Note: Descriptions are shown in the official language in which they were submitted.
CA 02655901 2008-12-22
Collagen product for medical or cosmetic applications, method for the
manufacture thereof
and use thereof
Field of invention
The invention relates to a collagen product for medical or cosmetic
applications, such
as healing or haemostatic dressings, implantable separation barriers,
sheets/foils and
membranes for guided tissue and bone regeneration, solid supports or
injectable carriers of
pharmacologically or cosmetically active substances or cosmetic products and
face masks.
Furthermore, the invention relates to the procedure of manufacture of said
collagen product
and uses thereof.
Background and prior art
Cosmetic, healing and haemostatic products, separating barriers and substrates
for
pharmacologically active substances are produced by using various polymers
which exhibit
appropriate compatibility in contact with live tissue and are biodegradable
within a reasonable
period of time.
Collagen is one of the important substances coming into consideration in this
respect:
it is very well tolerated by live tissue ¨ especially if the collagen has been
freed from
telopeptides (by the procedure described in Czech patent CZ 276891 for
instance) ¨ and
possesses a significant haemostatic activity and promotes the healing process.
In some cases,
however, the rate of resorption of native collagens is too high and should be
slowed down to
provide the desirable effect. To this end, collagen crosslinking through
transverse
intramolecular covalent bonds by using aldehydes was practised. Eventually,
however, such
procedures were abandoned because, as it turned out, implanted into live
tissue, collagens
crosslinked with aldehydes undergo calcification and are slowly converted to
unwanted hard
mineralized formations.
Crosslinking with chromium salts is a proven routine procedure used to extend
the
time of resorption of surgical collagen threads, such as cat gut. The use of
metal salts,
especially salts of chromium, aluminium, zirconium and titanium, for
crosslinking of leather
CA 02655901 2008-12-22
collagen is a well-known practice, widely used in the leather industry.
Ultraviolet (UV)
radiation is also sometimes used to achieve crosslinking of collagen, the
resistance of the
exposed collagen substance to degradation by collagenases, however, is thereby
only slightly
increased, and, as a drawback, free radicals which can degrade various active
substances
(depolymerize glucosaminoglycans for instance) are formed. Various reactive
organic
reagents, such as isocyanates, vinyl compounds, or carboimides, are also used
in
pharmacological practice. Such procedures, however, introduce foreign
substances into the
tissue, and such substances are alien to the body, their metabolism is not
quite clear, and side
effects are conceivable. Toxicity of the unreacted residues or products
emerging during a slow
release of the organic crosslinking agents is another adverse phenomenon.
Numerous attempts were made to introduce various bioactive substances into the
collagen matrix. Among important substances of this kind are
glucosaminoglycans,
hyaluronan in particular, which support wound healing. A product which
contains hyaluronan
and whose average molecular weight was increased by introducing chelated
polyvalent metal
cations is described in CS 264719.
A complex of gelatine and two glycosaminoglycans ¨ hyaluronan and chondroitin
sulfate ¨ has been prepared (see Bychov S.M., Nikolajeva S.S., Charlamova V.N.
in: Biull.
Eksp. Biol. Med., 1976; 82(10), p. 1211-1213). It is especially hyaluronan
that supports
migration and proliferation of skin fibroblasts.
Mixed products with hyaluronan and native collagen have been the subject of
many
patents. For example, Czech patent application PV 1991-2025 covers mixtures of
native
collagen and hyaluronan crosslinked by using metal complexes without any
coordination
bonds with collagen (although ionic bonds between the two types of biopolymer
are
conceivable). Native collagen contains antigenic determinants and thus gives
rise to immune
reactions in human body (human collagens being rarely used in practice). The
difference in
the diffuse mobility between the two components is another drawback.
Hyaltironan is a well-
soluble biopolymer and thus diffuses into wounds much faster that the low-
soluble collagen.
As a consequence, the composition of the product varies rapidly during the
healing process.
