Title of Invention

ANTIOXIDANT WOUND DRESSING MATERIALS

Abstract A wound dressing material comprising a solid bioabsorbable substrate wherein the substrate is selected from a freeze-dried and solvent-dried sponge and comprises solid bioabsorbable material which is dyed with an antioxidant dyestuff and which is selected from the group consisting of oxidized regenerated cellulose, collagen, chitosan or mixtures thereof.
Full Text ANTIOXIDANT WOUND DRESSING MATERIALS
The present invention relates to antioxidant wound dressing materials, processes
suitable for the preparation of such materials, and to the use of such wound
dressing materials.
Concentrations of reactive oxygen species such as hydroxyl radicals (-OH), singlet
oxygen (1O2), hydroperoxyl radicals (-OOH), superoxideradical anions (-O2-). and
hydrogen peroxide (H2O2) can rise in damaged tissues, producing a condition
known as oxidative stress. The presence of a low level of reactive oxygen species
may be advantageous in the early stages of wound healing by both attracting and
activating macrophages which engulf and kill bacteria and release cytokines and
growth factors. However, prolonged and more severe oxidative stress may delay
healing because it will produce chronic inflammation, divert available energy
supply towards antioxidant defence at the expense of tissue reconstruction, and
increase levels of matrix metalloproteinases which cause tissue breakdown. In
more severe cases, elevated levels of reactive oxygen species can give rise to
hydrogen peroxide-induced senescence or apoptosis (that is, programmed cell
death) or tissue necrosis (that is, uncontrolled cell death and therefore permanent
tissue damage).
Under mild oxidative stress, it is thought that hydrogen peroxide (H2O2) is the
dominant species present, being formed rapidly from superoxide by the enzyme
superoxide dismutase. This enzyme-mediated dismutation reaction also
minimises the production of singlet oxygen that can arise when superoxide is
produced too rapidly and therefore has the opportunity to dismutate spontaneously
without enzyme assistance. Rapid enzyme-mediated dismutation of superoxide
also minimises levels of hydroperoxyl radical, the unionised form of superoxide.
Levels of hydrogen peroxide are in turn kept low by the actions of catalase and
glutathione peroxidase. Thus, under mild oxidative stress conditions when
hydrogen peroxide levels are slightly raised (around 10-8 to 10-4 molar), it has been
found that the rate of cell proliferation in fibroblast cultures is stimulated.

Accordingly, the healing of chronic wounds may be assisted by the use of
antioxidant wound dressings that react specifically with excess reactive oxygen
species such as those listed above and hence reduce the level of oxidative stress.
• US-A-5667501 describes compositions comprising chemically modified polymers
grafted with chemical groups that confer antioxidant activity as measured by a
diphenylpicrylhydrazyl (DPPH) test and that also generate low levels of hydrogen
peroxide by reaction with molecular oxygen in the wound bed to stimulate
macrophage activity and fibroblast proliferation. The compositions may be used to
promote the healing of chronic wounds. Preferably, the polymer is a polymer
bearing hydroxyl, carbonyl or amide functional groups, or a polysaccharide bearing
hydroxyl functional groups, said functional groups having been converted to
derivatives that are persistent free radicals or precursors of persistent free
radicals, that is to say they are free radical scavenging antioxidant groups.
US-A-5612321 describes compositions comprising polysaccharides grafted with
antioxidants on at least one hydroxyl group of the polysaccharide. The
compositions may be used inter alia to promote the healing of chronic wounds.
Preferably, the polysaccharide is hyaluronic acid and the antioxidant group
comprises a phenol group.
The above antioxidant wound dressing materials are made by multi-step chemical
reactions to achieve covalent bonding of antioxidant moieties, such as
hydroquinones or benzimidazole derivatives, to the polymeric substrate materials.
A need remains for a more simple and inexpensive route to antioxidant wound
dressing materials.
Wound dressings that contain antimicrobial agents for the treatment of wound
infection are also known. Currently preferred antimicrobial agents for use in
wound dressings are certain antiseptics such as chlorhexidine and triclosan, and
silver, whether in the form of thin films, nanoparticles, or colloidal silver. Chemical
compounds of silver are also useful as antimicrobials. For example, the following
complex silver salts are favored for use against sensitive and resistant bacterial

strains: silver sulfadiazine, silver norfloxacinate, silver pipemidate, kaolin silver,
silver EDTAate, silver thiosalicylate and silver imidazolium chloride.
WO91/11206 describes the use of silver alginate salts in wound dressings.
WO87/05517 describes silver salts of hyaluronic acid that may be used as or in
antimicrobial wound dressings. These materials tend not to be stable in the
presence of light. The silver undergoes photochemical reduction to metallic silver,
causing a darkening of the materials over time.
WO02/43743 describes light-stabilized antimicrobial wound dressing materials in
which silver salts are stabilized by the addition of a photostabilizer selected from
the group consisting of ammonium salts, thiosulfates, metal chlorides and
peroxides. WO97/02038 describes light-stabilized antimicrobial wound dressing
materials in which silver salts are stabilized by the addition of a polyether polymer.
Such photostabilizers are of limited effectiveness, and will tend to be extracted
from the dressing material by wound fluid.
A need therefore remains for improved antimicrobial dressings containing light-
stabilized silver compounds.
GB-A-619165 describes calcium alginate fibers for use as wound dressings. A
bacteriostatic or antiseptic compound, such as an acridine derivative or eusol, may
be applied to the filaments after they are formed, or may be added to the solution
from which the filaments are drawn. Calcium alginate is not bioabsorbable.
EP-A-0368253 describes chitosan films, rods, sheets, medicated bandages,
patches and the like that have been fabricated from solutions or mixtures of certain
chitosan derivatives in combination with antimicrobial agents. There is no
disclosure of dyeing a bioabsorbable wound dressing material with an antioxidant
dye.
In a first aspect, the present invention provides a wound dressing material
comprising a solid bioabsorbable substrate dyed with an antioxidant dyestuff.

