Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Wound or tissue dressing comprising lactic acid bacteria
This patent application is a non-provisional patent application and claims the
benefit of
US 60/870,625 filed on December 19, 2006, which is hereby incorporated by
reference
in its entirety. All patent and non-patent references cited in US 60/870,625
as well as in
the present application, are also incorporated herein by reference.
Field of invention
The present invention relates to a wound or tissue dressing comprising lactic
acid
producing bacteria. Also provided are uses of such a wound or tissue dressing
in
healing wounds or in accelerating the healing of wounds.
Background of invention
Wounds to the skin and the underlying tissues of animals may be caused by e.g.
friction, abrasion, laceration, pressure, burning, or chemical irritation.
Tissue damage
may also result from internal metabolic or physical dysfunction, including,
but not
limited to, bone protrudence, diabetes, circulatory insufficiencies, or
inflammatory
processes.
Wounds can be classified as acute or chronic. Wounds that are not progressing
in 4
weeks and not healed within 8 weeks of formation are classified chronic.
Chronic
wounds are often found to be infected and the wound fluid in chronic wounds
have
been shown to have a higher pH and a higher level of protease activity than
wound
fluid from acute wounds. The higher level of proteases in chronic wounds can
either
origin from cells in the tissue or from bacteria in the wound exudate.
A wound to the skin and/or damage to the underlaying tissues significantly
reduce the
protective function of the skin. Consequently, damaged skin results in an
increased risk
of infection of the under laying tissue by infectious agents such as bacteria
and vira.
Areas of damaged skin are conventionally protected by the application of a
wound or
tissue dressing which facilitates wound healing. Wound or tissue dressings
generally
provide a suitable environment for wound healing by absorbing exudate, keeping
the
wound bed moist , and by protecting the wound and new tissue growth from
bacterial
contamination.
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Despite careful attention by medical personal in hospitals and health clinics,
many
wounds are often infected during the wound healing process - either in the
health care
environment or when out-patients are undergoing recovery outside the health
care
environment.
Several types of wound or tissue dressings exist. Most wound or tissue
dressings are
designed to maintain a moist wound bed. The most commonly used wound or tissue
dressing are briefly introduced below.
Gauze dressings are dressing made of cotton, available in a number of forms
including
sponges, pads, ropes, strips, and rolls, gauze can also be impregnated with
petroleum,
antimicrobials, and saline. With removal of a dried gauze dressing, there is a
risk of
wound damage to the healing skin surrounding the wound.
Hydrocolloid dressings are among the oldest and most frequently used wound or
tissue
dressings. Hydrocolloids are either occlusive (i.e. they do not allow air to
escape
through the dressing), or semi-occlusive (i.e. they do allow some air to
escape through
the dressing) and they are designed to seal the wound bed to retain and
interact with
exudate to promote healing. They used on dry wounds with necrotic tissue in
order to
get autolytic debridement and also as a protective layer on newly formed
epithelial
layer.
Hydrogel dressings are either sheets of cross-linked polymers or hydrogel
impregnated
gauze, or non-wowen sponge, used to cover a wound. The hydrogel dressing can
be in
the form of a hydrogel sheet dressing or in the form of an amorphous hydrogel
dressing. Hydrogel sheet dressings are indicated for, wounds with necrosis or
slough,
and burns. An amorphous hydrogel dressing is a soft, formless gel comprised of
either
polymers or copolymers and up to 95 percent water, whereas a hydrogel sheet
dressing is a firm sheet. Amorphous hydrogels carry the same indications as
hydrogel
sheets and they can also be used to lightly pack full-thickness wounds.
Alginate dressings are highly absorbent, biodegradable dressings derived from
seaweed. They are used for wounds with moderate to large amounts of exudate,
and
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for wounds requiring packing. These dressings work by combining with the wound
exudate to form a hydrophylic gel that creates a moist healing environment.
Foam dressings are semipermeable sheets of a polymer, such as polyurethane,
that
provide a specific, controlled moisture and temperature environment for wound
healing.
They are indicated for wounds with moderate to heavy exudate. Foam dressings
are
non-adherent and can repel contaminants.
Transparent film dressings are made of e.g. polyurethane, polyamide,
polyurethane or
gelatin. Although they are waterproof, transparent film dressings are somewhat
porous
allowing for oxygen and moisture to cross through their barriers. They are non-
absorptive so they must be changed often for wounds with exudate. They are
generally
effective on dry wounds with necrotic tissue in need of autolytic debridement.
Transparent film dressings are also used as a secondary material to secure
e.g. non-
adhesive gauzes and other types of dry dressings.
In addition to dressings meant to keep the wound bed moist, there are also
dressings
that act as a substrate for proteases and/or scaffolds for new tissue. These
dressings
can be based on collagen or gelatine.
Halper et al. (2003) have disclosed wound healing and angiogenic properties of
supernatants from Lactobacillus cultures (Exp. Biol. and Med., vol. 228, pp.
1329 -
1337.
EP 568 334 is directed to a collagen-containing sponge comprising an
absorbable
gelatin sponge, collagen, and an active ingredient. The absorbable gelatin
sponge can
be crosslinked.
US 5,399,361 is directed to a collagen-containing sponge comprising an
absorbable,
cross-linked gelatin sponge, soluble collagen, and a therapeutically effective
amount of
an active ingredient.
EP 1 082 964 is directed to a lactic acid bacteria-containing composition
comprising
lactic acid bacteria showing pharmacologic action in the digestive tract
and/or the
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urogenital organ, wherein the adhesion of said lactic acid bacteria to the
digestive tract
and/or the urogenital organ is enhanced to potentiate said pharmacologic
action.
US 6,716,435 and US 7,025,974 disclose an absorbent product comprising a
liquid
absorbent structure and a viable Bacillus coagulans. The Bacillus coagulans is
present
on an external surface of said product.
Summary of invention
The present invention in one aspect is directed to a wound or tissue dressing
comprising lactic acid producing bacteria. Further metabolites, in addition to
lactic acid,
are preferably also produced by said lactic acid producing bacteria. Examples
of such
metabolites are e.g. one or both of diacetyl and hydrogen peroxide. The lactic
acid
producing bacteria can further produce e.g. extracellular proteases and
bacteriocins
which have beneficial effects on the wound healing process.
The wound or tissue dressing according to the present invention is capable of
promoting wound healing in an individual in need thereof. The individual is
preferably
an animal, such as a human being.
Wound healing is a complex regeneration process, which is characterised by
intercalating degradation and re-assembly of connective tissue and epidermal
layer.
One reason for wounds not to heal can be infection with putrefactive bacteria
causing
an unbalanced wound environment. Most bacteria have a growth optimum at pH
between 6.5 and 7.5. It is believed that a lowering of the pH of the wound
environment
will reduce the number and/or growth of putrefactive bacteria to the extent
that the
reduction positively correlates with an improved wound healing.
The effect exerted by the dressing according to the present invention is
believed to be
also correlated with the fact that lactid acid producing bacteria present in
the dressing
express certain desirable bacteriocins in the form of toxins that inhibit
undesirable
bacterial growth.
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By lowering the pH in the wound environment, it is believed that one will also
reduce
the activity of proteases present in the wound. Especially in chronic wounds
are
protease activities believed to be out of balance.
5 While it is true that proteases are needed for the wound healing process,
high levels of
protease activities are likely to maintain the wound in a chronic state. Most
proteases
have a pH optimum at alkaline pH and a weakly acidic environment may therefore
promote healing of open wounds by inhibiting the action of undesirable
proteases. By
operably contacting the chronic wound with a bacteria capable of producing
certain
anti-bacterial and pH lowering components, putrefactive bacteria can be
neutralised
and/or eliminated and a weakly acidic wound environment can be obtained and
sustained over periods of time relevant for effectively promoting wound
healing.
Accordingly, in one embodiment there is provided a wound or tissue dressing
comprising a non-pathogenic bacteria capable of producing lactic acid by
fermentation
of polysaccharides or residues thereof. The bacteria can be spore forming - or
the
bacteria can be incapable of forming spores as the case may be. In one
preferred
embodiment, the lactic acid producing bacteria are not spore-forming bacteria.
The bacteria preferably belongs to the family of lactic acid producing
bacteria, more
preferably to a family of lactic acid producing bacteria capable of producing
further
metabolites, in addition to lactic acid. Examples of such further metabolites
are e.g. one
or both of diacetyl and hydrogen peroxide. The family of lactic acid producing
bacteria
is preferably one also capable of producing e.g. extracellular proteases and
bacteriocins which have beneficial effects on the wound healing process.
