Title: Drug and pharmaceutical composition for the
treatment of lesions of the nervous system and fractions enriched in
United States Patent: 6,573,251
Issued: June 3, 2003
Inventors: Barritault; Denis (4, rue Francaise, Paris, FR
75001); Caruelle; Jean-Pierre (32, rue Jules Joffrin, Saint Maur, FR 94100);
Aamiri; Ahmed (Block 22, N 7 Cite Hassan, Agadir, MA); Gautron; Jean (12,
rue Antoine Bourdelle, Vitry sur Seine, FR 94100)
Appl. No.: 725841
Filed: November 30, 2000
Use of at least one polymer or one biopolymer called HBGFPP, specifically
protecting the growth factors of FGFs and beta TGFs families from tryptic
degradation in the manufacture of a drug for the treatment of lesions of the
Description of the Invention
This invention relates to the use of polymers or biopolymers for the
preparation of a drug for the treatment of lesions of all origins affecting
the central or peripheral system in human or veterinary medicine. It also
concerns pharmaceutical compositions for this treatment. Lastly, it concerns
fractions enriched in heparan sulfate.
The synthesis of CMDBS polymers (dextrans substituted by carboxymethyl,
benzylamine and sulfonate) has been described in French Patent 2 461 724 and
U.S. Pat. No. 4,740,594. Some of these polymers mimic heparin and may be
used as plasma heparin replacement products, thanks to their anticoagulant
and anticomplement properties.
Some of these CMDBS polymers mimic another property of heparin consisting of
a stabilization, protection and potentialization of the in vitro biological
activity of the growth factors of the FGF family (Tardieu and coll., Journal
of Cellular Physiology, 1992, 150 pp. 194 to 203).
French patent 2 644.066 describes the use of certain CMDBSs associated with
FGFs for healing the skin and the cornea. Experiments have been conducted by
provoking a cutaneous wound with the help of a hollow punch 6 mm in diameter
in the rat. In this example, the CMDBS associated with the FGF 2 makes it
possible to obtain a clear effect on the speed and the quality of skin
Another biopolymer, dektran sulfate, has also been advanced in association
with FGFs as a stabilizer and protector, in Japanese Patent No. 13890.
Dextran sulfate, moreover, is widely used in skin healing ointments and
creams as well as in collyrium compositions, but, to the knowledge of the
applicant, has no reported effect on the healing and regeneration of lesions
of the nervous system.
A considerable number of factors whose activities facilitate the survival of
nervous cells, the repair of central or peripheral nervous lesions, or
muscular reinnervations have been described: polypeptide factors like Nerve
Growth Factors (NGF), factors of the FGF families, brain derived factors
like BDNFs, Ciliary Neurotrophic Factor (CNTF), Neurotrophin 3 (NT3), etc.
These factors have been used in experiments of muscular reinnervation,
repairs of cuts of peripheral nerves, motor nerves, in models of lesions of
cholinergic central nervous cells, and in numerous other models. For
purposes of reference, the reviews listed below describe a part of this
work: P. M. Richardson, Current Opinion in Neurobiology, 1991, 1: pp
401-406; T. Ebendal, Journal of Neurosciences Research, 1992, 32: pp.
461-470; P. G. Cordeiro, R. Brooke et al., Plastic and Reconstructive
Surgery, 1989, 86 (3): pp. 1013-1019; Q. Yan, J. Elliott et al., Nature
(Letters to Nature), 1992, 360: pp. 753-755; N. A. Seniuk, Journal of
Reconstructive Microsurgery, 8 (5): pp. 399-404; F. Hefti P. P. Michel et
al., Advances in Neurology, 1990, 53: pp. 123-127; A. C. Cuello, L. Garofalo
et al., Progress in Brain Research, 1990, 84: pp. 301-311; A. Tadeka, H.
Onodera et al., Brain Research, 1992, 569: pp. 177-180.
It thus emerges from an analysis of the state of the prior art that growth
factors and polymers in association with growth factors have already been
used in therapeutic applications.
However, none of the documents cited above shows that the polymers present
effects by themselves, that is to say without being associated with growth
Moreover, the activity of polymer-factor associations has been described
only on certain lesions of a very specific type of tissue, namely the
In view of the unpredictable nature of the therapeutic effects of a given
molecule, it was not clear whether these polymers could have an effect on
It is, in fact, well known that the different tissues of the human or animal
body present structural and functional specific features making it
impossible to predict the effect of a molecule, known for its effect on the
cutaneous tissue or another tissue.
Similarly, it is well known that it is impossible to predict the in vivo
activity of a molecule on a particular tissue from results obtained in vitro
on a specific experimental model.
