U.S. patent application number 11/482193 was filed with the patent office on 2007-01-11 for wound dressings containing complexes of transition metals and alginate for elastase sequestering.
Invention is credited to Kelman I. Cohen, Yousef Mohajer.
Application Number | 20070009586 11/482193 |
Document ID | / |
Family ID | 39314705 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009586 |
Kind Code |
A1 |
Cohen; Kelman I. ; et
al. |
January 11, 2007 |
Wound dressings containing complexes of transition metals and
alginate for elastase sequestering
Abstract
Transition metal (e.g., silver and copper) derivatized
phosphorylated polysaccharides (cellulose, starch, gauze) provide
antimicrobial and elastase sequestration properties to wound
dressings, and the wound dressing have enhanced water sorption and
elastase sequestration when used with alginates. Wound dressings
with alginates (e.g., silver alginate, crosslinked alginates, etc.)
provide enhanced wound fluid absorption as well as elastase
sequestration.
Inventors: |
Cohen; Kelman I.; (Richmond,
VA) ; Mohajer; Yousef; (Midlothian, VA) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON & COOK, P.C.
11491 SUNSET HILLS ROAD
SUITE 340
RESTON
VA
20190
US
|
Family ID: |
39314705 |
Appl. No.: |
11/482193 |
Filed: |
July 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10446806 |
May 29, 2003 |
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11482193 |
Jul 7, 2006 |
|
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09515172 |
Feb 29, 2000 |
6627785 |
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10446806 |
May 29, 2003 |
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Current U.S.
Class: |
424/445 ;
424/618; 424/638; 514/54 |
Current CPC
Class: |
A61L 2300/606 20130101;
A61K 31/715 20130101; A61L 15/44 20130101; A61K 31/734 20130101;
A61L 2300/102 20130101; A61L 15/28 20130101; A61K 33/34 20130101;
A61K 33/34 20130101; A61K 33/38 20130101; A61L 2300/232 20130101;
A61K 33/38 20130101; A61P 43/00 20180101; A61P 17/02 20180101; A61L
2300/104 20130101; A61K 31/734 20130101; C08L 1/28 20130101; A61L
15/28 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/445 ;
514/054; 424/618; 424/638 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61L 15/00 20060101 A61L015/00; A61K 33/34 20060101
A61K033/34; A61K 33/38 20060101 A61K033/38 |
Claims
1. A wound dressing, comprising: a phosphorylated polysaccharide
substrate complexed with at least one transition metal; and an
alginate coating.
2. The wound dressing of claim 1 wherein said phosphorylated
polysaccharide is selected from the group consisting of
phosphorylated cotton and phosphorylated gauze.
3. The wound dressing of claim 1 wherein said alginate coating is
crosslinked.
4. The wound dressing of claim 1 wherein said alginate coating is
silver alginate.
5. The wound dressing of claim 1 wherein the transition metal is
selected from silver and copper.
6. A wound dressing comprising: a polysaccharide substrate; and an
alginate coating mixed with at least one transition metal.
7. A method for treating a wound, comprising: contacting a wound
with a wound dressing comprising a polysaccharide substrate with an
alginate coating; absorbing wound fluid with said wound dressing;
and sequestering elastase from said wound.
8. The method of claim 7 wherein said polysaccharide substrate is
phosphorylated and is complexed with at least one transition
metal.
9. The method of claim 7 wherein said alginate coating is mixed
with at least one transition metal
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of application Ser. No.
10/446,806 filed May 29, 2003, which is a continuation-in-part of
U.S. patent application Ser. No. 09/515,172, entitled "Wound
Dressing with Protease-Lowering Activity," filed on Feb. 29, 2000,
that are incorporated herein in entirety by reference.
FIELD OF THE INVENTION
[0002] The invention generally relates to wound dressings and their
methods of use. In particular, the invention provides wound
dressings with associated active agents such as protease inhibitors
and sequestrants which enhance the healing of wounds, especially
chronic wounds.
BACKGROUND OF THE INVENTION
[0003] The normal response to tissue injury is a timely and orderly
reparative process that results in sustained restoration of
anatomic and functional integrity.(Lazarus, et al. 1994). In
contrast, in chronic ulcers, the healing process is prolonged,
incomplete and proceeds in an uncoordinated manner resulting in
poor anatomical and functional outcome. Clinically, wounds are
categorized as acute and chronic based on the timeliness of
healing.
[0004] Most chronic ulcers are associated with a small number of
well-defined clinical entities particularly chronic venous stasis,
diabetes mellitus, and pressure ulcers. These conditions are
responsible collectively for approximately 70% of all chronic
ulcers (Nwomeh et al. 1998). The incidence and prevalence of
chronic ulcers vary considerably but are especially high in spinal
cord injury patients as well as the elderly and nursing home
population. As our society continues to age it is predicted that
the incidence of chronic ulcers will continue to increase
dramatically. Patients with pressure ulcers also have a significant
socioeconomic impact on our society. For example, health care
expenditures for treating pressure ulcers alone have been estimated
to exceed $3 billion a year (Nwomeh, et al. 1998). Normal healing
involves a complex cascade of events involving interaction among
many cell types, soluble factors and matrix components. Healing can
be arbitrarily divided into overlapping temporal phases of
coagulation, inflammation fibroplasia and finally remodeling. Most
of the events are cytokine regulated. Normally, during the
inflammatory phase, polymorphonuclear leukocytes (PMNs) are the
first of the leukocytes to appear. They produce various proteases
such as MMP-8 (collagenase) and elastase, which help to remove
damaged matrix and aid in healing. In both the open acute and
chronic wound, various cytokines are important in contraction and
spontaneous closure of the wound as well as angiogenesis. Under
normal circumstances, closure of the open wound is aided further by
epithelization as these surface cells seal the final closure.
