U.S. patent application number 11/629579 was filed with the patent office on 2008-06-05 for sulfated depolymerized derivatives of exopolysaccharides (eps) from mesophilic marine bacteria, method for preparing same, and uses thereof in tissue regeneration.
This patent application is currently assigned to UNIVERSITE RENE DESCARTIES PARIS 5. Invention is credited to Sylvia Colliec-Jouault, Anthony Courtois, Florence Fioretti, Gaston-Jacques Godeau, Farida Gueniche, Jean Guezennec, Gerard Raguenes, Jacqueline Ratiskol, Karim Senni, Corinne Sinquin, Myriam Yousfi.
Application Number | 20080131472 11/629579 |
Document ID | / |
Family ID | 34946349 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131472 |
Kind Code |
A1 |
Senni; Karim ; et
al. |
June 5, 2008 |
Sulfated Depolymerized Derivatives of Exopolysaccharides (Eps) from
Mesophilic Marine Bacteria, Method for Preparing Same, and Uses
Thereof in Tissue Regeneration
Abstract
The invention relates to certain low-molecular weight sulphated
polysaccharide derivatives of marine native exopolysaccharides
(EPSs) excreted by mesophilic marine bacteria from a deep
hydrothermal environment, wherein said derivatives can be obtained
by means of a method which comprises a step of free radical
depolymerisation of said native EPSs followed by a step of
sulphating the resulting depolymerised derivatives. The present
invention further relates to the use of said low-molecular weight
sulphated polysaccharide derivatives as a wound-healing agent,
particularly for preparing pharmaceutical compositions suitable for
treating or preventing diseases of the connective tissues and
particularly skin and gum tissues. The figure demonstrates how
polysaccharide derivative GY 785 DRS according to the invention can
stimulate fibroblast proliferation in latticed or reconstructed
connective tissues at a concentration of 10 .mu.g(m)g/ml.
Inventors: |
Senni; Karim; (Aulnay Sous
Bois, FR) ; Gueniche; Farida; (Rueil Malmaison,
FR) ; Yousfi; Myriam; (Paris, FR) ; Fioretti;
Florence; (Paris, FR) ; Godeau; Gaston-Jacques;
(Antony, FR) ; Colliec-Jouault; Sylvia; (Nantes,
FR) ; Ratiskol; Jacqueline; (Sainte Luce Sur Loire,
FR) ; Sinquin; Corinne; (Nantes, FR) ;
Raguenes; Gerard; (Locmaria Plouzane, FR) ; Courtois;
Anthony; (Saint Renan, FR) ; Guezennec; Jean;
(Plouzane, FR) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
UNIVERSITE RENE DESCARTIES PARIS
5
12, rue de l'Ecole de Medicine Cedex 06
Paris
FR
75270
|
Family ID: |
34946349 |
Appl. No.: |
11/629579 |
Filed: |
June 6, 2005 |
PCT Filed: |
June 6, 2005 |
PCT NO: |
PCT/FR05/01379 |
371 Date: |
February 28, 2007 |
Current U.S.
Class: |
424/423 ;
424/400; 435/1.1; 435/72; 514/54 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 29/00 20180101; C12N 5/0662 20130101; A61P 9/10 20180101; C12N
5/0665 20130101; C12N 5/0666 20130101; A61P 43/00 20180101; A61P
17/00 20180101; C12N 5/0668 20130101; A61K 31/737 20130101; A61P
19/02 20180101; C12N 2501/10 20130101; A61P 35/00 20180101; A61P
1/04 20180101; C12N 5/0664 20130101; A61K 35/28 20130101; A61P
37/06 20180101; C08B 37/006 20130101; A61P 1/02 20180101; C12N
5/0663 20130101; C12N 2500/72 20130101; A61P 17/02 20180101; C12N
5/0667 20130101; A61P 19/04 20180101; A61P 31/00 20180101; C12N
5/0676 20130101 |
Class at
Publication: |
424/423 ;
424/400; 435/001.1; 435/072; 514/054 |
International
Class: |
A61K 31/737 20060101
A61K031/737; A01N 1/00 20060101 A01N001/00; A61K 9/00 20060101
A61K009/00; A61Q 19/00 20060101 A61Q019/00; C12P 19/04 20060101
C12P019/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 2004 |
FR |
0406405 |
Claims
1. A low-molecular-weight sulfated polysaccharide derivative of a
marine native exopolysaccharide (EPS) excreted by mesophilic marine
bacteria from a deep hydrothermal environment, it being possible
for said derivative to be obtained by means of the process
comprising the following steps: a step consisting of free-radical
depolymerization of said native EPS so as to obtain a depolymerized
derivative of low molecular weight, less than or equal to 100 000
g/mol, a subsequent step consisting of sulfation of the optionally
lyophilized, depolymerized derivative, comprising the addition of
at least one sulfation agent in an amount sufficient to obtain a
sulfated polysaccharide derivative having a degree of sulfate-group
substitution of between 10% and 45% by weight relative to the total
weight of the sulfated polysaccharide derivative, said sulfation
step being optionally followed by a dialysis step.
2. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 1, characterized in that the process also
comprises a reduction step that follows on from the free-radical
depolymerization step.
3. The low-molecular-weight sulfated polysaccharide derivative as
claimed in either one of the preceding claims, characterized in
that the process also comprises a step consisting of
N-deacetylation of the polysaccharide derivative obtained at the
end of the depolymerization step and/or at the end of the reduction
step.
4. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that
the process also comprises at least one step consisting of
lyophilization of the polysaccharide derivative, after the
depolymerization and/or after the reduction and/or after the
N-acetylation and/or before or after the sulfation.
5. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 1, characterized in that the first free-radical
depolymerization step is carried out by addition of a solution of
an oxidizing agent, preferably chosen from peroxides and peracids,
in the presence of a metal catalyst, preferably chosen from
Cu.sup.++, Fe.sup.++ and Cr.sup.+++ ions and the
Cr.sub.2O.sub.7.sup.2- anion.
6. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 5, characterized in that said oxidizing agent is a
solution of hydrogen peroxide, preferably having a concentration of
between 0.1% and 0.5% by weight, which is added at a flow rate of
between V1/1000 and V1/10 ml/minute, preferably from V1/50 to
V1/500 ml/minute, V1 being the volume of the reaction medium
containing a marine exopolysaccharide (EPS) excreted by mesophilic
marine bacteria from a deep hydrothermal environment, to which the
solution of hydrogen peroxide is added.
7. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that
the sulfation agent(s) used for the sulfation step is chosen from
complexes of pyridine sulfate, of triethylamine sulfate, of
dimethylformamide sulfate or of trimethylamine sulfate.
8. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that,
for carrying out the sulfation step, the chemical sulfation
agent(s) is (are) added in a weight amount representing from 4 to 6
times the mass of the polysaccharide derivative in solution and is
(are) added to the depolymerized EPS which is in dry form or in the
form of a solution in a solvent, preferably an anhydrous
solvent.
9. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that
it has a molecular weight of less than or equal to 100 000 g/mol,
preferably of between 5000 and 50 000 g/mol, a polydispersity index
of less than 5, preferably of between 1.5 and 4, and a degree of
sulfate-group substitution of between 10% and 45% by weight, and
preferably between 20% and 40% by weight, inclusive.
10. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 9, characterized in that it has a molecular weight
of less than or equal to 25 000 g/mol, a polydispersity index of
less than 2, and a degree of sulfate-group substitution of between
10% and 45% by weight, and preferably between 20% and 40% by
weight, inclusive.
11. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that
said mesophilic marine bacteria from a deep hydrothermal
environment belong to the Alteromonas or Vibrio genus.
12. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 11, characterized in that the bacteria of the
Alteromonas genus are of the GY785 strain.
13. The low-molecular-weight sulfated polysaccharide derivative as
claimed in claim 11, characterized in that the bacteria of the
Alteromonas genus are selected from the strains HYD 657, HYD 708,
HYD 721, HYD 1545, HYD 1644, ST 716 and MS 907.
14. The sulfated polysaccharide derivative as claimed in claim 11,
characterized in that the bacteria of the Vibrio genus are bacteria
of the HE 800 strain.
15. The low-molecular-weight sulfated polysaccharide derivative as
claimed in one of claims 11 to 13, characterized in that said
native exopolysaccharide (EPS) excreted by bacteria of the
Alteromonas genus has an oside composition comprising: from 20% to
70%, preferably from 30% to 60%, and more preferably from 38% to
57% by weight of neutral monosaccharides, from 5% to 60%,
preferably from 6% to 50%, and more preferably from 8% to 42% by
weight of acidic monosaccharides, and from 0% to 1% by weight of
amino sugars.
16. The low-molecular-weight sulfated polysaccharide derivative as
claimed in either one of claims 11 and 14, characterized in that
said marine exopolysaccharide (EPS) excreted by bacteria of the
Vibrio genus has an oside composition comprising: from 0% to 5%,
preferably from 0% to 1% by weight of neutral monosaccharides, from
20% to 50%, preferably from 25% to 40%, and more preferably from
30% to 32% by weight of acidic monosaccharides, from 20% to 50%,
preferably from 25% to 40%, and more preferably from 30% to 35% by
weight of amino sugars, and from 0% to 15%, preferably from 4% to
8%, and more preferably from 5% to 6% by weight of N-acetylated
groups.
17. The low-molecular-weight sulfated polysaccharide derivative as
claimed in any one of the preceding claims, characterized in that
said native EPS has a protein content of from 0% to 15%, preferably
from 0% to 5%, and more preferably from 0% to 1% by weight.
18. The use of low-molecular-weight sulfated polysaccharide
derivatives as claimed in any one of claims 1 to 17, as a
wound-healing agent for connective tissues, in particular for
dermal and gingival connective tissues.
19. The use as claimed in claim 18, in which said wound-healing
agent is an agent capable of stimulating the proliferation of
fibroblasts in two-dimensional cultures or in reconstructed
connective tissues, in particular dermal and gingival connective
tissues.
20. The use of low-molecular-weight sulfated polysaccharide
derivatives as claimed in any one of claims 1 to 17, for the
preparation of a pharmaceutical composition with wound-healing
activity, said composition making it possible in particular to
promote the reconstruction and the remodeling of connective
tissues, in particular of dermal and gingival connective
tissues.
21. The use as claimed in claim 18, in which said wound-healing
agent is an agent capable of inhibiting the secretion of
pro-inflammatory cytokines by the fibroblasts of connective
tissues, in particular of dermal and gingival connective tissues,
and in particular of inhibiting the secretion of interleukin
1.beta. and/or of TNF-.alpha..