Preferred lactic acid producing bacteria according to the present invention
are
preferably capable of one or more of 1) lowering the pH in an open wound
environment, 2) securing an intraspecies competitive exclusion, i.e.
preventing growth
of undesirable bacterial species, 3) exerting an immunomodulatory effect, and
4)
production of certain bacteriocins, such as toxins capable of sustaining a
wound
healing process.
The family of lactic acid bacteria according to the present invention
preferably refers to
any bacteria belonging to a genus selected from the group consisting of
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Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc,
Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and
Weissella. In one embodiment, the lactic acid bacteria employed do not belong
to the
genus Bacillus, including the species Bacillus coagulans.
The dressing according to the present invention can be regarded as "sterile" -
apart
from the fact that the dressing comprises lactic acid producing bacteria.
Accordingly,
the term "sterile" will be used herein to characterise the dressing as being
free from
viable microorganisms (i.e. "sterile") apart from the optionally freeze dried
and/or
encapsulated lactic acid bacteria forming part of the dressings.
The dressing according to the invention comprises an absorbant compound. The
absorbant compound can comprise the lactic acid producing bacteria, or the
lactic acid
producing bacteria can be present in the dressing in a compartment different
from the
absorbant compound. The lactic acid producing bacteria can be present on the
surface
part of the absorbant compound or the lactic acid producing bacteria can be
present
internally in the absorbant compound - in principle either by physical
entrapment, by
chemical bonding, or otherwise.
When being initially present in a compartment different from the absorbant
compound,
i.e. prior to the dressing contacting a wound or tissue, the lactic acid
producing bacteria
can be present in said different compartment either by physical entrapment
e.g. within
a matrix present in said different compartment, by chemical bonding to such a
matrix,
for example a gelatin and/or collagen matrix, i.e. an open cell structure,
such as e.g. a
sponge, or otherwise, e.g. by encapsulation with gelatin and/or collagen,
thereby e.g.
forming gel-like particles incapable of diffusing out of the matirx. The gel-
like particles
comprising the optionally lyophilized lactic acid producing bacteria can be
degraded
when coming into cantact with wound exudate, which degradation initially
releases the
lactic acid producing bacteria to the space of the different compartment, and
optionally
subsequently releases the lactic acid producing bacteria to the wound or to at
least part
of the absorbant compound.
The compartment different from the absorbant compound can be separated from
the
absorbant compound by a barrier or semi-permeable or permeable membrane. The
barrier or membrane can allow metabolites produced by the lactic acid
producing
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bacteria to diffuse over the membrane and e.g. into the wound area and/or the
barrier
or membrane can allow the lactic acid producing bacteria themselves to diffuse
over
the membrane and e.g. into the wound area.
The lactic acid producing bacteria are preferably lyophilized (freeze-dried)
by any state-
of-the-art method for lyophilizing lactic acid producing bacteria. The lactic
acid bacteria
in their optionally lyophilized state can further be encapsulated or contained
in
biodegradable microspheres, e.g. biodegradable microspheres which become
degraded when contacted by wound exudate.
The absorbant compound can be in contact with one or more pad(s) or
compartment(s)
comprising the lactic acid bacteria in their optional lyophilized state. The
pad or
compartment can be positioned on the proximal side of the absorbant compound,
i.e.
between the absorbant compound and the wound, and/or the pad comprising the
lactic
acid producing bacteria in their optional lyophilized state can be positioned
on the distal
side of the absorbant compound (in relation to the wound or tissue to be
treated).
The lactic acid producing bacteria in their optional lyophilized state can
generally be
contained in a pad or compartment defined by or comprising a permeable or semi-
permeable membrane allowing the lactic acid producing bacteria, or their
metabolites
only, as the case may be, to migrate from the pad or compartment to the
absorbant
compound and/or to the wound environment.
The pad or compartment can also comprise different membrane portions, wherein
each
portion has permeable or semi-permeable characteristics with respect to the
migration
of the lactic acid producing bacteria and their metabolites. One area or side
of the pad
or compartment can e.g. comprise a membrane portion allowing migration of the
lactic
acid producing bacteria to the wound, whereas another side or area of the pad
allows
migration to the absorbant compound of metabolites only. The metabolites can
be e.g.
lactic acid and/or bacteriocins.
Biodegradable microspheres used for encapsulation of the lactic acid producing
bacteria are disclosed herein below in more detail.
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In another embodiment there is provided a wound or tissue dressing comprising
an
absorbent compound for absorbing wound exudate, wherein said wound or tissue
dressing is attached to or comprises a lactic acid producing bacterium. The
lactic acid
producing bacterium is - in one embodiment - preferably present internally in
the wound
or tissue dressing, or internally in the absorbant compound, i.e. not on an
external
surface thereof.
In one embodiment, the above-cited wound or tissue dressing according to the
present
invention further comprises at least one adhesive surface suitable for
contacting a
wound and/or at least one topfilm for preventing leakage of wound exudate from
the
absorbant compound.
In yet another embodiment there is provided a wound or tissue dressing
comprising an
absorbent compound for absorbing wound exudate, wherein said absorbent
compound
is attached to or comprises a lactic acid producing bacterium, optionally in
lyophilized
and/or encapsulated form.
In yet another embodiment there is provided a dual compartment syringe
optionally
fitted with a plunger, wherein one compartment comprises e.g. a hydrogel as
disclosed
herein below in more detail (Figure 1, panel C) and wherein the other
compartment
comprises lactic acid producing bacteria, optionally in lyophilized and/or
encapsulated
form.
In a further aspect there is provided a method for manufacturing a wound or
tissue
dressing comprising an absorbent compound according to the present invention,
said
method comprising the steps of providing a lactic acid bacteria, mixing or
attaching said
lactic acid bacteria with the wound or tissue dressing or with the absorbent
compound
of the wound or tissue dressing, thereby obtaining a wound or tissue dressing
according to the present invention.
In a still further aspect there is provided a method for treating a wound in
an individual,
said method comprising the steps of contacting said wound with the wound or
tissue
dressing according to the invention, thereby treating the wound. The treatment
results
in healing of the wound or in accelerated healing of the wound.
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There is also provided the use of a lactic acid bacteria in the manufacture of
a wound
or tissue dressing for treating or accelerating the healing of a wound in an
individual.
In yet another aspect there is provided the use of a lactic acid bacteria in
the
manufacture of an absorbent compound for use in a wound or tissue dressing for
treating or accelerating the healing of a wound in an individual.
Definitions
"Wound" refers broadly to injuries to the skin and underlaying (subcutaneous)
tissue
initiated in different ways (e.g., pressure sores from extended bed rest and
wounds
induced by trauma) and with varying characteristics. Wounds may be classified
into
one of four grades depending on the depth of the wound: i) Grade I: wounds
limited to
the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade
III: wounds
extending into the subcutaneous tissue; and iv) Grade IV (or full-thickness
wounds):
wounds wherein bones are exposed (e.g., a bony pressure point such as the
greater
trochanter or the sacrum).
"Partial thickness wound" refers to wounds that encompass Grades I-III;
examples of
partial thickness wounds include burn wounds, pressure sores, venous stasis
ulcers,
and diabetic ulcers.
"Deep wound" is meant to include both Grade III and Grade IV wounds. The
present
invention contemplates treating all wound types, including deep wounds and
chronic
wounds.
"Chronic wound" refers to a wound that has not progressed within 4 weeks
and/or not
healed within 8 weeks.
"Positioning the wound or tissue dressing in or on the wound" means contacting
some
part of the wound with the wound or tissue dressing.
"Promote wound healing" and "enhance wound healing," and similar phrases,
refer to
either the induction of the formation of granulation tissue, of wound
contraction, and/or
the induction of epithelialization (i.e., the generation of new cells in the
epithelium).
Wound healing is conveniently measured by decreasing wound area.
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"Alginate" refers to a linear co-polymer with homopolymeric blocks of (1-4)-
linked B-D-
mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively,
covalently linked together in different sequences or blocks.
5
"Hydrocolloid" refers to a colloid system in which the colloid-forming
components are
dispersed in water, but not cross-linked. A colloid system is a system or
mixture in
which two substances are interspersed between each other. A hydrocolloid has
colloid
particles spread throughout water and depending on the quantity of water
available can
10 take on different states, e.g: gel-like consistency or a sol (liquid).
Hydrocolloids can be
either irreversible (single state) or reversible. Examples include
carrageenan, gelatin,
cellulose, and pectin.