[0005] Chronic wounds are very different. For example, pressure
ulcers are characterized by deep tissue necrosis with loss of
muscle and fat that is disproportionately greater then the loss of
overlying skin (Falanga, et al. 1998). These defects are common
among the immobilized and debilitated. There are approximately
225,000 spinal cord injury patients in the United States and
approximately 9,000 new cases per year. Approximately 60% of these
patients develop pressure ulcers and the annual cost is greater
then $25,000 per patient for medically related care.(Allman, 1998)
If the elderly nursing home population with pressure ulcers in
added to the spinal cord injury population then the figure for the
care of all pressure ulcers is enormous.
[0006] To date, the majority of the effort to improve rates of
healing of chronic wounds have focused on the use of exogenous
peptide growth factors and cell based products such as cytokines.
For the most part, these attempts have met with little notable
success. Another alternative approach has been the use of "skin
substitutes" such as Apligraf (matrix+cells) and Dermagraft
(matrix+cells). While this second approach has shown some promise,
its expense presently greatly limits its use to the richer
developed countries. Various modifications of the wound dressings
have also been suggested as a means to augment would healing.
[0007] Further examples include:
[0008] U.S. Pat. No. 5,098,417 to Yamazaki et al. teaches the ionic
bonding of physiologically active agents to cellulosic wound
dressings.
[0009] U.S. Pat. No. 4,453,939 to Zimmerman et al. teaches the
inclusion of aprotonin in composition for "sealing and healing" of
wounds. U.S. Pat. No. 5,807,555 to Bonte et al. teaches the
inclusion of inhibition for alpha-1-protease, collagenase, and
elastase in pharmaceutical compositions for promotion of collagen
synthesis.
[0010] U.S. Pat. No. 5,696,101 to. Wu et al., teaches use of
oxidized cellulose (e.g. Oxycel) as a bactericide and hemostat in
treatment of wounds.
[0011] World Patent WO 98/00180 to Watt et al. teaches complexation
of oxidized cellulose with structural proteins (e.g. collagen) for
chronic wound healing; and references the utility of
oligosaccharide fragments produced by the breakdown of oxidized
cellulose in vivo in the promotion of wound healing.
[0012] Neutrophils are a predominant infiltrating inflammatory cell
type present in the acute inflammatory response. Neutrophils
function primarily to destroy invading pathogens and to debride
devitalized tissue at the site of injury. The normal adult produces
approximately 10.sup.11 neutrophils per day. To function
effectively in host defense, they must migrate to the site of
inflammation and release selectively a large repertoire of lytic
enzymes, antimicrobial peptides, and potent oxidants from
cytoplasmic granules. Under other conditions, the neutrophil has
been implicated in causing disease by damaging normal host tissue.
Such inflammatory tissue injury are important in the pathogenesis
of a variety of clinical disorders including arthritis,
ischemia-reperfusion tissue injury and systemic inflammatory
response syndrome (SIRS) and the acute respiratory distress
syndrome(ARDS).(Weiss, 1989) There is strong evidence that
neutrophils also may have a significant role in the pathophysiology
of pressure ulcers.
[0013] Neutrophils are a prevalent cell type in pressure
ulcers.(Diegelmann, et al. 1999; Paloahti. et al. 1993; Rogers et
al. 1995) In addition, there is direct evidence correlating
neutrophil products with chronic pressure ulcers.(Yager, et al.
1996; Yager, et al. 1997). This includes neutrophil elastase,
gelatinase (MMP-9) as well as collagenase (MMP-8).(Wysocki, 1996;
Wysocki et al, 1993; Yager et al. 1997; Yager et al. 1996).
Therefore, these observations and the evidence that neutrophils
have been implicated in tissue destruction in other inflammatory
processes give strong credence to the hypothesis that neutrophil
products are involved in the pathogenesis of pressure sores and
subsequent failure to heal. Neutrophil-derived MMP-8 has been shown
to be the predominant collagenase in both acute and chronic
wounds.(Nwomeh, et al. 1999).
[0014] Neutrophils contain large amounts of elastase (1 pg /cell).
This serine protease has a broad substrate spectrum. As with
neutrophil-derived MM-8, elastase levels have also been found to be
significantly elevated in fluid derived from pressure ulcers.(Yager
et al. 1997) The presence of high levels of active elastase with a
wound site may have important implications for wound healing
therapies utilizing peptide growth factors. Elastase present in
chronic wounds can degrade peptide growth factors such as PDGF and
TGF-b.(Yager et al. 1997). Moreover, cell surface receptors for
peptide growth factors may themselves be functionally inactivated
by the actions of elastase. Elastase may also contribute to the
overall proteolytic environment of chronic wounds. It is known to
proteolytically inactivate the specific inhibitor, Tissue Inhibitor
of Metalloproteinases (TIMP). In addition, elastase itself may
participate in proteolytically activating collagenase and
gelatinase zymogens. Obviously, an unregulated proteolytic
environment can be a significant aspect of the pathophysiology of
chronic wounds.
[0015] It would be highly beneficial to have available additional
methods for enhancing wound healing. In particular, methods
directed to bringing the proteolytic environment of wounds under
control in order to promote wound repair would be desirable. Such
methods would be useful in the treatment of wounds in general, and
chronic wounds in particular. Further, it would be highly
beneficial if such methods were inexpensive and thus widely
accessible.
[0016] A number of patents and patent applications describe the use
of alginates in the treatment of burns or wounds. For example, U.S.
Pat. No. 6,696,077 to Scherr describes various silver alginate foam
compositions, and U.S. Pat. No. 6,809,231 to Edwards describes
cross-linked alginate formulations.
[0017] A number of patents and patent applications describe the use
of silver ions as antimicrobial agents. For example, U.S. Patent
Publication 2005/010900 to Qin describes polysaccharide fibers
formed with alginate that contain a silver compound as an
antimicrobial agent, and U.S. Patent Publication 2004/0241213 to
Bray describes carboxymethyl cellulose or alginate fibers in a
mixture that contains silver ions. Neither reference describes a
configuration where alginates are position in a wound dressing for
sequestration of elastase.
