Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
44~63~
1
(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
- 2 -
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.~'
- 3 -
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.
- 4 -
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
- 5 -
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.
6
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.
7
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
8
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
9
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.
11
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-
- 12 -
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
- 13 -
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
- 14 -
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~
- 15 -
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.
35
~.~. 2 ~ 4 ~ 6
- 16 -
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.
05 TABLE II
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
-18-
TABLE III
Dextran derivative % D % W % X % Y R/us
A 100 0 0 0 1
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-
GICAL STI1DIES OF THE HEALING EFFECT OF THE ASSOCIATION
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 .
- 19 -
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
- 20 -
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
35
2a~~s3
- 22 -
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
- 23 -
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
- 24 -
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
- 25 -
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~
- 26 -
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.
35
