dose-response curves with a maximum sti-mulating effect. By way of simplification, one unit of stimulation is defined as the dose of growth factor which, when added to one millilitre of culture medium on target cells, is capable of inducing an increase in the number of cells or in the incorporation of tri-tiated thymidine which corresponds to half (50°s) of the maximum value of this increase measured in the dose-response curve. This definition and the reproducibi-lity of these measurements are explained especially in PLOUET et al., 1984, Cellular and Molecular Biology, 30, p. 105.
EXAMPLE A: PROTECTIVE EFFECT OF SUBSTITUTED
D~ AGAINST THE INACTIVATION OF ACID AND BASIC FGFs BY ACID AND ALKALINE pH VALUES
In these experiments, the FGFs are in solution at a concentration of 100 ~cg per millilitre in an iso-tonic phosphate buffer (PBS) containing no dextran (control) or containing substituted dextran at 400 ~cg/
ml. 10 ~sl of these various solutions are taken and mixed with 1 ml of either PBS, or dilute acetic acid (CH3COOH) adjusted to pH 2 (about 1 N), or dilute sodium hydroxide (NaOH) adjusted to pH 9Ø These samples are incubated at 20°C for two hours and 1 ~1 is taken for determination of the biological activity.
Figure 1 shows the dose-response curve of bFGF

...~ 20~os~

on CCL39 fibroblasts.
In this Figure, the logarithm of the bFGF con-centration in pg/ml is plotted on the abscissa and the percentage stimulation on the ordinate.
05 Curve 1 corresponds to the control: curve 2 corresponds to bFGF by itself at pH 2; curve 3 corres-ponds to bFGF in the presence of dextran at pH 2; curve 4 corresponds to bFGF in the presence of dextran at pH
9: curve 5 corresponds to bFGF by itself at pH 9: and curve 6 corresponds to the control in.the presence of dextran.
The increase in the incorporation of tritiated thymidine represents the value of the number of counts per minute (cpm) obtained at the plateau of the dose-response curve of bFGF by itself minus the value in cpm for tritiated thymidine incorporated into the cells in the absence of FGF and determined in the same experi-ment.
Curves 3 and 4 show that bFGF in the presence of dextran preserves its stimulating power in both acid and basic media.
Table I summarizes the results obtained with acid and basic FGFs. The unit of stimulation is arbitrarily fixed at 1 for the starting aFGF or bFGF
incubated for two hours at 20°C.

~.~. 2 ~ 4 ~ 6 TABLE I
pH 2 pH 7 pH 9 FGFb (0C) 0.9 05 FGFb (2 h, 20C) 53 1 13 FGFb + FD (2 h, 20) 1 1 2.5 FGFb + HS (2 h, 20) 3 1 4 FGFa (0) 1 FGFa (2 h, 20) 6 1 6 FGFa + FD (2 h, 20) 0.5 0.4 2 FGFa + HS (2 h, 20) 1.5 0.8 4.5 FD - functionalized dextran, which in this Example is dextran E as defined in Table III below.
HS - heparan sulphate (from BIOVALORIS in Plouhermel (Ile-et-Villaine, FRANCE)).
This Table shows the protective effect of FD
(functionalized dextran) against the inactivation of acid and basic FGFs induced by acid and alkaline pH
values.
The incubation of basic FGF for two hours at 20°C in a buffer solution of pH 2 to 9 induces inacti-vation of the biological activity of .the basic FGF.
In fact, 53 times more product are needed at .acid pH and 13 times more at basic pH in order ,to induce.a biological effect in the initial product.
The addition of FD to this mixture totally pro tects the biological activity of the basic FGF against incubation at pH 2 or 9.
Similar results are observed in the case of acid FGF as far as the two types of treatment are con-cerned.
EXAMPLE B: EFFECT OF FUNCTIONALIZED DEXTRAN
(FD) ON THE INACTIVATION OF FGFs BY TEMPERATURE IN THE
SHORT AND LONG TERM
In this Example, FGF prepared as in Example A
is incubated at 4°C, 20°C, 37°C or 60°C for different times in the absence or presence of 400 ~g of func 04Ofi30 _ 17 _ tionalized dextran (FD), as defined in Table III below, and then determined.
The results are given in Table II below.