SUMMARY OF THE INVENTION
[0018] It is an object of this invention to provide novel wound
dressings for the treatment of wounds, especially for the treatment
of chronic, non-healing wounds. The wound dressings of the instant
invention are comprised of a support matrix which is preferably
cellulose, carboxymethylated cellulose, dialdehyde gauze,
sulfonated gauze, and phosphorylated gauze, in combination with
silver or copper, alginate or silver alginate. The wound dressings
combine antimicrobial activity together with elastase sequestration
and provide for enhanced wound healing in burns, surgical wounds,
ulcers and chronic wounds, etc. For example, in some embodiments,
the gauze may be derivatives with silver and then coated with
alginate. In other cases, the gauze may be coated with silver
alginate which may be bound to the support matrix or be able to
dissociate from the support matrix so as to allow migration in the
wound microenvironment. When phosphorylated gauze, sulfonated gauze
or dialdehyde gauze is used as a support matrix, the wound dressing
benefits from elastaste sequestration by the gauze itself as well
as by the alginate. The sequestrants bind proteases found in the
wound fluid and remove them from the wound microenvironment.
[0019] The invention also provides methods of use for the wound
dressings, including a method for sequestering elastase at a wound
site. This method comprises the step of contacting the wound site
with a wound dressing selected form the group consisting of
carboxymethylcellulose, dialdehyde gauze, sulfonated gauze, and
phosphorylated gauze together with transition metal such as silver
and alginate.
[0020] The dressings may be applied to wounds in order to enhance
would healing, especially the healing of chronic wounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B. Assessment of reduction in human neutrophil
elastase activity in samples of HLE after exposure to modified
cotton gauze. 3A: HLE samples were exposed to three different
oxidized cotton gauze samples corresponding to gauze Treatment
Methods 1, 2 and 3 (see Methods, Preparation of Dialdehyde Cotton
Gauze). 3B: HLE samples were exposed to 25 and 50 mg of two
different carboxymethylated cotton gauze samples, III and IV (see
Methods, Preparation of Carboxymethylated Cotton Gauze). Untreated
gauze was employed as a control. Data are mean.+-.S.D. of
triplicate determinations.
[0022] FIGS. 2A-C. Reaction progress curves for gauze-treated
solutions of elastase. Substrate hydrolysis was performed with a 60
.mu.M solution of MeOSuc-Ala-Ala-Pro-Val-pNA and reaction rates
monitored by spectrophotometric measurement of the release of
p-nitroaniline at 405 nm. 25, 50 and 75 mg samples of
phosphorylated cotton gauze (PSC, 5A), sulfonated cotton gauze
(SOC, 5B) and dialdehyde cotton gauze (DAG, 5C) were compared with
75 mg of untreated cotton gauze (UT).
[0023] FIG. 3 Initial velocities (v.sub.o) of residual elastase
activity in samples exposed to untreated gauze (UT), dialdehyde
gauze (DAG), sulfonated gauze (SOC), carboxymethylated gauze(CMC)
and phosphorylated gauze (PSC), compared to a sample that was not
treated with gauze (Bk).Weights of gauze samples were 75 (A), 50
(B), and 25 {circle over (C)} mg. Data are mean.+-.S.D. of
triplicate determinations. All are significantly different from
control, p<0.05, as determined by analysis of variance.
[0024] FIG. 4a-f are graphs of optimization experiments show the
amount of reagent used for phosphorylation of the substrate versus
the sequestering properties of the products at three different
temperatures (4a-c), and for the silver derivative of the
equivalent phosphorylated substrate at the same temperatures
(4d-f).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0025] The present invention is based upon the previously
unrecognized discovery that active agents such as inhibitors and
sequestrants of proteases may be used as healing accelerants of
wounds, and of chronic wounds in particular. These inhibitors and
sequestrants may be physically applied on wound dressings, or in
the alternative may be ionically or covalently conjugated to a
wound dressing material for purposes of sustained release of active
agent or sequestration of endogenous constituents from the wound
environment. In a preferred embodiment of the present invention,
the active agents inhibit or bind cationic, neutrophil-derived
proteases such as neutrophil elastase.
[0026] Specific pharmacological effects of proteases inhibitors and
sequences associated with wound dressings include inhibition of the
breakdown of growth factors that stimulate migration of cells to
the ulcer site of the wound, leading to the growth of new tissue
that heals the open wound. This technology is broadly applicable to
all forms of chronic wounds including diabetic ulcers and decubitus
bedsores. Both peripheral and central administration of the
compounds formulated on wound dressings accelerate wound healing of
chronic wounds. Silver complexed to matrix and silver alginate are
shown herein to have potent elastase sequestering properties which
were heretofore previously unrecognized and thereby inhibit
proteases such as human elastase and thus prevent growth factor and
tissue degradation. Alternatively, the silver alginate may be bound
(chemically (ionic or covalently) or physically) to the wound
dressing. As a component of such a matrix, they are able to
sequester destructive proteases from the microenvironment of the
wound, thus preventing the degradation of growth factors and
fibronectin that would otherwise occur.
[0027] The therapeutic administration of the modified wound
dressings containing inhibitors include a pharmacologically
effective dose of the inhibitor or sequestrant (e.g., silver
alginate, or silver alginate in combination with a phosporylated,
sulfonated or dialdehyde gauze) when used tin the treatment of a
patient in need thereof. The dose of inhibitor or sequestrant
required on the wound dressing to promote accelerated healing in
the patient ranges from about 0.2 mg/gram fiber to about 200
mg/gram fiber per day, with this in turn being dependent upon
specific factors including patient health, wound type, etc. The
term "patient" used herein is taken to mean mammals such as sheep,
horses, cattle, pigs, dogs, cats, rats, mice and primates,
including humans.
[0028] The term "wound dressing" used herein is taken to include
any pharmaceutically acceptable wound covering or support matrix
such as:
[0029] a) films, including those of a semipermeable or a
semi-occlusive nature such as polyurethane copolymers, acrylamides,
acrylates, paraffin, polysaccharides, cellophane and lanolin.