The term "dyed" refers to a solid material that has been surface-treated while in
the solid state with a dye to bind the dye to the surface thereof. That is to say, a
solid material that has been subjected to post-treatment with a dye after
solidification. It has been found that bioabsorbable substrate materials such as
oxidized regenerated cellulose have excellent avidity for antioxidant dyes such as
aniline and acridine dyes. This enables controlled amounts of the dyes to be fixed
onto the substrate materials in a simple and inexpensive dyeing step. It has
further been found that the resulting dyed materials retain the antioxidant
properties of the dyestuff, thereby making them excellent candidates for the
treatment of chronic wounds and other wounds characterised by elevated levels of
oxygen free radicals. The materials also have useful antimicrobial properties, in
particular against gram-positive and sometimes also gram-negative bacteria. The
gradual breakdown of the bioabsorbable material in the wound achieves sustained
release of effective amounts of antimicrobial.
The term "bioabsorbable substrate material" refers to a solid material that is fully
degraded and absorbed in vivo in the mammalian body. The term therefore does
not encompass cellulose or conventional textile materials. The substrate material
is usually not water soluble, but it may be water swellable. In certain
embodiments, the substrate comprises (and may consist essentially of) a solid
bioabsorbable material selected from the group consisting of collagens, chitosans,
bioabsorbable cellulose derivatives such as oxidized celluloses, galactomannans
such as guar/borate, glycosaminoglycans such as cross-linked hyaluronates,
polylactides/polyglycolides, polyhydroxybutyrates, and mixtures thereof.
In certain embodiments the substrate comprises (and may consist essentially of) a
solid bioabsorbable material selected from the group consisting of collagens,
chitosans, oxidized regenerated celluloses, and mixtures thereof.
Oxidized cellulose is produced by the oxidation of cellulose, for example with
dinitrogen tetroxide. This process converts primary alcohol groups on the
saccharide residues to carboxylic acid group, forming uronic acid residues within

the cellulose chain. The oxidation does not proceed with complete selectivity, and
as a result hydroxyl groups on carbons 2 and 3 are occasionally converted to the
keto form. These ketone units introduce an alkali labile link, which at pH7 or
higher initiates the decomposition of the polymer via formation of a lactone and
sugar ring cleavage. As a result, oxidized cellulose is biodegradable and
bioabsorbable under physiological conditions.
The preferred oxidized cellulose for practical applications is oxidized regenerated
cellulose (ORC) prepared by oxidation of a regenerated cellulose, such as rayon.
It has been known for some time that ORC has haemostatic properties, and that
application of ORC fabric can be used to reduce the extent of post-surgical
adhesions in abdominal surgery.
The oxidized regenerated cellulose (ORC) can be obtained by the process
described in US-A-3122479, the entire content of which is incorporated herein by
reference. This material offers numerous advantages including the features that it
is biocompatible, biodegradable, non-immunogenic and readily commercially
available. ORC is available with varying degrees of oxidation and hence rates of
degradation. The ORC may be used in the form of insoluble fibers, including
woven, non-woven and knitted fabrics.
In certain embodiments, the oxidized cellulose is in the form of particles, such as
fiber particles or powder particles, preferably dispersed in a suitable solid or
semisolid topical medicament vehicle. In particular, the materials preferably
contain ORC fibers, wherein a volume fraction of at least 80% of the fibers have
lengths in the range of 20pm to 1000pm. Such a size distribution can be
achieved, for example, by milling an ORC cloth, followed by sieving the milled
powder to remove fibers outside the range. Preferably, the average (mean by
volume) length of the ORC fibers is in the range 250pm to 450pm. The selection
of ORC fiber lengths in this range results in easy mixing of the ORC and other
components, and highly homogeneous products. The ORC is more thoroughly
complexed with the other components, which results in enhanced therapeutic
properties of the sponge.

Preferably, the oxidised cellulose has an average molecular weight greater than
50,000. Such oxidised cellulose is substantially insoluble in wound fluids, but will
undergo very gradual breakdown into bioresorbable fragments at physiological pH.
The oxidized cellulose may be in its free acid form, or it may be neutralized. For
example, the present invention encompasses the use of partially or completely
neutralised materials as described in EP-A-0437095, the entire content of which is
incorporated herein by reference. For example, the ORC may be partially
neutralised by a silver salt of a weak acid, such as silver acetate, as described in
more detail below.
In certain embodiments of the present invention, the oxidized cellulose is
complexed with collagen and/or chitosan to form structures of the kind described
in WO98/00180, WO98/00446, EP-A-1153622 or WO2004/026200, the entire
contents of which are expressly incorporated herein by reference. For example,
the oxidized cellulose may be in the form of milled ORC fibres that are dispersed
in a freeze-dried collagen sponge. This provides for certain therapeutic and
synergistic effects arising from the complexation with collagen.
Where used, the collagen in the materials of the present invention may be any
collagen, including Type I, Type II or Type III collagen, natural fibrous collagen,
atelocollagen, partially hydrolysed collagens such as gelatin, and combinations
thereof. Natural fibrous collagen, for example of bovine origin, is suitable. For
example, the collagen prepared from bovine hide is a combination of Type I
collagen (85%) and Type III collagen (15%).
Where used, the chitosan in the materials of the present invention is usually
derived from chitin. Chitin is a natural biopolymer composed of N-acetyl-D-
glucosamine units. Chitin may be extracted from the outer shell of shrimps and
crabs in known fashion. The chitin is then partially deacetylated, for example by
treatment with 5M-15M NaOH, to produce chitosan. Complete deacetylation of
the chitin is not a practical possibility, but preferably the chitosan is at least 50%
deacetylated, more preferably at least 75% deacetylated. Chitosan has been