The solution covered by French patent No. 2 585 576 uses a collagen-
hydroxyapatite-
hyaluronan mixture, where the occurrence of a very weak ionic bond between the
biopolymers, conveyed by calcium ions, is possible. However, due to the nearly
negligible
phosphate dissociation, this bond is actually unimportant. A procedure has
been proposed to
increase ionic interactions between collagen and glycosaminoglycans via
deacetylation of the
naturally occurring acetylated amino groups (see EP 0 640 647). Although
lowered to some
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CA 02655901 2008-12-22
extent by this procedure, the diffusability of hyaluronan still remains higher
than that of the
collagen components, because the weak ionic interactions are unable to prevent
changes in the
composition of the medical product during the healing process. And as an
adverse
consequence, the deacetylation of one of the two major saccharide components
brings about
occurrence of an unnatural component in the metabolism patterns of the
resorbed hyaluronan.
Similarly, deacetylation has been applied in support to ionic interactions in
a mixture
of collagen, chitosan and hyaluronan (EP 0 296 078). The drawbacks are the
same as in the
preceding procedure and moreover, another substance which is foreign to mammal
tissues is
thereby introduced into the metabolism (in fact, chitosan is a polysaccharide
prepared by
deacetylation of chitin, a substance present in the outer skeleton of
crustaceans).
US patent 5 470 911 relates to a conjugate of collagen and hyaluronan formed
by
bonds of synthetic polymers. This approach addresses the drawback of solutions
based on
mixtures of biopolymers, i.e. slow removal of glucosaminoglycans from the
collagen or
gelatine matrix due to which implants or dressings lose their activity. In
this solution,
however, the metabolism accompanying the wound healing process is contaminated
by
foreign substances, i.e. components of synthetic polymers and their reaction
products with
collagen aminoacids and also with saccharide components of hyaluronan.
Although side
effects of such compounds, if any, are not clear, the carcinogenic effect of
unsaturated
monomeric compounds is well known.
In order to improve the stability of the composites, coupling of the two
biopolymers
by chemical bonds has been proposed using various synthetic organic reagents,
such as
hexamethylene diisocyanate (see, e.g., Bakog D., Jorge-Herrero E., Koller J.
in Polim Med.
2000, 30 (3-4), 57-64), or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (see
Hong S.R.,
Chong M.S., Lee S.B., Lee Y.M., Song K.W., Park M.H., Hong S.H. in J. Biomater
Sci
Polym Ed. 2004; 15(2):201-214 and others). Although this results in an
increased stability of
the composites, the problem of potential side effects of the metabolites
emerging from the
foreign organic crosslinking agents remains open, and so does the problem of
toxicity of the
vinyl derivatives and carcinogenicity of the unreacted residues of the
crosslinking agents.
Description of the invention
The drawbacks described above are alleviated by use of the collagen product
for
medical or cosmetic applications claimed. In the invention, the product
contains biopolymers
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of the group of glycosaminoglycans, bonded to the collagen substance, partly
at least, by
strong non-ionic chemical bonds mediated by polyvalent metal cations.
The chemical bonds can be mediated with advantage by coordinately bonded
compounds of metals chosen from the group of transition metals, particularly
Cr, Zr, Al, Fe,
Mn or Ti, and/or by polyvalent cations of alkaline earth metals, especially
Ca, Mg, Zn, Ba or
Sr.
The chemical bonds of the collagen and biopolymeric substance mediated by
polyvalent metal cations can be combined with bonds of organic crosslinking
reagents, such
as monomers, isocyanates, aldehydes or tannins.
Such procedures aimed to achieve a strong link between biopolymers are
outcomes of
research into the mechanism of biopolymer crosslinking and grafting through
complexing
metal bonds. Polyfunctional cations of polyvalent metals have been found to be
able to form
strong non-dissociated coordination bonds and metal-complex bridges which bond
collagen to
glucosaminoglycans through strong and stable chemical bonds. Such combination
reactions
are referred to as interpolymeric crosslinking, grafting, or copolymerization.