"Hydrogel" refers to a gel wherein water is the dispersion medium and wherein
the gel-
forming components are cross-linked.
"Lactic acid bacteria" as used herein denotes any bacteria which has the
property of
producing lactic acid from sugars by fermentation. Accordingly, the taxonomy
of lactic
acid bacteria as used herein can be based on the Gram reaction and the
production of
lactic acid from various fermentable carbohydrates. "Lactic acid bacteria"
includes any
bacteria belonging to a genus selected from the group consisting of
Carnobacterium,
Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus,
Pediococcus,
Streptococcus, Tetragenococcus, Vagococcus and Weissella.
Reference is made to Stiles and Holzapfel; Int. J. Food Microbiol. (1997),
36(1), pp. 1-
29 and to the monograph "Genera of Lactic Acid Bacteria" by Holzapfel and
Wood,
published in 1998 as vol. 2 by Springer Verlag in the series "The Lactic Acid
Bacteria"
(ISBN: 0-7514-0215-X).
"Wound healing-promoting substance" is any substance capable of accelerating
the
wound healing process. Non-limiting examples include, PDGF (Platelet Derived
Growth
Factor), rhPDGF-BB (Becaplermin), EGF (Epidermal Growth Factor), PDECGF
(Platelet Derived Endothelial Cell Growth Factor), aFGF (Acidic Fibroplast
Growth
Factor), bFGF (Basic Fibroplast Growth Factor), TGF-alpha (Transforming Growth
Faxtor alpha), TGF-beta (Transforming Growth Factor beta), KGF (Keratinocyte
Growth Factor), VEGF( Vascular endothelial growth factor), IGF1/IGF2 (Insulin-
Like
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Growth Factor), ,coliagen, hyaluronic aoicl, protease ihhibitors such as;
alpha-1
antitrypsin and SLPI (Secretory Leukocyte Protease lnhibitor), and
antibacterial
proteins or pt;pfides direbted speoifically against undesirable, putrefactive
bacteria.
A'lvound healing-promoting substance is in preferred embodiments a bioaciive
agent
produced by a lactic acid bacteria. The wound healing-promoting substance can
sither
be a recombinantly expressed protein or an agent naturally expressed by the
lactic acid
bacteria. Examples of such naturally lactic aoid bacteria bioactive agents
include, but is
not limited to, any bloactive agent capable of inhibiting the growth of
putrefactive,
putrefactive microorganisms through other metabolic products such as e.g.
hydrogen
peroxide.
Preferred metabolites produced by lactie acid procuding bacteria according to
the
present invention that are bel9eved to exert antagonistic action(s) against
putrefaetive
microorganisms, as well as their mode of action, are summarized in the below
Table 1.
Table 1: Antagonistic activities caused by preferred lactic acid producing
bacteria
+~ . t ' h" .il '~ `*i { , , = ~ 1~
'~" M v^!. w+
, 1 i j ~4~, ~,Nt;. . ', ~ =,#jf ~ ~';~ ~ `~~~{~ 'i;jvrr ainf '~'~" .',7 9 ,;r
~` ~t "u `~ '~~;t ~ ~2M,`r y~lfi~h s,.,q~{~ ~, , , ( ~
. ,~
M v ~4~i
~+" a x ~ { ~ ~~i = " ~ ~~~~i
. , rr
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Yet another exampte of a lactic acid bacteria bioaciive agents believed to be
capable of
promoting wound healing is B-vitamins. Experiments on fermented milk products
have
revealed that lactic cuttures require B-vitamins for the'u metabolic
activities. However,
some lactic cultures synthesize B-vitamin 16 which - according to one
presently
preferred hypothesis - is believed to be involved in the promotion of wound
healing.
It should be noted that B-v'itamin contents of fermented milk prcducts are
believed to
be a function of both species as well as the strain of lactic acid bacteria
used in the
manufacture of the vitamin in question. Similarly, vitamins are synthesized by
the lactic
cultures in the gut microflora, In symbiosis with other flora.
"Treatment" as used herein refer equally to curative therapy, prophylactic
therapy, and
preventative therapy, The term includes an approach for obtaining beneficial
or desired
physiological resuits, which may be established clinically. For purposes of
this
invention, beneficial or desired clinical results include, but are not limited
to, alleviation
of symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening)
condition, delay or slowing of progression or worsening of condition/symptoms,
amelioration or palliation of the ccrndftion or symptoms, and remission
(whether partial
or total), whether detectable or undetectable. The term "palliation", and
variations
thereof, as used herein, means that the extent and/or undesirable
manifestations of a
physiological condition or symptom are Iessened and/or time course of the
progression
is slowed or lengthened, as compared to not administering compositions of the
present
tnvention.
A "treatment effect" or "therapeutic effect' is manlfested 'rf there is a
change in the
condition being treated, as measured by the criteria constituting the
definition of the
terms "treating" and "treatment " There is a"change" in the condition being
treated 'rf
there is at least 5% improvement, preferably 10% improvement, more preferably
at
least 25%, even more preferably at least 50%, such as at least 75%, and most
preferably at least 100% improvement The change oan be based on improvements
in
the severdy of the treated condifion in an individual, or on a difference in
the frequency
of improved conditions in populations of individuals with and without
treatment wifh the
bioactive agent, or with the bioaotive agent in combination with a
pharmaceutical
composition of the present invention.
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"Pharmacologically effective amount , "pharmaceutically effective amount" or
"physiologically effective amount of a"bioactlve agent" is the amount of an
active agent
present in a pharmaceutical composition as described herein that is needed to
provide
a desired level of active agent at the site of action in an lndividual to be
treated to give
an anticipated physiologioal response when such composition is administered.
The
precise amount will depend upon numerous factors, e.g., the active agent, the
activity
of the composition, the delivery device employed, the physical characteristics
of the
composition, intended patient use (i.e., the number of doses administered per
day),
patient considerations, and the like, and can readily be determined by one
skilled in the
art, based upon the information provided herein. An "effective amount" of a
bioactive
agent can be administered in one administration, or through multiple
administrattons of
an amount that total an effective amount, preferably within a 24-hour period.
It can be
determined using standard clinical procedures for determining appropriate
amounts
and timing of administration. It is understood that the "effective amount" can
be the
resuft of empirical and/or indlvidualized (case-by-case) determination on the
part of the
treatyng health care professional and/or individual.
The terms "enhancing" and "improving a beneficial effect, and variations
thereof, as
used herein, refers to the therapeutic effect of the bioactive agent against
placebo, or
an increase in the therapeutlc effect of a state-of-the-art medical treatment
above that
normally obtained when a pharrmaceutical composition is administered without
the
bioactive agent of this invention. "An increase in the therapeutic effects" is
man"rfested
when there is an aoceleration and/or increase in intensity and/or extent of
the
therapeutic effects obtained as a resuft of administering the bloactive
agent(s). It also
includes extension of the longevity of therapeutic beneffts. It can also
manifest where a
lower amount of the pharmaceutical composition is required to obtain the same
benefits and/or effects when it is co-administered wRh bloactive agent(s)
provided by
the present invention as compared to the administration in a higher amount of
the
pharmaceutical composition in the absence of bioactive agent. The enhancing
effect
preferably, but not necessarily, results in treatment of acute symptoms for
which the
phannaceutical composifion alone Is not effective or is less effective
therapeutically.
Enhancement is achieved when there is at least a 5"/a increase in the
therapeutic
effects, such as at least 10% increase in the therapeutic effects when a
bioactive agent
of the present invention is co-administered with a pharmaceutical composition
compared with administration of the pharmaceutical composition alone.
Preferably the
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increase is at least 25%, more preferably at least 50%, even more preferably
at least
75%, most preferably at least 3 00%.
"Co-administering or "co-administration" of bioac[ive agent(s), or bioactive
agents and
state-of-the-art medicaments, as used herein, refers to the administration of
one or
more bioactive agents of the present invention, or administration of one or
more
bioactive agents of the present invention and a state-of-the-art
pharmaceutical
composition within a certain time period. The time period is preferably less
than 24
hours, such as less than 12 hours, for example less than 6 hours- such as less
than 3
hours. However, these terms also mean that the bioacbive agent and a the-
dpeutic
composition can be administered together.
"individual" refers to vertebrates, particular members of the mammalian
species, and
includes, but is not limited to domestic animals, such as cattle, horses,
pigs, sheep,
mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as
horses, poly
ponies, dogs, camels, and primates, including humans.