employed for wound treatment in various physical forms, e.g. as a solution/gel;
film/membrane; sponge; powder or fiber. Chitosan in the free base form is
swellable but not substantially soluble in water at near-neutral pH, but soluble in
acids due to the presence of ammonium groups on the chitosan chain. The
solubility of the chitosan may be reduced by cross-linking, for example with
epichlorhydrin. Typically, the average molecular weight of the chitosan as
determined by gel permeation chromatography is from about 105 to about 106.
In particular embodiments, the substrate comprises (and may consist essentially
of) a mixture of: (a) collagen and/or chitosan; and (b) oxidized regenerated
cellulose, for example in a dry weight ratio range of from about 90:10 to about
10:90 of collagen/chitosan:ORC, preferably from about 75:25 to about 25:75, and
particularly from about 60:40 to about 40:60.
The wound dressing materials according to the present invention may also contain
a silver salt. Some of the silver may be present as metallic silver, but preferably a
major part of the silver is present as a silver salt, preferably as a substantially
colorless silver salt. Preferably, the silver in the material consists essentially of
silver salts, more preferably as substantially colorless silver salts. Preferably, the
amount of silver (as silver ions and metallic silver) in the materials according to the
present invention is from about 0.01 wt% to about 5wt.%, more preferably from
about 0.1 wt% to about 2wt.%, and most preferably about 0.1wt.% to about 1wt.%,
most preferably about 0.3wt.%. Lesser amounts of silver could give insufficient
antimicrobial effect. Greater amounts of silver could give rise to antiproliferative
effects on wound healing cells.
The silver may be introduced by treating a bioabsorbable substrate material with a
silver salt or compound dissolved or dispersed in water or an organic solvent such
as ethanol, for example as described in WO02/43743. Suitable compounds
include silver oxide, silver chromate, silver allantoinate, silver borate, silver
glycerolate, silver nitrate, silver acetate, silver chloride, silver sulfate, silver lactate,
silver bromide, silver iodide, silver carbonate, silver citrate, silver laurate, silver

deoxycholate, silver salicylate, silver p-aminobenzoate, silver p-aminosalicylate,
and mixtures thereof. Preferably, the silver is not present as silver sulfadiazine.
In preferred embodiments, the silver may be complexed to the substrate material.
The term "complex" refers to an intimate mixture at the molecular scale, preferably
with ionic or covalent bonding between the silver and the polymer. The complex
preferably comprises a salt formed between an anionic polymer and Ag+. Suitably,
the anionic polymer is a polycarboxylate or a polysulfated polysaccharide.
Suitably, the anionic polymer comprises an anionic polysaccharide. Suitable
anionic polysaccharides include hyaluronates, pectins, carrageenans, xanthan
gums, sulfated polysaccharides such as dermatan sulfate or sulfated dextrans,
and oxidized celluloses.
The complex of an anionic polymer and silver can be made by a method
comprising the step of treating an anionic polymer with a solution of a silver salt.
Preferably, the solution is an aqueous solution. Preferably, the anionic polymer is
substantially insoluble in water at pH7, and the treatment is therefore carried out
on the polymer in the solid state. For example, the polymer may be in the form of
solid fibers, sheet, sponge or fabric. In certain embodiments, the anionic polymer
is a salt and the treatment therefore can be regarded as an ion exchange. In other
embodiments, the anionic polymer is at least partly in free acid form, in which case
the silver salt is preferably a salt of a weak acid, for example silver acetate,
whereby the anionic polymer is at least partially neutralised by the silver salt.
Similar processes are described in EP-A-0437095, the entire content of which is
expressly incorporated herein by reference.
The neutralization reaction can be carried out in water or alcohol alone but is
preferably carried out in mixtures of water and alcohols. The use of a mixture of
water and alcohol provides good solubility for the weak acid salts via the water,
and the alcohol prevents the anionic polymer from excessively swelling, distorting
and weakening during the neutralization. Thus the physical properties of the
material are retained. Methanol is the preferred alcohol because many of the
above-mentioned salts have good solubility in this alcohol in combination with

water. Preferably, the alcohol to water ratio has a range of about 4:1 to 1:4 . If the
solution becomes too rich in alcohol, some salts may no longer be soluble
particularly if the alcohol is other than methanol. If the solution becomes too rich
in water, some swelling of the polymer will occur as neutralization takes place and
there will be some loss in physical properties such as in the tensile strength of the
polymer. Other useful alcohols include, for example, ethyl alcohol, propyl alcohol
and isopropyl alcohol.
The use of a mild neutralizing agent such as silver acetate allows for control of the
degree of neutralization. Use of stoichiometric and chemically equivalent amounts
of neutralizing agent and carboxylic acid on the anionic polymer does not produce
a 100% neutralized polymer as would be produced with strong irreversible
reactions with bases such as sodium hydroxide, sodium carbonate, sodium
bicarbonate and ammonium hydroxide.
Anionic polymers behave as an ion exchanger and will pull out of solution the
silver cation of any silver salt that is passed over them. The by-product of this
exchange is an acid from the salt and by using a salt of a weak organic acid, a
weak acid such as acetic acid is produced which does no damage to the polymer.
Using salts of strong acids such as sodium chloride or sodium sulfate produces
hydrochloric acid or sulfuric acid by-products respectively, and these strong acids
can cause damage such as depolymerization of the polymer
When using silver salts of weak acids, the silver ion is exchanged for a proton on
the polymer and part of the salt is converted to weak acid. The mixture of acid and
salt in the solution results in a buffered solution which maintains a fairly constant
pH and controls the degree of neutralization. An equilibrium reaction is established
whereby the silver ions are bound to the acid portion of the polymer and also to
the salt molecules. This partitioning of the silver ions prevents the neutralization of
the polymer from going to completion.
Using a stoichiometric amount of, for example, silver acetate brings about a 65-
75% degree of neutralization of the carboxylic acid groups on an oxidized cellulose