22. The use as claimed in claim 18, in which said wound-healing
agent is an agent capable of inhibiting the secretion of matrix
metalloproteases by the fibro-blasts of connective tissues, in
particular of dermal and gingival connective tissues, and in
particular of inhibiting the secretion of gelatinase A (MMP-2) and
of stromelysin 1 (MMP-3).
23. The use as claimed in claim 18, in which said wound-healing
agent is an agent capable of inhibiting the conventional complement
pathway.
24. The use as claimed in any one of claims 18 to 23, in which said
agent is used for the preparation of a pharmaceutical composition
for treating inflammatory pathologies, such as infectious,
neoplastic or autoimmune pathologies, affecting connective tissues,
in particular dermal and gingival connective tissues.
25. The use as claimed in claim 18, in which said wound-healing
agent is an agent capable of stimulating the proliferation of
fibroblasts to the detriment of myofibroblasts.
26. The use as claimed in claim 25, in which said agent is used for
the preparation of a pharmaceutical composition, said composition
making it possible in particular to prevent or treat hypertrophic
wound-healing processes or fibrotic pathologies of connective
tissues, in particular of dermal and gingival connective
tissues.
27. The use of the derivatives as claimed in any one of claims 1 to
17, as an agent capable of selectively stimulating the
proliferation of medullary cells intended to become mesenchymal
cells, to the detriment of other subpopulations in a heterogeneous
population of cells, in particular in tissue or cell therapy.
28. A pharmaceutical composition or medicament comprising
low-molecular-weight sulfated polysaccharide derivatives as claimed
in any one of claims 1 to 17.
29. The pharmaceutical composition or medicament as claimed in
claim 28, characterized in that it is used in combination with one
or more growth factors present in the pharmaceutical composition or
present in a different pharmaceutical composition, said growth
factors being in particular FGFs (fibroblast growth factors),
TGF.beta.s, BMPS (bone morphogenic proteins) or CTGF (connective
tissue growth factor).
30. The pharmaceutical composition or medicament as claimed in
either one of claims 28 and 29, characterized in that it is in an
injectable form, in which said low-molecular-weight sulfated
polysaccharide derivatives have a molecular weight of between 5000
and 50 000 g/mol, preferably less than or equal to 25 000 g/mol, a
polydispersity index of between 1.5 and 5, preferably less than or
equal to 2, and a degree of sulfate-group substitution of between
10% and 45% by weight, and preferably between 20% and 40% by
weight, inclusive.
31. A cosmetic or dermatological composition, characterized in that
it comprises low-molecular-weight sulfated polysaccharide
derivatives as claimed in any one of claims 1 to 17, in combination
with any suitable excipient.
32. The composition as claimed in any one of claims 28, 29 and 31,
characterized in that it is in the form of a gel, a cream, an
ointment, an emulsion or a solution.
33. The use of the composition as claimed in any one of claims 28,
29, 31 and 32 in substrates in situ, resorbable or nonresorbable
medical devices, including in particular delayed-release supports,
slowly disintegrating sponges, or surgical implants.
Description
[0001] The present invention relates to polysaccharide derivatives
obtained from native polysaccharides of a specific type, called
exopolysaccharides (EPSs) that are excreted by various strains of
mesophilic marine bacteria from a deep hydrothermal
environment.
[0002] More particularly, the present invention relates to
low-molecular-weight highly sulfated polysaccharide derivatives
that can be obtained by free-radical depolymerization and sulfation
of exopolysaccharides.
[0003] Highly sulfated low-molecular-weight polysaccharides that
are not bacterial exopolysaccharide derivatives are already known.
They have advantageous properties, in particular as therapeutic
substances. For example, heparin, a sulfated polysaccharide, is the
anticoagulant and antithrombotic agent most commonly used in the
prevention and treatment of venous thrombosis. The commercially
available heparins are currently extracted from porcine intestinal
mucosa. However, the use of these heparins of animal origin
presents a risk of contamination with pathogenic agents (for
example, prion). In order to reduce or avoid the risk of
contamination, the identification of novel polysaccharides from
origins other than animal origins appears to be a particularly
promising line of research.
[0004] Polysaccharides from bacteria, and more particularly from
marine bacteria, make it possible to satisfy this need. The study
and exploitation of polysaccharides excreted by marine bacteria, or
exopolysaccharides, therefore falls within this line of research,
in particular for the design of novel active ingredients or of
analogs of already existing molecules.
[0005] A screening of samples derived from the hydrothermal
environment of the ocean depths has made it possible to isolate a
large variety of mesophilic bacterial strains capable of producing
native EPSs at atmospheric pressure and at ambient temperature.
[0006] A small number of native EPSs has already been the subject
of patent filings and of publications: for example, European patent
EP 975 791 describes the strain Vibrio diabolicus, the native EPS
HE 800 and the use thereof as a medicament, including, in
particular, as a retroviral, antitumor and antithrombotic agent. In
the native state, the EPS HE 800, of 800 000 g/mol, is not
sulfated; it consists of approximately 30% by weight of amino
sugar, 32% by weight of acid monosaccharides and 1% by weight of
neutral monosaccharides; its protein content is close to 1% by
weight. European patent application No. 1 296 695 describes the use
of the EPS HE 800 in its native form as a material for facilitating
bone healing (Zanchetta et al., Calcif Tissue Int., 2003, 72:
74-79).
[0007] A second native EPS, called GY 785 and produced by the
bacterium Alteromonas infernos, has also been identified and
described in French patent No. 2 755 142. The native EPS GY 785
consists of a heterogeneous population of polysaccharide chains
having an average molar mass of greater than 10.sup.6 g/mol. The
native EPS GY 785 is slightly sulfated (amount of sulfate less than
10% by weight); it consists of 57% by weight of neutral
monosaccharides (predominantly glucose and galactose) and 42% by
weight of acidic monosaccharides (glucuronic acid and galacturonic
acid); it does not comprise any amino sugars or acetate, lactate,
pyruvate and succinate substituents; its protein content is
approximately 4% by weight (Guezennec J., Ind. Microb. Biotech.,
2002, 29: 204-208).
[0008] A third native EPS, called ST 716 and produced by the
bacterium Alteromonas macleodii subsp. fijiensis has also been
identified and described in European patent EP 1171625 (Rougeaux H.
et al., Carbohydr. Res., 1998, 312: 53-59). The native EPS ST 716
is slightly sulfated (its amount of sulfate is approximately 5% by
weight); it consists of 40% by weight of neutral monosaccharides
and 40% of acidic monosaccharides; its protein content is 2 to 4%
by weight.
[0009] Other novel native EPSs have also been identified and
described in the literature; in particular, the EPS HYD 721
produced by a Pseudoalteromonas (Rougeaux H. et al., Carbohydr.
Res., 1999, 315: 273-285 and Raguenes G. et al., 1997), the EPS HYD
657 (Cambon-Bonavita M. et al., J. Applied Microbiol, 2002, 93:
310-315) and the EPS MS 907 (Raguenes G. et al., Curr. Microbiol,
2003, 46: 448-52).
[0010] A subject of the present invention is sulfated
polysaccharide derivatives that come from the treatment of native
EPSs excreted by mesophilic bacterial strains from a deep
hydrothermal environment and that are of pharmaceutical or cosmetic
value or of value in tissue engineering. The sulfated
polysaccharide derivatives of native exopolysaccharides (EPSs)
excreted by mesophilic marine bacteria from a deep hydrothermal
environment according to the invention can be obtained by means of
the method comprising the following steps: [0011] a step consisting
of free-radical depolymerization of said native EPSs, so as to
obtain depolymerized derivatives of low molecular weight, less than
or equal to 100 g/mol, [0012] a subsequent step consisting of
sulfation of the optionally lyophilized, depolymerized derivatives,
comprising the addition of at least one sulfation agent in a
sufficient amount to obtain sulfated polysaccharide derivatives
having a degree of sulfate-group substitution of between 10% and
45% by weight relative to the total weight of the sulfated
polysaccharide derivative, said sulfation step being optionally
followed by a dialysis step.
[0013] During the first depolymerization step, the native EPS can
be used in a liquid form, i.e. as it is excreted by the bacteria
into the culture medium. Preferably, the culture medium is
centrifuged and only the supernatant containing the native EPS and
free of bacterial debris is conserved. The native EPS can be
collected by any suitable technique known to those skilled in the
art, in particular by membrane ultrafiltration, and can then
optionally be lyophilized as it is or in the form of an addition
salt.
[0014] The step consisting of free-radical depolymerization of the
native EPS is preferably carried out by addition of a solution of
an oxidizing agent to a reaction mixture comprising the native EPS,
preferably in the presence of a metal catalyst. The oxidizing agent
is preferably chosen from peroxides, in particular hydrogen
peroxide, and peracids, in particular peracetic acid and
3-chloroperbenzoic acid. The addition is preferably carried out
continuously and with stirring for a period of between 30 minutes
and 10 hours. Reaction mixture is preferably maintained at a pH of
between 6 and 8, for example by continuous addition of a basifying
agent such as sodium hydroxide, and at a temperature of between
approximately 30 and 70.degree. C. throughout the duration of the
free-radical depolymerization reaction.
[0015] According to a specific embodiment of the present invention,
in this step, the native EPS is present in the reaction mixture at
a concentration of between approximately 2 and 10 mg/ml of reaction
mixture.
[0016] According to a preferred embodiment of the invention, the
oxidizing agent is a solution of hydrogen peroxide.
(H.sub.2O.sub.2) preferably having a concentration of between
approximately 0.1% and 0.5% by weight, preferably of the order of
0.1% to 0.2% by weight, which is added at a flow rate of V1/1000 to
V1/10 ml/minute, preferably V1/50 and V1/500 ml/minute, very
preferably of the order of V1/100 ml/minute, V1 being the volume of
the reaction medium containing a marine exopolysaccharide (EPS) to
which a solution of hydrogen peroxide is added.
[0017] The metal catalysts that can be used during the
depolymerization step are preferably chosen from Cu.sup.++,
Fe.sup.++ and Cr.sup.+++ ions and the Cr.sub.2O.sub.7.sup.2- anion,
as described in particular in patent application EP 0 221 977.
According to a specific embodiment, the metal catalyst is present
in the reaction mixture at a concentration of between approximately
10.sup.-3 M and 10.sup.-1 M, and preferably at a concentration of
between approximately 0.001 and 0.05 M.
