U.S. patent application number 12/212093 was filed with the patent office on 2009-01-08 for biocompatible polymers, process for their preparation and compositions containing them.
Invention is credited to Denis Barritault, Jean-Pierre Caruelle.
Application Number | 20090012040 12/212093 |
Document ID | / |
Family ID | 9528857 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090012040 |
Kind Code |
A1 |
Barritault; Denis ; et
al. |
January 8, 2009 |
BIOCOMPATIBLE POLYMERS, PROCESS FOR THEIR PREPARATION AND
COMPOSITIONS CONTAINING THEM
Abstract
A process for treating fibroses including administering a
therapeutically effective amount of a pharmaceutical composition
which includes at least one biocompatible polymer of the following
general formula (I): A.sub.aX.sub.xY.sub.y wherein: A represents a
monomer selected from the group consisting of a sugar or
--(O--CH.sub.2--CH.sub.2--CO)--, X represents a carboxyl group
bonded to monomer A and is contained within a group according to
the following formula: --R--COO--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Y represents a sulfate or sulfonate
group bonded to monomer A and is contained within a group according
to one of the following formulas: --R--O--SO.sub.3--R',
--R--N--SO.sub.3--R', --R--SO.sub.3--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, a represents the number of monomers A
such that the mass of the polymers of formula (I) is greater than
approximately 5,000 da, x represents a substitution rate of the
monomers A by the groups X, which is between approximately 20 and
150%, and y represents a substitution rate of the monomers A by the
groups Y, which is between approximately 30 and 150%.
Inventors: |
Barritault; Denis; (Paris,
FR) ; Caruelle; Jean-Pierre; (Saint-Maur-des-Fosses,
FR) |
Correspondence
Address: |
IP GROUP OF DLA PIPER US LLP
ONE LIBERTY PLACE, 1650 MARKET ST, SUITE 4900
PHILADELPHIA
PA
19103
US
|
Family ID: |
9528857 |
Appl. No.: |
12/212093 |
Filed: |
September 17, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10695574 |
Oct 28, 2003 |
|
|
|
12212093 |
|
|
|
|
09765788 |
Jan 19, 2001 |
6689741 |
|
|
10695574 |
|
|
|
|
PCT/FR99/01774 |
Jul 20, 1999 |
|
|
|
09765788 |
|
|
|
|
Current U.S.
Class: |
514/54 ;
514/574 |
Current CPC
Class: |
A61P 17/16 20180101;
A61P 29/00 20180101; A61P 39/00 20180101; A61P 9/10 20180101; C08G
63/6882 20130101; A61P 25/00 20180101; A61P 17/00 20180101; A61P
39/06 20180101; A61P 9/12 20180101; C08B 37/0021 20130101; A61P
1/00 20180101; A61K 31/765 20130101; A61P 31/00 20180101; A61K
31/795 20130101; A61P 3/00 20180101; A61P 43/00 20180101; A61P
17/02 20180101; A61K 31/721 20130101; A61P 9/00 20180101; A61P
21/00 20180101; A61P 31/08 20180101 |
Class at
Publication: |
514/54 ;
514/574 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61K 31/194 20060101 A61K031/194 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 1998 |
FR |
98/09309 |
Claims
1. A process for treating fibroses comprising administering a
therapeutically effective amount of a pharmaceutical composition
which comprises at least one biocompatible polymer of the following
general formula (I): A.sub.aX.sub.xY.sub.y wherein: A represents a
monomer selected from the group consisting of a sugar or
--(O--CH.sub.2--CH.sub.2--CO)--, X represents a carboxyl group
bonded to monomer A and is contained within a group according to
the following formula: --R--COO--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Y represents a sulfate or sulfonate
group bonded to monomer A and is contained within a group according
to one of the following formulas: --R--O--SO.sub.3--R',
--R--N--SO.sub.3--R', --R--SO.sub.3--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, a represents the number of monomers A
such that the mass of said polymers of formula (I) is greater than
approximately 5,000 da, x represents a substitution rate of the
monomers A by the groups X, which is between approximately 20 and
150%, and y represents a substitution rate of the monomers A by the
groups Y, which is between approximately 30 and 150%.
2. The process according to claim 1, wherein the fibroses are
fibroses of smooth muscle tissue.
3. The process according to claim 2, wherein the fibroses are
fibroses of mesenchymal tissue.
4. The process according to claim 1, wherein the sugar is a
glucose.
5. A process for reducing fibroses comprising administrating a
therapeutically effective amount of a pharmaceutical composition
which comprises at least one biocompatible polymer of the following
general formula (I): A.sub.aX.sub.xY.sub.y wherein: A represents a
monomer selected from the group consisting of a sugar or
CH.sub.2--CH.sub.2--CO)--, X represents a carboxyl group bonded to
monomer A and is contained within a group according to the
following formula: --R--COO--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Y represents a sulfate or sulfonate
group bonded to monomer A and is contained within a group to one of
the following formulas: --R--O--SO.sub.3--R', --R--N--SO.sub.3--R',
R--SO.sub.3--R', in which R is a bond or an aliphatic hydrocarbon
chain, optionally branched and/or unsaturated, and which can
contain one or more aromatic rings except for benzylamine and
benzylamine sulfonate, and R' represents a hydrogen atom or a
cation, a represents the number of monomers A such that the mass of
said polymers of formula (I) is greater than approximately 5,000
da, x represents a substitution rate of the monomers A by the
groups X, which is between approximately 20 and 150%, and y
represents a substitution rate of the monomers A by the groups Y,
which is between approximately 30 and 150%.
6. A process for treating fibroses comprising administering a
therapeutically effective amount of a pharmaceutical composition
which comprises at least one biocompatible polymer of the following
general formula (II): A.sub.aX.sub.xY.sub.yZ.sub.z wherein: A
represents a monomer selected from the group consisting of a sugar
or --(O--CH.sub.2--CH.sub.2--CO)--, X represents a carboxyl group
bonded to monomer A and is contained within a group according to
the following formula: --R--COO--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Y represents a sulfate or sulfonate
group bonded to monomer A and is contained within a group according
to one of the following formulas: --R--O--SO.sub.3--R',
--R--N--SO.sub.3--R', --R--SO.sub.3--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Z represents at least one functional
chemical group, which is different from X and Y, selected from the
group consisting of a fatty acid, amino acid, fatty alcohol,
ceramide derivative thereof and nucleotide addressing sequences and
which confers supplementary biological or chemical properties, a
represents the number of monomers A such that the mass of said
polymers of formula (I) is greater than approximately -5,000 da, x
represents a substitution rate of the monomers A by the groups X,
which is between approximately 20 and 15.0%, y represents a
substitution rate of the monomers A by the groups Y, which is
between approximately 30 and 150%, and z represents a substitution
rate of the monomers A by the group Z, which is between
approximately 0 and 50%.
7. The process according to claim 6, wherein Z is an amino
acid.
8. The process according to claim 7, wherein the amino acid is
selected from the group comprising phenylalanine and leucine.
9. The process according to claim 6, wherein Z is a fatty acid.
10. The process according to claim 9, wherein the fatty acid is
acetate.
11. The process according to claim 6, wherein the fibroses are
fibroses of smooth muscle tissue.
12. The process according to claim 11, wherein the fibroses are
fibroses of mesenchymal tissue.
13. The process according to claim 6, wherein the sugar is a
glucose.
14. A process for reducing fibroses comprising administrating a
therapeutically effective amount of a pharmaceutical composition
which comprises at least one biocompatible polymer of the following
general formula (II): A.sub.aX.sub.xY.sub.yZ.sub.z wherein: A
represents a monomer selected from the group consisting of a sugar
or --(O--CH.sub.2--CH.sub.2--CO--)--, X represents a carboxyl group
bonded to monomer A and is contained within a group according to
the following formula: --R--COO--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Y represents a sulfate or sulfonate
group bonded to monomer A and is contained within a group according
to one of the following formulas: --R--O--SO.sub.3--R',
--R--N--SO.sub.3--R', --R--SO.sub.3--R', in which R is a bond or an
aliphatic hydrocarbon chain, optionally branched and/or
unsaturated, and which can contain one or more aromatic rings
except for benzylamine and benzylamine sulfonate, and R' represents
a hydrogen atom or a cation, Z represents at least one functional
chemical group, which is different from X and Y, selected from the
group consisting of a fatty acid, amino acid, fatty alcohol,
ceramide derivative thereof and nucleotide addressing sequences and
which confers supplementary biological or chemical properties, a
represents the number of monomers A such that the mass of said
polymers of formula (I) is greater than approximately 5,000 da, x
represents a substitution rate of the monomers A by the groups X,
which is between approximately 20 and 150%, y represents a
substitution rate of the monomers A by the groups Y, which is
between approximately 30 and 150%, and z represents a substitution
rate of the monomers A by the group Z, which is between
approximately 0 and 50%.
15. The process according to claim 14 wherein Z is an amino
acid.
16. The process according to claim 15, wherein the amino acid is
selected from the group comprising phenylalanine and leucine.
17. The process according to claim 14, wherein Z is a fatty
acid.
18. The process according to claim 17, wherein the fatty acid is
acetate.
Description
RELATED APPLICATION
[0001] This is a continuation of International Application No.
PCT/FR99/01774, with an international filing date of Jul. 20, 1999,
which is based on French Patent Application No. 98/09309, filed
Jul. 21, 1998.
FIELD OF THE INVENTION
[0002] This invention pertains to new biocompatible polymers, a
process for their preparation and compositions containing them.
BACKGROUND
[0003] Known in the prior art are polymer derivatives of dextrans
obtained by substitution by carboxymethyl,
carboxymethyl-benzylamide and carboxymethyl-benzylamide-sulfonate
residues. These polymers, the process for their preparation and
their properties, are described in French Patent No. 2,461,724 as
well as in U.S. Pat. No. 4,740,594. Among these polymers, certain
of them imitate the properties of heparin and can be used as plasma
substitution products because of their anticoagulant and
anticomplement properties. Others imitate a different property of
heparin which consists of a stabilization, protection and
potentiation of the in vitro biological activity of the growth
factors of the FGF family (Tardieu et al., Journal of Cellular
Physiology, 1992, 150, pages 194 to 203). Furthermore, French
Patent No. 2,644,066 describes the use of
carboxymethyl-benzylamide-sulfonate derivatives of dextran,
referred to as CMDBS, alone or, associated with FGFs, for
cicatrization.
[0004] More recently, French Patent Nos. 2,718,023, 2,718,024 and
2,718,026 proposed the use of polymers capable of protecting,
stabilizing and potentiating growth factors that have an affinity
for heparin, such as the fibroblast growth factors or FGF and
Transforming Growth Factor beta (TGF.beta.), as a drug for the
treatment of lesions of the gastrointestinal tract, nervous system
and muscle tissues, respectively. To illustrate this protective
effect of the CMBDS dextran derivatives, these patents present the
results of the proteolytic digestion by trypsin of FGF1, FGF2 or
TGF.beta.. These properties of protection, stabilization and
potentiation of the growth factors with an affinity for heparin
enabled characterization of a new class of polymers, designated as
HBGFPP to indicate "Heparin-Binding Growth Factor Protector and
Potentiator", that exhibit cicatrizing and repair activities in
relation to muscle, nervous and gastrointestinal tract tissues, and
which are devoid of anticoagulant activity at the doses
employed.
[0005] In addition to the above HBGFPP applications, French Patent
No. 2,718,025 proposes the use, of these polymers as drugs for the
treatment of inflammations. This anti-inflammatory activity is
illustrated by the in vitro inhibitory action against proteolytic
enzymes implicated in the inflammatory reaction such as leukocyte
elastase or plasmin and in vivo by histological studies
demonstrating a reduced inflammatory cellular reaction in tissues
treated by CMDBS dextran derivatives.
[0006] However, these CMDBS dextran derivatives are compounds which
are difficult to synthesize and present the risk of salting out
residues that are known for their toxic effects such as
benzylamine. Furthermore, the applications of the HBGFPP and thus
of the CMDBS proposed in the prior art are limited solely to the
repair and cicatrization of certain tissues.
SUMMARY OF THE INVENTION
[0007] The invention relates to a biocompatible polymer constituted
by a sequence of identical or different components of the general
formula (I): A.sub.aX.sub.xY.sub.y, in which A represents a
monomer, X represents a carboxyl group fixed on a monomer A, Y
represents a sulfate or sulfonate group fixed on a monomer A; a
represents the number of monomers A, x represents the substitution
rate of the set of monomers A by the groups X, y represents the
substitution rate of the set of monomers A by the groups Y. The
invention also relates to the pharmaceutical or diagnostic
compositions containing at least one polymer of general formula
(I).
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows formulas of selected .beta.-malic acids.
[0009] FIG. 2 shows selected structures of three
.beta.-lactones.
[0010] FIG. 3 shows the synthesis of alkyl malolactonate from
DL-aspartic acid.
[0011] FIG. 4 shows the synthesis of derivatives of
poly(.beta.-malic acid).
[0012] FIG. 5 shows structures of selected monomers A and monomers
type A-X, A-Y and A-Z.
[0013] FIG. 6 is a table representing selected polymers of type
CM.sub.aDS.sub.m and the percentages of three groups X, Y and
Z.
[0014] FIG. 7 shows the structure of CM.sub.2DPhSS.sub.1.
[0015] FIG. 8 shows the structure of CM.sub.2DES.sub.1.
[0016] FIG. 9 shows the structures of CM.sub.3DPheS.sub.2 and
CM.sub.3DTyrS.sub.2.
[0017] FIG. 10 shows the structures of CM.sub.1DPalmS.sub.1 and
CM.sub.1DOleicS.sub.1.
[0018] FIG. 11 is a table showing anti-coagulant activities of
selected polymers.
[0019] FIG. 12 is a table showing the stabilizing effects of
selected polymers on FGF.sub.1.
[0020] FIG. 13 is a table showing the potentiation effects of
FGF.sub.1 and FGF.sub.2 for selected polymers.
[0021] FIG. 14 is a table showing the percentage of FGF.sub.1 and
FGF.sub.2 and TGF.beta. not degraded by trypsin in the presence of
poly(.beta.-malic acid) polymers.
[0022] FIG. 15 is a table showing the percentage of FGF.sub.2 and
TGF.beta. not degraded by trypsin in the presence of polymers of
the invention derived from dextrans.
[0023] FIG. 16 is a table showing the inhibitory effects of
selected polymers of the invention on the activities of leukocyte
elastase and plasmin.
[0024] FIG. 17 is a table showing percentages of muscular
regeneration after injection of various doses of polymers.
[0025] FIG. 18 is a table showing modulation of the in vitro
activity of SOD by selected polymers of the invention.
[0026] FIG. 19 is a table showing the protective effect of selected
polymers of the invention on SOD after treatment by trypsin and
thermal shock.
[0027] FIG. 20 is a graph showing the potentiation effect of SOD
produced in vitro by activated monosites.
[0028] FIG. 21 is a table showing the inhibitory effects of
selected polymers of the invention on calpaine.
[0029] FIG. 22 is a table showing the inhibitory effects of
selected polymers of the invention on heparitinase.
[0030] FIG. 23 is a graph showing the actions of selected polymers
of the invention on the secretion of cologens in vitro by HISM
cells subjected to ionizing radiation of .sup.60Co.
[0031] FIG. 24 is a graph showing, the action of selected polymers
of the invention on the synthesis of Type I, II and V collagens by
HISM cells subjected to ionizing radiation of .sup.60Co.
[0032] FIG. 25 is a graph showing the protective effects of
selected polymers of the invention on the survival of cells
subjected to .sup.60Co irradiation.
[0033] FIG. 26 is a table showing the antifibrotic action of
selected polymers of the invention on pig aorta smooth muscle
cells.
[0034] FIG. 27 is a series of photographs showing the effects of
polymers of the invention on, cutaneous cicatrization.
[0035] FIG. 28 is a pair of photographs of the protective effects
of polymer RGTA 1005 against tissue injury in a muscle ischemia
model in a rat.
[0036] FIG. 29 is, a series of photographs showing; the effects of
a polymer of the invention RGTA 1015 on the regeneration of long
bones and histological and radiographic studies of femurs from rats
which were treated or not treated.