polymer. This control of pH by creating a self generating buffered solution and the
use of methanol to control the swelling of the material, leads to a partially
neutralized material in which the physical properties, e.g. tensile strength and
shape of the polysaccharide, are preserved.
The amount of silver salt used is generally about equal to or up to twice the
stoichiometric amount of acid content of the polymer. Alternatively, a second
charge of a stoichiometric amount of silver salt can be used if the reaction is
recharged with fresh solvent and salt after the first charge reaches a constant pH.
The material with elevated pH is then washed to remove the excess silver salt
and ions therefrom.
In some embodiments, at least a portion of the wound dressing material comprises
a collagen complexed with silver. This can be achieved by treating a collagen with
a solution of a silver salt. The silver salt may for example be silver acetate or
silver nitrate at a concentration of about 0.01 molar to about 1 molar. The
treatment is preferably carried out at a pH of from about 5 to about 9. It is thought
that the silver complexes primarily to the nitrogen-containing side chains of the
collagen amino acids, in particular to lysine, hydroxylysine, asparagine, glutamine
and arginine. The silver could also bind to the sulfhydryl groups of methionine and
cysteine residues, where present, and to carboxyl groups of aspartate and
glutamate.
Preferably the amount of silver in the collagen complex is from about 0.01 to about
30% by weight based on the weight of the collagen, more preferably from about
0.1% to about 20%, more preferably from about 2% to about 10% by weight.
Preferably, the amount of silver-collagen complex in the wound dressing material
is from about 0.1 to about 10 wt.%, more preferably from about 0.1 to about
2wt.%. In any case, the total amount of silver in the wound dressing material is
generally as specified above.
It will be appreciated that the complexes of silver with substrate materials
described above may be prepared with a relatively high silver content, for example

greater than 5wt.%, and then diluted with further substrate material (the same or
different) to achieve the desired overall silver content of from 0.01wt.% to 5wt.%,
preferably from about 0.2wt.% to about 2wt.%.
The materials according to the present invention may be provided in the form of
beads, flakes, powder, and preferably in the form of a film, a fibrous pad, a web, a

woven or non-woven fabric, a freeze-dried sponge, a foam or combinations
thereof. In certain embodiments, the solid bioabsorbabie substrate is selected
from the group consisting of woven fabrics, knitted fabrics, and nonwoven fabrics,
all of which may be made by conventional methods. In other embodiments, the
solid bioabsorbabie substrate may comprise (or consist essentially of) a freeze-

dried sponge or a solvent-dried sponge, for example as described hereinbefore.
The solid bioabsorbabie substrate is typically in sheet form, for example a sheet of
material having an area of from about 1cm2 to about 400cm2, in particular from
about 2cm2 to about 100cm2. The basis weight of the sheet is typically from about
100g/m2 to about 5000g/m2, for example from about 400g/m2 to about 2000g/m2.
The solid bioabsorbabie substrate material may make up at least 50% by weight of
the wound dressing material, for example at least 75% by weight or at least 90%
by weight.
The term "dyestuff" refers to a material that is useful as a colorant for textile
materials, that is to say an organic compound that is strongly light-absorbing in the
visible region 400-700nm. In certain embodiments, the antioxidant dyestuff is
selected from the group consisting of aniline dyes, acridine dyes, thionine dyes,
bis-naphthalene dyes, thiazine dyes, azo dyes, anthraquinone dyes, and mixtures
thereof. For example, the antioxidant dyestuff may be selected from the group
consisting of gentian violet, aniline blue, methylene blue, crystal violet, acriflavine,
9-aminoacridine, acridine yellow, acridine orange, proflavin, quinacrine, brilliant
green, trypan blue, trypan red, malachite green, azacrine, methyl violet, methyl
orange, methyl yellow, ethyl violet, acid orange, acid yellow, acid blue, acid red,
thioflavin, alphazurine, indigo blue, methylene green, and mixtures thereof.

The dyestuffs also stabilize silver salts present in the material against
photochemical decomposition, in particular against photochemical reduction to
metallic silver, by absorbing light near the surface of the material. The dyestuffs
further trap photochemically generated free radicals that could otherwise react with
the silver. In this way the dyestuffs can act as photochemical desensitisers. In
addition to the conventional dyestuffs listed above, medically acceptable organic
desensitisers of the kind used in photography may be suitable for use as the
dyestuffs in the materials of the present invention.
The dyestuff may be present in the wound dressing material according to the
invention in an amount of from about 0.05% to about 5wt.%, typically about 0.2 to
about 2wt.% based on the dry weight of the material.
The wound dressing material may also comprise up to 20% by weight, preferably
less than 10% by weight of water. The material may also contain 0-40% by
weight, preferably 0-25% by weight of a plasticiser, preferably a polyhydric alcohol
such as glycerol. The material may also comprise 0-10% by weight, preferably 0-
5% by weight of one or more therapeutic wound healing agents, such as non-
steroidal anti-inflammatory drugs (e.g. acetaminophen), steroids, antibiotics (e.g.
penicillins or streptomycins), antiseptics (e.g. silver sulfadiazine or chlorhexidine),
or growth factors (e.g. fibroblast growth factor or platelet derived growth factor).
All of the above percentages are on a dry weight basis.
The wound dressing material according to the present invention is preferably
sterile and packaged in a microorganism-impermeable container.
Preferably, the material according to the present invention has a free radical
activity, that is to say an antioxidant activity, of at least about 15% in the
diphenylpicrylhydrazyl (DPPH) test, measured as percentage reduction in
absorbance at 524nm after 4 hours of a 0.5%w/v dispersion of the polysaccharide
in 10-4M DPPH, as described further hereinbelow in Procedure 1. Preferably the
percentage reduction in absorbance in the DPPH test (after correction for any