[0018] The free-radical depolymerization process in accordance with
the invention and as described above makes it possible to obtain,
in a single step, without fractionation by stearic exclusion
chromatography, and with a good yield, homogeneous,
low-molecular-weight polysaccharide derivatives. The term
"low-molecular-weight polysaccharide derivatives" is intended to
mean derivatives with a molecular weight of less than or equal to
100 000 g/mol, preferably between 5000 and 50 000 g/mol, and more
preferably less than or equal to 25 000 g/mol. In the context of
the disclosure of the present invention, the term "homogeneous
derivatives" is intended to mean derivatives which, by high
performance stearic exclusion chromatography, exhibit a single main
peak representing a predominant population of polysaccharide chains
that are homogeneous with respect to size, characterized by a
polydispersity index I (Mw/Mn)<5, preferably of between 1.5 and
4, more preferably less than or equal to 2, with Mw=weight-average
molecular weight and Mn=number-average molecular weight.
[0019] When the depolymerization reaction has finished, according
to a specific embodiment of the invention, the process comprises a
step consisting of reduction of the polysaccharide derivatives
obtained, using a reducing agent, so as to stabilize the chains,
the reducing ends of which are very reactive, and in particular so
as to avoid chain hydrolysis by the "peeling" reaction. The nature
of the reducing agents that can be used to this effect is not
essential. It may in particular be sodium borohydride.
[0020] The metal catalyst used for the depolymerization can be
eliminated at the end of the depolymerization reaction, and in the
embodiment in which a reduction step is carried out, at the end of
the reduction, by ion exchange chromatography, preferably a weak
cation exchange resin passivated beforehand, or by treatment with
EDTA (ethylenediaminetetraacetic acid).
[0021] According to a specific embodiment of the process of the
invention, prior to the sulfation step, a step consisting of
N-deacetylation of the polysaccharide derivatives comprising
N-acetylated hexosamines and obtained at the end of the
free-radical depolymerization step and/or at the end of the
reduction step is carried out. This N-deacetylation step is carried
out according to a protocol adapted from Zou et al. (Carbohyd.
Res., 1998, 309: 297-301). Advantageously, the N-deacetylation step
is carried out by addition, to the reaction mixture comprising the
polysaccharide derivatives, of a solution of sodium borohydride,
with stirring. When the temperature of the reaction mixture reaches
approximately 80.degree. C., a basifying agent, preferably sodium
hydroxide, is added to the reaction medium. The mechanism of basic
hydrolysis of an amide in a basic medium, and preferably in the
presence of sodium hydroxide, is shown schematically below:
##STR1##
[0022] After reaction for one hour, the reaction medium is
neutralized by continuous addition of acetic acid until a pH of 5
is obtained. The polysaccharide derivatives obtained can be
recovered by membrane ultrafiltration and then can optionally be
lyophilized.
[0023] According to a preferred embodiment, the N-deacetylation
step is carried out on the polysaccharide derivatives originating
from the depolymerization of native EPSs excreted by hydrothermal
mesophilic marine bacteria of the Vibrio genus, preferably HE 800.
The native EPSs excreted by said bacteria of the Vibrio genus are
characterized in that they contain N-acetylated hexosamines.
[0024] The polysaccharide derivatives resulting from the
depolymerization and/or from the reduction and/or from the
N-deacetylation can, if necessary, be recovered by any suitable
technique well known to those skilled in the art, such as, for
example, by membrane ultrafiltration, and then can optionally be
lyophilized as they are or in the form of an addition salt with a
weak or strong base, that may, for example, be chosen from
pyridine, triethylamine, tributylamine, tetrabutylammonium
hydroxide and sodium hydroxide. This lyophilized salt may, for
example, be prepared by elution of an aqueous solution of the
polysaccharide derivatives at a concentration of between 1 and 8
mg/ml on an ion exchange resin column such as, for example, those
sold under the name Dowex.RTM. by the company Dow Chemical. The
eluate is collected as long as the pH remains acid, for example
less than 5, then the pH is subsequently adjusted to approximately
6.5 with the desired base as defined above. The polysaccharide
derivatives in the form of a salt are then ultrafiltered and
lyophilized.
[0025] The lyophilized polysaccharide derivatives, possibly in the
form of an addition salt, are preferably dissolved in an anhydrous
solvent at the beginning of the sulfation step; this solvent is
preferably chosen from dimethylformamide (DMF), dimethyl sulfoxide
(DMSO) and/or formamide. The amount of polysaccharide derivatives
present in the anhydrous solvent may be between approximately 1 and
10 mg/ml, preferably between approximately 1 and 5 mg/ml, and even
more preferentially this amount is approximately 2.5 mg/ml. The
dissolution of the EPS in the anhydrous solvent is preferably
carried out, with stirring, at ambient temperature for
approximately 1 to 2 hours and then at a temperature of between 40
and 50.degree. C., preferably at a temperature of approximately
45.degree. C. for approximately 2 hours under argon with molecular
sieve.
[0026] The chemical sulfation agent(s) used during the sulfation
step can be added to the depolymerized and/or reduced and/or
N-deacetylated EPSs that are in lyophilized form or in the form of
a solution.
[0027] The sulfation agents are preferably chosen from complexes of
pyridine sulfate (free or coupled to a polymer), of
dimethylformamide sulfate, triethylamine sulfate and of
trimethylamine sulfate. The chemical sulfation agent(s) is (are)
added to the solution of polysaccharide derivatives in a weight
amount preferably representing from approximately 4 to 6 times, and
even more preferably approximately 5 times, the mass of
polysaccharide derivatives in solution. The chemical sulfation
reaction is then preferably carried out with stirring for a period
of between approximately 2 and 24 hours depending on the desired
degree of sulfation. When the desired degree of sulfation is
reached, the sulfation reaction is stopped after cooling of the
reaction medium: [0028] either by addition of water in a proportion
preferably equal to 1/10 of the reaction volume and adjustment of
the pH of the reaction medium to 9 with a basifying agent such as,
for example, sodium hydroxide (3 M); [0029] or, and preferably, by
precipitation in the presence of sodium-chloride-saturated acetone
or of methanol, and then dissolution of the precipitate in
water.
[0030] According to a specific embodiment, the solution of sulfated
polysaccharide derivatives is preferably dialyzed in order to
remove the various salts, and then lyophilized.
[0031] In the context of the invention, the term "sulfated
polysaccharide derivatives" is intended to mean polysaccharide
derivatives that have been subjected to a chemical sulfation
treatment and comprise sulfate groups, irrespective of whether or
not they have sulfate groups before this sulfation treatment.
[0032] Preferably, the low-molecular-weight sulfated polysaccharide
derivatives in accordance with the invention have a molecular
weight of less than or equal to 100 000 g/mol, preferably of
between 5000 and 50 000 g/mol, a polydispersity index of less than
5, preferably of between 1.5 and 4, and a degree of sulfate group
substitution of between 10% and 45% by weight, and preferably of
between 20% and 40% by weight, inclusive.
[0033] More preferably, the low-molecular-weight sulfated
polysaccharide derivatives in accordance with the invention have a
molecular weight of less than or equal to 25 000 g/mol, a
polydispersity index of less than 2, and a degree of sulfate-group
substitution of between 10% and 45% by weight, and preferably of
between 20% and 40% by weight, inclusive.
[0034] According to a preferred embodiment of the invention, the
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention are obtained by treatment of native
EPSs excreted by mesophilic marine bacteria of hydrothermal origin
preferably belonging to the Alteromonas or Vibrio genus.
[0035] According to a variant of the invention, the bacteria of the
Alteromonas genus are selected from the strains GY 785, HYD 657,
HYD 721, HYD 1545, HYD 1644, ST 716 and MS 907.
[0036] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Alteromonas genus, said native EPSs having a
neutral monosaccharide content of from 20% to 70%, preferably from
30% to 60%, and more preferably from 38% to 57% by weight.
[0037] The invention also relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Alteromonas genus, said native EPSs having an
acidic monosaccharide content of from 5% to 60%, preferably of
between 6% and 50%, and more preferably of between 8% and 42% by
weight.
[0038] The invention also relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Alteromonas genus, said native EPSs having an amino
sugar content of from 0% to 1% by weight in their side
composition.
[0039] According to a specific embodiment, the low-molecular-weight
sulfated polysaccharide derivatives of the invention are obtained
from native EPSs excreted by bacteria of the Alteromonas genus,
said native EPSs having an side composition comprising: [0040] from
20% to 70%, preferably from 30% to 60%, and more preferably from
38% to 57% by weight of neutral monosaccharides, [0041] from 5% to
60%, preferably from 6% to 50%, and more preferably from 8% to 42%
by weight of acidic monosaccharides, [0042] from 0% to 1% by weight
of amino sugars.
[0043] According to another specific embodiment, the
low-molecular-weight sulfated polysaccharide derivatives of the
invention are obtained from native EPSs excreted by bacteria of the
Vibrio genus, preferably by the bacterial strain HE 800. The native
EPSs excreted by bacteria of the Vibrio genus are not sulfated.
[0044] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Vibrio genus, said native EPSs having a neutral
monosaccharide content of from 0% to 5%, preferably from 0% to 1%
by weight.
[0045] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Vibrio genus, said native EPSs having an acidic
monosaccharide content of from 20% to 50%, preferably from 25% to
40%, and more preferably from 30% to 32% by weight.
[0046] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Vibrio genus, said native EPSs having an amino
sugar content of from 20% to 50%, preferably from 25% to 40%, and
more preferably from 30% to 35% by weight.
[0047] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs excreted by
bacteria of the Vibrio genus, said native EPSs having an
N-acetylated group content of from 0% to 15%, preferably from 4% to
8%, and more preferably from 5% to 6% by weight.
[0048] According to a specific embodiment of the invention, the
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention are characterized in that they are
obtained from native EPSs excreted by bacteria of the Vibrio genus,
said native EPSs having an side composition comprising: [0049] from
0% to 5%, preferably from 0% to 1% by weight of neutral
monosaccharides, [0050] from 20% to 50%, preferably from 25% to
40%, and more preferably from 30% to 32% by weight of acidic
monosaccharides, [0051] from 20% to 50%, preferably from 25% to
40%, and more preferably from 30% to 35% by weight of amino sugars,
[0052] from 0% to 15%, preferably from 4% to 8%, and more
preferably from 5% to 6% by weight of N-acetylated groups.
[0053] The invention relates to low-molecular-weight sulfated
polysaccharide derivatives obtained from native EPSs that have a
protein content of from 0% to 15%, preferably from 0% to 5%, and
more preferably from 0% to 1% by weight.