[0037] FIG. 30 is a graph showing the effects of selected polymers
of the invention on the regulation of the osseous mass and on the
quality of its restructuring in chronic periodontal disease.
DETAILED DESCRIPTION
[0038] This invention resolves the drawbacks of the prior art by
providing new biocompatible polymers which are easy to prepare and
present the properties of the --HBGFPP but also, in an unexpected
manner, novel properties which enable very extensive fields of
application, especially in therapeutics, which are not limited to
just a few types of particular organs, tissues or cells. The
polymers of the invention are, therefore, referred to below as
"RGTA" for "ReGeneraTing Agents".
[0039] The polymers of the invention have a molar mass greater than
about 5,000 da and are constituted by a sequence of identical or
different components of the following general formula (I):
A.sub.aX.sub.xY.sub.y
[0040] in which:
[0041] A represents a monomer,
[0042] X represents a carboxyl group fixed on a monomer A and
contained within a group according, to the following formula:
--R--COO--R', in which R is a bond or an aliphatic hydrocarbon
chain, possibly branched and/or unsaturated, and which can contain
one or more aromatic rings, with the exception of benzylamine and
benzylamine sulfonate, and R represents a hydrogen atom or a
cation,
[0043] Y represents a sulfate or sulfonate group fixed on a monomer
A and contained within a group according to one of the following
formulas: --R--O--SO.sub.3--R', --R--N--SO.sub.3--R',
--R--SO.sub.3--R', in which R is a bond or an aliphatic hydrocarbon
chain, possibly branched and/or unsaturated, and which can contain
one or more aromatic rings, with the exception of benzylamine and
benzylamine sulfonate, and R' represents a hydrogen atom or a
cation,
[0044] a represents the number of monomers A, which is such that
the mass of the polymers of formula: (I) is greater than
approximately 5,000 da,
[0045] x represents the substitution rate of the set of monomers A
by the groups X, which is comprised between approximately 20 and
150%, preferably on the order of 50%,
[0046] y represents the substitution rate of the set of monomers A
by the groups Y, which is comprised between approximately 30 and
150%, preferably on the order of 100%.
[0047] The radical R of the groups X and Y in formula (I) is
advantageously selected from among an alkyl, allyl, aryl, linear or
branched group.
[0048] In the definition of the substitution rates cited above, a
substitution rate x of 100% is understood to mean that each monomer
A of the polymer of the invention contains statistically one X
group. Similarly, a substitution rate y of 100% is understood to
mean that each monomer of the polymer of the invention contains
statistically one Y group. Substitution rates higher than 100%
manifest the fact that each monomer bears statistically more than
one group of the type under consideration. Conversely, substitution
rates lower than 100% manifest the fact that each monomer bears
statistically less than one group of the type under
consideration.
[0049] The polymers offer the remarkable advantage of presenting in
vivo a slow degradability, which differentiates them from the
heparan sulfates which are products that are naturally and rapidly
degraded by heparinase or heparitinase. Furthermore, the polymers
of the invention neither contain nor release toxic products after
degradation, in contrast to the CMDBS, which have benzylamine
groups.
[0050] The monomers A which constitute the base elements of the
polymers of formula I can be identical or different, and are
selected from among all types of monomers such as, for example,
those proposed for the HBGFPP such as the sugars, esters, alcohols,
amino acids, nucleotides and the like. Among these, preference is
given especially to the -oses, and especially to glucose.
[0051] The number of monomers A is defined in a manner such that
the mass of the polymers of the invention is greater than
approximately 5,000 da. Consequently, the RGTA can be constituted
by diverse homomeric as well as heteromeric polymerizable
structures such as, for example: the polyester copolymers of
biosynthesis or chemical synthesis such as the aliphatic polyesters
or those of natural origin such as the polyhydroxylalcanoates. Also
applicable are polysaccharides and their derivatives of bacterial
origin such as cellulose, xanthan, dextran and the like,
unicellular or multicellular plant extracts such as starch, plant
cellulose or alginates or their derivatives, the fucanes and their
derivatives and the like, or animal extracts such as hyaluronic
acid or chitin and the like, or products of synthesis such as those
obtained by copolymerization. Synthesized proteins such as the
natural or chemically modified polyamino acids such as, for
example, collagen can also be employed in the constitution of the
polymers of the invention.
[0052] The selection of the polymer structure of the RGTA is based
on the various forms of presentation which can be used depending on
the tissue or the organ to be treated or the physicochemical
characteristics such as the solubility, the spatial conformation
with the solutions, suspensions, gels, powders, sponges, films,
compact moldable materials, porous, semiporous or nonporous
materials, materials that crumble or materials that do not crumble,
materials that erode and materials that do not erode, materials
that can be colonized or materials that can not be colonized,
etc.
[0053] Heparin or its fragments with weak anticoagulant properties
and natural heparan sulfates and their fragments are excluded from
the scope of the present invention because:
[0054] Their anticoagulant activity can be greater than 50 IU. In
fact, the polymers of the invention are devoid of significant
anticoagulant activity, i.e., they present an activity lower than
about 50 IU/milligram compared to that of heparin whose activity is
on the order of, 150 to 170 IU/milligram.
[0055] They exhibit biodegradability under the action of enzymes of
the heparinase or heparitinase type which is too rapid to enable
the biological effects obtained in the context of this
invention.
[0056] The polymers of the invention can also comprise functional
chemical groups, designated Z, which are different from X and Y,
and capable of conferring on the polymers of the invention
supplementary biological or physicochemical properties. Thus, the
groups Z are useful for conferring on the RGTA a better solubility
or lipophilic properties enabling better diffusion or tissue
penetration, for example, increasing the amphiphilic properties,
enabling crossing the blood-brain barrier and the like. As an
example, the groups Z, which can be identical or different, can be
amino acids, fatty acids, fatty alcohols, ceramides or derivatives
thereof, or nucleotide addressing sequences.
[0057] The groups Z can also represent identical or different
active agents, such as therapeutic or diagnostic agents, for
example, an anti-inflammatory, antimicrobial, antibiotic and the
like or an enzyme, a growth factor and the like.
[0058] The polymers of the invention in which Z is present have the
following formula II:
A.sub.aX.sub.xY.sub.yZ.sub.z
in which A, X, Y, a, x, and y have the same meanings as defined
above and z represents the substitution rate of the set of monomers
A by the groups Z, which is comprised between approximately 0 and
50%, preferably on the order of 30%. The groups X, Y and Z can be
fixed directly on the monomer A or fixed to each other with only
one of them being fixed to the monomer A. Thus, the groups Z can be
fixed by covalence directly on the monomers A or fixed by covalence
on the groups X and/or Y.
[0059] However, the groups Z can also be conjugated to the polymers
of formula (I) by bonds other than covalent bonds, such as ionic or
hydrophilic interactions, depending on the nature of A, X and Y.
The polymers of the invention can then constitute a vectorization
system of Z. Thus, the polymers of the invention are useful for
transporting an active agent Z, such as a growth factor or an
enzyme, to the level of the injured or diseased tissue as defined
below in the context of the applications of the polymers of the
invention.
[0060] The polymers in which the groups R of X and Y or the groups
Z are capable of inducing toxic effects directly or after
degradation are not included within the scope of this invention.
Among the groups Z excluded from the invention can be cited those
groups constituting mutagenic agents or those known to be
carcinogenic or exhibiting other toxic properties. This is the case
of benzylamine which can be precipitated out from the benzylamide
group contained in the CMDBS and the carcinogenic properties of
which are well known by the expert in the field (Wiessler M, et
al., Carcinogenesis 1983; 4(7): 867-871; Singer G M, et al., J.
Med. Chem. 1983; 26(3): 309-312). CMDBS is consequently excluded
from the polymers of the invention.
[0061] The invention also pertains to the pharmaceutical or
diagnostic compositions containing at least one polymer as defined
above associated in the composition with a pharmaceutically
acceptable vehicle. These compositions can be used in humans as
well as in animals. In fact, as with the HBGFPP described in the
prior art, the polymers of the invention present the following
properties:
[0062] They are devoid of significant anticoagulant activity, which
means that they present an activity lower than about 50
IU/milligram compared to that of heparin the activity of which is
on the order of 150 to 170 IU/milligram.
[0063] They stabilize and potentiate the growth factors that
exhibit an affinity for heparin and particularly, as an example,
FGF1 and/or FGF2 and TGF.beta..
[0064] They protect these factors against proteolytic agents such
as trypsin.
[0065] They inhibit the protease activities implicated in the
inflammatory process such as, for example, leukocyte elastase or
plasmin.
[0066] They exert a cicatrizing effect in at least the models
presented in the cited patents on muscles, nerves or the
gastrointestinal tract.
[0067] Thus, the polymers of the invention constitute a new class
of agents that promote the repair of muscle and nervous tissues,
and those of the gastrointestinal tract and which present, as
reported in U.S. Pat. No. 5,693,625 for the CMDBS, properties on
cutaneous cicatrization and that of the cornea or on flat bone as
described by Blanquaert F et al. (Bone, 1995; 17(6): 499-506).
However, the polymers of the invention also present unexpected
properties in relation to the CMDBS. As an example, we can cite the
effect of the CMDBS on cranial bone defects (Blanquaert F et al.,
Bone, 1995; 17(6): 499-506) which a priori is not transposable to
long bone. Flat bone and long bone are of a completely different
nature because:
[0068] They are of different embryological origin. Flat bone is a
derivative of the conjunctive tissue cells whereas long bone is a
derivative of the cartilaginous cells.
[0069] Flat bone is a spongy bone and does not contain a medullary
cavity in contrast to long-bone.
[0070] Flat bone does not cicatrize naturally when there is E a
loss of substance such as upon trepanation as can be seen by the
absence of cicatrization in the crania originating from prehistoric
civilizations, such as the Incas or Egyptians (see for example
Evidence for stone age cranial surgery, 7000 BC, Nature, 1997, 367,
360). Surprisingly, the polymers of the invention, present effects
on the repair of very severe bone defects on the order of a third
of the length of the diaphysis of a rat femur. Not only was repair
observed with reformation of an osseous shaft but also the shaft
was filled with marrow, i.e., the treatment with RGTA led to the
reformation of a true medullary cavity whereas in the absence of
RGTA the defect is simply packed with disorganized osseous
material. The same is true of cutaneous incisions because this
repair is of such high quality that there is practically no trace
of a scar.
[0071] The invention, therefore, pertains more particularly to the
previously described biocompatible polymers of formulas (I) and
(II) which are devoid of significant anticoagulant activity. These
polymers present an anticoagulant activity lower than about 10
IU/mg at single or daily doses comprised:
[0072] between about 0.001 mg and about 1 mg per cm.sup.3 when the
application is local,
[0073] between about 0.1 mg/kg and about 100 mg/kg when the
administration is via the systemic route, for example, via the
intravenous route,
[0074] between about 0.2 mg/kg and about 500 mg/kg when the
administration is via the intramuscular route,
[0075] between about 0.1 mg/kg to about 5 g/kg when administered
orally.
[0076] The anticoagulant activity of the polymers of the invention
at the doses indicated above is for a person weighing about 70-kg
lower than:
[0077] about 10 IU for a local application,
[0078] about 100 IU for an intravenous administration,
[0079] about 500 IU for an intramuscular administration.
[0080] Remarkably, the polymers of the invention are devoid of
significant anticoagulant activity at the doses specified above but
still present the capacity of preserving or restoring tissue
homeostasis. The invention, therefore, pertains particularly to the
use of the aforementioned polymers for the preparation of a drug
that is useful for the prevention or treatment of dysfunctions of
tissue homeostasis and which do not present significant
anticoagulant activity.
[0081] In a completely surprising manner, we found that the
polymers of the invention can be administered via routes other than
those described in the prior art for the CMDBS which only envisaged
local administration at the level of the injured tissue. Thus, it
is possible to select among the polymers of the invention those
which are suitable, for example, because of their volubility, for
the selected route of administration intravenous, intra-arterial,
intramuscular, intraperitoneal, intraocular, into the cerebrospinal
fluid or directly into the central nervous system to cross over the
blood brain barrier, as well as orally. All of these routes of
administration, possibly even combined, have been shown to be
particularly effective.
[0082] Thus, the invention concerns pharmaceutical compositions in
which the polymer of the invention is associated with a
pharmaceutical vehicle in a form enabling oral, cutaneous,
subcutaneous, topical, intramuscular, intravenous or intra-arterial
administration or administration into any of the fluid compartments
of the organism. Studies performed in the framework of this
invention revealed that the --RGTA act especially within the dosage
windows beyond which satisfactory biological effects were not
always obtained. These optimal effective doses are defined below
and depend on the route of administration and the type of
lesion.
[0083] Advantageously, in a manner to completely cover the surface
of a wound or to fill the volume of a tissue lesion, the
pharmaceutical compositions are dosed to allow administration of
about 0.001 to about 1 milligram of polymer per square centimeter
or of about 0.005 to about 1 milligram of polymer per cubic
centimeter of tissue to be treated. Thus, the compositions of the
invention preferably contain between about 0.01 and about 10
milligrams of polymer per milliliter of physiological solution of
dissolution.
[0084] For administration via the systemic route (venous;
arterial), via the intraperitoneal route or intraocular route, into
the cerebrospinal fluid, into the intracochlear fluid or into any
peritissular or intratissular fluids, the compositions of the
invention are dosed to allow administration of between about 0.1
and about 100 milligrams of polymer per kilogram of weight of the
human or animal to be treated.
[0085] For administration via the intramuscular route, the
compositions of the invention are dosed to allow administration of
between about 0.2 and about 500 milligrams of polymer per kilogram
of weight of the human or animal to be treated, preferably between
about 1 and about 50 mg per kg.
[0086] For oral administration, the compositions of the invention
are dosed to allow administration of between about 1 and about 5000
milligrams of polymer per kilogram of weight of the human or animal
to be treated, preferably between about 10 and about 500 mg per
kg.
[0087] Furthermore, the previously reported properties of the
HBGFPP, i.e., that they present a cicatrizing and repairing
activity on the muscle and nervous tissues and on those of the
gastrointestinal tract and that they can be used as drugs for the
treatment of inflammations, we have demonstrated the remarkable
effects of the polymers of the invention on the regulation of the
homeostasis of the mass and the functionality of the tissues and
organs, demonstrating that they act on tissular and cellular
regeneration, protection, preservation and aging in vivo and ex
vivo, as well as antifibrotic activities and protective activities
against the deleterious effects of ischemias, ionizing radiation
and oxidizing products induced by diseases or stress or stemming
from food. Consequently, the RGTA should be considered to be
regulators of tissular homeostasis in that they regulate the mass
of the regenerated tissues and they act on the reorganization of
the matrix in that they exert an antifibrotic and cicatricial
effect on the tissues and organs subjected to acute as well as
chronic destructive processes.
[0088] These properties of the RGTA are characterized by unique
effects on the quality and rate of the tissue repairs. These
effects are manifested by an almost complete reconstitution and
identity with the original tissue structure (prior to the lesion)
and by the almost complete absence of cicatricial traces such as in
particular signs of fibrosis or loss of its functional integrity.
More particularly, the research results which will be presented
below in the experimental part demonstrated the in vivo tissue
protection and regeneration properties of the RGTA in the following
models:
[0089] cutaneous wound lesion,
[0090] regeneration of osseous tissues such as long bones with the
example of the femur,
[0091] protection against the loss of osseous tissue and regulation
of its restoration in a model of chronic periodontal disease,
[0092] protection against the deleterious effects of ionizing
radiation,
[0093] protection against the deleterious effects of ischemias
irrespective of their location.
[0094] Thus, the properties below of the polymers of the invention
have been particularly demonstrated:
[0095] A protective effect against the deleterious effects linked
to oxidative stress and to oxidizing agents. The polymers of the
invention act in particular as protective agents and potentiators
of enzymes such as superoxide dismutase or SOD. This property makes
it possible to use the polymers of the invention for the
preparation of a drug intended for the treatment and/or the
prevention of lesions and disorders induced by oxidative stress and
oxidizing agents. But this property also makes it possible to use
the polymers of the invention alone or in association with another
compound as a preservative for foods or nutriments that naturally
contain antioxidants or to which antioxidants, such as animal or
plant SOD, were added.