absorbance by the dye) is at least about 25%, more preferably at least about 50%,
and most preferably at least about 75%.
Alternatively or additionally, the material according to the present invention may
exhibit antioxidant activity as measured by its ability to inhibit the oxidation of
ABTS (2,2'-azino-di-[3-ethylbenzthiazoline sulphonate]) by a peroxidase.
Preferably, the material according to the present invention will absorb water or
wound fluid and hence become wet, swell or become a gelatinous mass but will
not spontaneously dissolve or disperse therein. That is to say, it is hydrophilic but
has a solubility of preferably less than about 1g/liter in water at 25°C. Low
solubility renders such materials especially suitable for use as wound dressings to
remove reactive oxygen species from the wound fluid.
The antioxidant and antimicrobial properties of the materials according to the
present invention suggest applications in a range of medical applications, including
the treatment of acute surgical and traumatic wounds, bums, fistulas, venous
ulcers, arterial ulcers, pressure sores (otherwise known as decubitus ulcers),
diabetic ulcers, ulcers of mixed aetiology, and other chronic or necrotic wounds
and inflammatory lesions and disorders. The materials according to the present
invention are intended for the treatment of both infected and non-infected wounds
(that is to say wounds showing no clinical signs of infection).
Accordingly, in a second aspect, the present invention provides the use of a
material according to the present invention for the preparation of a medicament for
the treatment of a wound. Preferably, the wound is a chronic wound. More
preferably, the chronic wound is selected from the group consisting of ulcers of
venous, arterial or mixed aetiology, decubitus ulcers, or diabetic ulcers.
Preferably, the material is used as an antioxidant to reduce oxidative stress in the
wound environment and thereby to promote wound healing.
In a related aspect, the present invention provides a method of treatment of a
wound in a mammal comprising applying thereto a therapeutically effective

amount of a material according to the present invention. Preferably, the wound is
a chronic wound.
In a third aspect, the present invention provides a wound dressing comprising an
antioxidant wound dressing material according to the present invention.
The wound dressing is preferably in sheet form and comprises an active layer of
the material according to the invention. The active layer would normally be the
wound contacting layer in use, but in some embodiments it could be separated
from the wound by a liquid-permeable top sheet. Preferably, the area of the active
layer is from about 1cm2 to about 400 cm2, more preferably from about 4cm2 to
about 100cm2.
Preferably, the wound dressing further comprises a backing sheet extending over
the active layer opposite to the wound facing side of the active layer. Preferably,
the backing sheet is larger than the active layer such that a marginal region of
width 1mm to 50mm, preferably 5mm to 20mm extends around the active layer to
form a so-called island dressing. In such cases, the backing sheet is preferably
coated with a pressure sensitive medical grade adhesive in at least its marginal
region.
Preferably, the backing sheet is substantially liquid-impermeable. The backing
sheet is preferably semipermeable. That is to say, the backing sheet is preferably
permeable to water vapour, but not permeable to liquid water or wound exudate.
Preferably, the backing sheet is also microorganism-impermeable. Suitable
continuous conformable backing sheets will preferably have a moisture vapor
transmission rate (MVTR) of the backing sheet alone of 300 to 5000 g/m2/24hrs,
preferably 500 to 2000 g/m2/24hrs at 37.5 oC at 100% to 10% relative humidity
difference. The backing sheet thickness is preferably in the range of 10 to 1000
micrometers, more preferably 100 to 500 micrometers. It has been found that
such moisture vapor transmission rates allow the wound under the dressing to
heal under moist conditions without causing the skin surrounding the wound to
macerate.

Suitable polymers for forming the backing sheet include polyurethanes and poly
alkoxyalkyl acrylates and methacrylates such as those disclosed in GB-A-
1280631. Preferably, the backing sheet comprises a continuous layer of a high
density blocked polyurethane foam that is predominantly closed-cell. A suitable
backing sheet material is the polyurethane film available under the Registered
Trade Mark ESTANE 5714F.
The adhesive (where present) layer should be moisture vapor transmitting and/or
patterned to allow passage of water vapor therethrough. The adhesive layer is
preferably a continuous moisture vapor transmitting, pressure-sensitive adhesive
layer of the type conventionally used for island-type wound dressings, for example,
a pressure sensitive adhesive based on acrylate ester copolymers, polyvinyl ethyl
ether and polyurethane as described for example in GB-A-1280631. The basis
weight of the adhesive layer is preferably 20 to 250 g/mz, and more preferably 50
to 150 g/m2. Polyurethane-based pressure sensitive adhesives are preferred.
Further layers of a multilayer absorbent article may be built up between the active
layer and the protective sheet. For example, these layers may comprise an
absorbent layer between the active layer and the protective sheet, especially if the
dressing is for use on exuding wounds. The optional absorbent layer may be any
of the layers conventionally used for absorbing wound fluids, serum or blood in the
wound healing art, including gauzes, nonwoven fabrics, superabsorbents,
hydrogels and mixtures thereof. Preferably, the absorbent layer comprises a
layer of absorbent foam, such as an open celled hydrophilic polyurethane foam
prepared in accordance with EP-A-0541391, the entire content of which is
expressly incorporated herein by reference. In other embodiments, the absorbent
layer may be a nonwoven fibrous web, for example a carded web of viscose staple
fibers. The basis weight of the absorbent layer may be in the range of 50-
500g/m2, such as 100-400g/m2. The uncompressed thickness of the absorbent
layer may be in the range of from 0.5mm to 10mm, such as 1mm to 4mm. The
free (uncompressed) liquid absorbency measured for physiological saline may be