[0054] Surprisingly and unexpectedly, the inventors have
demonstrated that the low-molecular-weight sulfated polysaccharide
derivatives in accordance with the invention can be used as
wound-healing agents for connective tissues, and are in particular
capable of stimulating fibroblast proliferation, of inhibiting the
secretion of pro-inflammatory cytokines or soluble mediators by
fibroblasts, of inhibiting the secretion of matrix metalloproteases
by fibroblasts, of inhibiting the conventional complement pathway,
of stimulating the proliferation of fibroblasts to the detriment of
myofibroblasts, and of selectively stimulating the proliferation of
medullary cells intended to become mesenchymal cells to the
detriment of other subpopulations in a heterogeneous population of
cells.
[0055] Fibroblasts have a central role in the process of wound
healing of a damaged tissue with a view to the formation of a
replacement tissue so as to restore the functionality thereof.
Typically, the wound-healing process takes place in 3 phases during
which the fibroblasts cause the regeneration processes to progress
according to chronological sequences that are precise but
interlinked with one another:
[0056] (1) The first inflammatory and vascular wound-healing phase
is characterized by the release of a large number of growth
factors, of cytokines and of proteases and the migration of
inflammatory cells, fibroblasts and vascular cells at the level of
lesion. The influx of inflammatory cells and the production of
cytokines induce the production of hydrolases such as serine
proteases or matrix metalloproteases by the fibroblasts. When the
inflammatory phase, which is normally transient, is uncontrollably
prolonged, a chronic inflammatory pathology takes hold.
[0057] (2) The tissue reconstruction phase (proliferative phase or
granulation tissue) is reflected by the loss of tissue substances
that has occurred when there is a lesion being made up with an
extracellular matrix that is relatively unorganized and richly
vascularized. The fibroblasts proliferate rapidly under the effect
of growth factors. These fibroblasts perform a remarkable job of
reconst ruction by secreting extracellular matrix components such
as glycosaminoglycans (GAGs), fibronectin and collagen. Some of
these fibroblasts acquires a myofibroblastic phenotype, expressing
in particular smooth muscle .alpha.-actin. The cicatricial tissue
retracts by virtue of the contractile capacities of the
myofibroblasts.
[0058] (3) In the maturation phase, a large part of the
myofibroblasts disappears due to apoptosis and is replaced with
fibroblasts no longer expressing smooth muscle .alpha.-actin. At
this stage, persistence of the myofibroblasts, by virtue of their
activity, may lead to pathologies of fibrotic type. At the end of
this process, a dense fibrous cicatricial tissue has been formed
and can then remodel itself. The maturation of the cicatricial
tissue is characterized in particular by a modification of the
orientation of the matrix fibers, which tend to place themselves
along lines of greater tension as in a normal connective
tissue.
[0059] Tissue remodeling is a dynamic balance between the synthesis
of the extracellular matrix and the degradation thereof. When this
balance is in equilibrium, wound healing is normal. However, when
the balance leans for a lengthy period toward extracellular matrix
synthesis, the development of a fibrosis is witnessed. When the
balance leans toward excessive degradation of the extracellular
matrix, an inflammatory pathology takes hold.
[0060] The present invention relates to the use of the
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention, as wound-healing agents for
connective tissues, in particular dermal and gingival connective
tissues.
[0061] Given the fact that the low-molecular-weight sulfated
polysaccharide derivatives of the invention have fibroblast
proliferation-activating properties, it is therefore particularly
advantageous to use them as an agent capable of stimulating
fibroblast proliferation, or as a regenerating agent for the
preparation of a pharmaceutical composition with wound-healing
activity, said composition making it possible in particular to
promote the reconstruction and the remodeling of connective
tissues, in particular of dermal and gingival connective
tissues.
[0062] The inventors have also demonstrated that
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention can be used as agents capable of
inhibiting the secretion of pro-inflammatory cytokines or soluble
mediators by connective tissue fibroblasts, including in particular
interleukin 1.beta. (IL-1.beta.) and/or TNF-.alpha. (tumor necrosis
factor).
[0063] Certain chronic inflammatory pathologies, such as
periodontitis, chronic ulcers, delayed wound healing or rheumatoid
arthritis, are accompanied by an excessive and uncontrolled
degradation of matrix macromolecules. These pathologies are very
often associated with the deleterious secretion of cytokines, in
particular of pro-inflammatory cytokines, and with persistent
activation of complement, resulting, inter alia, in the prejudicial
overproduction of chemoattractant anaphylatoxins. In these
inflammatory pathologies, the excessive production of
pro-inflammatory cytokines disturbs the physiological cellular and
tissue functions, including in particular cell migration, cell
proliferation, and the expression, set n and activation of certain
proteases such matrix metalloproteases (MMPs). The expression of
these Mmps involved in matrix protein degradation is not generally
constitutive and is induced by pro-inflammatory cytokines such as
IL-1.beta. and/or TNF-.alpha. and/or growth factor weight sulfated
polyse derivatives with the invention can be used as e of
inhibiting ne secretior from metal as active tissue gival connectiv
issue.C. In , tives according to the invention are particularly
effective for inhibiting secretion of gelatinase A (MMP-2) The
inhibition of tgrated on are as IL-15. It is to ulate the sects
sible unconti atrix mac in cting deri -al ig in t- -,dontitis By
regul& pro-inflammatory cytokine and matrix metalloprotLase sec
-ion by connective tissue fibroblasts, and in -gival ive tissu
"fated charide de. .Sol withh the invent-on show good
anti-inflammatory activity.
[0064] Complement is a component of innate immunity (spontaneous
activation in response to an attack) which is defined as a complex
set of soluble or membrane proteins. When there is an inflammatory
response, these proteins become activated in a series of
proteolytic chain reactions that generates peptides with biological
activities. Complement activation, which is rapid and localized, is
subject to various particularly effective mechanisms of control.
However, some of these control mechanisms are disturbed in
inflammatory pathologies, such as autoimmune pathologies, leading
to persistent complement activation. Surprisingly and unexpectedly,
the inventors have demonstrated the use of the low-molecular-weight
sulfated polysaccharide derivatives of the invention as agents
capable of inhibiting the conventional complement pathway.
Advantageously, the low-molecular-weight sulfated polysaccharide
derivatives of the invention are used as anti-inflammatory agents
for the preparation of a pharmaceutical composition for treating
inflammatory pathologies in which complement is activated, in
particular autoimmune pathologies, for instance pemphigus.
[0065] Given their anti-inflammatory properties, it is advantageous
to use the low-molecular-weight sulfated polysaccharide derivatives
of the invention as anti-inflammatory agents for the preparation of
a pharmaceutical composition for in particular treating
inflammatory pathologies of connective tissues, in particular of
dermal and gingival connective tissues, for instance periodontitis,
chronic ulcerations or delayed wound healing.
[0066] Many pathologies can bring about the appearance of an
inflammatory process: not only autoimmune pathologies, but also
neoplastic or infectious pathologies. Advantageously, the
low-molecular-weight sulfated polysaccharide derivatives of the
invention are used as anti-inflammatory agents for the preparation
of a pharmaceutical composition for treating inflammatory
pathologies affecting dermal and gingival connective tissues, in
particular autoimmune, infectious or neoplastic pathologies, for
instance sarcomas.
[0067] The development of pathological fibroses appears to follow a
route similar to that followed during tissue regeneration. However,
the normal control of the cellular functions that occur during
tissue regeneration processes is disturbed. Specifically,
imbalances in the cellular component can be reflected, inter alia,
by an uncontrolled influx of inflammatory cells that maintain the
tissue degradation and/or by the persistence of cell subpopulations
such as myofibroblasts, leading to fibrotic pathologies. Whereas
myofibroblasts appear transiently during normal wound-healing
processes, this cell subpopulation persists when tissue
regeneration becomes pathological.
[0068] The inventors have shown that the sulfated polysaccharide
derivatives in accordance with the invention can be used as agents
capable of stimulating the proliferation of fibroblasts to the
detriment of myofibroblasts in connective tissues, in particular
dermal and gingival connective tissues. More particularly, the
sulfated polysaccharide derivatives in accordance with the
invention can be used as agents capable of stimulating the
proliferation of fibroblasts to the detriment of myofibroblasts in
two-dimensional cell cultures or in reconstructed dermal and
gingival connective tissues.
[0069] The use of the sulfated polysaccharide derivatives in
accordance with the invention makes it possible to stimulate the
proliferation of fibroblasts responsible for tissue homeostasis
while at the same time controlling the persistence of
myofibroblasts, two cellular events originating from the
stimulation of the nonpathological process of tissue
regeneration.
[0070] Given their property of selecting the fibroblast
subpopulation to the detriment of the myofibroblast subpopulation,
it is therefore particularly advantageous to use the
low-molecular-weight sulfated polysaccharide derivatives as defined
above, as anti-fibrotic agents for the preparation of a
pharmaceutical composition, said composition making it possible, in
particular, to prevent or treat hypertrophic wound-healing
processes or fibrotic pathologies of connective tissues, in
particular dermal and gingival connective tissues.
[0071] This property that the sulfated polysaccharide derivatives
in accordance with the invention have of selecting a specific cell
subpopulation within a heterogeneous population has also been
exploited so as to promote the obtaining of a subpopulation of
medullary cells intended to become mesenchymal cells. The medullary
cells intended to become mesenchymal cells are adult stem cells
capable of developing into differentiated mesenchymal cells
depending on the tissue in which they find themselves, in
particular into fibroblasts, chondrocytes, osteoblasts, adipocytes
and muscle cells having specialized morphological characteristics
and functions.
[0072] Thus, the inventors have demonstrated that the
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention are capable of selectively
stimulating the proliferation of medullary cells intended to become
mesenchymal cells to the detriment of other subpopulations in a
heterogeneous population of cells. This selective proliferative
effect is all the more advantageous since medullary cells intended
to become mesenchymal cells are rare since they are particularly
difficult to obtain and to maintain in culture. It is therefore
advantageous to complement cell culture media intended for the
obtaining and maintaining of medullary cells intended to become
mesenchymal cells, in the context of tissue or cell therapy.
[0073] Thus, the sulfated polysaccharide derivatives in accordance
with the invention therefore allow a selection-amplification by
proliferation of medullary cells intended to become mesenchymal
cells. These medullary cells intended to become mesenchymal cells
represent a source of pluripotent cells that can be used in tissue
therapy for transplant purposes in humans, insofar as they are
capable of differentiating into fibroblasts, chondrocytes,
osteoblasts, adipocytes and muscle cells depending on the tissue in
which they are implanted.
[0074] These cells are therefore advantageous in tissue and cell
therapy and more particularly when it is impossible to take samples
of skin fragments, as in individuals with third degree burns, or
when the tissue no longer has the ability to regenerate, such as
cartilage in adults.