Brief description of the drawing
Schematic drawings of different, non-limiting embodiment of the present
invention are
illustrated in Figure 1.
Figure 1, panel A: A wound dressing that allows the metabolites of the lactic
bacteria to
be delivered to the wound. Lyophilized lactic acid producing bacteria are
contained in a
membrane bag that allows for the passage of proteins but not for the bacteria.
On top
of the bacteria containing bag is attaChed an absorbing pad and a topfilm to
avoid
leakage of moisture. An adhesive surface is applied on the edges of the
topfilm.
Figure 1, panel 6: A wound dressing that allows for the lactic acid bacteria
to be
released into the wound. Capsules containing lyophilized lactic acid producing
bacteria
are distributed into an absorbing pad, As the pad absorbs fluid, the capsules
will
disintegrate and release the bacteria into the wound.
Figure 1, panel C: In a dual chamber syringe, one chamber is pre-filled with a
hydrogel
and the other chamber is pre-fflled with lyophilized lactic acfd producing
bacteria. When
the plunger is pressed the membrane closing the chambers are broken and the
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hydrogel and lactic acid bacteria from the two chambers are mixed in the
mixing
chamber before exiting the syringe.
Detailed description of the Invention
5 The present invention in one embodiment is directed to a wound or tissue
dressing
comprising lactic acid bacteria. The wound or tissue dressing can further
comprise an
absorbent compound for absorbing wound exudate, wherein the lactic acid
bacteria are
either attached to or comprised in the absorbent oompound.
10 Absorbent compound
The absorbent compound can be any compound capable of absorbing wound exudate.
In one embodiment the absorbent compound comprises or consists of a hydrogel
forming material. The hydrogel forming material can form an amorphous
hydrogel, but
15 the hydrogel forming material can also be in the form of e.g. a sheet - in
which case
the dressing will be a hydrogel sheet dressing.
In other embodiments, the absorbent compound of the wound or tissue dressing
comprises or consists of a hydrocolloid forming material.
The absorbent compound can comprise or consist of a porous polymer suitable
for
entry of wound exudate therein, i.e. the capilary force allows wound exudate
to enter
into the porous polymer. The porous polymer is often hydrophilic or
su'fficiently
hydrophilic to allow transpon of wound extrudate.
In a still further embodiment the absorbent compound comptises or consists of
a foam
forming material.
It is important that the absorbent compound is In fluid contact with the wound
e.g.
through a gel or a matrix, such as a scaffold, or, altematively, that the
absorbent
compound can contact the wound directly.
The porous material of the absorbent compound can be bioabsorbable and be
adapted
for serving as scaffold for new cells t0 migrate into and proliferate. Such
a"connective'
absorbent scaffold can remain In place on the wound bed throughout the healing
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process, and later be absorbed and replaced by new tissue. During the wound
healing
process, the connective absorbent compound will transmit wound exudate from
the
wound bed to the bioabsorbable and/or porous material of the absorbent
compound.
The lactic acid bacteria can be attached, covalently or non-covalently, to the
gel or
matrix of the bioabsorbable and/or porous material of the absorbent compound,
or the
lactic acid bacteria can be present in, i.e. mixed with, the bioabsorbable
and/or porous
material of the absorbent compound. In one embodiment the lactic acid bacteria
are
contained in a compartment separated from the bioabsorbable and/or porous
material
of the absorbent compound.
In one embodiment, when being prepared for use, a breakable barrier sealing
the
wound or tissue dressing from an external environment is broken and the lactic
acid
bacteria are brought into contact with the bioabsorbable and/or porous
material of the
absorbant compound - and over time the lactic acid bacteria are also brought
into
contact with wound exudate and the wound environment itself.
The absorbent compound can be a material that is absorbent to liquid while at
the
same time serves as a barrier for cell penetration. Such an absorbent compound
can
be referred to as an "absorbent barrier material". An absorbent barrier
material can e.g.
prevent lactic acid bacteria present in the bioabsorbable and/or porous
material of the
absorbent compound from entering the wound itself. However, bioactive agents
produced by the lactic acid bacteria and having wound healing promoting
abilities are
allowed to enter the wound area.
Besides absorbing wound exudate and preventing undesirable bacteria from
entering
the wound, the absorbent compound can also act as a reservoir for liquids to
hydrate
the wound. The features of non-adhesion and resistance to penetration by cells
provide
the important advantage that the absorbent barrier material - and any
subsequent
connective compound - is easily removed and/or replaced as needed without
causing
trauma to growing cells or tissue.
If desirable, the absorbent compound can be in contact with a further
compound, such
as a breathable film that can serve as a barrier to the entry of contaminants
into the
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17
wound bed. One example of such a barrier is a topfilm.
The absorbent compound can be any material approved for wound care. Materials
that
can be used as an absorbent compound include fabrics, foams or fibres of e.g.
polyester, polyurethans, polypropylenes and polyethylenes which are optionally
bonded
to polyester film (such as Kendall's Novenette). Other suitable materials
include, but
are not limited to, natural and synthetic polymeric absorbents, hydrocolloids,
superabsorbents, and cellulosic absorbents. Cellulosic materials include
cotton, rayon,
wood and cellulose.
The absorbent compound can be a superabsorbent material in any suitable form.
Typical superabsorbents include starch grafted copolymers of acrylate salts,
starch
grafted copolymers of acrylamide salts, polyacrylate salts and the like,
including
mixtures thereof.
Superabsorbent compounds and composites are easily prepared or commercially
available. Once such product is the composite air laid superabsorbent pad (dry
forming
process and the superabsorbent fiber flock SAFF) sold by Hanfspinnern Steen &
Company. The superabsorbent may also be a delayed released web superabsorbent.
Superabsorbent webs that may be used in the present invention to serve as, or
to be
incorporated into, the absorbent compound can also include carded or random
webs
made from, for example, cotton, rayon, polyethylene, polyester, or wool.
Another
suitable web is a spun-laced web made from polyester, polypropylene, or
polyethylene.
The superabsorbent webs may also be in the form of tissues either single ply
or
multiple ply and either creped or uncreped. Delnet, a product of Applied
Extrusion
Technologies which consists of a range of materials manufactured from
polyethylene or
polypropylene using extrusion embossing and orientation processes may also be
used
as a web for preparing a superabsorbent web.
Superabsorbent webs can be formed by any convenient means, e.g., by slightly
moistening or misting a web. After misting, a powdered superabsorbent may be
applied
followed by running the web through a dry oven or heating the roll. The powder
adjacent to the moistened web will become tacky and adhere to the adjacent
material
(fiber, surface), and the loose powder would then be vacuumed off.
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Alternatively, superabsorbent powder can be sandwiched between non-woven
webs/paper and subjected to moist steam which would make the superabsorbent
tacky
so that it would then stick to adjacent surfaces. The sandwiched
superabsorbent and
web would then be dried, creating a two-ply web with superabsorbent between
them.
The superabsorbent connective compound can also be heat bonded to the other
connective compounds.
The wound or tissue dressing according to the present invention can contain
from
about 5% to about 50% by weight of water, such as from about 5% to about 40%
by
weight of water, for example from about 5% to about 30% by weight of water,
such as
from about 5% to about 25% by weight of water, for example from about 5% to
about
20% by weight of water, such as from about 5% to about 15% by weight of water,
for
example from about 5% to about 10% by weight of water, such as from about 10%
to
about 40% by weight of water, for example from about 10% to about 30% by
weight of
water, such as from about 10% to about 25% by weight of water, for example
from
about 10% to about 20% by weight of water, such as from about 10% to about 15%
by
weight of water, such as from about 15% to about 40% by weight of water, for
example
from about 15% to about 30% by weight of water, such as from about 15% to
about
25% by weight of water, for example from about 15% to about 20% by weight of
water.
Adhesive surface
The absorbent compound can comprise at least one adhesive surface suitable for
contacting a wound or the absorbent compound can be attached to at least one
adhesive surface suitable for contacting a wound. When the absorbent compound
is
attached to at least one adhesive surface suitable for contacting a wound the
absorbent compound and the adhesive surface are most often manufactured
separately and only brought together during the manufacturing of the wound or
tissue
dressing according to the present invention. The adhesive surface can simply
be
positioned on or spread out over the corresponding surface of the absorbent
compound, such as the absorbent compound surface which is going to be aligned
with
the surface of a wound.