Surprisingly, it has been found, according to the invention, that certain
polymers have a very marked effect on the speed of healing and of
regeneration of tissue lesions of the central or peripheral nervous systems
as well as on the quality of this healing and/or regeneration, in such a way
that it may be measured by studying it using histological and physiological
methods. Muscular reinnervation with reformation of a functional junction of
the lesioned nerve with its muscle was observed.
This invention relates to the use of at least one polymer or one biopolymer,
caller HBGFPP, with the exception of mesoglycan, specifically protecting the
growth factors of the FGF and beta TGF families from tryptic degradation and
not significantly inhibiting coagulation, in the manufacture of a drug for
the treatment of muscular tissues.
In particular, such a polymer presents an anticoagulant activity of less
than 50 international units per mg of polymer measured, according to Maillet
et al. (Mol. Immunol, 1988, 25, 915-923). Preferentially, it does not
substantially activate the complement system, that is to say, it possesses
an anti-complement system of above 0.5 .mu.g for the CH50 (according to
Mauzac et al., Biomaterials, 6, 61-63, 1985).
Advantageously, the polymer potentializes the FGFs in vitro.
According to the invention, polymers are understood to mean any natural
substance, chemically modified natural substance or totally synthetic
substance responding to the definition given above.
The following polymers are therefore concerned:
polymers obtained from dextrans but modified by other types of substitutions
with other types of radicals,
natural polymers other than those deriving from dextrans but including
osidic residues (cellulose, chitin, fucans, etc.),
polymers obtained by polymerization of monomers of non-osidic nature
(modified or unmodified malic polyacid, oxalic polyacid, lactic polyacid,
polystyrene, polyethylene glycol).
Advantageously, the said polymer or biopolymer is a polysaccharide which may
be primarily composed of glucose residues.
Such a polysaccharide advantageously presents a molecular weight above 10 kD
and advantageously about 40 kD.
It may also comprise glucosamine and/or uronic acid residues, particularly
in the form of glucosamine dimer-uronic acid.
Particularly preferred polysaccharides are substituted dextrans,
glycosaminoglycans possibly in association with a lipid, a peptide or a
protide, or sulfates of these polymers.
This invention also relates to a pharmaceutical composition containing these
The polymers and/or biopolymers may be selected from natural substances
which may then be modified, if required, by additions of appropriate
chemical groups, or again be obtained entirely by synthesis. These natural,
semi- or wholly synthetic polymers are then selected on the basis of their
ability to interact specifically with several growth factors, notably those
of the FGF and the beta TGF families. They are also selected on their
ability to protect this (or these) factor(s) against proteolytic
degradations. These polymers will be referred to under the generic
abbreviation HBGFPP (heparin binding growth factor protectors and
Two prototypes of these polymers or biopolymers are given as examples
together with the processes and selection criteria of these polymers.
The first HBGFPP example belongs to the CMDBS family which are known
products, namely functionalized biospecific dextrans, substituted by
carboxymethyl, benzylamide and benzylamine sulfonate. These polymers
illustrate the yielding of HBGFPPs from natural products (dextrans) which
are subsequently chemically substituted.
The second example describes the selection of wholly natural products such
as purified sulfate proteoglycosaminoglycans from tissular extracts.
These two examples illustrate the ability of these HBGFPPs to interact,
stabilize, protect and potentialize the growth factors of the FGF and beta
TGF families, and their use in a pharmaceutical composition permitting a
healing and a regeneration of nervous liaisons and a protection and healing
of the nervous cells.
In this patent application, by <<treatment>> is meant any curative or
preventive operation carried out for the prophylaxis, the healing, the
protection or the regeneration of lesions touching the nervous system.
Thanks to the action of the HBGFPPs and in particular the CMDBSs, as the
examples below illustrate, the reinnervation of the EDL or Soleus type
muscle is accelerated. This reinnervation is manifested by the regeneration
of the nervous fiber and the speedy reformation of a functional synaptic
Not only is there an increase of axonal growth in lesions near the muscle
but also a control of this growth. Thus, with respect to sprouting, this
growth takes place in an organized and directed way resulting in an
acceleration of functional repair.
The properties of HBGFPPs are such as to make this family of molecules a
totally new and unique class of drugs which may be used to favor and improve
lesions of the central or peripheral nervous system, directly touching the
neuronal cells and their axonal and dendritic prolongations, cholinergic or
dopaminergic neurons or again touching cells associated with neurons such as
oligodendrocyte astrocyte glia cells and Schwann cells. These lesions may be
of any origin: traumatic, iatrogenic or chemical, due to the use of
radiation or induced by surgical operations, of bacterial, parasitic or
viral infectious origins, of auto-immune origin, or lesions and
deteriorations may be induced by bleedings such as ruptured vessels. These
new drugs are also used in the treatment of neurodegenerative diseases such
as Parkinson's or Alzheimer's disease, or of genetic origin. Lastly, these
drugs may with benefit be associated with cells used in treatments of
transplantations in the affected areas of the brain, using normal or
genetically modified cells.