4'C 20'C 37'C 60'C

bFGF t = 0' 1 bFGF t = 30' 1 1 3.5 >100 bFGF + FD t - 30' 1 1 1 9 aFGF t = 0' 1 aFGF t = 30' 1 1 2 >100 aFGF + FD t = 30' ~ 0.4 0.4 0.4 5 bFGF t = 24 h 1 1 6 bFGF + FD t = 24 h 1 1 1 aFGF 1 1 1 aFGF + FD 0.4 0.4 0.4 bFGF t = 7 days 2 5 >100 bFGF + FD t = 7 days 1 1 1 bFGF + HS t = 7 days 1 2 6 aFGF t = 7 days 2.5 8 >100 aFGF + FD t = 7 days 0.4 0.4 3 FD = functionalized dextran HS = heparan sulphate The initial unit of.-stimulation is arbitrarily fixed at a,~ value of 1.
This Table shows a strong inhibition of the activation of acid or basic FGF induced by treatment for one week at 37'C. The presence of FD in the incu bation medium protects both types of FGF against thermal denaturation.
Similar results are observed using HS (heparin sulphate), the biological equivalent of FD.
E~p~ C: EFFECT OF DIFFERENT FUNCTIONALIZED
DEXTRANS ON THE DOSE-RESPONSE EFFECTS OF FGF
The effect of different functionalized dextrans is measured as a ratio in Table III below.

Zp~~~3 TABLE III
Dextran derivative % D % W % X % Y R/us 05 B 0 106 0 0 1.6 C 0 84 21 0 1.7 D 10 76 0 14 2.6 E 0 89 6 5 2.36 F 0 74 16 10 3.1 G 65 30 1 4 2.54 H 29 42 24 5 2.1 Percentages:
D . dextran W . carboxymethyl X . benzylamide Y . benzylamidesulphonate R/us is the value of the ratio of the values of the units of stimulation of aFGF without functionalized dextran divided by the unit of stimulation in the pre-sence of functionalized dextran.
* STUDY OF THE EFFECT OF FUNCTIONALIZED BIO
SPECIFIC POLYMERS ON THE~PROTECTION OF THE BIOLOGICAL
ACTIVITY OF FGFs in vivo EXAMPLE D: KINETIC, PLANIMETRIC AND HISTOLO-FGF/FUNCTIONALIZED DEX'I'RAN
Experimental protocol:
The operations are carried out on male Wistar rats weighing 300 to 400 grams. Each experiment is performed on a group of 5 animals.
Types of wounds:
Two types of skin wounds are made on the pre-shaven dorsum of the animals.
- Skin removals are carried out with a punch (0.6 cm in diameter) down to the muscle floor.
Scarifications of 1 cm in length are made with a scalpel. They do not affect the dermo-epidermal 2~ ,~ ~ s 3 .

region.
Procedure:
According to the type of wound, the injuries are treated with different mixtures of products dis-05 solved in sterilized buffered isotonic solution (pH
7.4).
In the case of the punch wounds, these solu-tions are deposited in a collagen plug (GINGESTAT) pre-cut to the exact measurements of the tissue excision.
In the case of the scarifications, the products are deposited directly in liquid form on the wound.
The effects of the association of FGF (basic, acidic or a mixture in a solution of 1 ng to 10 ~sg/ml) and functionalized dextrans (in a solution of 100 ng to 1 mg/ml) are evaluated and compared with the action of a substituted functionalized dextran by itself and of each of the constituents considered as reaction con-trols (collagen, dissolving solution, FGF).
Each experimental group of animals is sacri ficed after an interval' of time defined by 24-hour periods and the injured regions are removed for two types of study:
- an external morphological analysis with planimetry of the wound:
- a histological study.
Results:
I - Stabilizing effects of functionalized dex-trans:
FGF radiolabelled with 1251 is deposited in a collagen plug in the presence or absence of functiona lized dextran.
The variation in the radioactivity in the impregnated collagen is assessed as a function of time.
The results are illustrated in Figure 2, in which the time in hours is plotted on the abscissa and o~~s3o the percentage radioactivity on the ordinate. Curve ?
corresponds to FGF in the presence of dextran and curve 8 corresponds to FGF by itself.
The radioactivity is measured in the collagen 05 gel and in skin removed at the periphery of the wound, 2 cm from the latter, by a punch equivalent to the one originally used.
II - Morphological and histological studies:
A) Morphological study Observation of the change in the wounds with the naked eye makes it possible to establish a very distinct action of the association FGF + functionalized dextran on the rate and quality of the superficial healing (epidermization + lysis of the clot).
1) After 24 hours, the collagen plugs impreg-nated with this association have totally adhered to the walls of the wound and can only be removed by lesion of the regenerated tissues. The control experiments only show total adhesion of the collagen plugs after 36 to 48 hours. ' 2) Re-epithelialization is visibl-a to the naked eye 'after the third day when the association FGF +
functionalizedwdextran is present, whereas an identical picture for the controls requires experimental periods of 5 to 7 days.
3) Planimetric analysis: Planimetric analysis of the external surface of the wounds shows the total absence of retraction of the regenerating tissues.
The degree of scar retraction is evaluated as a function of time by considering the ratio P/A, where P
is the perimeter of the wound and A the area of the scar.
The order of magnitude of this ratio P/A is of the type K/R, where K is a constant and R the radius of the original circular wound.