[0075] Each of the pharmaceutical compositions or medicaments
containing the low-molecular-weight sulfated polysaccharide
derivatives obtained in accordance with the invention can also be
used in combination with one or more growth factors present in the
pharmaceutical composition or present in a different pharmaceutical
composition that will then be administered separately, i.e. before,
simultaneously, or after the administration of the pharmaceutical
composition containing the sulfated polysaccharide derivatives.
Such growth factors are in particular chosen from FGFs (fibroblast
growth factors), TGF.beta.s, BMPs (bone morphogenic proteins) and
CTGF (connective tissue growth factor).
[0076] Given their properties on fibroblasts, the
low-molecular-weight sulfated polysaccharide derivatives as defined
above can be used for the preparation of a pharmaceutical
composition with wound-healing and/or antifibrotic and/or
anti-inflammatory activity.
[0077] The pharmaceutical compositions or medicaments of the
invention are intended to be administered via the appropriate
route. The pharmaceutical composition or the medicament of the
invention is preferably in an injectable form, in which the
sulfated polysaccharide derivatives have a molecular weight of
between 5000 and 50 000 g/mol, preferably less than or equal to 25
000 g/mol, a polydispersity index of between 1.5 and 5, preferably
less than or equal to 2, and a degree of sulfate group substitution
of between 10% and 45%, and preferably between 20% and 40%,
inclusive.
[0078] The present invention also relates to a cosmetic or
dermatological composition, characterized in that it comprises
low-molecular-weight sulfated polysaccharide derivatives in
accordance with the invention, in combination with any suitable
excipient.
[0079] Preferably, the pharmaceutical, cosmetic or dermatological
compositions can be administered locally and can be in the form of
a gel, a cream, an ointment, an emulsion or a solution.
[0080] They can also be used in situ by means of substrates, of
medical devices that are resorbable or nonresorbable, such as, for
example, delayed-release supports, slowly disintegrating sponges,
or surgical implants.
[0081] The present invention will be understood more clearly from
the examples that follow, which are read with regard to the
attached figures; these examples are given only by way of
illustration of the subject of the invention, of which they no way
constitute a limitation.
[0082] FIG. 1 is an infrared spectrum of the polysaccharide
derivatives HE 800 that have undergone a free-radical
depolymerization before (HE 800 DR) and after the N-deacetylation
reaction (HE 800 DRN);
[0083] FIG. 2 represents the infrared spectra of the HE 800
derivative that has undergone a free-radical depolymerization
(derivative HE 800 DR) or of the N-deacetylated and sulfated HE 800
derivative (HE 800 DRNS);
[0084] FIG. 3 is a graph which shows the effect of the free-radical
depolymerized and then sulfated GY785 derivative, according to the
invention, on the proliferation of fibroblasts in a reconstructed
dermis;
[0085] FIG. 4 is a set of 6 photographs a, b, c, d, e and f. This
figure relates to an immunodetection test, in a dermal fibroblast
culture, of .alpha.-actin filaments that are characteristic of the
myofibroblast subpopulation;
[0086] FIG. 5 is a graph which reflects a result obtained by
zymography, and shows the effect of HE 800 DRNS on the secretion of
MMP-2 by fibroblasts in culture: FIG. 5 shows that the secretion of
MMP-2 is inhibited;
[0087] FIG. 6 is a set of two photographs, a and b, which show the
effect of a derivative in accordance with the invention on the
secretion of a matrix protease, stromelysin (MMP-3).
EXAMPLE 1
Oside Composition of the Bacterial Native EPSs
Methods:
[0088] The protein content was determined according to the BCA
(bicinchoninic acid) method described by Wiehelman K. et al. (Anal.
Biochem. 1988, 175: 231-237).
[0089] The neutral monosaccharide content was determined by the
Tillmans and Philippi method (Analyt. Chem., 1929, 28: 350)
modified by Rimington (Biochem. J., 1931, 25: 1062-1071).
[0090] The uronic acid (GlcA) content was established using a
modification of the m-hydroxydiphenyl-H.sub.2SO.sub.4 method
(Filisetti-Cozzi and Carpitta, Anal. Biochem., 1991, 197: 157-162)
and using glucuronic acid as standard. Interference from neutral
hexoses was avoided by using potassium sulfamate and carrying out
controls comprising all the reagents with the exception of the
m-hydroxydiphenyl.
[0091] The neutral monosaccharide and acidic monosaccharide
contents were determined by gas chromatography. The analysis of the
glycoside residues in the form of trimethylsilylated derivatives
was carried out according to the method of Kamerling et al.
(Biochem. J., 1975, 151: 491-495) and modified by Montreuil et al.
(Glycoproteins In: Carbohydrate analysis, a practical approach,
1986, Chaplin M. F. and Kennedy J. F. (eds), IRL Press, Oxford,
143-204).
[0092] The hexosamine and N-acetylhexosamine (GalNAc and GlcNAC)
content is determined by the method of Belcher et al. (Analyst,
1954, 79: 201-208) adapted from that of Elson and Morgan (Biochem
J., 1933, 27: 1824-1828) and using N-acetylglucosamine and
glucosamine as standards.
[0093] The contents of total sulfates (free plus bound of the
native EPSs) were determined by elemental analysis of sulfur (S %),
and by applying the following relationship: percentage of sulfate
groups (%)=3.22.times.S %. The amount of free sulfates is
quantified by ion exchange chromatography on a Dionex.RTM. DX-500
system connected to a conductimeter, and according to the method
described by the manufacturer Dionex. The result obtained makes it
possible to calculate the amount of sulfates really bound to the
EPS derivative, which is equal to the amount of total sulfates
(obtained by elemental analysis) minus the amount of free sulfates
(obtained by ion exchange chromatography).
[0094] Description TABLE-US-00001 OSIDE COMPOSITION Amino Neutral
Acid sugars Sulfats Strain EPS % % % % HYD 1545.sup.1,2,3
1545.sup.1,2,3 49 34 0.2 11 Alteromonas sp HYD 721.sup.1,2,12
721.sup.1,2,12,16 55 11 <0.5 12 Pseudoalteromonnas sp HYD
1644.sup.2,7,8 1644.sup.2,7,8,16 55 35 <1 5 Alteromonas sp HYD
657.sup.2,14 657.sup.2,14 47 26 1.6 5 Alteromonas macleodii sp
subsp fijiensis biovar deepsane ST 716.sup.4,9,10 716.sup.9,10,16
40 40 <1 5 Alteromonas macleodii sp subsp fijiensis GY 785.sup.5
GY 55 40 <0.7 10 Alteromonas infernus 785.sup.5,11,15,16 sp MS
907.sup.16 MS 907.sup.16 50 37 0 0 Alteromonas macleodii sp subsp
fijiensis biovar medioatlantica HE 800.sup.6,9,13 HE 800.sup.6,9,13
1 32 30 0 Vibrio diabolicus sp .sup.1(Aymard et al., 1991 Food
Hydrocoll, 5, 167-169); .sup.2(Guezennec et al., 1994 Carbohydr.
Polym., 24, 287-294); .sup.3(Vincent et al., 1994 Appl. Environ.
Microb., 60, 4134-4141); .sup.4Raguenes et al., 1996 Appl. Env
Microbiol, 62, 67-73); .sup.5(Raguenes et al., 1997, Journal of
Systematic Bacteriology, 47, 989-995); .sup.6(Raguenes et al., 1997
J. Appl. Microbiol., 82, 422-430); .sup.7(Dubreucq et al., 1996
Carbohydr Res., 290, 175-81); .sup.8(Bozzi et al., 1996 Int J.
Biol. Macromol, 18, 9-17); .sup.9(Rougeaux et al., 1996 Carbohydr.
Polym. 31, 237-242); .sup.10(Rougeaux et al., 1998 Carbohydr. Res.,
312, 53-59); .sup.11(Guezennec et al., 1998 Carbohydr. Polym. 37,
19-24); .sup.12(Rougeaux et al., 1999 Carbohydr Res., 315,
273-285); .sup.13(Rougeaux et al., 1999 Carbohydr. Res., 322,
40-45); .sup.14Cambon-Bonavita et al., 2002 J Applied Microbiol,
93, 310-315); .sup.15(Guezennec, 2002 J Ind Microbiol Biol., 29,
204-208); .sup.16(Raguenes et al., 2003 Curr Microbiol., 46,
448-52); .sup.17(Roger et al., 2004, Res., 339, 2371-2380)
EXAMPLE 2
Preparation of Derivatives According to the Invention Based on the
Native EPS HE 800
[0095] (1) HE 800 DR corresponds to the low-molecular-weight
polysaccharide derivative,
[0096] (2) HE 800 DRS corresponds to the low-molecular-weight
sulfated polysaccharide derivative,
[0097] (3) HE 800 DRNS corresponds to the low-molecular-weight
N-deacetylated and sulfated polysaccharide derivative.
[0098] The low-molecular-weight sulfated polysaccharide derivatives
in accordance with the invention are obtained: (i) for the DRS
series, by applying a first step consisting of free-radical
depolymerization (DR) and a sulfation step (S), and (ii) for the
DRNS series, by applying a first step consisting of free-radical
depolymerization (DR) followed by an N-deacetylation step (N) and a
sulfation step (S).
2.1. Free-Radical Depolymerization of a Native EPS
[0099] 500 mg of EPS from the marine bacterium of hydrothermal
origin HE 800 are produced according to the process described in EP
975 791, and lyophilized. The native EPS is slowly rehydrated
overnight in 100 ml of water and is introduced into a jacketed
glass reactor. The metal catalyst is added to the reaction medium
in the form of a solution of copper acetate at 16 mg/ml. The
temperature of the medium is brought to 60.degree. C. Magnetic
stirring is maintained throughout the reaction. The reaction medium
is brought to a pH of between 7.5 and 8 with 10N concentrated
sodium hydroxide. The pH of the reaction medium is monitored and is
regulated through the addition of a sodium hydroxide solution.
[0100] Hydrogen peroxide (H.sub.2O.sub.2) is then added to the
reactor at a flow rate of 1 ml/min using a peristaltic pump. The
hydrogen peroxide is prepared extemporaneously from a concentrated
solution.
Reduction:
[0101] 1 g of sodium borohydride per 1 g of polysaccharide
derivative is dissolved in a small volume of water and then added
directly to a reactor. The reaction takes place at ambient
temperature and with stirring for a period of 2 to 20 hours. It is
stopped by the addition of 10 N acetic acid. A blackish precipitate
due to the copper forms during the reaction.