[0096] A preservative and protective effect against ischemias. This
property makes it possible to use the polymers of the invention for
the preparation of a drug intended for the treatment and/or the
prevention of pathologies associated with hypoxia, with cellular
degeneration such as the neuropathies, myopathies, hepatopathies,
nephropathies, the cardiopathies and the like and to the
peroxidations of molecules such as the lipids. This property also
makes it possible to use the polymers of the invention in
compositions for the preservation of the functionality of tissues
and organs especially for the purpose of their conservation, the
transport of ex vivo organs, organ transplants, their grafting as
well as prostheses.
[0097] An inhibitory activity against the enzymes of the calpaine
family. This property makes it possible to use the polymers of the
invention for the preparation of a drug intended for the treatment
and/or prevention of heart diseases and diseases of the nervous
system.
[0098] An inhibitory activity against the degradation enzymes of
heparin, or the heparan sulfates such as heparinase or
heparitinase. This property makes it possible to use the polymers
of the invention for the preparation of a drug intended for the
treatment and/or the prevention of the diseases associated with an
anarchic growth of the cells and the pathological processes in
relation with an angiogenesis.
[0099] An action of protecting cells against the effects of
ionizing radiation and of both the quantitative and qualitative
regulation of the constituents of the cell matrix such as, for
example, the collagens, making it possible to increase the survival
and functioning of the cells. This activity makes it possible to
use the polymers of the invention for the preparation of a drug
intended for the treatment and/or prevention of the deleterious
effects of ionizing radiation as well as the use of these polymers
in the preparation of cosmetic products.
[0100] An antifibrotic activity which is manifested in vitro and in
vivo by regulatory effects on the proliferation of mesenchymal
cells such as the smooth muscle cells, fibroblasts or hepatic-cells
and the quality of the type of collagen that they secrete, as well
as an activity on the phenotypic quality of the collagens
synthesized by these cells and by the notable reduction in the
fibrotic cicatricial sequelae. This activity makes it possible to
use the polymers of the invention for the preparation of a drug
intended for the treatment and/or prevention of pulmonary, renal,
hepatic, cardiac, vascular and dermatological pathologies as well
as pathologies of grafts and their functional integration, of
multiple derivatives linked to parasitism.
[0101] Thus, unexpectedly, the inventors discovered that the RGTA
presented the capacity to: [0102] protect, stabilize the enzymatic
activities of superoxide dismutase or SOD,
[0103] potentiate the enzymatic activities of superoxide dismutase
or SOD.
Thus, this enzyme can be protected either in situ and in vivo or ex
vivo. The RGTA can, therefore, be used alone as protective agents
of endogenous SOD and in this role protect SOD in all of its
functions or it can be used in association with SOD in the
indications known by those of ordinary skill in the therapeutic and
cosmetic fields. Due to this property, the RGTA also act as
preservatives of foods or nutrients or nutriments.
[0104] The RGTA act as protective and reparative agents of the
deleterious effects associated with tissue stress. Thus, in
ischemia, regardless of its origin our in response to a cellular or
tissular aggression, for example, under the effect of ionizing
radiation, or in response to an invasion by a pathogenic agent,
regardless of whether it be viral, microbial, parasitic or even of
the type causing pathologies of the TSSE (Transmissible Subacute
Spongiform Encephalopathies) type such as prions, or during
vascular rupture caused by hemorrhages whose effects are harmful by
causing functional losses, for example, in the case of hemorrhages
of the retinal vessels which can lead to blindness or in the brain
which can lead to loss of motor functions or others.
[0105] Consequently, the invention pertains to pharmaceutical
compositions containing at least one RGTA in both human and animal
fields of applications. These compositions are beneficial in
medical, veterinary and cosmetic fields as well as in alimentary,
fields as preservatives of foods or nutriments that naturally
contain antioxidants or to which are added antioxidants such as
animal or plant SOD.
[0106] The protective qualities of endogenous SOD or SOD that is
provided exogenously are reinforced by the RGTA. Thus, the RGTA can
be administered in all cases in which the beneficial effects of SOD
have been described.
[0107] For the treatment of diseases in which exogenously supplied
SOD has been shown to be effective, the therapeutic effects
resulting from the protective effect of endogenous SOD can then act
more effectively in the presence of RGTA. The RGTA limit or avoid
exogenous intakes from acting indirectly as antioxidant agents. The
following therapeutic effects result from these effects:
[0108] Protection against the aggressions of disease or
degeneration of the nervous tissues. For example in stress (Shahen
et al., Effects of various stressors on the level of lipid peroxide
antioxidants and Na.sup.+, K.sup.+--ATPase activity in brain,
Experentia, 1996, 52, 336-339) or of neuronal degeneration and the
neurodegenerative diseases associated with vascular accidents,
traumatic accidents or of pathologies such as Parkinsonism in which
the protective activity of endogenous SOD enables a more intensive
effect (Bostantjopoulos et al., Superoxide dismutase activity in
early and advanced Parkinson's disease, Funct. Neurol., 1997, 12,
63-68).
[0109] Aging due to an antiapoptosis effect (Fernandez Novoa et
al., Methods Find Exper Clin Pharmacol, 1997, 9, 99-106).
[0110] An antioxidant protection in ischemias of the limbs. The
RGTA can be administered by itself in this application. The
protective provided by treatment with SOD alone will be reinforced
by the administration of RGTA. In view of the properties described,
that the RGTA is beneficial when administered alone and/or in
association with SOD in the numerous applications describing the
effects of SOD (D'Agnillo and Chang, Reduction of hydroxyl radical
generation in a rat hind limb model of ischemia--reperfusion injury
using cross-linked hemoglobins--superoxide dismutase--catalase,
Artif. Cells Blood Substiti Immobil Biotechnol, 1997, 25,
163-80).
[0111] The administration of RGTA alone or in association with SOD
would be beneficial against disorders of the heart, brain and
central nervous system as in the case of lesions of the spinal cord
(Nakauchi et al., Effects of lecithinized superoxide dismutase on
rat spinal cord injury, J. Neurotrauma, 1996, 13, 573-82) or of the
retina.
[0112] In the treatment of respiratory insufficiencies associated
with diaphragmatic fatigue (Supinski et al., Effects of free
radical scavengers on diaphragmatic fatigue, Am. J. Respir. Crit.
Care Med., 1997, 155, 622). This could be notably of value in
patients with muscular dystrophy.
[0113] All tissues, especially those for which the endogenous
active SOD level is higher (vascular endothelial cells, in the
liver, the kidneys, the cardiac and skeletal muscles, the pancreas,
the epithelial cells of the intestinal mucosa, the colon, the
trachea, the esophagus, the conjunctive tissue and cartilage).
[0114] The treatment of the deleterious effects associated with
diabetes such as the diabetic retinopathies (Szabo et al., Direct
measurement of free radicals in ischemic/reperfused diabetic rat
retina, Clin. Neurosci., 1997, 4, 240-5).
[0115] The treatment of leprosy (Serum, zinc, copper, magnesium,
proteins and superoxide dismutase in leprosy patients on multidrug
therapy--a follow-up study, Indian J. Lepr., 1996, 68, 325-53).
[0116] In the treatment of endotoxic shock (Hepatic response to the
oxidative stress induced by E. coli endotoxin, Mol. Cell. Biochem.
1996, 159, 115-121).
[0117] In the treatment of the lesions induced by the stress caused
by the presence of pathogenic agents of all types, especially
viruses, including the AIDS virus, or the agents that induce TSSE
such as the prions.
[0118] In the preventive and/or curative treatment against
irradiations. Thus, the adverse effects of radiotherapy can be
reduced by a preventive and/or curative treatment with RGTA. The
use of the RGTA allows reduction of the dose of radiotherapy under
cancer treatment conditions (Prevention of radioinduced cystisi by
orgotein; a randomized study, Anticancer Res., 1996, 16, 2025-8) or
enable prevention of the clastogeric effects induced by the
irradiation. The hyperthermic effects associated with radiotherapy
can be diminished by the use of RGTA in the same manner as SOD
(Kandasamy et al., Involvement of superoxide dismutase and
glutathione peroxidase in attenuation of radiation-induced
hyperthermia by interleukin 1 alpha in rats, Brain Res., 1993, 606,
106-10).
[0119] In the protection against the effects induced by ultraviolet
radiation, for example on the retina. (Oguni et al., Chronic
retinal effects by ultraviolet irradiation, with special reference
to superoxide dismutase, Histol. Histopathol., 1996, 11, 695-702)
and the treatment of uveitis (Koch et al., Effects of different
antioxidants on lens-induced uveitis, Ger. J. Ophthalmol., 1996, 5;
1185-8). The treatment with RGTA can be alone or in association
with SOD and its; use can be medical as well as cosmetic.
[0120] In the treatment of hypertension. In fact, the activity of
endogenous SOD is, diminished in subjects with hypertension (Jun Ke
Yan and Catalano, Increased superoxide anion production in humans,
a possible mechanism for the pathogenesis of hypertension, J. Hum.
Hypertens. 1996, 10, 305-309). The intake of RGTA can have the
effect of augmenting this activity and reducing the
hypertension.
[0121] In the treatment of inflammatory diseases such as
arthritis.
[0122] The RGTA are also useful for the conservation of organs or
tissues as well as in the maintenance of biological fluids such
that the following applications can be envisaged:
[0123] In the biological fluids such as blood and its cellular
constituents, for example, in hemodialysis.
[0124] For the preservation of organs in the case of grafts or ex
vivo treatment or in reperfusion, (Razak et al., Cross-linked
hemoglobin-superoxide dismutase-catalase scavenges free radicals in
a rat model of intestinal ischemia-reperfusion injury, Artif. Cells
Blood Substiti Immobil. Biotechnol. 1997, 25, 181-192). The
addition of RGTA to solutions for organ conservation and those
administered in reperfusion enables preservation of these
organs.
[0125] The invention pertains to pharmaceutical compositions
containing at least one polymer as defined above and intended for
the treatment and/or prevention of tissular lesions and disorders,
such as those found in traumatology, requiring reparative or
plastic surgery of cutaneous or deeper floors such as those of
muscles, bone, brain, heart, viscera, etc., in the degenerative
diseases possibly associated with fibrosis, in the losses of tissue
mass such as osteoporosis or myopathies, in the cardiovascular and
neurological fields, in dermatology, etc.
[0126] A remarkable property of the RGTA is their capacity to fix
themselves on the injured tissues, which enables the use of the
RGTA as targeting vectors in a therapeutic and/or diagnostic
objective. Consequently, the polymers of the invention can be
coupled to molecules, represented above by the group Z, endowed
with therapeutic activities or to molecules that facilitate the
repair of the injured tissue.
[0127] Other advantages and characteristics of the invention will
become manifest from the examples below concerning, on the one
hand, the preparation of polymers of the invention in which the
polymer skeleton is of the polyester type or of a polysaccharide
nature and, on the other hand, the properties of the polymers of
the invention.
[0128] A) Preparation of the Polymers of the Invention
EXAMPLE 1
Synthesis of Poly(.beta.-Malic Acid) Derivatives from a
Non-Polysaccharide Skeleton
[0129] In this example, the polymer of the invention is a copolymer
of .beta.-malic acids of general formula (II), the components A of
which, substituted by X and/or Y and/or Z, are represented in FIG.
1. In FIG. 1:
[0130] A is --(O--CH.sub.2--CH.sub.2--CO)--
[0131] X is --COOH or --COO.sup.-Na.sup.+
[0132] Y is --CO--CH.sub.2--CHOH--CH.sub.2--SO.sub.3H or
--CO--CH.sub.2--CHOH--CH.sub.2--SO.sup.3-Na.sup.+
[0133] Z is --CO--OCH.sub.3--CH(CH.sub.2--CH.sub.3)--CH.sub.3
[0134] x, y and z correspond to: the percentages of the X, Y and Z
groups shown in Table I below in relation to the different polymers
synthesized.
TABLE-US-00001 TABLE I Type of Carboxylic Sulfonate Hydrophobic
Reference polymer groups = X groups = Y groups = Z RGTA 2010
Pcoo.sup.- 100% 0% 0% RGTA 2011 P1S 60% 10% 10% RGTA 2012 P2S 75%
11% 12%
[0135] Pcoo.sup.- corresponds to a polymer composed exclusively of
carboxylic or carboxylate groups X. P1S and P2S correspond to
polymers composed of sulfonate groups Y and hydrophobic butyl
groups Z in addition to the groups X.
[0136] The synthesis of this polymer proceeds from the preparation
of .beta.-substituted .beta.-lactones and is followed by an anionic
polymerization by ring opening. The three lactones synthesized are
shown in FIG. 2.
[0137] The monomers are obtained from DL-aspartic acid in four
steps prior to the polymerization as shown in FIG. 3.
[0138] The synthesis of the alkyl malolactonate is performed from
DL-aspartic acid in which R represents --CH.sub.2--C.sub.6H.sub.5
(benzyl malolactonate) or --CH(CH.sub.3)--CH.sub.2--CH.sub.3
(2-butyl malolactonate) or CH.sub.2--CH.dbd.CH.sub.2 (allyl
malolactonate).
I--Synthesis of the Monomers (Aspartic Acid Pathway)
1) Synthesis of (2R,S) Bromosuccinic Acid
[0139] Synthesis of (2R,S) bromosuccinic acid is obtained after a
diazotation reaction of DL-aspartic acid. For this reaction, the
carbon bearing the amine group undergoes a double inversion of
configuration (Guerin et al., Optically active poly(.beta.-malic
acid), 1985, Polymer Bulletin, 14: 187).
[0140] 100 g (0.75 moles) of DL-aspartic acid and 415 g (4.04
moles; 5.5 eq.) of sodium bromide are dissolved in 1620 milliliters
of 2N sulfuric-acid in an ice bath. Then 62 g (0.9 moles; 1.2 eq.)
of sodium nitrite are added in small quantities, under agitation.
Thirty minutes after the end of this addition, the reactional
medium is neutralized with 8 g (0.13 moles; 0.18 eq.) of urea for
30 minutes at room temperature. The bromosuccinic acid is extracted
with 1 liter of ethyl acetate and the aqueous phase washed with 1
liter of ethyl acetate. The organic phases are dried over magnesium
sulfate, filtered on a Buchner funnel then the ethyl acetate is
eliminated in the Rotavapor.
[0141] The bromosuccinic acid is then recrystallized 4 times in
acetonitrile, filtered on a Buchner funnel then dried for 4 h at
45.degree. C. under vacuum in a desiccator.
[0142] Characteristics: MW=197; m=52.8 g; Yield of 36%;
mp=168.degree. C.; appearance: white powder.
2) Synthesis of the Monoesters
[0143] The synthesis of the monoesters comprises the prior
preparation of the anhydride without racemization of the chiral
centers. The anhydride is synthesized from (2R,S) 2-bromosuccinic
acid under the action of a dehydrating agent, trifluoroacetic
anhydride (TFAA), under anhydrous conditions and under nitrogen.
The following step is the opening of the anhydride by an alcohol
which leads to two monoesters, only one of which will be
lactonizable. The choice of the alcohol is based on the nature of
the desired lactone.
[0144] a) Synthesis of Benzyl Bromosuccinate
[0145] Fifty g (0.25 moles) of bromosuccinic acid are degassed
under a nitrogen stream for 2 h. In an ice bath, 125 milliliters of
anhydrous tetrahydrofuran (THF) then 43.4 milliliters (0.30 moles;
1.2eq.) of trifluoroacetic anhydride (TFAA) are added drop by drop
by a dropping funnel. The solution is left for 2 h at room
temperature under agitation, then the THF, the TFA formed and the
TFAA in excess are eliminated in the Rotavapor. The bromosuccinic
anhydride is allowed to degas for 2 hours.
[0146] Then 27.7 milliliters (0.25 moles; 1 eq.) of benzyl alcohol
are added. The solution is agitated at 40.degree. C. under an inert
atmosphere for 12 h. The mixture of monoesters obtained in this
manner is dissolved in 150 milliliters of ether. The etherized
phase is washed 3 times with 100 milliliters of water, dried on
magnesium sulfate and filtered on a Buchner funnel.