[0030] b) hydrocolloids including carboxymethylcellulose protein
constituents of gelatin, pectin, and complex polysaccharides
including Acacia gum, guar gum and karaya. These materials may be
utilized in the form of a flexible foam or, in the alternative,
formulated in polyurethane or, in a further alternative, formulated
as an adhesive mass such as polyisobutylene.
[0031] c) hydrogens such as agar, starch or propylene glycol; which
typically contain about 80% to about 90% water and are
conventionally formulated as sheets, powders, pastes and gels in
conjunction with cross-linked polymers such as polyethylene oxide,
polyvinyl pyrollidone, acrylamide, propylene glycol.
[0032] d) foams such as polysaccharide which consist of a
hydrophilic open-celled contact surface and hydrophobic closed-cell
polyurethane.
[0033] e) impregnates including pine mesh gauze, paraffin and
lanolin-coated gauze, polyethylene glycol-coated gauze, knitted
viscose, rayon, and polyester.
[0034] f) cellulose-like polysaccharide such as alginates,
including calcium alginate, which may be formulated as non-woven
composites of fibers or spun into woven composites.
[0035] Preferred wound dressings are polysaccharide containing
support matrices which are derivatized with silver or copper and/or
which have silver alginate bound to or placed upon them, and it is
envisioned to include chitosans, alginates (e.g., cross-linked or
in a form other than silver alginate) and cotton or
carboxymethylated cotton in the form of gauze, films,
hydrocolloide, hydrogels, hydroactives, foams, impregnates,
absorptive powders and pastes,
[0036] Especially preferred wound dressings include cotton
cellulose formed as woven or non-woven gauze. This type of wound
dressing has the advantage of being readily available and
relatively inexpensive. In this case, the protease sequestrant or
inhibitor may be linked to the cellulose polysaccharide chain
through a chemical substituent such as amino, carboxylate, citrate,
phosphate, sulfonate, chloride, bromide, mono-carboxylic acid,
di-carboxylic acid, tri-carboxylic acid; or, any pharmaceutically
acceptable salt thereof. Exemplary salts are seen to include those
of acids such as acetic, glycolic, lactic, pyruvic, malonic,
succinic, glutaric, fumaric, malic, tartaric, ascorbic, maleic,
hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,
salicylic, and 2-phenoxyhenzoic; and sulfonic acids such as methane
sulfonic acid and hydroxyethane sulfonic acid. Salts of the carboxy
terminal amino acid moiety may include the nontoxic carboxylic acid
salts formed with any suitable inorganic or organic bases.
illustratively, these salts include those of alkali metals, as for
example, sodium and potassium; alkaline earth metals, such as
calcium and magnesium; light metals of Group IIA elements including
aluminum, and organic primary, secondary, and tertiary amines, as
for example, trialkylamines, including triethylamine, procaine,
dibenzylamine, 1-ethenamine, N,N'-dibenzylethylenediamine,
dihydroabietylamine, N-alkylpiperidine and any other suitable
amine.
[0037] The wound dressings of the instant invention may be used
alone or as an adjunct to other therapeutic measures. For example,
the wound dressings may be used together with the administration of
exogenous growth factors. Obviously, conditions that increase the
stability of an exogenous peptide growth factor or its receptor
will likely promote its efficacy. The wound dressings of the
present invention may also be used in conjunction with skin grafts,
in which case a proteolytic environment that is under control will
less likely cause the "rejection" or melting of a skin substitute
graft.
[0038] The wound dressings may also include other therapeutically
beneficial substances such as antibiotics, vitamins, and the
like.
[0039] The dressings and methods of the present invention may be
utilized to treat any type of appropriate wound. In a preferred
embodiment, the wound that is treated is a chronic, non-healing
wound.
[0040] The invention is illustrated by the following Examples which
are intended to be illustrative but should in no way be construed
as limiting.
EXAMPLES
Carboxymethylated and Dialdehyde Cotton Gauze
Methods
Preparation of Dialdehyde Cotton Gauze
[0041] Dialdehyde cotton gauze (also referred to as 2,3
dialdehyde-anhydroglucos-cellulose, oxidized cellulose,
oxycellulose, or periodate-oxidized cellulose) was prepared as
follows: cotton gauze (12 ply-4 in..times.4 in.), USP type VII,
were treated under three different reaction conditions in lots of
50 gauze sponges as follows: Treatment 1: a 0.07 M solution of
sodium periodate for 1 h at 45.degree. C. with a solution pH of
4.2. Treatment 2: a 0.2 M solution of sodium periodate for 1.5 h at
45.degree. C. with a solution pH of 4.5. Treatment 3: a 0.2 M
solution of sodium periodate for 3 h at 45.degree. C. with a
solution pH of 4.5. Following the treatment excess periodate was
removed by rinsing the gauze through a screen under running tap
water. Following the rinse cycle the gauze samples were passed
through a conventional ringer to remove excess moisture. The
samples were then separated and placed on a wire rack to air dry
overnight. The dried gauzes were placed in Chex all II..TM..
instant sealing pouches (5 in..times.10 in.) and sterilized with
ethylene oxide gas by Micro Test Laboratories, Agauam, Mass.
Preparation of Carboxymethylated Cotton Gauze
[0042] Carboxymethylation was completed as outlined previously
(Liyanage et al, 1995). A solution was made by mixing 24 parts of
dichloroacetic acid with 24 parts of water, and while cooling in an
ice bath, stirring in 75 parts of sodium hydroxide solution. This
solution was used to pad a sample of cotton gauze to a wet pickup
of 135%. The wet sample was then placed in an oven at 100.degree.
C. and dried/cured for 10 minutes.
Determination of Dialdehyde Content and Degree of Substitution of
Carboxymethylcellulose
[0043] Previously outlined procedures were employed to determine
the dialdehyde content (Hofreiter et al. 1995) and the degree of
substitution for the carboxymethylated gauze (Reinhardt et al.)