in the range of 5 to 30 g/g at 25°. Preferably, the absorbent layer or layers are
substantially coextensive with the active layer.
The wound facing surface of the dressing is preferably protected by a removable
cover sheet. The cover sheet is normally formed from flexible thermoplastic
material. Suitable materials include polyesters and polyolefins. Preferably, the
adhesive- facing surface of the cover sheet is a release surface. That is to say, a
surface that is only weakly adherent to the active layer and the adhesive on the
backing sheet to assist peeling of the adhesive layer from the cover sheet. For
example, the cover sheet may be formed from a non-adherent plastic such as a
fluoropolymer, or it may be provided with a release coating such as a silicone or
fluoropolymer release coating.
Typically, the wound dressing according to the present invention is sterile and
packaged in a microorganism-impermeable container.
In a further aspect, the present invention provides a method of manufacture of an
antioxidant wound dressing material, comprising the step of dyeing a
bioabsorbable substrate material with a suitable dye. The method preferably
further comprises treating the substrate material with a silver salt dissolved or
dispersed in water or an organic solvent.
The method according to the present invention may be used to prepare a wound
dressing according to the present invention.
The method of the present invention may comprise dyeing a substrate material in
sheet form, for example a woven, nonwoven or knitted fabric or sponge sheet of
the substrate material by immersing it in a dye bath, followed by washing to
remove unbound dye and drying. In other embodiments, the substrate material
may be dyed while it is in fibrous or particulate form, followed by forming the
material into a sheet. For example, a slurry of fibers or particles of the substrate
material may be treated with dye, and then freeze-dried to form a dyed sponge.
The silver treatment may be carried out after the step of removing unbound dye.

It will be appreciated that any feature or embodiment that is described herein in
relation to any one aspect of the invention may also be applied to any other aspect
of the invention equally.
Certain specific embodiments of the present invention will now be described
further in the following examples.
Example 1
An antioxidant wound dressing material based on a collagen/ORC freeze-dried
sponge material is prepared as follows.
The collagen component is prepared from bovine corium as follows. Bovine
corium is split from cow hide, scraped and soaked in sodium hypochlorite solution
(0.03% w/v) to inhibit microbial activity pending further processing. The corium is
then washed with water and treated with a solution containing sodium hydroxide
(0.2% w/v) and hydrogen peroxide (0.02% w/v) to swell and sterilize the corium at
ambient temperature. The corium splits then undergo an alkali treatment step in a
solution containing sodium hydroxide, calcium hydroxide and sodium bicarbonate
(0.4% w/v, 0.6% w/v andO.05% w.v, respectively) at pH greater than 12.2, ambient
temperature, and for a time of 10-14 days, with tumbling, until an amide nitrogen
level less than 0.24mmol/g is reached. The corium splits then undergo an acid
treatment step with 1% hydrochloric acid at ambient temperature and pH 0.8-1.2.
The treatment is continued with tumbling until the corium splits have absorbed
sufficient acid to reach a pH less than 2.5. The splits are then washed with water
until the pH value of corium splits reaches 3.0-3.4. The corium splits are then
comminuted with ice in a bowl chopper first with a coarse comminution and then
with a fine comminution setting. The resulting paste, which is made up in a ratio of
650g of the corium splits to 100g of water, as ice, is frozen and stored before use
in the next stage of the process. However, the collagen is not freeze-dried before
admixture with the ORC & other components in the next stage.

The ORC component of the freeze-dried pad is prepared as follows. A
SURGICEL cloth (Johnson & Johnson Medical, Arlington) is milled using a rotary
knife cutter through a screen-plate, maintaining the temperature below 60°C.
Methylene blue, an acidic dye, was incorporated by dissolving an appropriate
amount of the dye in 0.05M acetic acid and adding to the collagen paste with the
milled ORC powder to obtain a final solids concentration of 1%. Samples were
made in which the dye was incorporated at the following concentrations in the
slurry: 0% (reference example), 1mg/ml, 0.5mg/ml and 0.1mg/ml.
The resulting slurries were poured to a depth of 3mm in petri dishes, placed onto
freezer shelves where the temperature has been preset to -40°C. The freeze-drier
programme was then initiated to dry and dehydrothenmally cross-link the collagen
and ORC to form sponge pads. On completion of the cycle, the vacuum was
released, sponge samples were then packaged, and sterilized by cobalt 60
gamma-irradiation.
Example 2
The procedure of Example 1 was followed, but replacing the methylene blue dye
by crystal violet, a basic dye. The crystal violet was incorporated at the following
concentrations in the slurry: 0% (reference example), 1mg/ml, 0.5mg/ml and
0.1mg/ml.
Example 3
The procedure of Example 1 was followed, but replacing the methylene blue dye
by flavin 3,6-Diaminoacridine hemisulfate, a basic dye. The flavin was
incorporated at the following concentrations in the slurry: 0% (reference example),
1mg/ml, 0.5mg/ml and 0.1mg/ml.
Example 4
The procedure of Example 1 was followed, but replacing the methylene blue dye
by flavin 3,6-Diaminoacridine hemisulfate, a basic dye. The flavin was

incorporated at the following concentrations in the slurry: 0% (reference example),
1mg/ml, 0.5mg/ml and 0.1mg/ml.
Example 5
The procedure of Example 1 was followed, but replacing the methylene blue dye
by a mixture of methylene blue and flavin 3,6-Diaminoacridine hemisulfate, each
dye being incorporated in the slurry at a concentration of 0.5mg/ml.
Example 6
The procedure of Example 1 was followed, but replacing the methylene blue dye
by a mixture of crystal violet and flavin 3,6-Diaminoacridine hemisulfate, each dye
being incorporated in the slurry at a concentration of 0.5mg/ml.
Example 7
The procedure of Example 1 was followed, but replacing the methylene blue dye
by a mixture of crystal violet and methylene blue, each dye being incorporated in
the slurry at a concentration of 0.5mg/ml.
Examples 8-14
Silver was incorporated into the wound dressing materials made as described in
Examples 1-7 by dissolving silver acetate in 0.05M acetic acid and adding the
solution to the ORC/colIagen slurry in an amount sufficient to produce a final slurry
containing 1wt.% silver on a total solids basis.
The sponges according to the invention obtained in Examples 1 to 14 all showed
stable absorption of the dyes. The sponges could be soaked in serum at 25°C for
a number of days and remained coloured at all times. Depending on
concentration of dye added there was an initial release of the excess dye and then
a gradual release as the sponges began to degrade.