[0147] Characteristics: MW=287; m=71.3 g; Yield of 95%; appearance:
pale yellow oil.
b) Synthesis of Allyl Bromosuccinate
[0148] The same protocol as previously employed is used but 17.86
milliliters (0.25 moles; 1 eq.) of allyl alcohol are added. The
reactional medium is agitated for 22 h at 60.degree. C. under an
inert atmosphere rather than for 12 h at 40.degree. C. The
monoesters are purified in identical manner.
[0149] Characteristics: MW=237; m=17.6 g; Yield of 72.8%;
appearance: viscous oil.
[0150] c) Synthesis of 2-butyl Bromosuccinate
[0151] The same protocol as above in 2)b), but with 18.8 g (1 eq.)
of (RS)2-butanol that has been distilled in advance. The reactional
medium is agitated for 12 hours at, 60.degree. C. under an inert
atmosphere.
[0152] Characteristics: MW=253; m=45 g; Yield of 90%; appearance:
orangish yellow oil.
3) Synthesis of the Lactones
[0153] The lactonization reaction is performed on the sole
lactonizable monoester and presents an inversion of configuration.
It is performed directly on the mixture of monoesters after
neutralization at pH 7.2 with 2N soda. The reaction takes place in
a dichloromethane/water biphasic medium. The lactone is purified by
silica column chromatography. The nature of the eluent varies
depending on the nature of the lactone. This lactone is then
distilled on an appropriate column.
[0154] a) Synthesis of Benzyl Malolactonate
[0155] 71 g (of which 70% is lactonizable monoester, i.e., 0.173
moles) of the mixture of monoesters are dissolved in 300
milliliters of ether and 250 milliliters of water in a balloon
flask. A 2N solution of sodium hydroxide is added drop by drop
until reaching pH 7.2, then 450 milliliters of dichloromethane are
added. The flask is placed on a refrigerant system and the biphasic
system is strongly agitated for 3 h at 40.degree. C.
[0156] After decantation, the organic phase is washed 2 times with
250 milliliters of water then 2 times with 250 milliliters of
brine, dried over magnesium sulfate and filtered. The solvent is
eliminated in the Rotavapor. This lactone is purified on silica
column (eluent: 8/2 dichloromethane/petroleum ether) and distilled
3 times under vacuum.
[0157] Characteristics: MW=206; m=20.7 g prior to purification,
i.e. a yield of 40.5%; m=3.41 g after purification, i.e., a yield
of 6.7%; bp=116-118.degree. C. under 310.sup.-2 mbar; IR (n,
cm.sup.-1): .sup.n(CO lactone)=1825 cm.sup.-1; .sup.n(CO ester)
1740 cm.sup.-1; appearance: colorless oil.
[0158] b) Synthesis of Allyl Malolactonate
[0159] According to the same protocol as above in 3)a), but the pH
of the aqueous phase is 7.8 rather than 7.2 and the solution is
agitated for 5 h rather than 3 h. The lactone is purified on silica
column (eluent: 4/6 petroleum ether/ethyl ether) and is distilled 3
times under vacuum.
[0160] Characteristics: MW=156; m=15.3 g prior to purification,
i.e., a yield of 53.4%; m=4 g after purification, i.e., a yield of
13%; bp=62-65.degree. C. under 310.sup.-2 mbar; IR (n, cm.sup.-1):
.sup.n(CO lactone)=1825 cm.sup.-1; .sup.n(CO ester)=1740 cm.sup.-1;
appearance: colorless liquid.
[0161] c) Synthesis of 2-butyl Malolactonate
[0162] The same protocol is employed as in 3)a) above. The lactone
is purified on silica column (eluent: 8/2 dichloromethane/petroleum
ether) and is distilled 3 times under vacuum.
[0163] Characteristics: MW=172; m=26.7 g prior to purification,
i.e., a yield of 55%; m=14.4 g after purification, i.e., a yield of
28%; bp=80-82.degree. C. under 310.sup.-2 mbar; IR (n, cm.sup.-1):
.sup.n(CO lactone)=1825 cm.sup.-1; .sup.n(CO ester)-1740 cm.sup.-1;
appearance: colorless viscous oil.
II--Synthesis of the Polymers
[0164] The lactones were polymerized in the presence of tetramethyl
ammonium benzoate (10.sup.-3 eq.) at 37.degree. C. for 15 days. The
polymerization was monitored by infrared analysis with observation
of the disappearance of the lactone band at 1850 cm.sup.-1.
1) Synthesis of Allyl Butyl-Co-Malate Benzyl-Co-Malate
Polymalate
[0165] Five g (24.2 mmoles) of benzyl malolactonate, 1.4 g (9.3
mmoles) of allyl malolactonate and 0.7 g (4.1 mmoles) of butyl
malolactonate are degassed for 2 h and transferred via cannula into
a balloon flask containing, 471 milliliters of a primer solution
(tetraethylammonium benzoate: 10.sup.-3 eq.; 37.7210.sup.-6) at
8010.sup.-3 M which was degassed in advance for 2 h under a
nitrogen stream. The copolymerization was performed at 37.degree.
C. for 15 days under an inert atmosphere and under agitation. The
polymer was then dissolved in a minimum of chloroform. The chains
were terminated by addition of a drop of concentrated hydrochloric
acid and the polymer was precipitated with ethanol then dried under
vacuum at 40.degree. C. for 48 h.
[0166] Characteristics: m=4.37 g; yield of 61.5%; Tv=-5.degree. C.;
IR (m, cm.sup.-1): .sup.n(C.dbd.O)=1748 cm.sup.-1; SEC (THF,
polystyrene standard): Mn=6600; MW=9200; Ip=1.4; MSEC=10,000; S 134
(CH.dbd.); 168 (C.dbd.O lateral chain); 170 (C.dbd.O principal
chain); appearance: transparent vitreous polymer.
2) Epoxidation of Allyl Butyl-Co-Malate Benzyl-Co-Malate
Polymalate
[0167] 1.12 g (7.91 mmoles of unsaturated units) of polymer are
dissolved in 3 milliliters of anhydrous dichloromethane in a
balloon flask. A solution containing 466.34 milligrams (4.69
mmoles; 6 eq.) of metachloroperbenzoic acid (MCPBA) in 2
milliliters of dichloromethane is added by cannula. The mixture is
agitated for 24 hours at room temperature. The polymer is then
precipitated with ethanol and dried in a desiccator under
vacuum.
[0168] Characteristics: m=4 g; precipitation yield of 92%;
epoxidation reaction yield of 100%.
[0169] The molar mass did not change upon epoxidation because MCPBA
does not induce a modification of the chain length.
3) Hydrogenolysis of Allyl Butyl-Co-Malate Benzyl-Co-Malate
Polymalate
[0170] Four g of the polymer are dissolved in 5 milliliters of
freshly distilled dioxane on sodium in a balloon flask. 800
milligrams (20% by weight) of palladium on active charcoal are
added and the hydrogenolysis is begun. When the volume of hydrogen
consumed no longer increases (24 h after the beginning of the
reaction), the hydrogenolysis is stopped. The solution is filtered
on Celite and the dioxane is eliminated in the Rotavapor.
[0171] Characteristics: m=2 g; hydrogenolysis yield of 100%.
[0172] 4) Sulfonation of Allyl Butyl-Co-Malate Benzyl Co-Malate
Polymalate
[0173] Four g of polymer (i.e., 5.64-10.sup.-3 moles of epoxide)
are dissolved in 20 milliliters of water. 2.14 g of sodium
bisulfite (2 eq., 11.2810.sup.-3 moles) are added. The pH of the
solution is adjusted to 7.4 (Housse-Ferrari, "Preparation and
characterization of porous silicas modified by polymers and
copolymers of N,N'-dimethyl acrylamide", Thesis, University of
Paris VI, Jan. 30, 1990). The solution is left under agitation for
7 h in ice then ultrafiltered for 24 h at 4.degree. C. against
water and lyophilized.
[0174] FIG. 4 summarizes the synthesis steps of the
poly(.beta.-malic acid) derivatives.
EXAMPLE 2
Synthesis of Polysaccharide Polymers Constituted by Substituted
Glucose Motif
I--Synthesis of Carboxymethyl Dextran Sulfates Designated CMDS
[0175] In this example, the polymer of the invention is constituted
of substituted dextran in which the glucose A motifs substituted by
X and/or Y and in which Z is nothing are represented in FIG. 5. The
different types of grafting are shown in FIG. 5 in which:
[0176] A is a glucose monomer on which X, Y and Z are grafted by
the intermediary of the hydroxyl functions in position 2 and/or
position 3 and/or position 4 and/or by the intermediary of the Y
groups for Z,
[0177] X is --CH.sub.2COOH or --CH.sub.2COO.sup.-Na.sup.+
[0178] Y is --SO.sub.3H or SO.sub.3-Na.sup.+
[0179] Z is a variable group of which several examples are
presented below.
[0180] The polymers of type CMDS in which Z=nothing contain
multiple types of monomers. The first types of substituted monomers
are the carboxymethyl glucose of type A-X substituted in position 2
and/or 3 and/or 4 (motifs presented in FIG. 5). The addition of the
group Y=(motifs A-Y represented in FIG. 5) corresponds to an
O-sulfation and becomes, with R=nothing and R'=H.sup.+ or Na.sup.+,
Y=O--SO.sub.3.sup.-H.sup.+/Na.sup.+. If Y is fixed on X, R of Y
becomes CH.sub.2--CO and the lost functionality of X(COO.sup.-) is
no longer considered to exist. It then becomes part of Y and enters
into the measurement of the percentages of substitution of the
active groups Y.
[0181] The different monomers constituting the CMDS polymer are
thus either unsubstituted glucose or glucose carboxymethyl or
glucose sulfate or glucose carboxymethyl sulfate. The different
isomeric forms are diagramed in FIG. 5. Thus the polymers
correspond to all of the possible combinations of the different
monomeric forms and are defined by the residual rate of free X and
Y groups.
[0182] The monomers obtained in this manner are either glucose
sulfate in position 2, 3 and/or 4 and/or glucose carboxymethyl
sulfate. The sulfate is fixed either on the glucose or on the
carboxylic group.
[0183] Thus, in FIG. 5, in which the bonds of the groups
schematized by a dotted line represent the monomers in which all of
the circular combinations can be envisaged.
1) Synthesis of Carboxymethyl Dextran Sulfate Designated
CM.sub.nDS.sub.m
[0184] a) Carboxymethylation
[0185] The first dextran carboxymethylation step is performed
according to the protocol described in Mauzac et al. (Mauzac et
al., Anticoagulant activities of dextran derivatives. Part I:
Synthesis and characterization, 1984, Biomaterials 6/61-63). It
comprises an etherification of the hydroxyl functions of the
glucose residue of the dextran in order to obtain a carboxymethyl
dextran. This reaction can be reproduced multiple times and results
in products referred to as CM.sub.nD in which n represents the
number of carboxymethylation steps. The various products referred
to as CM.sub.nD are characterized by an increasing percentage of
COOH. This process thus makes it possible to obtain different rates
of carboxymethylation of the dextran as indicated in the table of
FIG. 6.
[0186] Thus, in a refrigerated 250-milliliter balloon flask,
Dextran T40-(37.37 g) 9.34 10.sup.-4 mole), from Sigma and of
molecular weight 40,000 D, is solubilized (182 milliliters of
distilled water) at, 4.degree. C. A soda solution (74 g, 1.85 mole
in 124 milliliters of distilled water), also, cooled to 4.degree.
C., is poured slowly into the Dextran solution while maintaining
constant the temperature of 4.degree. C. Monochloroacetic acid
(76.2 g, 0.806 mole), reduced to a fine powder, is added slowly
while maintaining the same reaction temperature. However, the
temperature of the reactional medium rises at the end of the
reaction from 4.degree. C. to 21.degree. C. within several minutes.
The mixture is then brought to 50.degree. C. in a thermostated oil
bath for 40 minutes. During heating, the reactional medium acquires
a yellow coloration. After cooling, the pH is neutralized to 7.2
with glacial acetic acid (Takakura, 1990). The carboxymethyl
dextran is collected by precipitation in cold absolute ethanol (5
to 6 times the reactional volume). It is then dried in an oven
under vacuum. The polymer CM.sub.1D is purified by ultrafiltration
then lyophilized (mass=55 g gross).
[0187] The presence of the carboxylic ions is quantified by reverse
acid-basic quantification using nitric acid. The base used is 1 N
soda. The result is expressed in % of COOH groups. % COOH of
CM.sub.1D=48.98%. This, percentage means that statistically
approximately one out of every two glucose units was
carboxymethylated.
[0188] In practice, this reaction can be performed multiple times
to attain the desired substitution rates. The same protocol was,
therefore, applied for the synthesis of CM.sub.2D from CM.sub.1D
and of CM.sub.3D from CM.sub.2D:
[0189] % COOH of CM.sub.2D=91.8% and % COOH of
CM.sub.3D=118.3%.
[0190] b) Sulfatation
[0191] The sulfatation reaction of the residual hydroxyl functions
after the carboxymethylation steps is performed with chlorosulfonic
acid. It produces the compounds referred to as CM.sub.nDS.sub.m
which are presented in FIG. 6, in which m corresponds to the
chlorosulfonic acid equivalents as defined in the example
below.
Example of Sulfatation of CM.sub.1D
[0192] Five hundred milligrams (MW=7.8 mmol/g) of
carboxymethyl.sub.1dextran (CM.sub.1D) are dispersed in 40
milliliters of dry dichloromethane. The number of hydroxyl residues
that remain free and capable of reacting in a sulfatation reaction
is nOH=410.sup.-3 mole. The reaction was performed in the presence
of one chlorosulfonic acid equivalent (nClSO.sub.3H=410.sup.-3
mole) or approximately=0.5 g or a volume of 0.3 milliliters, the
density of the chlorosulfonic acid solution being 1.75. The 0.3
milliliters of chlorosulfonic acid are diluted in 4 milliliters of
dehydrated dichloromethane. The CM.sub.1DS.sub.1 obtained in this
manner is recovered by filtration of the reactional medium under
vacuum on frit.
[0193] The same reaction can be performed in the presence of 0.5 or
1.5 or 2 or 3 equivalents of acid chlorosulfonic acid or an excess
so as to graft the increasing quantities of O-sulfate groups. The
RGTA polymers thereby obtained are referred to as CM.sub.nDS.sub.m,
with n=1, 2, etc. and m=0.5, 1, 2, etc.
[0194] Using the same principle and with the goal of comparing
these polymers with other sulfated molecules, we considered as
comparison products with the CM.sub.nDS.sub.m either commercial
dextran sulfates (Pharmacia Biotech product, code 17-0270-01) or
dextrans sulfated from dextran T.sub.40 in the presence; of m
equivalents of chlorosulfonic acid, i.e., under reaction conditions
comparable to those employed for producing the
CM.sub.nDS.sub.m.
[0195] The results of different quantitative determination of the X
groups by titration and the Y groups by elemental analysis of the
levels of sulfur atoms make it possible to specify the
corresponding values x and y for each of the CM.sub.nDS.sub.m
compounds synthesized. These data are presented in FIG. 6. This
figure also indicates this percentage for commercial dextran
sulfate and for the dextrans sulfated under the same conditions as
the CM.sub.nDS.sub.m.
2 Synthesis of the Carboxymethyl Dextran-Phenyl Sulfonate Polymers
Indicated as CM.sub.2DPhS and of a Carboxymethyl Dextran Sulfate
Phenyl Sulfonate Designated CM.sub.2DPhSS
[0196] These polymers are constituted by a sequence of motifs shown
in FIG. 7 and correspond to those of FIG. 4 in which Z was nothing.
These polymers are constituted by a sequence of motifs of type A-X,
A-Y and A-Z as shown in FIG. 6.
[0197] In this example, the group Z is phenyl sulfonate indicated
as PhS. FIG. 7 shows a monomer A-Z in the case in which Z received
as an addition product a carboxyl radical grafted in position 2 of
the original glucose, which was itself sulfated in positions 3 and
4.