Assay of Treated Gauze for Elastase Activity
[0044] Treated and untreated gauze samples were submerged in 1
milliliter of buffer containing 0.1 units/ml of human neutrophil
elastase. The samples were allowed to incubate for one hour at room
temperature, and the gauze samples were removed and placed in a
press to drain unbound buffer and enzyme. The unbound buffer and
enzyme fractions were combined and assayed for elastase activity as
described below.
Enzyme Assays
[0045] Enzyme assays of the solutions containing unbound human
neutrophil elastase were conducted in pH 7.6 buffer composed of 0.1
M sodium phosphate, 0.5 M NaCl, and 3.3% DMSO and subjected to
spectrophotometric measurement of the release of p-nitroaniline at
410 nm from the enzymatic hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA
(Sigma). The spectrophotometric kinetic assays were performed in a
BioRad Microplate Reader (Hercules, Calif.) with a 96-well format.
200 microliter aliquots of a elatase solution (0.2 units) were
assayed to initiate the enzyme reaction.
Results
[0046] The gauze finishes employed in this study were prepared to
assess the effect of 1) both sterilization and variation of the
sodium periodate finishing conditions on the activity of dialdehyde
cotton gauze in reducing elastase activity; and 2) the effect of
the degree of substitution of carboxymethylated gauze in reducing
elastase activity.
[0047] As shown in FIG. 1A, variation of the oxidation conditions,
and hence percent aldehyde incorporation, effects elastase-lowering
activity of the dialdehyde cotton gauze. The results of these
studies suggests that Treatment #1 is optimal for retaining
efficacy of the dialdehyde cotton gauze. Prolonged exposure and
higher periodate concentration, which is correlated with fewer
dicarbonyl units in the cotton cellulose, appears to decrease the
efficacy of the gauze in reducing elastase activity in
solution.
[0048] Two different degree of substitution (DS) levels of carboxy
methylated cotton cellulose were also compared. As shown in FIG.
1B, higher substitution levels of carboxylate on cotton resulted in
an increased reduction in elastase activity in solution.
[0049] Correlation of decreased enzyme activity with number of
carboxylate or aldehyde sites on cellulose observed within a narrow
range of enzyme rates of activity suggests that the cotton
derivatized aldehyde and carboxylates bind elastase into readily
accessible binding sites in the modified cotton fiber of the
gauze.
[0050] These results suggest that dialdehyde cotton gauze and
carboxymethylated gauze can be used to effect the sequestration of
the protease elastase from solutions of the enzyme.
Oxidized, Sulfonated, and Phosphorylated Cotton Gauze Dressings
Selectively Absorb
Neutrophil Elasase Activity in Solution
Methods
Preparation of Periodate-Oxidized, Sulfonated, and Phosphorylated
Cotton
[0051] 2,3 dialdehyde-anhydroglucose-cellulose (i.e. periodate
oxidized) cotton. Cotton gauze (12 ply-4 in..times.0.4 in.), USP
type VII, was treated in lots of 50 gauze sponges in a 0.07 M
solution of sodium periodate for 1 h at 45.degree. C. with a
solution pH of 4.2. Alternatively, cotton gauze was oxidized with
0.2M sodium metaperiodate (pH 5) at 40.degree. C. for 3 hours.
Following the treatment excess periodate was removed by rinsing the
gauze through a screen under running tap water. Following the rinse
cycle, the gauze were passed through a conventional ringer to
remove excess moisture. The samples were then separated and placed
on a wire rack to air dry overnight. The dried gauze are placed in
a Chex all II..TM.. instant sealing pouch (5.times.10 in.) and
sterilized with ethylene oxide gas by Micro Test Laboratories,
Agauam, Mass.
[0052] Sulfonated cotton. The cotton gauze may be sulfonated by
washing the dialdehyde oxycellulose with 5% sodium bisulfite
(NaHSO.sub.3) under pH 4.5, liquor ratio 1:60 for 3 hours. Excess
sodium bisulfite may be removed by rinsing with water under running
tap water. Following the rinse cycle the gauze are passed through a
conventional ringer to remove excess moisture. The samples are then
separated and placed on a wire rack to air dry overnight.
[0053] Phosphorylated cotton. Phosphorylation of cotton gauze is
accomplished by applying inorganic phosphate salt (sodium
hexametaphosphate) to cotton gauze in 4-16% composition. Urea is
usually included in the formulation on a 2:1 weight ratio of urea
to phosphate. All formulations contained 0.1% Triton X-100 as a
wetting agent. The cotton gauze is padded to 80-90% wet pickup and
then dried at 60.degree. C. The samples are cured at 160.degree. C.
for 7 min.
[0054] The phosphorylated and sulfonated cotton cellulose D.S.
levels were 0.035 and 0.011 respectively, as measured by elemental
analysis.
Carboxymethylated Cotton Gauze
[0055] Carboxymethylation was completed as outlined previously
(Reinhart et al. 1957). A solution was made by mixing 24 parts of
dichloroacetic acid with 24 parts of water and while cooling in an
ice bath stirring in 75 parts of sodium hydroxide solution. This
solution was used to pad a sample of cotton gauze to a wet pickup
of 135%. The wet sample was then placed in an oven at 100.degree.
C., and dried/cured for 10 minutes.
Free-Swell Absorbency and Wicking Test
[0056] A free-swell absorbency test was performed as follows: A 0.5
gram sample of the cotton gauze was placed in 30 mL of a 0.9% by
weight aqueous saline solution and left for 5 minutes. The cotton
textile was then filtered through a sintered Mark 1 funnel of pore
size 100-160 microns and is left for 5 minutes, or until it stops
dripping. The water filtered through the funnel was weighed and the
weight of water absorbed by the filaments is calculated by
subtraction. A wicking test was made by immersing the cotton gauze
in deionized water containing foxboro red dye such that the gauze
was just touching the water surface. The time required for the dye
solution to migrate 1.5 cm on the gauze strip was measured.