~w~ 2p~,~~3 21 _ As a function of time, the lower and more con-stant this ratio, the more the scar retains a plani-metry similar to that of the original lesion. Conse-quently, the lower the ratio P/A, the more limited is 05 the degree of scar restructuring. The healing quality can thus be reflected by the absence of contraction.
The results obtained are illustrated in Figure 3, in which the time in days is plotted on the abscissa and the ratio P/A on the ordinate. The degree of retraction is represented by ~ for the control, by for bFGF, by ~ for FGFs in the presence of heparan sulphate and by $~ for FGFs associated with functiona-lized dextrans.
The results are also shown in Tables IV and V
below; Table IV gives the percentage healing area as a function of the amount of functionalized dextran (FD) in the presence or absence of bFGF: Table V gives the ratio P/A under the same conditions.
TABLE IV
P/A CONTROL FD FD FD bFGF bFGF 1 ~g + FD at 500 50 beg S ~g '~. different concen-~cg ' ~g trations:

500 ~g 50 ~g 5 ~g 2 2 0.17 0.20 0.13 0.08 0.13 0.19 0.09 0.10 5 d 4 0.20 0.18 0.19 0.11 0.15 0.13 0.11 0.08 d 8 0.58 0.41 0.30 0.28 0.24 0.38 0.2I 0.19 d 2a~~s3 TALE V
FD FD FD bFGF bFGF 1 ~g + FD at 500 ug 50 ug 5 1 ~cg different concen-~g trations:

05 500 ~.g 50 ~g 5 ~cg 2 d 125 104 173 147 112 160 128 4 d 105 213 160 169 182 231 260 8 d 280 128 145 386 329 237 253 The effects of functionalized dextrans on this retraction are particularly visible on the fourth and eighth days after the operation.
The Tables above clearly show the inherent healing effect of dextrans; in fact, in Table IV, the percentage area in the presence of 5 ~cg. of FD after 8 days is similar to that in the presence of 1 ~g of bFGF
by itself, these percentages themselves being less than the control.
The retraction islvery small in comparison with those observed in the control experiments or those observed in the presence of FGFs by; themselves or . associated with heparan sulphates, these conditions already being distinctly more favourable than those of the control.
B) Histological study The treated regions are removed, fixed and impregnated with paraffin. The histological study is carried out on 7 ~cm sections. The stains used permit topographical and histochemical studies.
The histological analysis shows that the asso-ciation FGF + FD accelerates the traditional steps of dermo-epidermal healing and enhances the quality of the reconstituted tissues.
Impregnated collagen permits a very rapid ~04~63 colonization (1 day) of the surrounding categories of cells (fibroblasts, smooth muscle cells) from the healthy surrounding tissues and in particular from the connective tissue of the subjacent striated muscle 05 floor.
At the same time, neoangiogenesis enables the tissue which is being formed to be colonized by a very high density of blood capillaries. After three days (as opposed to five to six for the controls), the re-epithelialization which had started from the epidermis of the lips of the wound joins up the edges. On the fourth day, the epidermis is totally reconstituted and the subjacent tissues, which are totally reorganized, have a normal density compared with the controls, for which the density is much lower. These same illustra tions reveal the absence of retraction of the edges of the wound in the case of the punch wounds treated with the association FGF + functionalized dextrans, in con trash to the controls, where the excised tissues are made up by extraction.
The effects of the association of bFGFs and functionalized dextrans on the healing quality, com-pared with natural healing without the addition of products, are shown in Figures 4 and 5.
Figure 4 shows a photograph of a histological section of a control scar (absence of treatment) four days after the wound was made (X 40). Figure 5 shows a photograph of a histological section of a scar after treatment with a collagen plug impregnated with a solution of bFGF and 1 ~,g/ml and functionalized dex-trans at 50 ~g/ml, four days after the wound was made and at the same magnification of 40.
Figure 5 shows the epidermis (E) entirely re constituted, whereas in Figure 4 it is not reformed. A
retraction of the surrounding tissues on the control zo~s3 wound is not recorded on the treated wound. This cica-tricial space, which is relatively anarchic in Figure 4, has organization and a satisfactory cell density in the case of the treated wound (Figure 5). It is cha-05 racterized by the presence of blood vessels represen-ting the local angiogenic effect of the association of the products of this invention.
It is therefore apparent that the association bFGF + functionalized dextran is a powerful healing agent in vivo, which on the one hand accelerates the natural regenerative processes and on the other hand permits an enhanced healing quality through the absence of any retraction phenomenon such as the rapid mobili zation of the different categories of cells necessary for tissue restoration.
EXAMPLE E: PLANIMETRIC AND HISTOLOGICAL STUDIES
OF THE HEALING EFFECT OF FUNCTIONALIZED DEXTRANS
The experimental protocol, which is identical in every respect to that employed in the context of Example D, is carried out in order to assess the healing effects of functionalized dextrans. .The dex-trans studied are listed in Table III of Example C.
The healing effects of these functionalized dextrans or of their association were assessed relative to two con-trol experiments in the presence of vehicle by itself, a collagen plug or a collagen plug impregnated with unsubstituted dextran (product designated by A).
A - Morphological study Observation of the change in the wounds with the naked eye makes it possible to establish a very distinct action of functionalized dextrans on the rate and quality of superficial healing.
Compared with the control experiments, the adhesion of the vehicle is accelerated in the case of the wounds treated with functionalized dextrans.

,~ ~04~63 The re-epithelialization follows kinetics com-parable to those observed under the action of FGFs.
The ratio P/A, where P is the perimeter of the wound and A the area of the scar, represents a totally 05 significant decrease in the degree of scar retraction.
The results obtained are illustrated in Table VI.
These experiments confirm the specific role of functionalized dextrans in the inhibition of scar retraction and deformation of the surrounding area of l0 skin, as already specified above in Example D.
B - Histological study The analysis is identical to that performed in the previous Example.
It reveals, compared with the observations of 15 the control experiments, a more rapid and more intense colonization of the collagen impregnated with func tionalized dextrans from the various types of cells - surrounding the wound.
The neoangiogenesis is distinct but less sus-20 tained than that observed'in the presence of FGFs.
The extensions of the epidermis join up edge to edge at around day 4, which is at least 24 hours ahead of the re-epithelialization observed in the controls.
It is therefore apparent that there is a 25 healing effect inherent in the action of functionalized dextrans which manifests itself' as healing at the harmonious contours, resulting in a decrease in the natural contraction of the sides of the wound and an increased and rapid mobilization of cells colonizing 30 the collagens, culminating in a denser and more vas-cularized regenerating tissue than that observed in the case of the control experiments. Such an effect might perhaps be explained by the fact that substituted functionalized dextrans potentiate, on the tissues, the 35 action of FGFs secreted in situ by the surrounding Zp~~63~

tissues.
TABLE VI
The degrees of retraction P/A are shown for the 05 collagen controls by themselves, collagen impregnated with type A dextran and the various dextrans listed above. All these molecules act at a dilution of 3 gig/
ml.
P/A 2 d 4 d 8 d T(1) 0.21 0.20 0.61 T(2) 0.20 0.25 0.55 B 0.16 0.18 0.30 C 0.15 0.18 0.26 D 0.13 0.13 0.20 E 0.14 0.16 0.22 F 0.10 0.11 0.20 G 0.15 0.18 0.29 H 0.13 0.14 . 0.30 A+D 0.18 0.20 0.28 D+H 0.11 0.15 0.22 (1): collagen (2): collagen + A
As is apparent from the foregoing description, the invention is in no way limited to those modes of execution, embodiments and modes of application which have now been described more explicitly; on the con-trary, it encompasses all the variants thereof which may occur to those skilled in the art, without devi-ating from the framework or the scope of the present invention.