Elimination of the Catalyst:
[0102] In order to eliminate the precipitate formed during the
reduction reaction, the solution containing the polysaccharide
derivative is filtered through a buchner funnel equipped with
filters made of 3 .mu.m glass micro-fibers. The residual copper is
then eliminated by passing the solution containing the
polysaccharide derivative over a Chelex.TM. resin, with a capacity
of 0.4 meq/ml. The solution containing the polysaccharide
derivative percolates, at a flow rate of 4 to 5 ml/min, through a
column (25.times.400 mm) of 200 ml of resin passivated beforehand.
At the outlet, the solution containing the polysaccharide
derivative has a basic pH of 10.
Diafiltration, Concentration by Ultrafiltration and
Lyophilization:
[0103] The solution containing the polysaccharide derivative is
ultrafiltered through a Pellicon 2 ultrafiltration system
(Millipore) equipped with a Pall membrane of 1000 g/mol. The
conductivity of the solution (4 to 5 mS) is measured throughout the
diafiltration. When a stable value below 100 .mu.S is reached on
the filtrate, the solution is brought back to a neutral pH. It is
then concentrated then lyophilized (CIRP lyophilizer). Once
lyophilized, the polysaccharide derivative obtained is
characterized.
2.2. N-Deacetylation
[0104] Principle: With the aim of at once substituting the oside
units in terms of N- and O-sulfate groups, the polysaccharide HE
800 DR is N-deacetylated. The process for N-deacetylation of the HE
800 derivative is carried out on large amounts of product.
Method:
[0105] 259 mg of EPS HE 800 DR are solubilized in 10 ml of water
and placed in a round-bottomed flask with magnetic stirring. 263 mg
of NaBH.sub.4 are solubilized in 1.25 ml of water and then added to
the solution of EPS HE 800 DR. When the temperature of the mixture
reaches 80.degree. C., 1.25 ml of 10 N NaOH are added. The final
concentration of the solution is then 1 N with respect to sodium
hydroxide and 2% with respect to NaBH.sub.4, for a total volume of
12.5 ml.
[0106] After reaction for one hour, the solution is neutralized
with 10 N acetic acid until the effervescence has stopped. The
volume added is 1.5 ml and the pH is 5. The solution is then
ultrafiltered through a 1000 g/mol membrane and then lyophilized.
167 mg of N-deacetylated EPS HE 800 DR (HE 800 DRN) are obtained,
reflecting a yield of 65%.
2.3. Sulfation of the EPS HE 800 DR or of HE 800 DRN
Preparation of the Polysaccharide in the Form of a Salt:
[0107] 50 mg of EPS are solubilized in 20 ml of H.sub.2O. The
polysaccharide derivative is placed in the H+ form by elution on a
Dowex resin column. The elution is carried out with water, and the
eluate is collected as long as the pH remains acidic, preferably
less than 5. The pH is immediately adjusted to 6.5 with the desired
base (pyridine, triethylamine, tributylamine, sodium hydroxide).
The polysaccharide derivative in the form of a salt is then
lyophilized.
Sulfation of the Polysaccharide:
[0108] The polysaccharide derivative in the form of a salt is
dissolved in 100 ml of anhydrous DMF with gentle stirring (250 rpm)
for 2 hours at ambient temperature, and then for 2 hours at a
temperature of 45.degree. C.
[0109] When dissolution is complete, 2.5 g of pyridine-SO.sub.3
complex are added to the reaction medium, i.e. 5 times the mass of
the polysaccharide. The temperature of the mixture is then
maintained at 45.degree. C. for 2 hours with stirring. The reaction
is terminated by adding 40 ml of water and sodium hydroxide in
order to obtain a pH at 9. The reaction mixture is then dialyzed in
water with a dialysis bag having a cutoff threshold of 3500 Da.
[0110] After dialysis, the solution containing the sulfated EPS is
filtered through filters of 2.7 .mu.m and 0.7 .mu.m and
ultrafiltered through a 1000 g/mol membrane and then
lyophilized.sup.2.
2.4. Characterization of the Various Derivatives:
[0111] (1) HE 800 DR; (2) HE 800 DRS and (3) HE 800 DRNS.
[0112] The molecular weights (Mc: chromatographic molecular weight
determined at the summit of the peak; Mw: weight-average molecular
weight and Mn: number-average molecular weight) and the
polydispersity (I=Mw/Mn) of the various EPS HE 800 derivatives
obtained were determined by high performance steric exclusion
chromatography (HPSEC) on a Biotech system, in 0.1 M aqueous
ammonium acetate at a flow rate of 0.1 ml/min using a Superdex.RTM.
200 column or a Superdex.TM. Peptide column (AMERSHAM). The column
was calibrated with polysaccharide standards as follows: pullulans:
758 000-5900 g/mol (Polymer Laboratories, Interchim), noncommercial
standard polysaccharides: 4000; 3000 and 1500 g/mol; melezitose:
522 g/mol (FLUKA), sucrose: 342 g/mol; glucose: 180 g/mol (SIGMA).
The results are analyzed using the Aramis.RTM. software (Varian,
France).
[0113] The neutral monosaccharide content was determined by the
method of Tillmans and Philippi (Analyt. Chem., 1929, 28, 350-)
modified by Rimington (Biochem. J., 1931, 25: 1062-1071).
[0114] The uronic acid (GlcA) content was established using a
modification of the m-hydroxydiphenyl-H.sub.2SO.sub.4 method
(Filisetti-Cozzi and Carpitta, Anal. Biochem., 1991, 197: 157-162)
and using glucuronic acid as standard. Interference from neutral
hexoses was avoided by using potassium sulfamate and carrying out
controls comprising all the reagents with the exception of the
m-hydroxydiphenyl.
[0115] The hexosamine and N-acetylhexosamine (GalNAc and GlcNAc)
content is determined by the method of Belcher et al. (Analyst,
1954, 79: 201-208) adapted from that of Elson and Morgan (Biochem
J., 1933, 27: 1824-1828) and using N-acetylglucosamine and
glucosamine as standard.
[0116] The total sulfates (free plus bound) contents were
determined by elemental analysis of sulfur (S %), and by applying
the following relationship: percentage of sulfate groups
(%)=3.22.times.S %.
[0117] The amount of free sulfates is quantified by ion exchange
chromatography on a Dionex.RTM. DX-500 system connected to a
conductimeter and according to the method described by the
manufacturer Dionex. The result obtained makes it possible to
calculate the amount of sulfates really bound to the EPS
derivative, which is equal to the amount of total sulfates
(obtained by elemental analysis) minus the amount of free sulfates
(obtained by ion exchange chromatography).
[0118] Fourier transform infrared spectroscopy (FT-IR) was carried
out on a Vector 22 having a resolution of 4 cm.sup.-1. The infrared
spectra of the polysaccharides were determined using KBr pellets (2
mg of polysaccharide are mixed with 200 mg of dry KBr), all the
infrared spectra were recorded between 4000 and 400 cm.sup.-1.
2.5. Results
[0119] Composition of an HE 800 DR Derivative: TABLE-US-00002
Characteristics HE 800 DR derivative Neutral monosaccharides 0
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 40
Hexosamines (g/100 g).sup.1 40 Total --SO.sub.3Na (g/100 g).sup.2 0
Mc (g/mol).sup.4 15 000 Mw (g/mol).sup.4 29 000 Mn (g/mol).sup.4 13
000 I (Mw/Mn).sup.4 2.2 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
[0120] Composition of an EPS HE 800 DRS Derivative: TABLE-US-00003
Characteristics HE 800 DRS derivative Neutral monosaccharides 0
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 30
Hexosamines (g/100 g).sup.1 30 Total --SO.sub.3Na (g/100 g).sup.2
25 Mc (g/mol).sup.4 4800 Mw (g/mol).sup.4 5800 Mn (g/mol).sup.4
4500 I (Mw/Mn).sup.4 1.3 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
[0121] Composition of an EPS HE 800 DRNS Derivative: TABLE-US-00004
Characteristics HE 800 DRNS derivative Neutral monosaccharides 0
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 20
Hexosamines (g/100 g).sup.1 20 Total --SO.sub.3Na (g/100 g).sup.2
34 Mc (g/mol).sup.4 22 000 Mw (g/mol).sup.4 27 000 Mn (g/mol).sup.4
19 000 I (Mw/Mn).sup.4 1.4 .sup.1Colorimetric assays. .sup.2Assay
by elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
[0122] FIG. 1 shows the infrared spectra of the polysaccharide
derivatives, before (HE 800 DR) and after the N-deacetylation
reaction (HE 800 DRN). The FT-IR spectra in FIG. 1 were recorded on
a Bruker Vector 22 spectrophotometer (resolution of 4 cm.sup.-1). 2
mg of EPS HE 800 derivative were treated with 200 mg of KBr during
the N-deacetylation step. The analysis of the infrared spectra of
the derivatives, before and after the N-deacetylation step, shows,
at the frequency of 1550 cm.sup.-1, the loss of an absorption band
characteristic of N-acetylated groups (FIG. 1).
[0123] FIG. 2 shows the infrared (IR) spectra of the non-sulfated
HE 800 polysaccharide derivative (HE 800 DR) and of the
N-deacetylated and sulfated HE 800 poly-saccharide derivative (HE
800 DRNS). The appearance of the bands corresponding to the
presence of a sulfate ester (1250, 820 and 600 cm.sup.-1) is
observed for the polysaccharide derivative HE 800 DRNS.
EXAMPLE 3
Preparation of Derivatives According to the Invention Based on the
Native EPS GY 785
[0124] (1) GY 785 DR corresponds to the low-molecular-weight
polysaccharide derivative obtained after a depolymerization
step.
[0125] (2) GY 785 DRS corresponds to the low-molecular-weight
polysaccharide derivative obtained after a depolymerization step
followed by a sulfation step.
1) Free-Radical Depolymerization and Reduction with Sodium
Borohydride
[0126] 400 mg of sulfated EPS GY 785 obtained above in the
preceding step were dissolved in 95 ml of water. After dissolution,
2 ml of a catalytic solution containing 36 mg of copper acetate
monohydrate (10.sup.-3 M) were added. The temperature of the
reactor is then brought to 60.degree. C. and the pH is adjusted to
7.5 by the addition of 1 M sodium hydroxide. A 0.115% (v/v)
solution of hydrogen peroxide was then added at a flow rate of 1 ml
per minute, and the pH was regulated at around 7.5 by the addition
of 1 M sodium hydroxide. The reaction was stopped after 1 hour.