Patients and Wound Fluid
[0057] Informed consent was obtained for all procedures, and
approval was received from the Virginia Commonwealth University
Committee on the Conduct of Human Research, in accordance with the
1975 Declaration of Helsinki. Fluids were harvested from a grade
III trochanteric pressure ulcer of a patient with spinal cord
injury using a sub-atmospheric device (V.A.C.R.TM.., KCl, San
Antonio, Tex.). Fluids were clarified by centrifugation at 14,000 g
for 15 min at 4.degree. C. The protein concentration was determined
with the Bio-Rad Protein assay (Richmond, Calif.) with bovine serum
albumin as a quantitation standard.
Assay of Wound Fluid
[0058] The patient wound fluid was diluted (1:100; wound fluid:
buffer; v:v) at a volume of 3 mL with buffer (0.1M sodium
phosphate, 0.5 M NaCl, and 3.3% DMSO) and incubated with weighed
samples of gauze ranging from 75 mg to 700 mg. The gauze samples
were soaked in the wound fluid solutions for one hour whereupon the
solutions were filtered from the gauze under pressure applied to
the gauss using a Whatman Autovial (0.45 micron PFTE membrane).
Recovery of the wound fluid solution from the gauze was judged to
be 90%. The wound fluid solution was assayed for elastase activity
in a manner similar to the elastase enzyme assay described below.
Rates of substrate hydrolysis were measured on a reaction progress
curve of absorbance versus time.
Sequestration and Inhibition of Elastase Activity by Finished
Cotton Gauze
[0059] The effect of a variety of cotton gauze finishes was tested
to assess extraction of elastase from solution. Carboxymethylated,
sulfonated, phosphorylated, and oxidized cotton gauze were assayed
as 50 and 75 milligram samples of type VII cotton gauze (used
typically in patients with chronic wounds). Treated and untreated
gauze samples were submerged in 1 milliliter of buffer containing 1
unit/mL of human neutrophil elastase. The samples were allowed to
incubate for one hour at room temperature, and each individual
gauze sample was removed and placed in an Autovial press filter
(Whatman,) to extract unbound buffer and enzyme. The filtered
fraction of each individual sample was re-combined with solution
not taken up by the gauze and assayed for elastase activity.
[0060] The modified gauze containing bound elastase was assessed
for recoverable enzyme activity by pooling gauze samples and
extracting bound elastase with 20% acetic acid solution. Samples of
1-2 grams of modified gauze were soaked in acetic acid solutions,
filtered and the solutions lyophilized to dryness. The lyophilized
pellet was resuspended in buffer, filtered on a sintered glass
filter funnel and the resulting solution was assayed in 200
microliter aliquots. Elastase activities recovered from the gauze
were 43 milliunits per gram in untreated gauze and 160 milliunits
per gram from dialdehyde cotton gauze.
Enzyme Assays
[0061] Enzyme assays of the solutions containing unbound human
neutrophil elastase were conducted in pH 7.6 buffer composed of
0.1M sodium phosphate, 0.5 M NaCl, and 3.3% DMSO and subjected to
spectrophotomeric measurement of the release of p-nitroaniline at
410 nm from the enzymatic hydrolysis of
N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitoranilide (Sigma) (Nakajima
et al. 1979). The spectrophotometric kinetic assays were performed
in a Bio-Rad Microplate Reader (Hercules, Calif.) with a 96-well
format. Two hundred microliter aliquots of an elastase solution
(0.2 units) were assayed per well, and 20 microliters of a 60
micromolar substrate solution was added to initate the enzyme
reaction.
Inhibition of Elastase Activity with Dialdehyde Starch
[0062] Elastase activity was measured in dialdehyde starch
solutions. Solutions of dialdehyde starch (Sigma) were prepared in
the buffer described above at concentrations of 100 to 0.1
micromolar. The dialdehyde starch solutions were incubated with
stirring in Reacti-Vials with 0.2 units/mL of elastase for an hour.
The solutions were centrifuged at 1200.times.g for five minutes and
the supernatant was assayed for elastase activity as described
above.
Results
[0063] Cotton gauze was subjected to phosphorylation, oxidation,
and sulfonation. The degree of substitution (D.S.) was determined
by a standard degree of substitution relationship for cellulose
(based on the per cent of total phosphorous and sulfur for the
phosphorylated and sulfonated samples). Base titration of free
carboxyls was employed to determine D.S. levels on
carboxymethylated cotton cellulose (CMC). The phosphorylated and
sulfonated cotton cellulose D.S. levels were 0.035 and 0.011
respectively. This corresponds to one phosphate for every 28
anhydroglucose units and one sulfate for every 91 anhydroglucose
units. The degree of substitution for the dialdehyde was also 0.011
since the bisulfite addition reaction is utilized to determine D.S.
levels for dialdehyde cotton. The degree of substitution for CMC
was 1.4.
Effect of Modified Gauzes on Elastase Activity
[0064] Initial experiments examined the ability of the modified
cotton celluloses to absorb purified neutrophil elastase.
Twenty-five, fifty and seventy-five milligram quantities of gauze
were soaked to saturation for an hour in one milliliter of buffered
solution containing 0.2 units of elastase. Unbound enzyme was
removed by filtration followed by pressing under high pressure. The
recovery of buffer from the filtration process was found to be
90%.
[0065] The assessment of elastase activity in solution exposed to
the treated gauze was performed on the unbound enzyme.
Acid-extractable elastase activity was assayed in a 96-well format
using MeOSuc-Ala-Ala-Pro-Val-pNa for substrate hydrolysis. The
kinetics of elastase activity is based on the relative initial
velocity (v.sub.o) values for enzyme solutions exposed to cotton
gauze. In this study 0.2 units of elastase were tested per sample.