Analytical
evidence of such a process is provided by decrease in the content of
extractable
glucosaminoglycan from collagen composites grafted by the metal-complex
mechanism
(Table 1 in the Examples). It is also evident from the data that the strength
of the coordination
bond is affected appreciably by the cation used and that the choice of this
cation can be
vehicle to control the time of resorption of the product in the live tissue.
For instance, the
stability of the composites was highest and the extractable glucosaminoglycan
content was
lowest when using basic chromium compounds.
Since the polyvalent metal cations which constitute the new inorganic grafting
reagents are also constituents of compounds which are natural components of
animal cells and
tissues, their metabolism is largely known and can be analytically monitored.
It was also
found within the studies that in certain circumstances, glycosaminoglycans in
collagen inhibit
ossification of thigh tendons in turkeys (see Fig. 1 in the Examples). Thus
the collagen-
glucosaminoglycan complex helps address the problem of unwanted calcification,
encountered when using aldehydically crosslinked collagens.
Making use of those findings, particularly the ability of carboxy groups in
collagen
and carboxy groups of glucuronic acid in hyaluronan to form strong
coordination bonds with
atoms in hydrated basic compounds of polyvalent metals, both of the two major
drawbacks of
the procedures known so far can be addressed.
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The new procedure eliminates the unwanted calcification tendency of
aldehydically
crosslinked collagen in implants and, in particular, represents a new way to
obtain
physiologically safer transverse bonds, both intermolecular and
intramolecular; this is of
importance especially in the case of pharmaceutical and cosmetic composites.
Instead of by
organic reagents which are foreign to the tissue, the crosslinking and
grafting reactions are
mediated by inorganic ions which are naturally encountered in animal
physiology and
metabolisms. Polyvalent metal ions which the body needs, i.e. essential ions,
and/or ions
which are often deficient in the metabolism are the preferred choice for this
purpose.
Example of embodiment of the invention
In this model embodiment of the invention, the product contains collagen and
biopolymeric glucosaminoglycan which is bonded to the collagen protein
structure by a
coordination bond involving metal ions via carboxy groups. The term
glucosaminoglycan is
understood as encompassing a group of biopolymers which repeatedly contain
glucosamine
bonded by a glycoside bond to a simple acidic sugar (glucuronic acid in the
case of
hyaluronan). The product can contain auxiliary substances to control
viscosity, moisturizing
substances, binders, softeners, penetration accelerators, preservatives,
disinfectants, pH
buffers, antioxidants, active substances stabilizers, oils, fats, waxes,
emulsifiers, fragrances,
dyes and/or inert fillers.
In this model embodiment, in a product is collagen type I, freed from
telopeptides
(partly at least), and possessing a structure which is not crosslinked to a
significant extent. It
contains bonded sodium hyaluronan and the coordination bond involves magnesium
salts.
The collagen product can be in the form of a powder, microparticles, fibres,
flakes,
foam, felt-like matter, sponge, needles, rods, tablets, gel, viscous solution,
creams, films or
laminates.
The collagen product manufacturing process in this model embodiment includes
drying in a spray dryer, lyophilization, coating or casting followed by
drying, steps of
separation and phase precipitation, filling into vessel including pressure
vessels for obtaining
foams or aerosols.