Claims

1. A stabilized composition comprising an agent having a cell and tissue regenerating activity, said agent comprising at least one functionalized, substituted dextran containing functions which are selected from the group consisting of carboxy-methyl, benzylamide and benzylamide sulphonate in association with at least one of the following: an acid FGF; a basic FGF; a derivative thereof; an analogue thereof; and a fragment thereof, said fragment thereof having a biological activity; said agent being capable of at least partially restoring the biological activity of the acid and/or basic FGF/FGFs which have been inactivated by prolonged storage or temperature.

2. A stabilized composition comprising an agent having a cell and tissue regenerating activity, said agent comprising at least one functionalized, substituted dextran, said substituted dextran being selected from the group consisting of soluble dextrans and insoluble dextrans, said subsituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate in association with at least one of the following: an acid FGF; a basic FGF;
a derivative thereof; an analogue thereof; and a fragment thereof, said fragment thereof having a biological activity; said agent being capable of at least partially restoring the biological activity of the acid and/or basic FGF/FGFs which have been inactivated by prolonged storage or temperature.

3. The composition as claimed in claim 1 or claim 2, comprising from 0.1 to 1000µg/ml of at least one said agent having a cell and tissue regenerating activity, and from 0.01 ng to 300 µg of at least one FGF which is selected from the group consisting of acid FGFs, basic FGFs and their derivatives, their analogues and their fragments, said fragments having a biological activity.

4. The composition as claimed in claim 1, claim 2, or claim 3, also including other associated active principles, said associated active principles being selected from the group consisting of local anaesthetics, anti-infectious agents, serum proteins and collagen.

5. The composition as claimed in any one of claims 1 to 4, which also contain at least one appropriate pharmaceutically-acceptable vehicle and/or physiologically-acceptable support.

6. The composition as claimed in claim 5, wherein said vehicle is water, and wherein said composition also includes buffers and/or salts so as to keep the mixture at a pH of between 6.8 and 7.4 and at an ionic strength of between 0.1 and 0.2 in NaCl equivalents.

7. The composition as claimed in claim 5 or claim 6, which is incorporated into appropriate liposomes.

8. The composition as claimed in any one of claims 5 to 7, wherein said support is selected from the group comprising dressings and biomaterials.

9. The composition as claimed in any one of claims 5 to 8, which is in the form of an aerosol, wherein said vehicle is an appropriate gas.

10. The composition as claimed in any one of claims 1 to 9, in the form of an ointment, cream, paste or lotion.

11. The composition as claimed in claims 1 to 9, which is impregnated in a gel.

12. The composition as claimed in claim 11, wherein said gel is a collagen gel.

13. The composition as claimed in claims 1 to 9, which is included in a dressing or biomaterial, which directly or indirectly favours cell repair.

14. The composition as claimed in claims 1 to 9 which is impregnated in a dressing or biomaterial, which directly or indirectly favours cell repair.

15. Use of at least one functionalized substituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for obtaining a composition intended for a therapeutic activity of cell and tissue regeneration.

16. Use of at least one functionalized substituted dextran which is selected from the group consisting of soluble dextrans and insoluble dextrans, said selected dextrans containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for obtaining a drug intended for a therapeutic activity of cell and tissue regeneration.

17. Use of at least one functionalized substituted dextran containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate for a therapeutic activity of cell and tissue regeneration.

18. Use of at least one functionalized substituted dextran which is selected from the group consisting of soluble dextrans and insoluble dextrans, said selected dextrans containing functions which are selected from the group consisting of carboxymethyl, benzylamide and benzylamide sulphonate, for a therapeutic activity of cell and tissue regeneration.

19. Use of a composition as claimed in any one of claims 1 to 12 for a therapeutic activity of cell and tissue regeneration.