Procedure 1
The ability of the wound dressing materials to react with and remove oxygen
containing free radicals is assessed by the DPPH test described in W094/13333,
the entire content of which is expressly incorporated herein by reference. The test
is adapted from that described by Blois M.S. in Nature 181: 1199 (1958), and
Banda P.W. et al., in Analytical Letters 7: 41 (1974).
Briefly, the wound dressing material under test (2.5mg; 5mg; & 25mg sample
sizes) was suspended in 2.5ml of 0.1 M pH 7.0 phosphate buffer. A solution of
diphenylpicrylhydrazyl (DPPH) in methanol (10-4 M) was added in an amount of
2.5 ml and the mixture was shaken and stored in the dark at 20°C. The samples
were assessed by measurement of their light absorbance at 524nrh over 6 hours
in comparison with a control, particular attention being paid to the figure after 4
hours. The percentage reduction of absorbance relative to the control after 4
hours gives the DPPH test value, with a reproducibility generally of ±5%. This
value may conveniently be expressed in terms of a simple reduction in absorbance
units (AU) relative to the control.
Ascorbic acid, a well-known antioxidant, provides a useful positive control
substance for comparative purposes. Freeze-dried sponges of chitin/chitosan and
hydroxyethyl cellulose were used as negative controls.
Application of this test to the materials according to the present invention of
Examples 1-7 resulted in DPPH test values of 80 - 90% for the positive control
(10-4M). In contrast, the negative controls chitin/chitosan and hydroxyethyl
cellulose exhibited much lower DPPH values of less than 15%. The collagen/ORC
without any added dye exhibited some activity in the DPPH test, indicating that
ORC itself has some antioxidant properties. The dyed materials according to the
present invention exhibited significantly higher activity in the DPPH test than
collagen/ORC alone, consistent with antioxidant activity of the dyes.
Procedure 2

The bactericidal activity of the sponges prepared in Examples 8 to 14 is tested on
pseudomonas Aeruginosa and staphylococcus Aureus by looking at zone of
inhibition.
Six 2cm x 2cm squares of each sample are cut out in sterile conditions. On day
one of the experiment, cultures of both Pseudomonas aeruginosa (ATCC 27853
and various PSI strains) and Staphylococcus aureus (provided by the Dept of
Clinical Microbiology and Pathology) are incubated aerobically at 37°C for 24
hours on Diagnostic Sensitivity Agar (DSA). After 24 hours test samples are each
placed on a DSA plate and immediately wetted with 0.5mls of a buffer solution.
Three squares of sample are placed on plates inoculated with Pseudomonas
aeruginosa and three are placed on plates inoculated with Staphylococcus aureus.
The plates are then incubated at 37°C for 24 hours. The zone of inhibited growth
around the sample is then measured using calipers, and the test sample is placed
on a new inoculated DSA plate. A swab test is carried out on the area beneath the
sample to determine if the sample is bacteriostatic if not bactericidal by smearing
the swab on a DSA plate and incubating it for 24 hours and then examining the
growth. The samples are transferred onto fresh inoculated plates with the above
procedure being carried out every 24 hours for 72 hours as long as the samples
remain intact.
As a negative control, a freeze dried sponge of 45%ORC/55%collagen without any
silver or dye was tested. A commercially available silver-containing antimicrobial
dressing (ACTICOAT, registered trade mark of Smith & Nephew) and silver nitrate
solution (0.5%) were used as positive controls and zones of inhibition were
observed for both over the test period.
It is found that significant bactericidal effects are observed against Staphylococcus
aureus and Pseudomonas Aeruginosa for the materials according to the invention.
The performance of the materials containing 1% silver and above is expected to
be comparable to that of the ACTICOAT dressing.

The above embodiments have been described by way of example only. Many
other embodiments falling within the scope of the accompanying claims will be
apparent to the skilled reader.

WE CLAIM:
1. A wound dressing material comprising a solid bioabsorbable substrate
wherein the substrate is selected from a freeze-dried and solvent-
dried sponge and comprises a solid bioabsorbable material which is
dyed with an antioxidant dyestuff and which is selected from the
group consisting of oxidized regenerated cellulose, collagen, chitosan
or mixtures thereof.
2. The wound dressing material as claimed in any preceding claim,
wherein the antioxidant dyestuff is selected from the group consisting
of aniline dyes, acridine dyes, thionine dyes, bis-naphthalene dyes,
thiazine dyes, azo dyes, anthraquinones, and mixtures thereof.
3. The wound dressing material as claimed in any preceding claim,
wherein the antioxidant dyestuff is selected from the group consisting
of gentian violet, aniline blue, methylene blue, crystal violet,
acriflavine, 9-aminoacridine, acridine yellow, acridine orange,
proflavin, quinacrine, brilliant green, trypan blue, tryan red, malachite
green, azacrine, methyl violet, methyl orange, methyl yellow, ethyl
violet, acid orange, acid yellow, acid blue, acid red, thioflavin,
alphazurine, indigo blue, methylene green, and mixtures thereof.