The at least one adhesive surface can be separated from the absorbent compound
by
a permeable or semi-permeable barrier allowing wound extrudate to be diverted
from
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19
the wound to the absorbent compound. Alternatively, the at least one adhesive
surface
can itself comprise a barrier acting as a permeable or semi-permeable barrier
that
allows wound extrudate to be diverted from the wound to the absorbent
compound.
The absorbent compound can also be attached to a topfilm at least partly
sealing the
absorbent compound from the external environment. Alternatively, the absorbent
compound itself comprises a functionality acting as a topfilm at least partly
sealing the
absorbent compound from the external environment.
The topfilm is often porous and the topfilm can comprise an oxygen- and vapor-
permeable layer permitting transpiration of liquid from the absorbent
compound.
Gelatin and collaaen absorbent compounds
In some embodiments the wound or tissue dressing according to the present
invention
comprises an absorbent compound comprising or consisting of gelatin and/or
collagen,
including a combination of gelatin and collagen.
When the absorbent compound comprises or consists of gelatin, the gelatin can
be
cross-linked and form a matrix, such as a matrix in the form of a hydrogel.
Alternatively, the wound or tissue dressing can comprise or consist of gelatin
which is
not crosslinked. The gelatin can be in granulated or particulated form and
most often
such dressings employ hydrocolloids.
When the absorbent compound comprises or consists of collagen the collagen can
be
cross-linked and form a matrix, such as a matrix in the form of a hydrogel.
Alternatively, the wound or tissue dressing can comprise or consist of
collagen which is
not crosslinked. The collagen can be in granulated or particulated form and
most often
such dressings employ hydrocolloids.
Hyaluronic acid can be present in the dressing in combination with any or both
of
gelatin and collagen. In one embodiment, there is provided an absorbant
compound
comprising a biologically absorbable material and hyaluronic acid, or a
derivative
thereof.
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Hyaluronic acid is a natural heteropolysaccharide consisting of alternate
residues of D-
glucuronic acid and N-acetyl-D-glucosamine. It is a linear polymer having a
molecular
weight ranging from about 50 to about 13,000 kDa, depending on the source it
is
5 obtained from and on the method of preparation. Hyaluronic acid is the main
component of the extracellular matrix, but it has other functions such as
hydration of
tissues, lubrication as well as cell migration and differentiation. A suitable
molecular
weight for hyaluronic acid for the purposes described herein will be in the
range of from
50 to 5,000 kDa, such as in the range of from 50 to 4,000 kDa, e.g. in the
range of from
10 100 to 3,000 kDa. In a particular preferred embodiment of the invention,
the hyaluronic
acid, or a derivative thereof, has a molecular weight in the range of from 250
to 3,500
kDa, more preferably in the range of from 500 to 2,500 kDa, such as in the
range of
from 500 to 2,000 kDa.
15 Optionally, the hyaluronic acid molecule may be cross-linked, e.g. by
chemical or
physical means. In a preferred embodiment of the invention the employed
hyaluronic
acid is pH neutral, i.e. an aqueous solution of the employed HA exhibits a pH
value in
the range of from 5 to 9, preferably in the range of from 6-8, in particular
in the range of
from 6.5 to 7.5, such as about 7. The hyaluronic acid used in the present
invention may
20 be extracted from any source, for example from rooster comb. Alternatively
hyaluronic
acid may be obtained by fermentation.
Derivatives of hyaluronic acid include, for example, esters of hyaluronic
acid, as well as
the derivatives described in US 5,356,883; US 6,548,081; US 4,851,521; US
6,027,741; US 2003 181689; EP 1 095 064; EP 0 341 745; WO 02/18450 and WO
2004/035629. In addition, the term "derivative" is also intended to cover
hyaluronate
salt, including, but not limited to, sodium hyaluronate, potassium
hyaluronate,
magnesium hyaluronate and calcium hyaluronate.
Alciinate absorbent compounds
In one embodiment the absorbent compound comprises an optionally cross-linked
alginate compound, such as an alginate ester, for example an alginate ester
comprising propylene glycol alginate.
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The degree of esterification of the alginate ester is typically from from 35%
to 95% and
the absorbent compound can contain from 10% to 25% by weight of the alginate
ester.
Reference is made to US 6,022,556 and US 5,735,812, both of which are
incorporated
herein by reference.
Wound healing-promoting substance(s)
The invention in some embodiments is directed to dressings and methods further
comprising one or more wound healing-promoting substance(s) either added as
proteins or supplied by recombinantly expression by the lactic acid bacteria.
Such
substances are e.g. capable of promoting the healing of slow-healing or
chronic
wounds. Examples include alpha-1 antitrypsin, SLPI (Secretory Leukocyte
Protease
Inhibitor), PDGF (Platelet Derived Growth Factor), rhPDGF-BB (Becaplermin),
EGF
(Epidermal Growth Factor), PDECGF (Platelet Derived Endothelial Cell Growth
Factor),
aFGF (Acidic Fibroplast Growth Factor), bFGF (Basic Fibroplast Growth Factor),
TGF-
alpha (Transforming Growth Faxtor alpha), TGF-beta (Transforming Growth Factor
beta), KGF (Keratinocyte Growth Factor), IGF1/IGF2 (Insulin-Like Growth
Factor),
VEGF (Vascular endothelial growth factor).
Lactic acid bacteria
Lactic acid bacteria produce lactic acid from lactose by means of enzymes such
as
beta-galactosidases, glycolases and lactic dehydrogenases (LDH).
A lactic acid bacteria in accordance with the present invention can be any
bacteria
capable of producing lactic acid - and actually producing lactic acid and/or
another
desirable bioactive agent, such as a protease, under practical circumstances
when
employed in connection with the claimed wound or tissue dressing.
Lactic acid producing bacteria according to the present invention are
preferably
selected from the group consisting of Carnobacterium, Enterococcus,
Lactobacillus,
Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus,
Tetragenococcus, Vagococcus and Weissella. In one embodiment, Bacillus
coagulans
is excluded from the above definition of lactic acid producing bacteria.
The skilled person will be aware that the genus Lactobacillus remains
heterogeneous
with over 60 species (ymol% G+C content ranging from 33 to 55). Lactobacilli
are gram
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22
positive and vary in morphology from long, slender rods to short coccobacilli,
which
frequently form chains. Their metabolism is fermentative; some species are
aerotolerant and may utilize oxygen through the enzyme flavoprotein oxidase,
while
others are strictly anaerobic. While spore bearing lactobacilli are
facultative anaerobes,
the rest are strictly anaerobic. The growth is optimum at pH 5.5 to 5.8 and
the
organisms have complex nutritional requirements for amino acids, peptides,
nucleotide
bases, vitamins, minerals, fatty acids and carbohydrates.
The genus Lactobacillus is generally divided into three groups based on
fermentation
patterns:
1. homofermentative: produce more than 85% lactic acid from glucose.
2. heterofermentative: produce only 50% lactic acid and considerable amounts
of
ethanol, acetic acid and carbon dioxide.
3. less well known heterofermentative species which produce DL-lactic acid,
acetic acid
and carbon dioxide.
Particularly preferred species of Lactobacillus include L. sporogenes; L.
acidophilus; L.
plantarum; L. casei; L. brevis; L. delbruckii and L. lactis.
According to one presently preferred hypothesis, the lactic acid forming
bacteria
according to the present invention are capable of counter-acting a delayed
wound
healing process caused by bacterial infection of a wound, as described in more
detail
herein below.
Apart from causing pain, swelling and odour problems for the patient,
putrefactive
bacteria also affect the wound healing process through the production of
proteases and
toxins, as well as by promoting a chronic inflammatory state. In the
inflammatory state,
cells of the tissue (e.g. neutrophils) release numerous proteases. Proteases
from said
putrefactive bacteria as well as from inflammatory cells can be expected to
digest
growth factors and other desirable proteins that would otherwise have been
able to
promote the wound healing process.
The activity of a protease is likely to be dependent on the pH of the
environment in
which the protease is to exert its activity (Schultz et al, 2005). Many
proteases (e.g.
elastase and plasmin) have a pH optimum at about pH 8. Open wounds tend to
have a
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23
neutral or alkaline pH, predominantly in the range of from 6.5 to 8.5
(Dissemond et al,
2003). Chronic wounds are known to have permanently elevated protease levels
and
one strategy in accordance with the present invention for promoting the wound
healing
process is to modulate and preferably reduce the proteolytic activity of
undesirable
proteases by using a pH modulator, such as the lactic acid produced by the
lactic acid
forming bacteria present in the wound or tissue dressing according to the
present
invention.