[0198] The polymer CM.sub.2D (2 g; 3.90 mmol) is dissolved in 13
milliliters of distilled water. The pH of the solution is adjusted
to 3.5 with 3 M HCl. The coupling agent EEDQ (1.93 g, 2 eq.) is
dissolved in 16 milliliters of ethanol (0.12 g of EEDQ/milliliter)
at 40.degree. C. The EEDQ solution is added progressively to the
polymer and the reactional mixture is strongly agitated for 30
minutes. The phenylsulfanilic acid salt (NH.sub.2PhSO.sub.3Na,
3.045 g, 2 eq.) is then added in small amounts. The pH of the
reaction is adjusted to 9 with soda. The mixture is agitated at
room temperature for 4 hours and then neutralized with dilute HCl.
The product CM.sub.2DPhS is then ultrafiltered, evaporated under
vacuum and then lyophilized (1.66 g).
[0199] The elemental and acid-base analyses of the CM.sub.2DPhS
yielded: % C=33.9; % H=5.28; % N=0.3; % S=0.67; % COOH=81%.
[0200] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.2DPhSS*, yielded % C=23.16; % H=3.72; % N=0.27; %
S=9.60; % COOH=52%.
3) Synthesis of a Carboxymethyl Dextran N and O Sulfate
Derivative
[0201] These polymers are constituted by a sequence of motifs of
type A-X, A-Y and A-Z as shown in FIG. 6, in which one of the
characteristic motifs A-Z is presented in FIG. 8. In this example,
the group Z which is ethylenediamine, indicated as DE, is grafted
on the carboxyl radical of the carbon 2 of the original glucose
monomer, which is itself sulfated in positions 3 and 4.
[0202] The same protocol as employed above was applied to CM.sub.2D
with, in a first stage, the addition of an ethylenediamine group Z,
indicated as DE, so as to yield the product designated
CM.sub.2DE.
[0203] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.2DE showed: % C=37.67; % H=6.25; % N=5.35; %
COOH=19%.
[0204] The sulfatation protocol was performed on a fraction of this
polymer using 1 chlorosulfonic acid equivalent. The products
obtained correspond to the CM.sub.2DES.sub.1 represented in FIG. 7.
In this case, according to the general formula of the RGTA, Z is
diethylamine.
[0205] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.2DDES, showed: % C=36.83; % H=5.82; % N=4.67; %
S=1.82; % COOH=10.7%.
[0206] These syntheses make it possible to graft the N-sulfate
groups in addition to the O-sulfate groups of the CMDS. These
different polymers enable evaluation of the specific roles of the
N-sulfate and O-sulfate groups of the CM.sub.2DES in relation to
the phenyl sulfonate groups, indicated as PhS, of the compounds
CM.sub.2DPhS, the sulfate and sulfonate compounds of the
CM.sub.2DPhSS or to the compounds. In this type of polymer, it
would seem that the sulfate groups are essentially linked to the
properties of the RGTA.
4) Synthesis of the Polymers of Carboxymethyl Dextran-Phenylalanine
Sulfate Designated CM.sub.3DPheS and of Carboxymethyl
Dextran-Tyrosine Sulfate Designated CM.sub.3DTrS Represented in
FIG. 9
[0207] These polymers are constituted of a sequence of motifs of
type A-X, A-Y and A-Z as shown in FIG. 6 in which these
characteristic motifs are represented in FIG. 9. In this example,
the groups Z are respectively either phenylalanine for Phe or
tyrosine for Tyr. They were subjected to the addition process on a
carboxyl radical grafted on position 2 of the original glucose
which is itself sulfated on positions 3 and 4.
[0208] In these polymers, Z is an amino acid with either
phenylalanine (Phe) or tyrosine (Tyr) and Y is an --OSO.sub.3.sup.-
group.
[0209] These syntheses were performed so as to evaluate the
importance of the aromatic structures of these two amino acids Phe
and Tyr by replacing the benzylamine indicated as B as is known in
the CMBDS of the prior art, but the salting out of which in in vivo
applications can be detrimental by generating risks of
tumorigenicity.
[0210] The polymer CM.sub.3D (% COOH=136%, 0.6 g, 3.014 mmol) is
dissolved in 6 milliliters of distilled water. The pH of the
solution is adjusted to 3.5 with 3M HCl. The coupling agent EEDQ
(745 milligrams, 1 eq.) is dissolved in 6.2 milliliters of ethanol
at 40.degree. C. The EEDQ solution is added to the polymer drop by
drop and the reactional mixture is agitated vigorously for 30
minutes at room temperature. The phenylalanine methyl ester (1.3 g,
2 eq.) is added very slowly to the mixture and the pH is brought
from 5 to 9 with soda. The reaction is maintained at room
temperature for 4 hours. The solution is then neutralized with
dilute HCl. The polymer CM.sub.3DPhe is then ultrafiltered,
evaporated under vacuum and then lyophilized (524 milligrams).
[0211] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.3DPh showed: % C=36.9; % H=5.03; % N=0.44; %
COOH=101.05%.
[0212] The same protocol was performed on the CM.sub.3D with the
tyrosine methyl ester CM.sub.3DTyr.
[0213] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.3DTyr showed: % C=34.41; % H=5.12; % N=0.28; %
COOH=101.76%.
[0214] The sulfatation protocol was performed on these two polymers
using 2 chlorosulfonic acid equivalents.
[0215] The elemental and acid-base titrimetric analyses of the
polymer CM.sub.3DPheS*.sub.2 showed: % C=18.08; % 14=-2.89; %
N=0.30; % S=14.71; % COOH=28.79.
[0216] The elemental and, acid-base titrimetric analyses of the
polymer CM.sub.3DTyrS.sub.2 showed: % C=17.99; % H=3.13; % N=0.3; %
S=14.35; % COOH=19.85.
5) Synthesis of the Lipidic Polymers: Example with Carboxymethyl
Dextran-Oleic Acid Sulfate (CM.sub.1DoleicS) and Carboxymethyl
Dextran-Palmitic Acid Sulfate (CM.sub.1DpalmS) Represented in FIG.
10
[0217] The polymers are constituted by a sequence of motifs of type
A-X, A-Y and A-Z as represented in FIG. 6 in which the
characteristic motifs are represented in FIG. 10. In this example,
the groups Z, which are respectively either oleic acid indicated as
oleic or palmitic acid indicated as palm, were added on the
hydroxyl of the carbon 3 of a carboxymethyl glucose in position 2
sulfated in position 4.
[0218] These compounds respond to formula (I) in which
Y=-OSO.sub.3.sup.- and Z is oleic or palmitic acid. These fatty
acids were grafted to evaluate the importance of the
hydrophobicity/hydrophilia balance of the polymers in addition to
the role itself of these fatty acids.
[0219] To the polymer CM.sub.1D (1 g, 2.43 mmol) dissolved in DMSO
(16 milliliters) was added triethylamine (0.8 milliliters) and then
oleic chloride (1.6 milliliters) was added drop by drop. The
reactional mixture was agitated at room temperature for 2 hours.
The polymer was precipitated in 120 milliliters of ethyl acetate,
centrifuged and then dried under vacuum. The precipitate was
dissolved in 20 milliliters of 2M sodium acetate and the salt
formed was precipitated in ethanol (160 milliliters), filtered,
dissolved in 20 milliliters of distilled water then finally
dialyzed against water. After dialysis (24 h), the solution
evaporated under vacuum-yielded the lipid dextran CM.sub.1Doleic
(751 milligrams).
[0220] Elemental analysis of the product CM.sub.1Doleic: % C=34.25
and % H=5.91.
[0221] The same protocol was performed with the palmitic chloride
polymer CM.sub.1Dpalm.
[0222] The elemental analyses of the polymer CM.sub.1Dpalm showed:
% C=35.13, % H=5.96.
[0223] The sulfatation protocol was performed on these two polymers
using 1 chlorosulfonic acid equivalent.
[0224] The elemental analyses of the polymer CM.sub.1DoleicS*.sub.1
showed: % C=28.28; % H=5.04; % S=5.26.
[0225] Elemental analyses of the product CM.sub.1DpalmS*.sub.1
showed: % C=29.17; % H=4.88; % S=5.14.
[0226] The different examples presented which involve grafting a
group Z yield the compounds, the definitions of several of which
are presented in Table II below.
TABLE-US-00002 TABLE II Reference Groups X Y Z RGTA 1110
CM.sub.2DPhSS.sub.1 52.1 43.8 8.9 RGTA 1111 CM.sub.2DES.sub.1 10.7
42.4 21.2 RGTA 1112 CM.sub.2DPheS.sub.2 28.9 56.2 17.9 RGTA 1113
CM.sub.3DTyrS.sub.2 19.8 65.9 28.9 RGTA 1114 CM.sub.1DpalmS.sub.1
39.8 47.4 3.8 RGTA 1115 CM.sub.1DoleicS.sub.1 36.0 43.9 2.2
[0227] Table II above indicates the percentages of substitution of
the polymers containing a group Z.
6) Purification Steps for the Different. Dextran Derivatives of the
Above Examples
[0228] a) Dialysis to Equilibrium
[0229] After each synthesis step the polymers are collected in
solid form (precipitation or filtration followed by
lyophilization). The polymers are then resolubilized in the minimum
volume of distilled water and then introduced into dialysis tubing
(Spectrapor) with a cut-off threshold of 6000 to 8000
g/mole.sup.-1. The dialysis is performed against twice-distilled
water (MilliQ) in a ratio of 1 volume of product per 50 volumes of
water for 4 to 5 days with two changes of water per day.
[0230] b) Chromatography
[0231] The preceding step is associated with HPLC chromatography on
molecular sieve (Column TSK) in order to establish the molar masses
of the purified polymers.
[0232] c) Tangential Ultrafiltration
[0233] After the dialysis, the content of the tubing is
ultrafiltered in an ultrafiltration cell (Pellikon, Millipore) on a
cellulose membrane with a cutoff threshold of 10,000 g/mole.sup.-1.
The quality of the purification was monitored with a conductimetry
cell. When the conductivity of the water eliminated at the outlet
of the cell had returned to the conductivity of pure distilled
water (2 .mu.S), the purification was stopped and the solution was
concentrated prior to lyophilization.
7) Determination of the Percentages of Substitution
[0234] For the two groups of examples presented above, the
percentages of substitution of the groups X, Y and possibly Z were
determined in the following manner.
[0235] a) Poly .beta.-Malic Acid Polymers (Examples 1)
[0236] For these polymers, the percentages of, substitution are
defined a prion; in relation to the proportion of the different
monoesters subjected to polymerization.
[0237] a) Dextran Polymers (Examples 2)
[0238] Two cases must be envisaged for these polymers obtained from
dextran depending on the definition of the group Z.
[0239] The first case corresponds to the case of the
CM.sub.nDS.sub.m in which Z=nothing; the second case depends on the
chemical nature of Z.
[0240] On each glucose residue, three hydroxyl functions are
capable of reacting. A relative molar mass of 54 g/mole.sup.-1 is
attributed to each hydroxyl function, i.e., one third of the
molecular mass of 162 g/mole.sup.-1 of a constitutive residue of
dextran. It is assumed that each hydroxyl has the same reactivity
and that the substitutions first affect each glucose unit once
prior to a possible second substitution on the same residue.
[0241] A dextran T40 of 40,000 g/mole.sup.-1 thus contains 247
glucose residues of molar mass 162 g/mole.sup.-1.
[0242] The substitution rates attained during the
carboxymethylations are determined by acid-base determination with
an automatic titrimeter (Tacussel). This determination finds a
value x.sub.1 corresponding to the number of moles of acid fixed
per gram of polymer.
[0243] Thus, when a hydroxyl is substituted, there appears on the
glucose a motif: --OCH.sub.2COON.sub.a. Each of these substituted
subunits has a relative molecular mass of 240 g/mole.sup.-1.
[0244] Multiple motifs appear after sulfatation.
[0245] The rates of free carboxylic groups determined by acid-base
determination gives a value X.sub.2 which is always lower than the
initial value X.sub.1. The difference X.sub.1-X.sub.2 corresponds
to the motifs --OCH.sub.2COO--SO.sub.3Na. Each of these substituted
subunits has a molecular mass of 320 g/mole.sup.-1.
[0246] NMR analysis revealed that the S corresponds to a
sulfatation of the free hydroxyls of the glucose residues in
addition to the preceding reaction. In this case, a motif
--OSO.sub.3Na appears. Each of these sulfated glucose subunits has
a relative molecular mass of 200 g/mole.sup.-1. The microanalyses
provide the rates of S as a percentage of the mass of the
polymer.
[0247] It is, therefore, possible at the end of synthesis to obtain
the percentages of free carboxyl radicals X.sub.1 and X.sub.2
determined respectively before and after the sulfatation step,
taking into account that the polymer contains:
[0248] a unsubstituted glucose residues of mass 162 g.
[0249] X.sub.2 free carboxylic residues of mass 240 g.
[0250] X.sub.1-X.sub.2 sulfated carboxylic residues of mass 320
g.
[0251] Y sulfated glucose residues of mass 200 g.
[0252] Based on these data, it is possible to establish the
percentage of substitution of the groups X and Y.
[0253] Thus, the percentage of sulfur provided by the microanalysis
results (S %) make it possible to determine the number of atoms of
sulfur (.SIGMA..sub.S) grafted on the polymer. This number of atoms
is .SIGMA..sub.S=(S %.times.MM)/32.times.100, in which 32 is the
atomic mass of S and MM is the molar mass of the synthesized
polymer.
[0254] It is possible to obtain from this the percentage Y of
radicals SO.sub.3.sup.- as equal to: (100.times.S
%.times.MM)/247.times.3200.
[0255] In the second case in which the grafted group Z is for
example tyrosine, the same reasoning is applicable with the value
of nitrogen given by the elemental analysis results.
B) Properties of the Polymers of the Invention
[0256] The properties that are common to the RGTA and the
HBGFPP
[0257] 1) They are devoid of significant anticoagulant activity,
i.e., they present an activity lower than 50 IU/milligram compared
to that of heparin whose activity is on the order of 150 to 170
IU/milligram.
[0258] 2) They stabilize and potentiate the growth factors that
present an affinity for heparin, particularly as examples FGF1
and/or FGF2 and/or TGF.beta..
[0259] 3) They protect these factors against proteolytic agents
such as trypsin.
[0260] 4) They inhibit the protease activities implicated in the
inflammatory process such as for example leukocyte elastase or
plasmin.
[0261] 5) They exert a cicatrizing effect in at least one of the
models presented in the cited patents, i.e., the muscles, the
nerves or the gastrointestinal tract.
[0262] The Novel Properties of the RGTA
[0263] 6) They protect and potentiate the enzymatic activities
implicated in combating oxidative stress such as for example
superoxide dismutase or SOD. Due to this property, they act as
antioxidant agents and can be used alone or associated with SOD in
the therapeutic and/or cosmetic indications of SOD or as an
antioxidant protective agent especially in the protection of foods
and nutrients.
[0264] 7) They inhibit the activity of enzymes such as
calpaine.
[0265] 8) They inhibit the activity of enzymes such as heparitinase
or heparinase.
[0266] 9) They increase the survival of cells subjected to ionizing
radiation and regulate the secretion on both the quantitative and
qualitative levels of the constituents of their matrix as well as
the collagens for example.
[0267] 10). They act as antifibrotic agents by modulating the
growth of the mesenchymal cells such as the smooth muscle cells,
the fibroblasts or the hepatic cells and the quality of the type of
collagen that they secrete.
[0268] 11) They present a slow degradability, a criterion which
enables their differentiation from the heparan sulfates which are
products that are naturally degraded by heparinase or
heparitinase.
[0269] 12) They neither contain nor liberate after degradation
products that are known to be toxic, such as for example can occur
with the CMDBS With the grafted groups Z=benzylamine, which
therefore rules out the CMDBS.
[0270] 13) They present in vivo capacities of protection and
tissular regeneration in the following different models:
[0271] a) cutaneous wound lesion,
[0272] b) regeneration of osseous tissues such as the long bones,
with the example of the femur,
[0273] c) protection against the loss of osseous tissue and
regulation of its, reorganization such as in the case of
osteoporosis or periodontal disease. They are, therefore, regulator
agents of tissular homeostasis and of tissular masses such as
osseous or muscular mass.