[0127] The reduction is carried out at the end of depolymerization
by the addition to the reactor of sodium borohydride (270 mg of
NaBH.sub.4 dissolved in 10 ml of water). The reduction is carried
out with stirring for 2 hours at ambient temperature. The reduction
is stopped by the addition of 10 N acetic acid, which makes it
possible to eliminate the excess NaBH.sub.4 remaining in the form
of hydrogen gas that is given off. The solution was then filtered
through a Buchner funnel with filters made of glass microfibers
(porosity 3 .mu.m). The filtered solution was eluted on a
CHELEX.RTM. 20 column (BIORAD) in order to eliminate the residual
copper. The decontaminated solution was then ultra-filtered through
a cassette (cutoff threshold 1000 Da) and then lyophilized.
2) Chemical Sulfation of the EPS GY 785
[0128] 500 mg of EPS GY 785 lyophilizate produced by the marine
bacterium of hydrothermal origin Alteromonas infernus according to
the process described in example 1 of patent FR 2 755 142, were
dissolved in 100 ml of anhydrous DMF with gentle stirring (250 rpm)
for 2 hours at ambient temperature, and then for 2 hours at a
temperature of 45.degree. C.
[0129] When dissolution is complete, 2.5 g of pyridine-SO.sub.3
complex sold under the reference 84737 by the company Fluka (i.e. 5
times the mass of the GY 785 polysaccharide) were added to the
reaction medium. The temperature of the mixture was then brought to
and maintained at 45.degree. C. for 2 hours with stirring. The
reaction mixture was transferred into a beaker. The reaction was
then stopped by the addition of 40 ml of water, and the pH was then
brought to 9 with 3 M sodium hydroxide. The reaction mixture was
then dialyzed in a dialysis bag having a cutoff threshold of
between 12 000 and 16 000 Da, against tap water (overnight with
running water), and then 3 times for 24 hours against Milli-Q
water.
[0130] After dialysis, the solution containing the sulfated EPS GY
785 was frozen and lyophilized.
[0131] The characteristics of the EPS DR and EPS DRS derivatives
were determined according to the methods described above in example
1 and are summarized in the table below: TABLE-US-00005
Characteristics EPS DR EPS DRS Total monosaccharides 51 nd (g/100
g).sup.1 Acidic monosaccharides 38 nd (g/100 g).sup.1 Total
--SO.sub.3Na (g/100 g).sup.2 10 42 Mc (g/mol) 7800 13 000 Mw
(g/mol) 17 300 23 600 Mn (g/mol) 6000 8800 I (Mw/Mn) 2.8 2.7
Anticoagulant activity.sup.3 inactive 7 .sup.1Colorimetric assays.
.sup.2Assay by elemental analysis. .sup.3Amount of polysaccharide
in .mu.g/ml of human plasma necessary to double the control
coagulation time, ACT (control time = 40 seconds); nd: not
determined
[0132] Composition of Another GY 785 DR Derivative: TABLE-US-00006
Characteristics GY 785 DR derivative Neutral monosaccharides 40
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 15
Hexosamines (g/100 g).sup.1 0 Total --SO.sub.3Na (g/100 g).sup.2 10
Mc (g/mol).sup.4 16 000 Mw (g/mol).sup.4 40 000 Mn (g/mol).sup.4 13
000 I (Mw/Mn).sup.4 3 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
[0133] Composition of Another GY 785 DRS Derivative: TABLE-US-00007
Characteristics GY 785 DRS derivative Neutral monosaccharides 20
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 10
Hexosamines (g/100 g).sup.1 0 Total --SO.sub.3Na (g/100 g).sup.2 45
Mc (g/mol).sup.4 23 000 Mw (g/mol).sup.4 29 000 Mn (g/mol).sup.4 21
000 I (Mw/Mn).sup.4 1.4 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
EXAMPLE 4
Preparation of Derivatives According to the Invention Based on the
Native EPS HYD 721
[0134] (1) HYD 721 DR corresponds to the low-molecular-weight
polysaccharide derivative obtained after a depolymerization
step,
[0135] (2) HYD 721 DRS corresponds to the low-molecular-weight
polysaccharide derivative sulfated by chemical sulfation according
to the invention.
[0136] The sulfated HYD 721 polysaccharide derivative is obtained
by carrying out the process described in example 2. However, since
the native EPS HYD 721 does not contain N-acetylated hexosamines,
the process for preparing the sulfated derivative does not comprise
an N-deacetylation step.
[0137] Composition of an HYD 721 DR Derivative: TABLE-US-00008
Characteristics HYD 721 DR derivative Neutral monosaccharides 68
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 17
Hexosamines (g/100 g).sup.1 0 Total --SO.sub.3Na (g/100 g).sup.2 11
Mc (g/mol).sup.4 10 000 Mw (g/mol).sup.4 12 000 Mn (g/mol).sup.4
7000 I (Mw/Mn).sup.4 1.7 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
[0138] Composition of an HYD 721 DRS Derivative: TABLE-US-00009
Characteristics HYD 721 DRS derivative Neutral monosaccharides 35
(g/100 g).sup.1 Acidic monosaccharides (g/100 g).sup.1 5
Hexosamines (g/100 g).sup.1 0 Total --SO.sub.3Na (g/100 g).sup.2 43
Mc (g/mol).sup.4 17 500 Mw (g/mol).sup.4 20 000 Mn (g/mol).sup.4 10
000 I (Mw/Mn).sup.4 2 .sup.1Colorimetric assays. .sup.2Assay by
elemental analysis. .sup.3Assay by ion exchange chromatography.
.sup.4Determined by HPSEC chromatography in pullulan
equivalents.
EXAMPLE 5
Effect of the Derivatives According to the Invention on the
Proliferation of Dermal and Gingival Fibroblasts
[0139] The sulfated polysaccharide derivatives used in the
proliferation assays were prepared and characterized according to
the protocols of examples 2 and 3.
5.1. Cells in Two-Dimensional Culture
[0140] The cells are seeded in two culture dishes at a rate of 10
000 cells per well, in Dulbecco's MEM Glutamax I culture medium
containing 100 U/ml of penicillin, 100 .mu.g/ml of streptomycin, 2
.mu.g/ml of fungizone and supplemented with 10% of fetal calf serum
(FCS).
[0141] After adhesion and spreading of the cells for 12 hours, the
culture medium is replaced with a culture medium that may or may
not be supplemented with various concentrations of a
low-molecular-weight sulfated polysaccharide derivative: (i) the
EPS GY 785 (GY 785 DRS) or (ii) the EPS HE 800 (HE 800 DRNS) HE
800. The cells are then counted after 2, 4, 7 and 10 days of
culture. The controls correspond to cell cultures in the absence of
derivatives in accordance with the invention (*).
[0142] Effect of the GY 785 DRS Derivative on the Proliferation of
Dermal Fibroblasts in Two-Dimensional Culture: TABLE-US-00010 Day 2
Day 4 Day 7 Day 10 Control* 100 100 100 100 0.1 .mu.g/ml GY 785 DRS
94.5 100.0 145.3 151.4 1 .mu.g/ml GY 785 DRS 108.8 113.7 173.0
160.1 10 .mu.g/ml GY 785 DRS 87.6 157.3 156.9 151.2 100 .mu.g/ml GY
785 DRS 77.4 121.2 115.6 123.4
[0143] Effect of GY 785 DRS on the Proliferation of Gingival
Fibroblasts in Two-Dimensional Culture: TABLE-US-00011 Day 2 Day 4
Day 7 Day 10 Control* 100 100 100 100 0.1 .mu.g/ml GY 785 DRS 112.0
121.8 135.0 139.1 1 .mu.g/ml GY 785 DRS 99.2 101.5 122.6 133.0 10
.mu.g/ml GY 785 DRS 90.2 124.3 160.4 183.3 100 .mu.g/ml GY 785 DRS
77.7 95.4 118.4 161.2
[0144] Effect of HE 800 DRNS on the Proliferation of Dermal
Fibroblasts in Two-Dimensional Culture: TABLE-US-00012 Day 2 Day 4
Day 7 Day 10 Control* 100 100 100 100 10 .mu.g/ml HE 800 DRNS
109.00 159.96 157.70 146.56 100 .mu.g/ml HE 800 DRNS 95.49 191.12
163.60 167.60
[0145] The sulfated polysaccharide derivatives in accordance with
the invention are capable of stimulating the proliferation of
dermal and gingival fibroblasts in two-dimensional culture.
5.2. Reconstructed or Latticed Tissue (FIG. 3)
[0146] Reconstructed or latticed connective tissues consist of
acid-soluble collagen type I fibers which, after neutralization,
polymerize and form a gel containing fibroblasts. The preparation
of a lattice is carried out under cold conditions in order to have
better control of the polymerization of the collagen fibers.
[0147] This lattice under the influence of the cells will undergo
numerous rearrangements that are noticeable in particular by virtue
of its retraction, which is observed during the first two weeks of
culture. This type of model makes it possible to study the behavior
of the cells within an extracellular environment closer to the
physiological environment than simple culturing on a dish.
[0148] FIG. 3 reveals that the low-molecular-weight sulfated
polysaccharide derivative GY 785 DRS in accordance with the
invention is capable, at the concentration of 10 .mu.g/ml, of
stimulating the proliferation of fibro-blasts in reconstructed
connective tissues.
EXAMPLE 6
Demonstration of the Effect of the Derivatives According to the
Invention on the Selection of the Fibroblast Subpopulation
[0149] As for the proliferation assays in two-dimensional cultures,
the cells are seeded into culture dishes at a rate of 10 000 cells
per well, in Dulbecco's MEM Glutamax I culture medium containing
100 U/ml of penicillin, 100 .mu.g/ml of streptomycin, 2 .mu.g/ml of
fungizone and supplemented with 10% of fetal calf serum (FCS).
[0150] After adhesion and spreading of the cells for 12 hours, the
culture medium is replaced with a culture medium that may or may
not be supplemented with various concentrations of a sulfated
derivative obtained from the EPS GY 785 (GY 785 DRS). The cultures
are then fixed with alcohol after 2, 4, 7 and 10 days of culture,
and the immunodetection on these cell cultures is carried out as
follows:
[0151] The fixed cells are re-permeabilized in 70% ethanol (20
min), and then rehydrated in PBS (10 min). Endogenous peroxidases
are blocked with a methanol (30%), H.sub.2O.sub.2 (0.3%) solution.
This process is followed by rinsing with PBS (2 min) then blocking
of the nonspecific antigenic sites with a PBS/1% skimmed milk
solution (1 h). The cultures are then incubated with a primary
antibody (mouse IgG) directed against human .alpha.-actin (1/30; 50
min) and then rinsed with PBS (3.times.10 min). The cells are then
incubated, in the dark for 60 min, with a biotinylated anti-mouse
IgG antibody (1/200), rinsed with PBS (3.times.10 min), and then
incubated with peroxidase-coupled streptavidin (1/200).