Measurement of elastase activity remaining in solution upon
treatment with the gauze was accomplished by monitoring the
reaction rate within a thirty-minute time frame. The reaction
progress curves for the treated samples are shown in FIG. 2a-c. A
decrease in active enzyme sites is apparent from the decreasing
dose response relation of the treated gauze samples with
dialdehyde, sulfonated, and phosphorylated cotton. The decreased
rate reflects a decrease in units of elastase activity retained in
the eluted buffer. A plot of v.sub.o values shown in FIG. 3 for the
samples also demonstrates this dose response relationship. The plot
of v.sub.o values was within the same range for the dialdehyde,
sulfonated and phosphorylated cotton. A similar decrease in
velocity was demonstrated with increasing weight of treated
gauze.
[0066] The lower v.sub.o values for the treated samples when
compared with the untreated cotton gauze suggests that the elastase
activity is retained in the treated cotton gauze due to selected
modifications on the gauze. Retention of elastase activity in
treated gauze was found to be four-fold higher than in untreated
gauze.
[0067] To assess whether the dialdehyde cotton gauze may act
through active site uptake of elastase, dialdehyde starch was
employed as a soluble aldehydic polysaccharide that may bind
elastase. The results demonstrated that inhibition of elastase by
dialdehyde starch is observed within a low micromole range, which
is an inhibitory concentration within the titer of aldehydes per
gram of dialdehyde cotton used in the current study. Thus,
inhibition of elastase activity by a soluble form of a high
molecular weight aldehydic carbohydrate suggests that the
dialdehyde cotton gauze may function as a serine protease
sequestrant through active site access to elastase.
[0068] Non-specific binding of the enzyme by the dialdehyde cotton
gauze is an alternative explanation for elastase inhibition by
dialdehyde cotton gauze. Since aldehydes can form Schiff bases with
protein amino groups the potential for Schiff base formation
between the protein amino groups of elastase and the aldehydes of
dialdehyde cotton (DAG) was a concern. To mimic the effect of
protein amines a high molecular weight polylysine was employed.
Polylysine is a single amino acid biopolymer containing only
epsilon amines as the side chains of the primary amino acid
structure. To test for a potential non-specific Schiff base
reaction effect between the elastase and the DAG, the dialdehyde
cotton was incubated in a polylysine solution and elastase added to
the solution to test for retention of elastase-lowering activity.
DAG retained its inhibitory effect on elastase in the presence of
polylysine. Based on this result it may be inferred that
proteinaceous amines do not interfere with the observed
elastase-lowering effects of the dialdehyde cotton gauze.
Elastase-Lowering Activity in Wound Fluid
[0069] The dialdehyde cotton gauze (DAG) was selected for further
evaluation using human wound fluid. To assess the ability of the
modified gauze to lower wound fluid-containing elastase activity in
comparison to untreated gauze (UT), DAG samples and UT were placed
in wound fluid in a range of 2.5 to 20 milligrams of gauze per
microliter of patient wound fluid. After exposure to the DAG or UT,
the solutions of chronic wound fluid were assessed for residual
elastase activity using a known elastase substrate
[0070] The results showed that the chronic wound fluid which had
been exposed to DAG possessed less elastase activity than that
which had been exposed to UT at each quantity of gauze tested. This
suggests that more elastase has been sequestered by DAG than by UT.
Increasing the quantity of DAG resulted in a dose dependent
decrease in the amount of retained elastase activity.
[0071] These results reflect the superior ability of the DAG
samples to remove elastase activity from wound fluid as compared to
untreated cotton gauze. Dialdehyde cotton gauze extracted 2-5 fold
more elastase activity with increased gauze loading per volume of
wound fluid when compared with untreated gauze.
[0072] Measurement of protein levels remaining in the wound fluid
following incubation with the gauzes was performed to compare the
relative amounts of protein taken up by treated and untreated
gauze. Lower levels of protein were found in the wound fluid soaked
with DAG than with the untreated cotton. This is consistent with
the lower activity of elastase found in the wound fluid soaked with
DAG samples.
[0073] The results obtained demonstrate that dialdehyde cotton
effects the sequestration of the protease elastase from wound
fluid.
[0074] We have optimized phosphorylation of cellulosic materials to
achieve products with exceptional sequestering properties for
elastase and other proteases. These optimization involved
utilization various phosphorylation reagents, achieving products
with great sequestering properties at high product throughput and
lower reagent utilization. FIGS. 4a-d for example show
phosphorylation of cotton gauze with monoammonium phosphate (MAP)
in the presence of urea (U) at different loading at mild
temperatures. In these figures the X-axis represent the % solid
content of fabric before curing (MAP+U) and the Y-axis is the %
elastase sequestration (using pure neutrophil elastase at 20 mu/ml
and fabric to liquid ratio of 50 mg/1 ml soln). All the fabrics had
been cured for 6 minutes. It is interesting to note that (FIG. 4A,
B, C) we can carry out these reactions at temperature as low as 130
C to achieve products with good elastase sequestering performance
(Elastase Sequestering for cotton is about 25% using pure
elastase). But it is more interesting to note that silver
derivatives of these phosphorylated cotton gauzes have outstanding
elastase sequestering properties (see, FIG. 4C and D) whereas
silver derivative of product formed at 80 C does not show this
performance. Silver derivatization was achieved by bringing 1 g of
phosphorylated product with 10 ml of silver nitrate solution (200
ppm silver nitrate) for about 2 minutes followed by rinsing with
excess deionized (DI) water. No free silver nitrate remains on the
fabric which is white. It is apparent that phosphorylation at 80 C
is not adequate enough to make good performing silver derivative
(FIG. 4E). Additional benefits of these transition metal
derivatives, such as silver or copper, is that in addition to their
great elastase sequestering performance they also possess
antimicrobial properties.