[0274] d) hepatic regeneration or protection against the
degeneration of the central and peripheral nervous systems,
[0275] e) protection against the deleterious effects, of ischemia
regardless of its localization.
EXAMPLE 3
Measurement of the Anticoagulant Activities of the Polymers of
Examples 1 and 2
[0276] The coagulation tests were performed using the Activated
Cephalin Time technique or A.C.T. (Biggs, 1972, In: Human Blood
Coagulation, Oxford Blackwell Scientific Publications). One hundred
microliters of a polymer solution at different concentrations in
Owen Koller buffer are incubated for 5 minutes at 37.degree. C.
with 100 microliters of plasma poor in platelets and 100
microliters of a solution of rabbit brain cephalin. 100 microliters
of 0.25 M calcium chloride are added and the time until appearance
of the coagulum is referenced by chronometry.
[0277] As shown in FIG. 11, the polymers of Examples 1 and 2 do not
present anticoagulant activities greater than 50 IU/milligram,
especially with respect to heparin which was used as a; positive
control. It should be noted that all of the values for the
anticoagulant activities of the products presented here as examples
are lower than 10 IU/milligram of product.
EXAMPLE 4
Stabilization and Potentiation of the Polymers of Examples 1 and 2
on Growth Factors Presenting an Affinity for Heparin and
Particularly as Examples FGF1 and/or FGF2 and/or TGF.beta.
[0278] This example considers the effect of the polymers on the
stabilization of FGF1 and the effect on the potentiation of FGF1
and FGF2. These effects are evaluated on the growth of 3T3. BALB/c
or CCL39 cells. The conditions employed for these experiments are
those described in the prior art in the patents relative to the
HBGFPPs which are incorporated in the present invention by
reference.
1) Effects of the Polymers of the Invention on the Stabilization of
FGF1 or FGF2
[0279] FIG. 12 shows the effects of the RGTA against thermal
degradation at 20.degree. C. and at 37.degree. C. in relation to
the incubation time of FGF2 conserved alone or in the presence of
the tested products. The ED.sub.50 represents the concentration in
micrograms/milliliter of FGF1, here 6 nanograms/milliliter, that
must be inoculated in a culture of fibroblastic cells, the cells
CCL 39, in order to obtain 50% of the maximum rate of incorporation
of tritiated thymidine.
[0280] The results obtained show that all of the polymers tested
exert at 20.degree. C. as well as at 37.degree. C. protective
effects comparable to those of heparin, and with greater efficacy
in some cases.
2) Effects of the Polymers of the Invention on the Potentiation of
FGF.sub.1 or FGF2
[0281] The protocol is the same as described previously with a
variable quantity of FGF possibly associated with a constant
quantity of polymer. The concentration of polymer employed
corresponds to that which potentiates to the maximum the mitogenic
effect of the FGF. These tests are performed on 3T3 cells for FGF1
and FGF2. The controls are the same as those previously cited with
the exception of a systematic determination for each test of the
ED.sub.50 value.
[0282] The molecules of these two families of tested polymers
potentiate the actions of FGF1 and FGF2 because ED.sub.50 values
are obtained for FGF values that are lower or comparable to that
obtained, in the presence of heparin (FIG. 13).
EXAMPLE 5
Protection of the Factors Against Proteolytic Agents Such as
Trypsin
[0283] Trypsin is a proteolytic enzyme with a broad spectrum of
action which is used in in vitro tests and which is one of the
primordial functional enzymes in the digestive process.
[0284] The test for protection against trypsin was therefore
performed. One nanogram/final milliliter of iodinated FGF2 or 5
micrograms/final milliliter of trypsin are incubated in a first
step for 15 minutes at 37.degree. C. with different concentrations
of polymers (0.5 to 500 micrograms/milliliter) in a 100 mM Tris HCl
buffer, 0.18 M NaCl, Brij 0.03% pH 7.6. Five micrograms/f
milliliter of trypsin or 1 nanogram/final milliliter of iodinated
FGF2 are then added respectively. The total reactional volume is 30
.mu.l. The enzymatic reaction is stopped after 2 h of incubation at
37.degree. C. by addition of Laemmli buffer and heating for 5
minutes at 90.degree. C. (Laemmli U.K. Cleavage of structural
proteins during assembly of the head of the bacteriophage T4,
Nature, 1970, 227: 680-685).
[0285] Each sample is placed on a 15% polyacrylamide gel. Migration
is performed for 1 h at 200 V in ice. The gels are dried for 2 h at
80.degree. C. under vacuum and exposed at -80.degree. C. in the
presence of an autoradiographic film. The intensity of the bands is
measured by image analysis and the percentage of FGF2 protected in
relation to percentage of FGF2 degraded is calculated.
Protection of FGF1 and TGF.beta.
[0286] FIG. 14 shows the protective effects expressed as % of
protection of the poly-.beta.-malic acid polymers on FGF1, FGF2 and
TGF.beta. in relation to an attack by trypsin. FIG. 15 presents the
protective effects expressed in % of protection of the polymers
derived from dextran on FGF2 and TGF.beta. in relation to an attack
by trypsin.
[0287] Most of these polymers exert a protective effect which is
comparable to that of heparin which was used as positive
reference.
EXAMPLE 6
Inhibition of the Protease Activities Implicated in the
Inflammatory Process Such as for Example Leukocyte Elastase or
Plasmin
[0288] Leukocyte elastase and plasmin are key proteases in the
installation and unfolding of the inflammatory tests. These tests
are intended to establish whether the polymers protect the growth
factors from degradations by human leukocyte elastase.
[0289] The tests for protection against leukocyte elastase are
therefore performed. The protocol employed is the same as that used
with trypsin with 30 nanograms/final milliliter of elastase and 0.5
to 500 micrograms/milliliter of polymers. The buffer is 100 mM Tris
HCl, 0.18 M NaCl, 0.03% Brij pH 8. The intensity of the bands is
evaluated by image analysis and the percentage of nondegraded FGF2
is calculated. The positive control for these tests is heparin.
Dextran T40 and dextran sulfate were used as internal
references.
[0290] FIG. 16 presents the inhibitory effects of the different
polymers expressed by their IC.sub.50 values.
EXAMPLE, 7
Example of the Effects of the RGTA on the Regeneration, Protection
and Functional Restoration of the Tissues: Case of the Regeneration
of Skeletal Muscle
[0291] The model employed for evaluating most effectively the
cicatrizing properties of the RGTA was that of the crushed muscle
as defined and presented in French Patent No. 2 718 026.
[0292] After crushing the EDL (Extensor Digitorum Longus) of the
rear paw of the rat and injection of the crushed muscle into a
solution of physiological serum containing or not containing the
test substances, the muscles treated in this manner are recovered 8
days after the operation. Analysis of the weights as well as a
histological study make possible quantification of the effects of
the polymers on muscular regeneration. The results are expressed in
% in relation to the characteristics of a muscle that only received
an injection of the physiological serum without polymer under the
same experimental conditions. FIG. 17 presents the results obtained
which demonstrate that the new polymers derived from the dextran
skeleton as well as the copolymers of .beta.-malic acid exert the
claimed effects.
[0293] It is important to note that these effects on muscular
regeneration are obtained not only by in situ injection of the
polymers but also by intravenous, intra-arterial or intramuscular
injection as long as the doses injected are selected in relation to
the routes of administration.
EXAMPLE 8
Effects of the RGTA on the Regeneration of Flat Bone
[0294] The model employed for evaluating the cicatrizing properties
of the RGTA is the method already known in the prior art and
described by Blanquaert F, et al., Bone, 1995; 17(6): 499-506.
[0295] This model comprises performing a circular trepanation of 5
millimeters in diameter in the calvaria of an adult rat. The defect
is filled with a collagen buffer that has been cut to the same
dimensions and impregnated with or not impregnated with a solution
containing the RGTA. In the example presented here, the polymers
studied are the type CMS polymers (RGTA 1005 and 1012) and the
.beta.-malic acid copolymers (RGTA 2011). Table III below presents
the percentages of osseous filling established by image analysis of
the radiographs taken 35 days after treatment.
TABLE-US-00003 TABLE III Type of treatment % of osseous filling
control 18 .+-. 4.8 RGTA 2011 56 .+-. 7.0 RGTA 1005 54 .+-. 6.9
RGTA 1012 72 .+-. 8.9
[0296] Thus, both types of polymers stimulate osseous regeneration
since under the cicatrization conditions the sagittal suture forms
ad integrum.
EXAMPLE 9
Protection and Potentiation of the Enzymatic Activities Implicated
in Combating Oxidative Stress Such as for Example Superoxide
Dismutase or SOD
[0297] The production of O.sub.2.sup.- ions and that of hydrogen
peroxide (H.sub.2O.sub.2) represent radicals that exert especially
destructive cytotoxic effects. SOD or superoxide dismutase are
enzymes engaged in the detoxification of these radicals. They are
agents that preserve the organism from oxidative stress.
[0298] The RGTA present various types of activity in relation to
SOD:
[0299] They exert a potentiating effect on the catalytic activity
of SOD at neutral pH and a protective and potentiating effect at
acid and basic pH values.
[0300] They present the property of protecting SOD in relation to
enzymatic degradations such as for example trypsin and also in
relation to heat treatment.
[0301] On models of activated monocyte cultures, they stimulate the
catalytic activity of the endogenous SOD and enable diminishment of
the production of the superoxide ions.
[0302] In all cases, the quantitative determination of SOD activity
is performed using, Pick's technique (Freund M and Pick E, The
mechanism of action of lymphokines. IX. The enzymatic basis of
hydrogen production by lymphokine-activated macrophages. J Immunol
1986, 15; 137(4): 1312-1318). This technique is based on the
determination of the O.sub.2.sup.- ions by reduction of cytochrome
c. MnSOD (Sigma ref. S 8151) at 30 U/milliliter is dissolved in the
presence of different. RGTA at the concentration of 10
micrograms/milliliter. These, mixtures are subjected to different
treatments.
[0303] The SOD activity is evaluated in the presence of different
concentrations of polymers under normal reactional conditions.
[0304] The mixtures of SOD and polymers are either subjected at
room temperature to the action of trypsin (same conditions as for
the protection tests of Example 5) or they are subjected to thermal
treatment at 60.degree. C. for 30 minutes. The residual catalytic
activity of the SOD of these mixtures is then evaluated by
conventional enzymatic techniques or on a cellular system.
[0305] The samples treated in this manner are incubated in a
suspension of monocytes (2.510.sup.6 cells/milliliter) stimulated
by 200 nM of PMA. This condition induces the production of
superoxide, anion by these monocytes activated into macrophages.
The stimulation by PMA induces an increase in the production of
superoxide ions normally produced at a basal level in the absence
of activation. The addition of active MnSOD used as positive
control diminishes the quantity of superoxide ions produced.
[0306] Under these conditions, the lower the production of
superoxide ions, the higher will be the residual catalytic activity
of the SOD contained in the mixtures. Thus, these tests make it
possible to evaluate the protective and potentiator effects of the
polymers on the activity of exogenous as well as endogenous
SOD.
[0307] FIG. 18 illustrates the protective and potentiator effects
of the RGTA on the catalytic activity of SOD in vitro at different
pH values. FIG. 19 illustrates the protective effects of the RGTA
on SOD subjected to an attack by trypsin or after a thermal shock.
FIG. 20 illustrates the potentiator effects of the SOD activity on
the production of superoxide ions by the activated macrophages.
[0308] Conclusion: Thus, the polymers exert potentiator and
protective effects in both acellular and cellular systems. Thus,
the addition of different polymers modulates the catalytic activity
of SOD added endogenously as well as SOD produced endogenously by
the cells.
EXAMPLE 10
Inhibition of the Activity of Enzymes Such as Calpaine by the
RGTA
1) Introduction
[0309] Calpaine 1 (Sigma P 4533) is used in this example. Thus,
0.78 enzyme unit (57.2 nM) are incubated in 1 milliliter of 50 mM
Tris-HCl buffer, pH 7.4, 0.5% Brij, 2 mM CaCl.sub.2, 2 mMDT and
0.15M NaCl. The RGTA solutions to be tested are added over 5
minutes at 27.degree. C. The substrate (250 micromolar of
N-succinyl-leu-tyr-amido-7-methyl coumarin, from Sigma (S 1153) is
then added to a quartz tank to which the mixture described above is
then added. Measurements are then performed every 3 to 5 minutes by
excitation at 380 nm and detection in fluorescence at 460 nm of the
transformation of the substrate into 7-amino-4-methyl coumarin
(according to the protocol described by Sasaki, Kikuchi, Yumoto N,
Yoshimura and Murachi T, 1984, in J. Biol. Chem. 259 (20), p
12489-12494).
[0310] The results obtained are summarized in FIG. 21.
2) Conclusion
[0311] We can predict from the results obtained in the inhibition
of the activity of calpaine by the RGTA that the RGTA have a
protective activity against the lesions induced by cerebral
ischemia as could be anticipated from the publications of Markgraf
C G et al. (Stroke, 1998, 29, 152-158) or Saido et al.
(Neuroscience Letters, 1997, 16; 227: 75-78) which, describe the
effect of calpaine inhibitors such as MD L28170 or the protein
calpastatine in the treatment of postischemic lesions of the cortex
or hippocampus. Administration of RGTA via the local or
intravenous, route has the effect of inhibiting the calpaines and
promoting the repair of the nervous-tissue which has been injured
especially by lack of oxygen supply as is the case in ischemia.
EXAMPLE 11
Inhibition of the Activity of Enzymes Such as Heparitinase by the
RGTA
[0312] a) Inhibitory Activity of Heparinase and Heparitinase
[0313] Measurement of the inhibition by the RGTA of heparinase or
heparitinase activities is performed using as substrate heparan
sulfates radio-tagged with sulfur 35 and present in an
extracellular matrix synthesized by endothelial cells cultured in
the presence of Na2 35SO4 for 7 days. The protocol used is the one
described by Ishal-Michaeli R et al. (Importance of size and
sulfation of heparin in release of basic fibroblast growth factor
from the vascular endothelium and extracellular matrix.
Biochemistry 1992; 31(7): 2080-2088). The extracellular matrix
obtained after elimination of the endothelial cells is then
incubated for 24 h at 37.degree. C. in the presence or absence of
heparinase and 0.5 microgram/ml of RGTA. The incubation medium is
collected and deposited on a Sepharose 6B column in accordance with
the protocol described in Ishai-Michaeli (Biochemistry 1992;
313(7): 2080-2088) to measure the degradation of radio-labeled
heparan sulfates. FIG. 22 illustrates the results obtained. The
heparan sulfates of high molecular weight corresponding to the
material not degraded by the enzyme heparinase were eluted first
(fractions 3 to 20). The heparinase treatment caused the
disappearance of this peak and the appearance of a peak
corresponding to low molecular weight fractions of degraded heparan
sulfates, (fractions 20 to 40). In the example presented, the
effect of RGTA 1005 at 50 micrograms/ml induces 50% inhibition and
100 micrograms/ml induces 100% inhibition of the heparinase
activity.
EXAMPLE 12
Effects of the RGTA on the Protection of Human Intestinal Smooth
Muscle Cells Subjected to Ionizing Radiation: Effects on their
Survival and on their Antifibrotic Effects Evaluated by Means of
the Quantity and Quality of the Secreted Collagens
[0314] The formation of a fibrous or fibrotic tissue is an
essential physiological step associated with the processes of
tissue repair and restructuring. The fibrotic tissue is a filling
tissue, normally transitory, intended to conserve the structural
and functional integrity of the tissues and organs. It is
characterized by its richness in collagens of the extracellular
matrix.
[0315] When this condition persists or develops, it corresponds to
a pathology that illustrates a disturbance of the structural and
functional homeostasis of the tissues and is manifested by an
abnormally high accumulation of extracellular matrix which
generates a fibrosis. A fibrosis, regardless of its origin, is
characterized by: [0316] the presence of a permanent inflammatory
infiltrate, the existence of a disequilibrium in the balance
between a proliferative and a quiescent state of the conjunctival
or mesenchymal cells such as fibroblasts or smooth muscle cells,
the progressive destruction of the invaded tissue which is renewed
only slightly or in a defective manner, the existence of a
disequilibrium of the balance between the synthesis and the
degradation of the extracellular matrix.