[0152] After rinsing (PBS 3.times.10 min), the visualization of the
peroxidase activity with 3,3'-diaminobenzidine is carried out in a
Tris/HCl buffer (100 mM, pH 7.2-7.4) containing 0.1% of
H.sub.2O.sub.2 (15 min, in the dark). The peroxidase activity
reveals a brown fibrillar material that corresponds to the
.alpha.-actin microfilaments in the cytoplasm of the positive
cells. The products used come from the company DAKO under the name
DAKOImmuno-detection.
[0153] FIG. 4 demonstrates that the low-molecular-weight sulfated
polysaccharide derivative GY 785 DRS in accordance with the
invention is capable of stimulating the proliferation of
fibroblasts to the detriment of myofibroblasts. Photographs a, c
and e correspond to cultures supplemented with 10 .mu.g/ml of
sulfated derivative, in which the dominant population is made up of
fibroblasts to the detriment of myofibroblasts. Photographs b, d
and f correspond to control cultures without sulfated derivative,
in which numerous myofibroblasts are visible.
[0154] The control cultures not treated with the derivatives in
accordance with the invention comprise a large number of
myofibroblasts (.alpha.-actin-positive), while fibroblasts not
expressing .alpha.-actin form the dominant cell type in the treated
cultures.
EXAMPLE 7
Effect of the Derivatives According to the Invention on the
Secretion of Matrix Proteases
7.1. Protocol
[0155] The cells are seeded into culture dishes at a rate of 40 000
cells per well and are brought to confluence in Dulbecco's MEM
Glutamax I culture medium containing 100 U/ml of penicillin, 100
.mu.g/ml of streptomycin, 2 .mu.g/ml of fungizone and supplemented
with fetal calf serum (FCS).
[0156] At confluence, the culture medium is replaced with medium
that does not contain any FCS and may or may not be supplemented
with IL-1.beta. at a final concentration of 100 U/ml, in the
presence or absence of sulfated derivatives.
[0157] The culture media are sampled after 48 hours, in order to
study the MMP secretion by the fibroblasts, by zymography or by
Western blotting. For each experimental condition carried out in
quadruplicate, two wells are fixed with ethanol and the other two
are trypsinized in order to detach the cells so as to count
them.
[0158] The metalloprotease secretion is detected and quantified by
zymography. It is a particularly sensitive method based on SDS-PAGE
electrophoresis carried out under reducing conditions. The MMP-2
substrate, gelatin, copolymerizes with the acrylamide. After
migration, the SDS is eliminated by washing in a solution of Triton
X-100, allowing restoration of the MMP-2 activity. The gel is then
incubated at 37.degree. C. in an incubation buffer (0.1 M Tris/HCl,
pH 7.4, 30 mM CaCl.sub.2, 0.001% NaN.sub.3, 0.0015% Brij, 0.1 .mu.M
ZnCl.sub.2).
[0159] After staining (coomassie blue (0.5%), acetic acid (10%),
isopropanol (30%)), and then destaining (acetic acid (10%),
methanol (40%), distilled water (50%)), the bands illustrating the
MMP-2 metalloprotease activity appear destained. The shade of gray
and the surface area of these bands are quantified on an image
analyzer, the [(shade of gray.times.surface area)/number of cells]
ratio allows a quantification of the gelatino-lytic activities and
comparison between the control cultures and the cultures containing
the exopolysaccharide.
7.2. Effect of HE 800 DRNS on the Secretion of MMP-2 by Fibroblasts
in Culture, Result Obtained by Zymography (FIG. 5)
[0160] The graph in FIG. 5 shows that the MMP-2 secretion is
inhibited when the fibroblasts are cultured in the presence of
low-molecular-weight sulfated polysaccharide derivatives, in the
presence or absence of IL1.beta..
7.3. Effect of HE 800 DRNS on the Secretion of Stromelysin 1
(MMP-3) by Fibroblasts in Culture, Result Obtained by Western
Blotting (FIG. 6)
[0161] FIG. 6 shows that the addition of HE 800 DRNS greatly
decreases the secretion of MMP-3 by the fibroblasts, whether this
secretion is baseline (photograph a) or induced by an inflammatory
cytokine IL-1.beta. (photo-graph b). Photograph (a) shows confluent
fibroblasts incubated for 48 h in a serum-free medium (lane 1), and
in a serum-free medium containing 10 .mu.g/ml of EPS derivative
(lane 2); photograph (b) shows confluent fibroblasts incubated for
48 h in a serum-free medium containing 100 U/ml of IL-1.beta. (lane
1), and in a serum-free medium containing 10 .mu.g/ml of EPS
derivative and 100 U/ml of IL-1.beta. (lane 2).
1: control=untreated culture
2: culture treated with 100 U/ml of HE 800 DRNS.
[0162] In conclusion, the sulfated polysaccharide derivatives in
accordance with the invention are capable of inhibiting the
secretion of matrix proteases, gelatinase A (MMP-2) and stromelysin
1 (MMP-3), by fibroblasts in two-dimensional cultures.
EXAMPLE 8
Effect of the Derivatives According to the Invention on
Complement
8.1 Principle
[0163] Complement can be activated by 3 different activation
pathways: the conventional pathway, the alternative pathway and the
mannose-binding lectin (MBL) pathway resulting, via different
mechanisms, in the formation of enzymes having identical
substrates: the C3/C5 convertases capable of activating C3 and C5.
The activation reactions take place as a cascade: one component
acquires an enzymatic activity that induces the activation of the
next component, and so on.
[0164] Insofar as the final activation of complement is common to
the three pathways, only the conventional pathway is studied in
this example. The activation of the conventional pathway occurs
when the C1 complex interacts with antigen-antibody complexes or
immune aggregates containing IgGs or IgMs. The system used to study
the conventional pathway is based on the complement activation by
an immune complex consisting of rabbit antibody-coated sheep red
blood cells. The rabbit antibodies having recognized as foreign the
red blood cells, in the presence of human serum, trigger mainly the
activation of the conventional pathway. This activation leads to
the formation of C3 convertase, which then triggers the activation
of the alternative pathway. The activation of these two pathways
results in the formation of a membrane attack complex at the
surface of the red blood cells. This complex induces rupturing of
the red blood cells and the release of hemoglobin. The activation
of the complement system is measured by assaying the amount of
hemoglobin released, by measuring the spectrophotometric absorption
at 414 nm. The activator (sheep red blood cells) is also used as a
revealer of the activation by virtue of the hemoglobin released
during the cell lysis. The dilution of the human serum is adjusted
for a given amount of blood cells, such that 50% of the cells are
lysed.
[0165] The antibody-coated red blood cells are incubated in the
absence (control) and in the presence of various sulfated
polysaccharide derivatives. The amount of hemoglobin released
decreases (decrease in the absorption at 414 nm), reflecting a
decrease in the number of red blood cells lysed and an inhibitory
effect of the sulfated polysaccharide derivatives on the activation
of the conventional complement pathway.
[0166] 350 .mu.l of normal human serum (NHS) (diluted to 1/100th in
VBS2+ buffer) are incubated with 450 .mu.l of VBS2+, and 200 .mu.l
of rabbit antibodies (10.sup.8 cells/ml), in the presence or
absence of sulfated polysaccharide derivatives. After an
approximate reaction time of 45 min at 37.degree. C., a solution of
cold NaCl (0.15 M) is added and the cells are centrifuged at 2400
rpm for 10 min. The absorption of the supernatants is measured at
414 nm.
8.2. Result
[0167] Percentages of Complement Inhibition (Conventional Pathway)
in the Presence of Various Amounts of EPS Derivatives GY 785 DRS
and HE 800 DRNS: TABLE-US-00013 Amount of derivatives Percentage
inhibition 0.5 .mu.g 5 .mu.g 10 .mu.g 2.5 .mu.g GY785DRS 42 100 100
100 GY785DR 0 0 0 0 1 .mu.g HE800DRNS 18 38 48 HE800DR 0 0 0
[0168] The low-molecular-weight sulfated polysaccharide derivatives
in accordance with the invention are capable of inhibiting the
conventional complement pathway.
EXAMPLE 9
Effect of the Derivatives According to the Invention on the
Proliferation of Medullary Cells Intended to Become Mesenchymal
Cells
9.1. Protocol
[0169] Stromal cells are obtained from ground bone marrow material,
and this ground material is centrifuged so as to recover a cell
pellet. The cells are resuspended in culture medium made up, for
500 ml, of medium containing 10% of horse serum, 10% of fetal calf
serum, 200 nM L-glutamine (4 ml), 100.times.MEM vitamin (Gibco brl)
(4 ml), 7.5% Na.sub.2HCO.sub.3 (4 ml), 50.times. essential amino
acids with L-glutamine (Gibco brl) (4 ml), 100.times. sodium
pyruvate (4 ml), 100.times. nonessential amino acids (Gibco brl)
(1.6 ml), the whole being diluted in a 1.times. McCoy's medium
(Gibco brl).
[0170] After 24 hours of culture, only the cells having adhered to
the bottom of the dish are kept.
[0171] The proliferation assays are carried out under the same
conditions as those described for the proliferation assays on the
cultures of human gingival and dermal fibroblasts. The cell
countings are carried out after 2, 4, 7 and 10 days of culture. The
immunodetections directed against certain phenotypic markers (see
.alpha.-actin protocol) showed that these cells do not express a
marker characteristic of macrophages, CD68; a minority of these
cells express a leukocyte marker, CD45, but, on the other hand,
they express a cyto-skeletal protein characteristic of mesenchymal
cells, vimentin. Moreover, some of these cells are capable of
expressing, without stimulation, collagen type I and collagen type
III, which are characteristic of mesenchymal cells in culture.
9.2. Result
[0172] Effect of GY 785 DRS on the Proliferation of Medullary Cells
Intended to Become Mesenchymal Cells: TABLE-US-00014 Day 2 Day 4
Day 7 Day 10 Control (culture 100 100 100 100 without derivatives)
0.1 .mu.g/ml GY 785 DRS 93.6 129.4 127.8 130.4 1 .mu.g/ml GY 785
DRS 95.5 170.3 170.0 164.8 10 .mu.g/ml GY 785 DRS 79.2 134.5 155.9
179.8 100 .mu.g/ml GY 785 DRS 86.2 129.3 139.4 167.9
[0173] The proliferation of the medullary cells intended to become
mesenchymal cells is greatly stimulated in the presence of the EPS
GY 785 DRS.
* * * * *