Accordingly, one beneficial action of the lactic acid bacteria according to
the present
invention is their ability to modulate the activity of undesirable
polypeptides present in a
wound which has been infected by putrefactive bacteria.
The levels of optical isomeric forms of lactic acid produced by lactic acid
forming
bacteria depend upon the nature of the culture. The structural configurations
of these
isomers are as follows:
COOH COOH
H---C-y OH H O- C-y H
CH CH
3 3
D(-) levorotatory lactic acid L(+) dextrorotatory lactic acid
Both forms may be relevant in connection with the present invention. In
humans, both
isomers are absorbed from the intestinal tract. Whereas L(+) lactic acid is
completely
and rapidly metabolized in glycogen synthesis, D(-) lactic acid is metabolized
at a
lesser rate, and the unmetabolized acid is excreted in the urine. As an
example, L.
acidophilus produces the D(-)-form, whereas L. sporogenes produces only L(+)-
lactic
acid.
It is furthermore believed that some of the more volatile acids produced
during lactic
acid bacteria fermentation may also possess some antimicrobial activity, e.g.
under
conditions of low oxidation-reduction potential.
Lactococcus as used herein refer collectively to a lactic acid bacterial genus
of five
major species. They are typically spherical or ovoid, 0.5 to 1.2 pm by 0.5 to
1.5 pm,
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24
and occur in pairs and short chains. They are non-spore forming and are not
motile.
The type species for the genus is L. lactis. Lactococcus differ from other
lactic acid
bacteria as they have pH, salt and temperature tolerances for growth.
The new genus Weissella has been established to include one member of the
genus
Leuconostoc (Leuc. paramesenteroides) and heterofermentative lactobacilli with
unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut
division of
the streptococci, morphological and physiological features of Weissella do not
directly
support this grouping which now incorporates species that produce D(-)-lactate
as well
as DL-lactate.
The new genus Carnobacterium is morphologically similar to the lactobacilli,
but it
shares some physiological similarities (e.g. growth at pH 9.5) and a common
phylogenetic branch with the genus Enterococcus.
The wound or tissue dressing according to the present invention can contain -
in one
embodiment - from 100 to 109 lactic acid producing bacteria per cm3, such as
from 101
to 106 lactic acid producing bacteria per cm3, for example from 102 to 104
lactic acid
producing bacteria per cm3.
Biodearadable microspheres
The optionally lyophilized lactic acid producing bacteria can be encapsulated
or
formulated as a gel - e.g. in gelatin or collagen - with a view to stabilizing
the lactic acid
producing bacteria. In one embodiment, the optionally lyophilized lactic acid
producing
bacteria are formulated as a lipid and/or polymer-comprising microsphere which
includes at least one biodegradable polymer.
The biodegradable polymers can be homopolymers, such as polylactides,
polyglycolides, poly(p-dioxanones), polycaprolactones, polyhydroxyalkanoates,
polypropylenefumarates, polyorthoesters, polyphosphate esters, polyanhydrides,
polyphosphazenes, polyalkylcyanoacrylates, polypeptides, or genetically
engineered
polymers. At the same time, the biodegradable polymers can be copolymers
(random
or block) such as poly(lactide-glycolides), poly(p-dioxanone-lactides), poly(p-
dioxanone-glycolides), poly(p-dioxanone lactide-glycolides), poly(p-dioxanone-
caprolactones), poly(p-dioxanone-alkylene carbonates), poly(p-dioxanone-
alkylene
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oxides), poly(p-dioxanone-carbonate-glycolides), poly(p-dioxanone-carbonates),
poly(caprolactone-lactides), poly(caprolactone-glycolides),
poly(hydroxyalkanoates),
poly(propylenefumarates), poly(orthoesters), poly(ether-esters), poly(ester-am
ides),
poly(ester-urethanes), polyphosphate esters, polyanhydrides, poly(ester-
anhydrides),
5 polyphosphazenes, polypeptides and genetically engineered copolymers. The
lipids of
the microspheres, when present, can be zwitterionic lipids, acidic lipids,
cationic lipids,
sterols, or triglycerides of many types, including many phospholipids.
A biodegradable polymer according to the present invention is one that can be
10 degraded to a low molecular weight and may or may not be eliminated from a
living
organism. The products of biodegradation may be the individual monomer units,
groups of monomer units, molecular entities smaller than individual monomer
units, or
combinations of such products. Such polymers may also be metabolized by
organisms.
Biodegradable polymers can be made up of biodegradable monomer units. A
15 biodegradable compound is one that can be acted upon biochemically by
living cells or
organisms, or parts of these systems, or reagents commonly found in such
cells,
organisms, or systems, including wound exudates and similar aqueous
compositions,
including water, and broken down into lower molecular weight products. An
organism
can play an active or passive role in such processes.
The biodegradable polymer chains useful in the present invention preferably
have
molecular weights in the range of from 500 to 5,000,000. The biodegradable
polymers
can be homopolymers, or random or block copolymers. The copolymer can be a
random copolymer containing a random number of subunits of a first copolymer
interspersed by a random number of subunits of a second copolymer. The
copolymer
can also be block copolymer containing one or more blocks of a first copolymer
interspersed by blocks of a second copolymer. The block copolymer can also
include a
block of a first copolymer connected to a block of a second copolymer, without
significant interdispersion of the first and second copolymers.
Biodegradable homopolymers useful in the invention can be made up of monomer
units selected from the following groups: hydroxy carboxylic acids, such as
alpha-
hydroxy carboxylic acids, including lactic acid, glycolic acid, lactide
(intermolecularly
esterified dilactic acid), and glycolide (intermolecularly esterified
diglycolic acid); beta-
hydroxy carboxylic acids, including beta-methyl-beta-propiolactone; gamma-
hydroxy
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26
carboxylic acids, delta-hydroxy carboxylic acids; and epsilon-hydroxy
carboxylic acids,
including epsilon-hydroxy caproic acid; lactones, such as: beta-lactones;
gamma-
lactones; delta-lactones, including valerolactone; and epsilon-lactones, such
as
epsilon-caprolactone; benzyl ester-protected lactones, such as benzyl
malolactone;
lactams, such as: beta-lactams; gamma-lactams; delta-lactams; and epsilon-
lactams;
thiolactones such as 1,4-dithiane-2,5-dione; dioxanones; unfunctionalized
cyclic
carbonates such as: trimethylene carbonate, alkyl substituted trimethylene
carbonates,
and spiro-bis-dimethylene carbonate (2,4,7,9-tetraoxa-spiro[5.5]undecan-3,8-
dione);
anhydrides; substituted N-carboxy anhydrides; propylene fumarates;
orthoesters;
phosphate esters; phosphazenes; alkylcyanoacrylates; aminoacids;
polyhydroxybutyrates; and substituted variations of the above monomers.
Hydrocolloids
The wound or tissue dressing can comprise a hydrocolloid, but in some
embodiments
the hydrocolloid can be omitted. In embodiments wherein a hydrocolloid is
used, the
hydrocolloid comprises about 20 to about 60 weight percent of the wound or
tissue
dressing, based on total weight.
The hydrocolloid can comprise e.g. from about 25 to about 55 weight percent of
the
composition, such as from about 30 to about 50 weight percent of the
composition. In
one embodiment, the hydrocolloid comprises about 40 weight percent of the
composition.
The hydrocolloid used in the present invention can be synthetically prepared
or
naturally occurring. Varieties of hydrocolloids within the scope of the
present invention
include synthetic polymers prepared from single or multiple monomers,
naturally
occurring hydrophilic polymers, or chemically modified naturally occurring
hydrophilic
polymers. It is preferred that the hydrocolloid is dermatologically acceptable
and non-
reactive with the skin of the patient or other components of the composition.
Preferred
examples are hydrocolloids comprising gelatin and/or collagen.
Further specific examples include hydrocolloids comprising e.g.
polyhydroxyalkyl
acrylates and methacrylates, polyvinyl lactams, polyvinyl alcohols,
polyoxyalkylenes,
polyacrylamides, polyacrylic acid, polystyrene sulfonates, natural or
synthetically
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modified polysaccharides, alginates, gums, and cellulosics and modified
celluloses.
Representative polysaccharides include e.g. starch, glycogen, hemicelluloses,
pentosans, celluloses, pectin, chitosan, and chitin.
Representative gums include e.g. Arabic, Locust Bean, Guar, Agar, Carrageenan,
Xanthan, Karaya, tragacanth, Ghatti, and Furcelleran gums.