[0317] A fibrosis can be induced either subsequent to a trauma of
various origins (infectious, mechanical, toxic, etc.) or subsequent
to ionizing radiation (notably by .gamma. rays). In this case, we
are dealing with radioinduced fibroses as is frequently the case in
patients undergoing radiotherapy.
[0318] The collagens are the major components of the extracellular
matrix of normal tissues as well as of fibrotic tissues. The
collagens are essentially synthesized by the mesenchymal cells such
as the fibroblasts and the smooth muscle cells. In a fibrous
tissue, the total collagen is quantitatively increased due to
reasons affecting on both the quantitative and qualitative levels
the synthesis and/or degradation of the collagens, i.e., the
dynamic of the restructuring. Fibroses are characterized by an
increase in type III collagen, with this taking place
preferentially in the case of radioinduced fibroses. This increase
in type III collagen is associated with an increase but to a lesser
degree in the ratio between type III collagen and type I collagen.
Another collagen, type V collagen, is associated with the quality
of the organization of the collagen-fibers in the matrix, i.e., the
fibrillogenesis. In fibrotic tissues, the decline in the levels of
type V collagen is one of the origins of the loss of structure of
the collagen fibers of the extracellular matrix.
[0319] The cellular model employed in this example is that of HISM
cells, Human Intestinal Smooth Muscle cells (American Type Culture
Collection, Rockville, Md. ATCC CRL 192), stemming from the
muscularis propria of human jejunum (Graham M., Diegelmann R.,
Elson C., Bitar K. and Ehrlich H., Proc. Soc. Exp. Biol. Med. 176
(1984) 503). This line of human intestinal smooth muscle cells was
used to evaluate the effects of radiation on cellular survival and
the induction of fibrotic phenomena analyzed by means of the
quantity and quality of the types of collagen-secreted by these
normal cells or in an inflammatory situation or in the repair
process by a fibrosis.
[0320] The cells are cultured in DMEM medium containing 1 g/l of
glucose, 1% of L-glutamine, 1% penicillin-streptomycin and 10% of
fetal, calf serum and conserved in an incubator at 37.degree. C.
under an atmosphere saturated at 5% CO.sub.2 and 95% relative
humidity, in 75-cm.sup.2 plates. The HISM cells are seeded on
plates with 24 flat-bottom wells at the rate of 20,000 cells per
well. The volume of each well is brought up to 2 milliliters of
medium. Various growth kinetics are implemented in the presence or
lack of presence of RGTA at different concentrations ranging from
0.4 to 400 micrograms/milliliter.
[0321] Irradiation is performed from a .sup.60cobalt irradiation
source in an incubator in which the culture plates are arranged.
Two plates are subjected simultaneously to this irradiation the
source of which is vertical and, the doses are evaluated in
relation to the surface irradiated. The irradiator flow rate is 1
Gy/min. The doses absorbed are 10 Gy for an irradiation time per
plate of 10 minutes.
[0322] Different protocols are used to evaluate the role of the
RGTA depending on whether they are added to the culture medium
before, during or after the irradiation, i.e., at the preventive or
curative level, or both at the same time. Table IV below provides
more specific information on the different protocols.
TABLE-US-00004 TABLE IV Sequence RGTA Irradiation RGTA Control --
-- -- Irradiation -- 0 -- Curative -- 0 0 Preventive 0 0 --
Preventive and curative 0 0 0
[0323] The preventive effect is evaluated by addition of RGTA (+)
at the dose of 400 micrograms/ml in the culture medium 48 hours
prior to the irradiation. The curative effect is evaluated by
addition of RGTA 2 hours after irradiation at the same doses as for
the preventive effect. For the cumulative preventive and curative
effects, the cells are continuously cultured in the presence of the
same doses of RGTA.
[0324] 72 hours after the irradiation, the cells are incubated in a
medium free of serum in the presence of tritiated proline (10
microCi/ml) and ascorbic acid (50 micrograms/ml) for 24 hours. The
supernatant is then collected and the cellular layer is recovered
using a robber-policeman in a final volume of 18 ml. The culture
media and the recovered cellular layers are extensively dialyzed
(cutoff threshold of 6 to 8 kDa) against flowing water (24 hours at
4.degree. C.) to eliminate the small molecules from the
macromolecules. After dialysis, aliquot fractions are collected and
hydrolyzed (6M HCl, 105.degree. C., 24 h) for determination of the
radioactivity of the hydroxy(3H)proline, specific marker of the
collagens. At this stage, an aliquot fraction is used, for
quantification by counting of the radioactivity of the total
synthesis of the collagens.
[0325] The remaining volume is dialyzed again in the presence of
pepsin and collagen I against 0.5.degree. M acetic acid for 24 h at
4.degree. C. The pepsin will digest the noncollagenic contaminants
which will be eliminated in the dialysate in the form of peptides.
Type I collagen is added to augment the proportion of collagen
which is low in each sample and to trend towards an
enzyme/substrate ratio of 1:5. The reaction conditions, are as
follows: 1 ml of pepsin solution (0.5 M)+1 ml of solution of
collagen I(0.5 M)+0.514 ml of acetic acid to have a final
concentration of acetic acid of 0.5M in an 18-ml sample. Each
dialysate obtained in this manner is lyophilized in the cold state
(-50.degree. C.) and conserved at -20.degree. C. until use.
[0326] The different collagen .alpha. chains are separated by
polyacrylamide gel electrophoresis in the presence of SDS (Sodium
Dodecyl Sulfate) in accordance with Leammli's method, after
reduction with .beta.-mercaptoethanol. The radioactivity
incorporated in each collagen is determined by hydrolysis of the
.alpha. chains obtained by cutting off the gel bands corresponding
to each collagen chain of a specific type. These bands are then
dissolved in oxygenated water at 60.degree. C. and the
radioactivity contained in the different dissolved bands is counted
with the liquid scintillation .beta. counter or by direct
autoradiography on the gels.
[0327] The electrophoretic separation is performed with 5
microliters of collagen V and 50 microliters of each reduced
sample.
[0328] These gels allow various types of processing:
[0329] Determination of the different types of collagen by analysis
of the incorporated radioactivity.
[0330] Quantification of these different types of collagen by
densitometric analysis after autoradiography.
[0331] The aliquot fractions collected after the dialysis against
flowing water are hydrolyzed (6M HCl, 105.degree. C., 24 h) in
sealed ampoules. The acetic acid is then evaporated, and then the
content of the ampoule is resuspended in 1 ml of distilled water.
The proline and hydroxyproline are separated by the method of
Rojkind and Gonzales (Rojkind. M and Gonzalez E, An improved method
for determining specific radioactivities of proline-14C and
hydroxyproline-14C in collagen and noncollagenous proteins.
Analytical Biochemistry 1974; 57(1): 1-7). The principle is based
on oxidizing the proline and hydroxyproline with chloramine T. The
proline is transformed into pyrroline carboxylate which is soluble
in toluene whereas the hydroxyproline is transformed into
water-soluble carboxylate. After treatment, the proline and
hydroxyproline fractions are recovered for each sample and
quantified by measurement of the radioactivity.
[0332] Under normal conditions, the HISM cells are characterized by
the quantity of collagen synthesized (FIG. 23) and especially on
the qualitative level by the phenotype of the collagens secreted
(FIG. 24).
[0333] Under normal conditions, the collagen that comprises the
majority of the collagen is type I collagen. Type II and type V
collagens are present in smaller proportions. The quantity of type
V collagen is associated with the quality of the organization of
the fibrillogenesis.
[0334] Type III collagen is a "warning" collagen, theoretically
synthesized in a transitory manner in the case of reaction to a
stress but in a permanent manner in the case of a fibrosis. The
proportional quantity of type III collagen in relation to type I
collagen increases in a noteworthy manner in situations involving
response to a tissular lesion, stress or aggression such as for
example ionizing radiation. Type III collagen becomes preponderant
in the matrices of tissues presenting an acute as well as a chronic
fibrotic reaction. This collagen can be considered to be a
signaling component of the fibrotic reaction.
[0335] When the HISM cells are cultured under control conditions,
i.e., without RGTA, they synthesize these three types of collagen
(FIG. 24). Irradiation modifies the quantity (FIG. 23) and the
quality (FIG. 24) of the collagens produced. The overall synthesis
of collagen increases by close to 50% (FIG. 23). Secretion of this
type I collagen and especially of type III collagen increases
considerably (respectively by 50% and close to 500%). In contrast,
synthesis of type V collagen diminishes by more than 507%.
[0336] The presence of different RGTA (RGTA 1005 and RGTA 1025)
restores almost exactly the control behavior of the cells despite
their exposure to the irradiation. FIG. 23 shows that the overall
synthesis of collagen returns to normal values especially for the
conditions of preventive or cumulative (preventive+curative)
treatments. FIG. 24 confirms this return to a reference homeostasis
especially in the case of cumulated treatments. The curative as
well as the preventive treatments exert the same types of effects,
especially with regard to the values of the ratios of secretion of
type I and type III collagens which return to values comparable to
those of the control cells. These RGTA restore the functioning of
the HISM cells in relation to the collagens.
[0337] The RGTA also act on the survival of the cells (FIG. 25). In
fact, irradiation induces a high cellular mortality rate, of more
than 50% of the irradiated population. In the presence of the
polymers, a clear protective effect is recorded because the
mortality rate is reduced to approximately 25% with the most
pronounced effect being obtained for the cumulated
preventive+curative treatments.
EXAMPLE 13
Action as Antifibrotic Agents in Modulating the Growth of
Mesenchymal Cells Such as Smooth Muscle Cells. Fibroblasts or
Hepatic Cells and the Quality of the Type of Collagen that they
Secrete
[0338] As discussed in example 12 above, fibrosis manifests an
alteration in growth of the mesenchymal cells associated with a
modification of the quantity and quality of the collagens
synthesized.
[0339] This example uses another cellular model which comprises
smooth muscle cells from the pig aorta. These cells are obtained by
means of the aorta explant method. The aorta is composed of three
cellular layers: the adventitia (external layer) which contains the
fibroblastic cells, the media (central layer) which contains the
smooth muscle cells (SMC) and the intima (internal layer) which
contains the endothelial cells.
[0340] The media is removed and torn up into tiny pieces. The
explants are cultured on a 25-cm.sup.2 plate containing DMEM 1 g/l
of glucose with phenol red, with the addition of 20% of fetal calf
serum (FCS), 1% of L-glutamine and 1% of penicillin-streptomycin.
The cells become detached from these explants and colonize the
medium (stage P0). Two weeks after the beginning of the culture
phase, the cells are congealed at a cellular density of 2 million
cells in 10% DMSO and DMEM 20% FCS.
[0341] The growth kinetics are determined by evaluating the number
of cells per well by means of an automatic particle counter
(Coulter Counter ZM, Coultronics) which was previously calibrated
by an evaluation of the cell diameter by means of a Malassez cell.
Each counting was performed on an average of 4 wells from which the
cells were detached by means of 500 microliters/well of a
trypsin-EDTA (10 mM) solution.
[0342] The smooth muscle cells were seeded under the same
experimental conditions as the HISM cells. The growth kinetics were
determined in the presence of or lack of presence of polymers of
the series RGTA 1000 to 1025 for the polymers in which Z is
nothing, and of the series RGTA 1110 to 1115 for the polymers in
which Z exists, and of heparin as control; at different
concentrations ranging from 0.4 to 400 mg/ml.
[0343] The percentage of inhibition of the proliferation is
calculated according to the following formula:
I%=100.times.(1-net growth with polymers/net control growth)
[0344] in which net growth represents the difference between the
quantity of cells counted when they merge with the number of cells
seeded at the beginning of culturing.
[0345] The control corresponds to the smooth muscle tissue cultures
in the absence of polymers. Heparin and the RGTA represent the
different effectors tested which are capable of modifying the
biological activity of the cell.
[0346] Under the same conditions as employed for the HISM cells,
the synthesis and typing of the collagen by the smooth muscle cells
was determined.
[0347] FIG. 26 shows the results obtained. The RGTA exert an
inhibitory effect on the proliferation of the smooth muscle cells
which is comparable to that of heparin which was used as control
reference molecule. This effect can be seen from the values in the
two first columns. However, and in contrast to heparin, these
polymers reestablish the collagen secretion phenotype. In summary,
the overall synthesis rate of collagens is significantly diminished
in the presence of RGTA 1005, 1012 and 1013. At the qualitative
level, these same polymers, again in contrast to heparin, diminish
the rate of synthesis of type I and type, III collagens while
increasing the secretion of type V collagen.
[0348] These effects confirm the antifibrotic properties of the
polymers of the invention.
EXAMPLE 14
Effects of the RGTA on Regeneration of the Skin in the Rat
[0349] This example illustrates the effect of the RGTA on deep
cutaneous cicatrization after suture in the rat.
[0350] Male Hairless rats weighing 250 grams were anesthetized by
an IM injection of ketamine and Largactil. Two 3-cm excisions were
made laterally in relation to the dorsal axis of symmetry of the
animals on each side of the spinal column (FIG. 27). In relation to
the control animals which were not treated with RGTA 1012, the
treated animals received via the intramuscular route an injection
of a solution of RGTA 1012 at 1 milligram per kilogram 30 minutes
prior to: the excision and 100 microliters of a solution at 100
micrograms per milliliter as topical application on the wound just
prior to suturing. The suture was performed in a single plane with
an intradermal continuous suture with Prolene 2-0. The treated rats
and the control rats were macrophotographed on days 7, 21 and 60
after the operation. At each of these time points, three rats from
each experimental series were euthanized so as to be able to
perform a histological study of the cutaneous cicatricial
samples.
[0351] FIG. 27 shows the effects of the RGTA on the cutaneous
cicatrization:
[0352] FIG. 27-A: Deep cutaneous incision performed down to the
subjacent muscular floor, 3 cm long, on both sides of the spinal
column.
[0353] FIG. 27-B: Dorsal view of the animal after suturing of the
wound edges by continuous suture.
[0354] FIG. 27-C: Appearance of the cicatrices ten days after the
treatment. C1 and C2 correspond to a control animal treated with
physiological serum without RGTA 1012. In C1 the threads have not
been removed in contrast to photograph C2. The cicatrix is visible
and still presents scabs of coagulated blood especially where
traces can be seen of the needles used for the suture. C3 and C4
correspond to an animal treated with physiological serum containing
RGTA 1012. At the same fire, the cicatrices are no longer visible.
The suture threads are visible in figure C3 whereas they were
removed in figure C4. In this photograph, only a fine border marks
the original incision.
[0355] FIG. 27-D: Histological analysis. D1 and D2 present the
histological sections of the skin that was incised 60 days earlier.
D1 corresponds to a control animal that was treated with
physiological serum-without RGTA 1012; figure D2 corresponds to an
animal treated with physiological serum containing RGTA 1012. In
D1, the dermis subjacent to a still imperfectly mature epithelium
has not returned to a structure identical to that of normal skin.
The restructuring is very partial. The opposite is true of D2 in
which the skin has returned to a structure and an organization
identical to that of skin that was never injured. The only trace of
a cicatrix is visible in the depth of the dermis where an
incompletely restructured zone can be detected. In this later case,
there is complete absence of external cicatrix.
[0356] The photographs of FIG. 27 show at the end of 7 days the
disappearance of the superficial cicatrix whether the threads had
(C4) or had not (C3) been removed whereas in the control animals a
cicatrix can be seen under both of these conditions (C1 and
C2).
[0357] A histological analysis-performed at 60 days revealed that
the skins of the untreated animals (D1) presented a dermis that was
absolutely not mature whereas the animals treated with the RGTA
revealed only a slight dermal trace at the deep level. The
histology of the skin of the treated animals (D2), has an
appearance comparable to the skin of a control animal not subjected
to any cicatricial processes whatsoever. In this model, the RGTA
not only accelerate, the cicatrization rate of the cutaneous floor
but also and especially enable a restoration of the cicatricial
tissue which results in a regenerated tissue in which no trace of
fibrosis can be detected. This example shows that the RGTA are
especially powerful regulators of tissular homeostasis.