So far, collagen products containing bonded glycosaminoglycans have been
usually
prepared by crosslinking and covalent linking of the biopolymeric components
by using
organic agents containing isocyanate, vinyl, or carboimide groups, or by
applying UV
radiation. In the product based on the invention, on the contrary, the linking
and/or
crosslinking of the biopolymers is achieved through a metal-complexing
reaction where the
CA 02655901 2008-12-22
carboxy groups in the biopolymers act as donor ligands and hydrated ¨
preferably
hydroxylated ¨ polyvalent metal cations act as acceptors of the electron pairs
of the carboxy
group, whereby a highly stable coordination bond is formed. Since proteins
contain a large
number of free carboxy groups belonging to side chains of the aminoacid parts
of aspartic and
glutamic acids, the reaction proceeds so rapidly that it is appropriate to
retard it by lowering
the pH, which tends to suppress the dissociation and reactivity of the carboxy
groups. This
pathway gives rise to transverse intermolecular bonds as well as to
extramolecular "grafting",
i.e. mutual linking of polymeric chains of the various biopolymers. The degree
of transverse
collagen crosslinking can be controlled in a simple manner through the amount
of the bonded
metal complexes and acidity of the reaction medium. If the reaction pH is
below approx. 2.0,
a single-point bond of the metal ions is largely formed and collagen
crosslinking is nearly nil;
this is also evident from the fact that its denaturation temperature does not
increase. A low
degree of crosslinking, resulting in an increase in the denaturation
temperature of collagen
conversion to gelatine by 1 C to 5 C, is suitable for pharmaceutical and
cosmetic purposes.
Decisive in the formation of coordinately bonded glucosaminoglycan is the
linking
bond of the polyftinctional acceptor cation of the polyvalent metal via
carboxy groups of the
acidic saccharide units and aminoacids, glutamic and aspartic acids, in
collagen. The metal
salt solution should be high purity and low in neutral salts. The reaction
usually occurs in a
weakly acid medium and is accompanied by increase in viscosity and, in case of
high-degree
crosslinking, by the formation of a fibrous precipitate. The reaction of
collagen containing
bonded glucosaminoglycans with an additional excess of collagen is a next
important step.
This is preferably conducted by slowly adding a suspension of the complex
already formed to
a stirred colloid solution of native collagen. At the beginning, precipitation
of the reaction
mixture takes place. Additional portions, however, bring about gradual
dispersion leading to a
viscous colloid solution.
Differences in the degree of collagen crosslinking, bonded hyaluronan content
and free
collagen (if any) affect markedly the properties of the collagen product. For
instance, they
affect the stability of a gel or foam and rate of implant resorption. The
properties of the
product can be controlled smoothly within wide limits (taking into account
requirements
dictated by the intended use of the product) by a suitable choice of the type
of complexing
metal ions, hyaluronan content, and degree of crosslinking.
The collagen product in the model embodiment of the invention finds use
primarily as
a resorbable haemostatic with an extended resorption time, as implantable
resorbable barriers
for guided bone and tissue regeneration, for tissue separation and for forming
adhesion
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barriers during surgeries, as healing dressings for the treatment of skin
lesions and as
superficial or implantable supports for pharmacologically active substances,
enabling a
gradual and guided control of the active substance release kinetics.
Active substances administered by using the collagen product according to the
invention include but are not limited to: antibiotics, antiseptics,
anaesthetics, analgetics,
cytostatics, hormones, steroids, cytokines and stimulators, hormone-releasing
and release-
inhibiting factors, prostaglandins, enzymes, and growth and osteoinductive
factors.
Fields of application of the invention
The collagen product claimed is applicable to skin in the form of cosmetic
products,
foam masks, or films for aged, wrinkled or unclean skin care, and for care and
protection of
skin exposed to harsh environments or radiation.
The collagen product can contain the following ingredients:
- moisturizing agents, such as glycerine, sorbitol, polyetylene glycol,
polypropylene glycol
and free g lyco sam in og lycans;
- softening agents, such as citric acid esters, tartaric acid esters and
glycerinic acid esters;
- preservatives, such as cresol derivatives, phenyl ethyl alcohol, phenoxy
ethyl alcohol,
chlorobutanol, hydroxybenzoic acid methyl and ethyl or propyl ester,
benzalconium chloride,
cetylpyridinium chloride, chlorhexidine diacetate or digluconate, ethanol,
isopropanol or
propylene glycol.