The drug and the pharmaceutical composition according to the invention may
contain an effective quantity of HBGFPP, for example CMDBS associated with
one or more compatible and pharmaceutically acceptable vehicles. It may also
be associated with pharmaceutical agents such as anti-inflammatory agents
The vehicle may be physiological serum or buffers such as PBS containing
0.15M NaCl or any other compatible solution which does not irritate the
damaged nervous tissues. Formulations providing thick or gel solutions
according to standard techniques known to the person of ordinary skill in
the art may be proposed depending on the type and the accessibility of the
Advantageously, such a composition is designed to be injectable directly on
the site of the lesion at a dose of 2.5 to 2500 mg/ml of HBGFPP as
exemplified by CMDBS, or like natural HBGFPP biopolymers such as mesoglycan,
but the intravenous or intramuscular route may be preferred in the case of
de-innervation of de-innervated muscle. Injection in the spinal cord may
also be preferable in the case of lesions of said spinal cord or of lesions
of branches of prolongations of nervous fibers being motor and/or sensitive
fibers. The injection volume is estimated in function of the size of the
lesion. Doses corresponding to 100 .mu.l often prove sufficient.
In addition to the "Heparin Binding" growth factor protection qualities, the
HBGFPPs selected according to the tests described below present a very low
anticoagulant activity compared to that of heparin, too weak to hamper
coagulation in the case of a trauma. In the case of an injection by
intravenous route, the injected dose must be adjusted to the blood volume of
the man or animal treated in this way so that the dose of HBGFPP in the
blood also lies between 2.5 and 2,500 mg/ml.
In the examples described in the following pages, concerning the
reinnervation of the skeletal muscle of the male EDL-type rat (Extensor
Digitorum Longus) or the postural slow muscles (soleus), a single injection
of 100 .mu.l of a solution of CMDBS at 50 .mu.g/ml on the site of the wound
induces a complete reinnervation in 17 days, whereas, in the case of the EDL
muscle, this innervation is observed only after 60 days when no treatment is
given. With regard to the soleus muscles, the CMDBS effect is even more
pronounced since, after injection of CMDBS, reinnervation is total and
functional in 17 days whereas the reinnervation of the controlateral muscle
is defective even after 60 days. These effects are specific to the HBGFPPs
and notably certain CMDBSs responding to selection criteria with respect to
FGFs and beta TGF family growth factor protection against proteolytic
degradations induced by the action of trypsin. This specificity may be
illustrated by comparing the effects of repairing nervous tissue lesions by
HBGFPPs and related products such as heparin, dextran, dextran sulfate or
sucrase (sucrose octyl sulfate). Although these molecules interact with the
FGFs, and at any rate as far as heparin is concerned with beta TGF, neither
sucrase, nor heparin nor dextran sulfate protect the beta TGF against the
proteolysis induced by the action of trypsin, as is shown by the application
of screening and selection tests of the HBGFPPs described in the examples
below. These products have no effect on lesions of the nervous tissues.
Thus, by carrying out in vitro screening on the basis of a double protection
of the FGFs and beta TGFs against the proteolysis induced by Trypsin, it is
possible to select HBGFPPs, like certain CMDBSs including those given in
these examples. These same selection criteria applied to natural biopolymers
such as mesoglycan or sulodexide have shown that mesoglycan, which presents
a double protection and stabilization activity for both FGFs and beta TGFs,
has a beneficial activity in nervous repair and regeneration, and, as such,
belongs to the HBGFPP family, whereas sulodexide, which protects the FGFs
against the proteolysis induced by the activity of trypsin, has no
significant protective action against the action of trypsin on beta TGFs.
This invention also relates to fractions of mesoglycan or sulodexide
enriched in heparan sulfate, presenting advantageously 80%, and preferably
95%, of heparan sulfate.
Such fractions may be obtained by a heparan sulfate enrichment process of a
glycosaminoglycan composition, comprising the following stages:
ion exchange chromatography of the composition,
elution on a DEAE gel,
treatment by ABC chondroitinase,
chromatography on molecular sieve, and
heparan sulfate elution.
Claim 1 of 4 Claims
What is claimed is:
1. A method of treating an amenable lesion of the nervous system, which
comprises administering, to a subject in need of such treatment, an
effective amount of an active ingredient which consists of a polymer or
biopolymer, called heparin binding growth factor protector and promoter (HBGFPP),
specially protecting the growth factors of families of FGFs and beta TGFs
from tryptic degradation and having an anticoagulant activity of less than
50 International units per mg of polymer, wherein said polymer consists of a
polymer obtained from a dextran which has been chemically modified, and
wherein said polymer is associated with at least one pharmacologically
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