EXAMPLE 15
Protective Effects Against Ischemia Manifested by the RGTA
[0358] This example demonstrates, using the polymer RGTA 1005, the
protective effects of the RGTA against tissular damage which
enabled conservation of 80% of the mass of an organ compared to the
untreated organs (FIG. 28). These protective effects of the RGTA
against the deleterious effects induced by the stress caused by a
lack of oxygen supply stemming from an ischemia of the muscles are
presented in the experimentation described below.
[0359] The model employed is inspired by the model described by
Hansen-Smith, F. M., Carlson, B. M. & Irwin, K. L. (1980,
Revascularization of the freely grafted extensor digitorum longus
muscle in the rat. Am. J. Anat. 158, 65-82). The experimental
procedure consists of sectioning the neurovascular trunk of the EDL
muscles (Extensor Digitorum Longus) on the two rear paws of adult
Wistar rats (350 g) at the level of its entry in the muscle and of
completing the ischemia by a ligature of the two tendons. An
injection of 100 microliters of an RGTA 1005 solution at 50
micrograms per milliliter in physiological serum was then made
directly into an EDL muscle. The same volume of physiological serum
without RGTA was injected into the other contralateral muscle.
[0360] Seven days after the injection, the muscles were removed and
examined with a microscope after histological preparation. In each
group of treated or untreated muscles, groups of parameters such as
the mean diameter of the muscle, the thickness of the epimysium, of
the peripheral zone and the mean diameter of the ischemic zone were
measured using a 10.times. objective and a micrometric scale. The
number of layers of muscle fibers that survived the ischemia in the
peripheral zone was counted in thirty different fields selected at
random and observed with a 20.times. objective.
[0361] FIG. 28 shows the protective effects of the RGTA (RGTA 1005)
against tissue injury in a muscle ischemia model in the rat. FIG.
28-A and FIG. 28-B show, respectively, histological muscle sections
from a control rat (28-A) and from a rat treated (28-B) with a
solution of RGTA 1005. In the control, the ischemia caused the
degeneration of the muscle fibers with the exception of a corona of
peripheral fibers in contact with the epimysium. Administration of
RGTA 1005 limits to a considerable degree the degeneration of the
deep muscle fibers just as it diminishes in a very significant
manner the inflammatory reaction and the degradation that this
reaction induces. Thus, RGTA 10015 protects the cells from the
deleterious effects induced by ischemia.
[0362] It can be seen in FIG. 28 that after one week, the mean
diameter is unchanged after treatment with RGTA (5.2.+-.0.3 mm)
compared to the diameter (5.1.+-.0.2 mm) of an uninjured control
muscle. The inflammatory reaction in the epimysium is diminished in
the muscles treated with the RGTA in a very significant manner
since the thicknesses of the epimysia are 10.+-.5 micrometers with
treatment by the RGTA compared to 85.+-.10 micrometers without
treatment with the RGTA (p<0.01). The central ischemic zone of
the EDL muscles not treated with RGTA presents a mean diameter of
4.4000.+-.100 micrometers in which the muscle fibers have
completely disappeared. This zone is surrounded by a peripheral
zone of 270.+-.50 micrometers containing an average of 3.2.+-.0.5
layers of muscle fibers. The treatment with the RGTA is
characterized by a clear decrease in the size of the central
degenerated zone whose mean diameter is 300.+-.200 micrometers
(p<0.05) and an augmentation of the peripheral zone the
thickness of which is 700.+-.40 micrometers (p<0.05) and which
contains 8.3.+-.1.8 layers (p<0.01) of fiber.
EXAMPLE 16
Effects of the RGTA on the Regeneration of Long Bone
[0363] This example illustrates the reconstruction of an osseous
defect created in the diaphysial shaft of a rat femur, restored to
the original state after 8 weeks and better at 12 weeks, with
reconstitution of a medullary cavity identical to the original one
and mature cortices as in the original unfractured bone (FIG.
29).
[0364] Male Wistar rats (Ico: WI (IOPS AF/Han), Iffa Credo)
weighing from 275 to 325 grams were used. The study was performed
in accordance with the EEC recommendations on animal
experimentation (decree 87-848-Apr. 19, 1987). The animals were
anesthetized by injection of sodium pentobarbital. The femur was
approached laterally. The muscle and periosteal tissues were
separated from the diaphysial shaft. A high-density polyethylene
plate was fixed to the surface of the femur with Kirschner pins. A
segmentary defect of 5 millimeters was implemented in the middle of
the femoral diaphysis. An implant was inserted in the place of the
osseous defect prior to suturing the tissues. This implant
corresponds to a demineralized allogenic osseous matrix prepared
from femoral diaphyses from other rats according to the procedure
described by F. Blanquaert et al. (1995, Bone, 17: 499-506), which
were impregnated or not impregnated with RGTA 1012 by incubation in
a saline solution comprising 100 micrograms per milliliter of this
product. The animals were then maintained in cages without
ambulatory restraints. The femurs of the animals were radiographed
every two weeks for 12 weeks before being euthanized. The femurs
were then collected and subjected to the treatments required for
histological study. The radiographs were studied especially in
their densitometric aspects by image analysis.
[0365] FIG. 29 shows the effects of the RGTA on the regeneration of
the long bones and shows especially the histological and
radiographic studies of femurs from rats which were either treated
or not treated by RGTA 1015.
[0366] FIG. 29-A shows the model defect created in the femoral
diaphysis.
[0367] FIGS. 29-B, 29-D, 29-F and 29-H represent radiographs of
femurs from different experimental groups. FIGS. 29-C, 29-E and
29-G represent histological sections of operative pieces
corresponding to the specimens presented in 29-D, 29-F and 29-H,
respectively.
[0368] FIGS. 29-C and 29-D show the femoral defect which did not
receive any particular treatment. During the 12-week period there
was no cicatricial phenomenon and the osseous shaft was not
reconstituted.
[0369] FIGS. 29-E and 29-F show that the osseous defect was filled
by the demineralized osseous matrix. In this case, filling took
place but no reorganization can be seen. The structure of the
filling does not present the organization of a traditional long
bone.
[0370] In FIGS. 29-G and 29-H, it can be seen that the osseous
defect was filled by the demineralized osseous-matrix impregnated
in a solution of RGTA 1015. In this case, the structure of the
filling tissues corresponds to that of normal bone. The compact
cortical bone is comparable to the uninjured zone in which the mark
of the fixation screw can be seen. This part delimits a cavity
filled with bone marrow in continuity with the original medullary
cavity.
[0371] FIGS. 29-I1, 29-I2, 29-J1 and 29-J2 show the effect of RGTA
1015 on the reformation rate of the long bone. Radiographs I1 and
I2 were taken at 8 weeks; the radiographs J1 and J2 were taken at
12 weeks. I1 and J1 correspond to the treatment presented in 29-E
and 29-F in which the osseous defect was filled solely by the
demineralized osseous matrix without RGTA. I2 and J2 correspond to
the treatment presented in 29-G and 29-H in which the osseous
defect was filled by the demineralized osseous matrix impregnated
in a solution of RGTA 1015. These radiographs show an acceleration
of the cicatrization and especially of the maturation of the
reformed bones and in terms of a pronounced corticalization (I2 and
J2) which can not be detected in I1 and J1. RGTA 1015 acts as a
regeneration agent which enables accelerated reconstitution of the
osseous structure of long bone with a structure identical to that
of the original bone.
[0372] Thus, in a surprising manner, RGTA 1012 impregnated in the
demineralized osseous matrix, compared to a matrix impregnated in
physiological serum without the polymer, induces an extremely
significant acceleration in the restructuring and maturation
processes of the bone. This effect is manifested in the appearance
of new cortices after only 8 weeks whereas this phenomenon did not
take place under the control condition. After 12 weeks, six of the
seven animals treated by the association with, RGTA 1012 presented
in the radiological study the evidence of a complete union of the
defect with the reformation of thick and delimited cortices whereas
without CM.sub.1DS.sub.2 only the union is observable with
radiological images of immature bone without corticalization. A
quantitative image analysis study of the radiographs confirmed
these observations. This study also showed that the profile of the
bones treated by RGTA 1012 is comparable to that of normal bone,
with the only difference being that the osseous material has a
relatively low density at the experimental time point of 12 weeks.
Projection of the density of the bone that is newly formed in the
original defect shows that the treatment by the polymer RGTA 1012
induces a tissular restructuring via the cortical reformation and a
medullary cavity.
[0373] The histological studies correlate with and confirm the
results established on the basis of image analysis of the
radiological data. These histological studies demonstrate the
presence of new cortices constituted of compact bone with still
several regions of marrow and the presence of a medullary cavity in
continuity with the original diaphysial shaft, filled with new bone
marrow. The femurs treated without RGTA 1012 do not show any figure
of maturation. The reconstituted bone does not present any specific
organization. It is constituted by a heterogeneous mixture of
compact bone and medullary tissue without specific territorial
delimitation.
[0374] These results illustrate the osteoinductive effects of the
polymers of the invention on their capacity to induce not only the
repair of the long bones but also and especially their potential to
regulate the homeostasis of the regenerated tissues at the level of
their mass as well as their reorganization.
[0375] The properties of the RGTA as regulatory agent of the
homeostasis of the osseous tissues, i.e., of the tissular mass, its
functionality and its restructuring are confirmed by Example 17
below.
EXAMPLE 17
Effects of the RGTA on the Restructuring and the Protection of the
Osseous Mass in a Model of Acute Periodontal Disease in the
Hamster
[0376] The model used in this example pertains to the osseous
restructuring of the mandible of the hamster.
[0377] Periodontal disease is induced in golden Syrian hamsters
(Depre breeding center, references HSM 41/50) after two weeks of a
hyperglucidic diet. The feed administered was composed of sucrose
(56%), powdered skimmed milk (28%), whole wheat flour (6%),
brewer's yeast (4%), powdered alfalfa (3%), liver powder (1%) and
sodium chloride (2%).
[0378] The animals were distributed into experimental groups.
Fourteen animals constituted the control groups which received a
normal diet comprised of dry feed. Twenty-four animals constituted
the experimental groups subjected to the hyperglucidic diet. They
developed chronic periodontal disease which was established at the
end of two months. After this time point, the experimental groups
received each week for 3 weeks an intramuscular injection of RGTA
1005 at different doses comprised between 0.1 and 15 milligrams per
kilogram in a volume of 0.5 milliliters of buffered-physiological
serum. The control groups received an injection of physiological
serum without RGTA 1005 (SHAM). The animals' weights were measured
each week. One month after each type of treatment, the animals were
sacrificed, the mandibles were collected and prepared for
histological study. The inclusions of the operative pieces were
implemented in stabilized methyl methacrylate.
[0379] In this model of periodontal disease, only 200 micrometers
in height could be processed for each hemimandible. The system is
standardized to always study the same sequence of sections at a
depth that is defined and referenced by the osseous tissues between
the roots of the two first molars of the lingual side.
[0380] The periodontal disease at the level of these molars is
manifested by the appearance of a periodontal pouch which delimits
a volume filled with bacterial plaque. This disease leads to a
notable destruction of the periodontal bone which is characterized
by a notable osteoclastic resorption and a reduction in the osseous
surfaces in apposition, i.e., to an osteosynthesis phase.
[0381] The osseous resorption is quantified by measuring the zones
of contact between the bone and the osteoclasts, (Oc). These are
giant cells that are stained blue by toluidine blue. The apposition
in turn is characterized by a band of osteoid tissue, identified by
an attenuated blue coloration covered by osteoblasts. In contrast
to the osteoclasts, the osteoblasts are cells of small size, of
cubic form and mononuclear. The quantifications of these phenomena
are performed with an imaging system that uses an image-processing
program.
[0382] FIG. 30 presents the quantifications obtained under the
various experimental conditions. In the control animals who did not
develop the disease, the resorption activity is practically
nonexistent whereas the apposition activity is noteworthy. In the
untreated animals (SHAM), the disease was strongly developed and
characterized by an intense resorption and a low degree of
apposition. In the animals treated by the CM.sub.1DS.sub.2,
particularly at the dose of 1.5 milligrams per kilogram, the
resorption rate is greatly diminished and is associated with a very
large apposition rate which reaches close to 80% of the rate
observed in the control animals.
[0383] These effects were obtained by intramuscular injection. They
show that the RGTA regulate the osseous tissue mass by
re-equilibrating the balance of the restructuring between
resorption and apposition.
EXAMPLE 18
Pharmacokinetic Data Demonstrating the Properties of the RGTA for
Vectorizing Molecules Towards Injured Tissues
[0384] This example illustrates the capacity presented by the
polymers of the invention for fixing themselves in a specific
manner and concentrating at the level of the sites of tissular
injury.
[0385] In the model of the regeneration of crushed muscle described
in Example 7, a lesion is implemented on the EDL of the left paw of
the animals. Each animal receives an intravenous injection of
2-10.sup.6 cpm of RGTA 1012 radiolabeled with tritium by the
company SibTech, Inc. (NY, USA). The specific activity of this
tracer is 20 millicurie per milligram.
[0386] At different postoperative times, the injured EDL muscles of
the left paws and the uninjured muscles of the right paws were
collected and frozen in liquid nitrogen. The implementation of
frozen histological sections enabled, by means of a beta imager
(Societe Biospace), measurement, of the quantity of radiolabeled
product fixed at the level of the tissue sections studies. The
results presented in Table V below show that the injured muscles
concentrated after 24 hours a quantity of radioactive product
approximately 5 times greater than the uninjured muscles the
labeling level of which did not differ from the device's background
noise.
TABLE-US-00005 TABLE V cpm of RGTA 1012 fixed at the level of the
tissues Postoperative time 24 hours 48 hours 96 hours Injured
muscle tissue 11,800 .+-. 890 10,150 .+-. 10120 9000 .+-. 790
Contralateral control 2060 .+-. 530 1980 .+-. 390 1870 .+-. 640
muscle tissue Background noise of 1800 .+-. 160 1780 .+-. 210 1950
.+-. 180 registration
[0387] These results demonstrate the autotargeting capacities of
the polymers of the invention which concentrate themselves
specifically at the level of the tissues presenting a disorder or a
lesion. Thus, a particularly interesting property of these polymers
resides in their capacity to vectorize a medical or diagnostic
principle.
EXAMPLE 19
Effects of RGTA in Periodontics and on the Osseous Mass
[0388] In the field of periodontics, a macroscopic study of the
loss of alveolar bone was performed on the periodontitis of the
hamster.
[0389] After a 2-month period for induction of the disease, the
animals (n=20) were treated via the IM route for an additional 2
months without acting on the initial cause of the disease, i.e.,
the bacterial component. Other animals were left untreated (n=20).
These two groups were compared with healthy hamsters (no
periodontitis) (n=12).
[0390] At the end of the experimental period, all fleshy tissues
were removed from the superior maxillaries so as to enable
determination of the bone loss. The zone of the first molar was
photographed in a standardized manner. A reference line was traced
on each photograph which corresponded to the enamel-cement
junction. Then a second line was traced which ran along the
contours of the osseous ridge. These two lines were reunited in
front of and behind the first molar. This surface was measured.
[0391] It should be noted that in the controls, there is a zone of
denudation of the root which corresponds to:
[0392] a zone of physiological fibrous insertion which anchors the
gum on the root,
[0393] a loss of bone height which is produced in relation to the
aging of the animals.
[0394] In our ails this denudation represented 0.96 mm.sup.2.
[0395] In the diseased animals, the bone loss was 1.34 mm.sup.2,
which includes the initial physiological zone (which was destroyed
over the course of the periodontitis) and a part of the loss due to
aging. Nevertheless, we could conclude that the disease induced a
bone loss on the order of 0.38 mm.sup.2 (difference:
p<0.0001).
[0396] In the treated animals, there is always an incompressible
zone (the same is true of the controls). The denudation of the root
represents 1.02 mm.sup.2 in these animals. Thus, there is a net
deficit in relation to the controls of 0.06 mm.sup.2 (difference
not significant) and an improvement by 0.32 mm.sup.2 in relation to
the untreated animals (p=0.0005).
* * * * *