4. The wound dressing material as claimed in any preceding claim, wherein
the antioxidant dyestuff is present, in an amount of from 0.2 to 2wt.%
based on the dry weight of the material.
5. The wound dressing material as claimed in any preceding claim, wherein
the material further comprises a silver salt, whereby the dyestuff
photostabilizes the silver salt.
6. The wound dressing material as claimed in claim 5, wherein the polymeric
substrate comprises an anionic polymer, and said silver salt comprises a
salt of Ag+ with the anionic polymer.
7. The wound dressing material as claimed in claim 5 or 6, wherein the
composition comprises from 0.01wt.% to 5wt.% of silver, based on the dry
weight of the composition.
8. The wound dressing material as claimed in any preceding claim, wherein
the material is in sheet form.

9. The wound dressing material as claimed in any preceding claim, wherein the
material is sterile and packaged in a microorganism-impermeable container.
10. The wound dressing material as claimed in any preceding claim wherein
the material has a free radical activity in the diphenylpicrylhydrazyl (DPPH)
test for antioxidant activity as herein defined of at least 15%.
11. A method of manufacture of an antioxidant wound dressing material,
comprising the step of dyeing a bioabsorbable substrate material with an
antioxidant dye, wherein the substrate is a freeze-dried or solvebt-dried
sponge and comprises a solid biobsorbable material selected from the group
consisting of oxidized regenerated cellulose, collagen, chitosan or mixtures
thereof.
12. The method as claimed in claim 11, for the manufacture of a wound
dressing material as claimed in any of claims 1 to 10.
13. The wound dressing comprising an antioxidant wound dressing material
as claimed in any of claims 1 to 10.

14. The wound dressing as claimed in claim 13, wherein the material is sterile
and packaged in a microorganism-impermeable container.


A wound dressing material comprising a solid bioabsorbable substrate
wherein the substrate is selected from a freeze-dried and solvent-dried
sponge and comprises solid bioabsorbable material which is dyed with an
antioxidant dyestuff and which is selected from the group consisting of
oxidized regenerated cellulose, collagen, chitosan or mixtures thereof.

Documents:

00003-kolnp-2006-abstract.pdf

00003-kolnp-2006-claims.pdf

00003-kolnp-2006-description complete.pdf

00003-kolnp-2006-form 1.pdf

00003-kolnp-2006-form 2.pdf

00003-kolnp-2006-form 3.pdf

00003-kolnp-2006-form 5.pdf

00003-kolnp-2006-international publication.pdf

00003-kolnp-2006-international search authority.pdf

00003-kolnp-2006-pct forms.pdf

3-KOLNP-2006-(02-08-2012)-FORM-27.pdf

3-KOLNP-2006-ABSTRACT 1.1.pdf

3-KOLNP-2006-ABSTRACT.pdf

3-KOLNP-2006-CANCELLED PAGES.pdf

3-KOLNP-2006-CLAIMS 1.1.pdf

3-KOLNP-2006-CLAIMS.pdf

3-kolnp-2006-correspondence-1.1.pdf

3-KOLNP-2006-CORRESPONDENCE.pdf

3-KOLNP-2006-DESCRIPTION (COMPLETE) 1.1.pdf

3-KOLNP-2006-DESCRIPTION (COMPLETE).pdf

3-KOLNP-2006-EXAMINATION REPORT REPLY RECIEVED 1.1.pdf

3-kolnp-2006-examination report.pdf

3-KOLNP-2006-FORM 1 1.1.pdf

3-KOLNP-2006-FORM 1.pdf

3-kolnp-2006-form 18.pdf

3-KOLNP-2006-FORM 2 1.1.pdf

3-KOLNP-2006-FORM 2.pdf

3-kolnp-2006-form 26.pdf

3-KOLNP-2006-FORM 3 1.2.pdf

3-kolnp-2006-form 3-1.3.pdf

3-KOLNP-2006-FORM 3.1.1.pdf

3-KOLNP-2006-FORM 3.pdf

3-kolnp-2006-form 5-1.1.pdf

3-KOLNP-2006-FORM 5.pdf

3-KOLNP-2006-FORM-27.pdf

3-kolnp-2006-granted-abstract.pdf

3-kolnp-2006-granted-claims.pdf

3-kolnp-2006-granted-description (complete).pdf

3-kolnp-2006-granted-form 1.pdf

3-kolnp-2006-granted-form 2.pdf

3-kolnp-2006-granted-specification.pdf

3-kolnp-2006-others-1.1.pdf

3-KOLNP-2006-OTHERS.pdf

3-KOLNP-2006-PA.pdf

3-KOLNP-2006-PETITION UNDER RULE 137.pdf

3-kolnp-2006-reply to examination report-1.1.pdf

3-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf


Patent Number 250991
Indian Patent Application Number 3/KOLNP/2006
PG Journal Number 07/2012
Publication Date 17-Feb-2012
Grant Date 15-Feb-2012
Date of Filing 02-Jan-2006
Name of Patentee JOHNSON & JOHNSON MEDICAL LIMITED
Applicant Address AIREBACK MILL, GARGRAVE, SKIPTON BD23 3RX
Inventors:
# Inventor's Name Inventor's Address
1 CULLEN, BREDA, MARY 7 CONSORT STREET, SKIPTON, NORTH YORKI-SHIRE BD23 1HR
2 GREENHALGM, DAVID 22 TILE CLOSE, SKIPTON NORTH YORKSHIRE BD 23 2LG
3 ADDISON, DEBORAH 2, DOVENANTER COTTAGE, KEASDEN, CLAPHAM, VIA LANCASTER LA2 8HB
PCT International Classification Number A61L 15/28, 15/22
PCT International Application Number PCT/GB2004/002636
PCT International Filing date 2004-06-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0314454.0 2003-06-20 U.K.
2 60/491,991 2003-08-04 U.K.
3 0326844.8 2003-11-18 U.K.