- disinfectants, such as iodine, bromine, polyvidone-iodine, halo compounds
such as
sodium hypochlorite, sodium tosyl chloride, oxidants such as hydrogen
peroxide, potassium
permanganate, arylmercury and alkylmercury compounds, organotin compounds such
as tri-n-
butyl-tin-benzoate, silver compounds such as silver acetylstannate, alcohols,
phenols, and
organic nitrogen compounds such as 8-hydroxyquinoline, chloroguinadol,
clioquinol,
ethacridine, hexetidine, chlorhexidine and ambazone;
- pH buffers, such as citrate buffer, borate buffer, phosphate buffer and
mixtures thereof;
- antioxidants, such as ascorbic acid, ascorbyl palmitate, tocopherol acetate,
propyl
galactate, butylhydroxyanisol and butylhydroxytoluene;
- active substance stabilizers, such as mannitol, glucose, lactose,
fructose and sucrose;
- emulsifiable ingredients such as oils, fats and waxes;
- fragrances, dyes, cleansing agents, skin care products;
- emulsion stabilizers, such as non-ionic emulsifiers, amphoteric, cationic
and anionic
surfactants;
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- fillers, such as microcrystalline cellulose, aluminium oxide, zinc oxide,
titanium oxide,
talc, silicon oxide, magnesium silicate, aluminium magnesium silicate, kaolin,
starch
derivatives, zinc or calcium stearate and calcium phosphate.
The active substances and other ingredients can be introduced into the claimed
product
by dissolution or dispersion in the final dispersion of collagen with the
bonded hyaluronan
prior to the forming process. If more than one substance is used, some of the
substances can
be introduced following the forming process, viz. by coating, spraying,
impregnation,
submerging, or by other absorption processes.
If the collagen product claimed is used for dermal, intradermal or transdermal
application of pharmaceutically active substances or cosmetically active
substances, flat forms
are preferred, such as foils/sheets, membranes, foams or sponges. Such flat
forms can possess
a laminate structure, consisting of separating layers with no active
substance, permeable
separating layer, guiding membrane, and a layer of an adhesive. Such single-
layer or
multilayer collagen products are preferably provided with a back layer and, on
the other side,
with a removable protective layer. Both the back layer and protective layer
can consist of
conventional materials such as are used in the manufacture of sticking
plasters and sticking
tapes.
In the embodiment of the collagen product according to the invention, intended
for
superficial wounds or for in-body applications, the product is porous, e.g.
foamy, felt-fibrous
or spongy. The pore size and structure of the product is controlled so as to
enable cells, e.g.
fibroblasts or osteoblasts, to penetrate into the structure of the product and
to take a
orientation similar to that of the collagen ligament tissue.
8
CA 02655901 2011-06-10
=
Table 1: Amount of water-extractable hyaluronan in 500 mg of the collagen
composite.
(The table documents the decrease in the extractable glucosaminoglycan content
from metal-
complex grafted collagen composites)
Amount of hyaluronan (mg)
__________________________________________________ r -
Amount of hyaluronan added to 0 25 50 100
a gel with 500 ui_g_919.2.1.1Ageja __
Extraction yield from a non- 0 22 47 - 93
cross-linked sample
Extraction yield from a -sample
cross-linked with Mg2+ (10 mg 0 0 8 14
MgO)
Extraction yield from a ,sample
cross-linked with Cr3+ (10 mg 0 0 0 7
Cr203)
Fig, 1: Colorimetrically determined hyaluronan content along a turkey thigh
tendon
(The figure documents how significantly the region of inhibited ossification
correlates with the
. presence of hyaluronan in the collagen ligament).
= c 6: T
es
"' = V7,4
t'
= 43
T-7 2
a.
ij,M.JF;':=,.'4ftch" %.:,P1:111i,LILL.,õ.%'011%.14;;Yt SIVA
Non ()SS I tied p t
=
9