Representative modified celluloses include methyl cellulose, hydroxypropyl
methyl
cellulose, carboxymethylcellulose, and hydroxypropyl cellulose.
Hydrocolloids which are water soluble or swellable hydrocolloids can be
selected e.g.
from the group consisting of polyvinyl alcohols, powdered pectin, methyl
cellulose,
hydroxypropyl methyl cellulose, carboxymethylcellulose, hydroxypropyl
cellulose and
mixtures thereof.
Further examples of suitable hydrocolloids include synthetic polymers that may
be
either linear or crosslinked. Non-limiting examples of synthetic hydrocolloids
include
e.g. polymers prepared from N-vinyl lactams, e.g. N-vinyl-2-pyrrolidone, 5-
methyl-N-
vinyl-2-pyrrolidone, 5-ethyl-N-vinyl-2-pyrrolidone, 3,3-dimethyl-N-vinyl-2-
pyrrolidone, 3-
methyl-N-vinyl-2-pyrrolidone, 3-ethyl-N-vinyl-2-pyrrolidone, 4-methyl-N-vinyl-
2-
pyrrolidone, 4-ethyl-N-vinyl-2-pyrrolidone, N-vinyl-2-valerolactam, and N-
vinyl-2-
caprolactam.
Other monomers useful to prepare a synthetic hydrocolloid include hydroxyalkyl
acrylates and methacrylates, (such as 2-hydroxyethyl acrylate, 2-hydroxyethyl
methacrylate, 2-hydroxypropyl acrylate, 2-hydroxypropyl methacrylate, 2,3-
dihydroxypropyl methacrylate), acrylic acid, methacrylic acid and a tertiary
amino-
methacrylimide, (e.g. trimethylamino-methacrylimide), crotonic acid, and
pyridine.
Additional monomers useful to prepare a synthetic hydrocolloid include water
soluble
amides, (such as N-(hydroxymethyl)acrylamide and -methacrylamide, N-(3-
hydroxpropyl)acrylamide, N-(2-hydroxyethyl) methacrylamide, N-(1,1-dimethyl-3-
oxabutyl)acrylamide N-[2-(dimethylamine)ethyl]acrylamide and -methacrylamide,
N-[3-
(dimethylamino)-2-hydroxylpropyl]methacrylamide, and N-[1,1-dimethyl-2-
(hydroxymethyl)-3-oxabutyl]acrylamide); water-soluble hydrazine derivatives,
(such as
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trialkylamine methacrylimide, and dimethyl-(2-hydroxypropyl)amine
methacrylimide);
mono-olefinic sulfonic acids and their salts, (such as sodium ethylene
sulfonate,
sodium styrene sulfonate and 2-acrylamideo-2-methylpropanesulfonic acid); and
the
following monomers containing nitrogen in the non-cyclic or cyclic backbone of
the
monomer: 1-vinyl-imidazole, 1-vinyl-indole, 2-vinyl imidazole, 4(5)-vinyl-
imidazole, 2-
vinyl-l-methyl-imidazole, 5-vinyl-pyrazoline, 3-methyl-5-isopropenyl-pyrazole,
5-
methylene-hydantoin, 3-vinyl-2-oxazolidone, 3-methacrylyl-2-oxazolidone, 3-
methacrylyl-5-methyl-2-oxazolidone, 3-vinyl-5-methyl-2-oxazolidone, 2- and 4-
vinyl-
pyridine, 5-vinyl-2-methyl-pyridine, 2-vinyl-pyridine-1 -oxide, 3-isopropenyl-
pyridine, 2-
and 4-vinyl-piperidine, 2-and 4-vinyl-quinoline, 2,4-dimethyl-6-vinyl-s-
triazine, and 4-
acrylyl-morpholine.
Hvdroaels
Cross-linking of the linear polymer chains of the hydrocolloid may be desired
to
improve cohesive properties of the gel dispersed in the pressure sensitive
adhesive
matrix. When such crosslinking is desired for polymers made from vinyl
monomers
discussed above, a multi-ethylenically unsaturated compound with the ethylenic
groups
being vinyl, allyl, or methallyl groups bonded to nitrogen, oxygen or carbon
atoms can
be used.
Non-limiting examples of cross-linking agents for vinyl containing polymers
include
divinyl, diallyl, or dimethallyl esters (e.g. ethylene glycol dimethacrylate,
divinyl
succinate, divinyl adipate, divinyl maleate, divinyl oxalate, divinyl
malonate, divinyl
glutarate, diallyl itaconate, diallyl maleate, diallyl fumarate, diallyl
diglycolate, diallyl
oxalate, diallyl adipate, diallyl succinate, diallyl azelate, diallyl
malonate, diallyl
glutarate, dimethallyl maleate, dimethallyl oxalate, dimethallyl malonate,
dimethallyl
succinate, dimethallyl glutarate, and dimethallyl adipate); divinyl, diallyl
or dimethallyl
ethers (e.g. diethyleneglycol divinyl ether, butane diol divinyl ether,
ethylene glycol
divinyl ether, ethylene glycol diallyl ether, diethylene glycol diallyl ether,
butane diol
diallyl ether, ethylene glycol dimethallyl ether, diethylene glycol
dimethallyl ether, and
butane diol dimethallyl ether); divinyl, diallyl or dimethallyl amides
including bis(N-vinyl
lactams), (e.g., 3,3'-ethylene bis(N-vinyl-2-pyrrolidone) and methylene-bis-
acrylamide);
and divinyl, diallyl and dimethallyl ureas.
Preferred cross-linking agents include ethylene glycol dimethacrylate,
methylene-bis-
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acrylamide, diallyl maleate, and 3,3'-ethylidene bis (N-vinyl-2-pyrrolidone).
For n-vinyl
lactams, the preferred crosslinking agents are diallyl maleate and 3,3'-
ethylidene bis
(N-vinyl-2-pyrrolidone). For acrylates and methacrylates, the preferred
crosslinking
agents are ethylene glycol dimethacrylate and methylene-bis-acrylamide.
Humectants
The dressing can also contain a humectant to reduce the partial vapor pressure
of the
water in the wound or tissue dressing or to reduce the rate at which the wound
or
tissue dressing dries out. Suitable humectants are miscible with water to a
large extent
and are generally suitable for application to the skin.
The humectant can be e.g. glycerol and propylene glycol and the absorbent
compound
typically contains from about 10% to about 90% by weight of the humectant.
Polyols are especially suitable for the purpose and suitable polyols may
include
monopropylene glycol or glycerin (glycerol). The polyol may be present in
proportions
of 20 to 50% (by weight) of the total formulation; alternatively the range is
30 to 40%.
This relatively high proportion of polyol also ensures that if the paste
should dry out to
any degree, the resulting paste remains soft and flexible because the glycerin
may act
as a plasticiser for the polymer. When the paste is applied on a bandage, for
example,
it may therefore still be removed easily from the skin when the paste has lost
water
without the need to cut the bandage off. The polyol also has the advantage of
functioning to prevent the proliferation of bacteria in the paste when it is
in contact with
the skin or wound, particularly infected wounds.
Further bioactive compounds
The formulation can include other ingredients such as antibacterial agents,
antifungal
agents, anti-inflammatory agents, and the like. Other ingredients may also be
found
suitable for incorporation into the formulation.
Outer packaging comprising a sterile barrier seal
The "sterile" dressings according to the present invention can be protected
from
undesirable contamination by an outer packaging comprising a sterile barrier
seal in
the form of an unbroken seal separating the "sterile" dressing from an
external, non-
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sterile environment. The sterile barrier seal is only broken immediately prior
to use in
order to retain the "sterility" of the dressing according to the present
invention.
The outer packaging may be pelable or otherwise removable from the outer
surface of
5 the dressing. The dressing is preferably enclosed in an outer packaging of a
flexible,
semi-rigid, or rigid plastic and/or metallic film providing a sterile barrier.
The outer
packaging typically consists of materials selected from the group consisting
of plastics,
aluminium foils and plastic laminates, where the plastic is preferably
selected from the
group consisting of PET, PE, LLDPE, CPP, PA, PETP, METPET, Tyvek, said plastic
10 being optionally bonded with an adhesive (e.g. Polyurethane) or co-
extruded. The outer
packaging preferably forms a complete barrier to moisture.
A particularly interesting embodiment of the outer packaging includes a pouch
of
laminated foil. The laminate may be PET, such as PET having a thickness of
approximately 12 microns.
15 Method for manufacturing wound or tissue dressings according to the
invention
The present method is also directed to a method for manufacturing the wound or
tissue
dressing according to the invention, said method comprising the steps of
providing an
optionally encapsulated and/or lyophilized lactic acid producing bacteria,
mixing or
attaching said lactic acid producing bacteria with the wound or tissue
dressing, or with
20 the absorbent compound of the wound or tissue dressing, thereby obtaining a
wound or
tissue dressing according to the invention.
The method can comprise the further step of providing the absorbent compound
with at
least one adhesive surface suitable for contacting a wound, or the further
step of
25 attaching at least one adhesive surface suitable for contacting a wound to
the
absorbent compound.
In another further step there is provided a permeable or semi-permeable
barrier for
separating the at least one adhesive surface from the absorbent compound by
30 introducing said permeable or semi-permeable barrier between the absorbent
compound and the at least one adhesive surface, wherein said permeable or semi-
permeable barrier allows wound exudate to be diverted from the wound to the
absorbent compound.
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In a yet further step the method comprises providing a permeable or semi-
permeable
barrier capable of partly separating - during use - the at least one adhesive
surface
from the wound by introducing said permeable or semi-permeable barrier on the
surface of the adhesive surface, wherein said permeable or semi-permeable
barrier -
during use - allows wound exudate to be diverted from the wound to the
absorbent
compound through the adhesive surface.
In yet another step, the permeable or semi-permeable barrier will allow
proteins and
exudate to pass, but will retain the lactic acid producing bacteria in the
dressing.
In yet another further step a topfilm can be provided and attached to the
absorbent
compound, wherein said topfilm seals at least partly the absorbent compound
from the
external environment. The absorbent compound can also comprise a topfilm as an
integrated part, wherein said topfilm at least partly seals the absorbent
compound from
the external environment. The topfilm can be porous or non-porous. In one
embodiment, the topfilm comprises an oxygen- and vapor-permeable layer
permitting
transpiration of liquid from the absorbent compound.
In order to kept the moisture of the dressing low, the dressing can be packed
with a
desiccant, such as silica.
Wound treatment methods
Various uses of the wound or tissue dressings according to the present
invention are
envisaged. In one embodiment there is provided a method for treating a wound
in an
individual, said method comprising the steps of contacting said wound with the
wound
or tissue dressing according to the present invention, and treating the wound.
The treatment can in principle result in healing of the wound or in
accelerated healing
of the wound. The accelerated healing can be a result of e.g. administration
of a
wound-healing promoting substance, or a bioactive agent produced by the lactic
acid
bacteria. The wound-healing substance and/or the bioactive agent can be
produced
naturally or recombinantly by the lactic acid bacteria. Alternatively, the
wound healing
can be promoted by preventing bacterial or viral infection, or by reducing the
risk of
such an infection which would otherwise have prolonged the wound treatment
process.
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In another embodiment there is provided a method for treating damaged tissue
in an
individual, said method comprising the steps of contacting said damaged tissue
with
the wound or tissue dressing according to the invention, and treating the
damaged
tissue.
Likewise, the treatment can in principle result in healing of the damaged
tissue or in
accelerated healing of the damaged tissue. The accelerated healing can be a
result of
e.g. administration of a tissue-healing promoting substance, or a bioactive
agent
produced naturally or recombinantly by the lactic acid bacteria.
Alternatively, the
healing of damaged tissue can be promoted by preventing bacterial or viral
infection, or
by reducing the risk of such an infection which would otherwise have prolonged
the
treatment of the damaged tissue.
The tissue damage can e.g. be caused by bone protrudence, by diabetes, by
circulatory insufficiencies or by undesirable inflammatory processes in an
individual.
There is also provided a method for preventing or reducing the risk of wound
or tissue
infection in an individual having suffered a wound or damaged tissue, said
method
comprising the steps of contacting said wound or tissue with the wound or
tissue
dressing according to the invention, and treating the wound or tissue at risk
of being
infected. The infectious agent at risk of infecting the wound or tissue can be
a
pathogenic bacteria.
As e.g. gelatin and hyaluronic acid independently and in combination have a
haemostatic effect, there is also provided a method for promoting haemostasis
in a
wound in an individual, said method comprising the steps of contacting said
wound with
the wound dressing according to the invention, and promoting haemostasis in
the
wound.
In addition to contacting a wound or damaged tissue with the wound or tissue
dressing
according to the invention, there is also provided combination methods wherein
one or
more wound or tissue healing-promoting substance(s) are administered
simultaneously
or sequentially in any order one or more at the same time as the wound or
tissue to be
treated is contacted with the wound or tissue dressing according to the
invention. This
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may be of particular importance when treating slow-healing wounds, partial
thickness
wound, deep wounds and chronic wounds.
Accordingly, at least one wound healing-promoting substance selected from the
group
consisting of alpha-1 antitrypsin, SLPI (Secretory Leukocyte Protease
Inhibitor), PDGF
(Platelet Derived Growth Factor), rhPDGF-BB (Becaplermin), EGF (Epidermal
Growth
Factor), PDECGF (Platelet Derived Endothelial Cell Growth Factor), aFGF
(Acidic
Fibroplast Growth Factor), bFGF (Basic Fibroplast Growth Factor), TGF-alpha
(Transforming Growth Faxtor alpha), TGF-beta (Transforming Growth Factor
beta),
KGF (Keratinocyte Growth Factor), VEGF (Vascular endothelial growth factor),
and
IGF1/IGF2 (Insulin-Like Growth Factor), can be administered simultaneously or
sequentially in any order with the bioactive agent produced by the lactic acid
bacteria of
the absorbent compound.
In further embodiments the present invention is directed to the following
uses:
Use of a lactic acid bacteria in the manufacture of a wound or tissue dressing
for
treating a wound or tissue or accelerating the healing of a wound or tissue in
an
individual.
Use of a lactic acid bacteria in the manufacture of an absorbent compound for
use in a
wound or tissue dressing for treating a wound or tissue or accelerating the
healing of a
wound or tissue in an individual.
Use of a lactic acid bacteria in the manufacture of a wound or tissue dressing
for
preventing or reducing the risk of wound or tissue infection in an individual
having
suffered a wound.
Use of a lactic acid bacteria in the manufacture of a wound or tissue dressing
for
promoting haemostasis in a wound in an individual.
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Examples
Example 1:
Lyophilized Lactobacillus GG are resuspended in saline to a final
concentration of
109/ml. Gelatine sponges (2x2 cm) are kneaded in 0,8 ml of the lactobacillus
suspension or in 0,8 ml saline, and transferred to LB plates previously plated
with 100
ul of an 1/10 diluted o.n culture of Staphylococcus aureus, Psuedomonas
aeruginosa
or Escherichia coli. The plates are incubated at 37 C. After 3 days a growth
inhibitory
(clear) zone is visible around the gelatin sponges kneaded in the
lactobacillus
suspension but not around the sponges kneaded in saline.
Example 2:
In a two compartment syringe the one compartment is filled with 10 ml
hydrogel, sealed
and subsequently autoclaved. After the sterilization process, the other
compartment is
filled with 1010 lyophilized lactic acid bacteria formulated with suitable low
moisture
exhibiants (carboxymethyl cellulose and maltodextrin) and prebiotica
(trehalose) .
Staphylococcus aureus, Psuedomonas aeruginosa or Escherichia coli cultures are
grown to in exponential growth and 100 ul of the cultures and plated on LB
plates
when the cultures have reached an OD600 of 0,5. Wells are made in the agar
plates
and these wells are filled with a plain hydrogel or with the lactobacillus
containing
hydrogel using the two compartment syringe. The plates are incubated at 37 C.
After 3
days a growth inhibitory (clear) zone is visible around the wells containing
the hydrogel
with lactobacillus. No inhibitor zone is visible around the well containing
the plain
hydrogel.
Example 3:
Lyophilized Lactobacillus GG are resuspended in saline to a final
concentration of
109/ml and 1 ml of this suspension is filled in a dialysis tubing with a
molecular weight
cut offs at 300 kDa allowing molecules up to 300 kDa to pass, but retaining
the bacteria
in the tube. In another tube 1 ml of saline is added. The tubes are placed on
LB plates
previously plated with 100 ul of an 1/10 diluted o.n culture of Staphylococcus
aureus,
Psuedomonas aeruginosa or Escherichia coli. The plates are incubated at 37 C.
After
3 days a growth inhibitory (clear) zone is visible around the tubings
containing the
lactobacillus suspension but not around the tubings containing saline.