[0075] Moreover, by incorporating alginates and starches into
cellulosic products we have made products with high liquid sorption
and liquid retention capabilities. In experiments described below
we used ammonium alginate, Protamon S obtained from FMC Biopolymer,
Philadelphia Pa. Other types of alginates such as sodium alginate,
potassium alginate, magnesium alginate or alginic acid could be
used as well in the wound dressing applications contemplated by
this invention. Alginates are biopolymers of mannuronic (M) and
glucoronic (G) acids. Alginate occurs in the seaweed as a mix of
calcium, magnesium, sodium and potassium salts. The process of
manufacturing of this product from the seaweed involves extraction
of the alginic acid from the weed followed by neutralization by
different salts to prepare appropriate product. Depending on the
source of the seaweed and the extraction process different grades
of product with different M and G ratio and sequence and molecular
weight are obtained. Depending on the molecular weight of the
alginate, different viscosity is obtained at the same
concentration. Above 2.5% wt/wt solution the ammonium alginate we
used produced solutions with viscosity over 200 centipoise at room
temperature so we kept concentration below this value.
Elastase Sequestering Performance:
[0076] The elastase sequestering performance of our products was
measured by placing 50 mg of the product in 1.00 ml of Simulated
Wound Fluid (SMFL). SMFL was prepared by dissolving leukocyte
elastase into buffer at concentration of 20 m. unit/ml. Included in
SMFL is 0.1% bovine serum albumin (BSA) to simulate average chronic
wound. We have found that this formulation has activity similar to
"average" chronic wound and allows us to compare performance of
different wound care product to each other and to controls. The
elastase sequestering performance of the product is obtained by
comparing the activity of the SMFL fluid before and after
equilibration (2 hr) of the fluid with the product.
Competitive Liquid Sorption and Retention
[0077] This procedure is to simulate the performance of products
according to the invention when they are in touch with another
liquid absorber. These absorptive materials retain fluid better
than the standard cotton dressing. Therefore there is not only
absorption of fluid, but he absorb fluid is retained within the
dressing. Clinically this means that dressing could be left in the
wound for longer period of time and the normal skin adjacent to the
wound would be less likely to be macerated. This procedure is to
simulate fluid sorption capacity of wound care products according
to the invention as compared to average liquid sorption capacity of
cotton gauze or paper towel. The experiment is carried out in the
following manner. A piece of the product (0.1 to 1.00 gram) is
allowed to come in contact with water for 60 seconds. The amount of
the water used in this experiment is in excess of total sorption
capacity of the product and we keep it to 1.5 ml water per 0.1 g of
the product. So if the wound care product is 0.2 gram, we use 3.0
ml and if 0.5 gram, we use 7.5 ml of water.
[0078] After the product has soaked the water, it is removed and
placed flat on 10 folds of dry paper towels allowing excess water
to drain away for 90 seconds. The product is then weighed
accurately and the % water retained under this competitive
condition is calculated as described below; % Competitive Water
Sorption/Retentive=100.times.(Wt.sub.90
sec-Wt.sub.Original)/Wt.sub.Original Where Wt.sub.90 sec is the
weight of the dressing after 90 second of contact with the dry
paper towel and Wt.sub.Original is the original weight of the
dressing. This property will be referred to % water retention
throughout this patent (% WRC). Alginate and Silver Alginate
Solution Preparation Preparation of 2.2% Alginate Solution:
[0079] Dissolved 10.0 gram of ammonium alginate in 449.0 grams of
water and mixed overnight to achieve uniform solution. Solutions
above 3% had too high viscosity and were difficult to handle
without heating.
Preparation of 1.1 wt % Alginate 10% Neutralized with Silver
Nitrate
[0080] Alginate solution was diluted by mixing 40 gram of the 2.2%
alginate solution with 30 gram of DI water. Silver nitrate (Reagent
Grade) from Baker was dissolved in DI water (77.4 mg in 5 ml) and
added drop wise into the alginate solution with rapid agitation. A
semi-clear solution resulted after overnight agitation. This
"solution" will settle if left standing indicating gel formation.
When we attempted to make higher concentration solutions, insoluble
gels were formed which were hard to handle. Also when we attempted
making silver alginate solutions above 10% neutralization (molar),
gel formation made this product difficult to handle. Therefore a
major drawback of making silver alginate and using them for making
products is difficulty of handling gels. Despite this difficulty,
in some applications, silver alginates may be preferred.
[0081] Highly liquid retentive dressings can be formed by coating
of cotton or phosphorylate cotton with alginate and silver
alginates as shown in Table 1. TABLE-US-00001 TABLE 1 % Elastase
Seq. Examples Description SWFL % CWR Comparative 1 Cotton Gauze
16.0 100 Comparative 2 PhosCot 44.2 100 Example 1 PhosCot w/3.7%
Alg 45.3 513 Example 2 PhosCot w/3.7% Silver 77.7 540 Alg (10%)
Comparative 3 Cotton w/3.7% Alg 26.3 583 Example 3 Cotton w/3.7%
Alg 79.4 436 (10% Silv)
In Table 1, PhosCot stands for phosphorylated cotton. Comparative
Example 1 shows the behavior of common cotton which has low
elastase sequestering performance and also low competitive liquid
retention capacity. PhosCot (Comparative Example 2) has
significantly higher elastase sequestering property, but it has low
% CWR. Cotton coated with alginate (Competitive Example 3) has high
% CWR, but it has low % elastase sequestering properties. On the
other hand when phosphorylated cotton is coated with alginate
(Example 1) or with silver alginate (Example 2) we have products
with both good % Elastase sequestrations and % CWR. We can also
achieve good performance when coating cotton with silver alginate
(Example 3). As we have mentioned in the silver alginate solution
preparation the viscosity and gel formation is a difficulty that
could be avoided by other approaches of preparing these products by
different processes as described below.
[0082] Desirable products (Table 2) were prepared by coating cotton
(Example 4) or phosphorylated cotton (Example 5) with ammonium
alginate in the first step, and then exposing the alginate coated
fabric to silver nitrate solution to convert alginate into silver
alginate to get product with high elastase sequestering properties
(% CWR was not measured for these samples but were high). We used
1000 ppm silver nitrate solution for this derivatization. The
advantage of this process is that one does not have to deal with
gels of silver alginate, but the disadvantage of this process is
that during the silver conversion a portion of the alginate is
transferred into the silver nitrate solution (in form of gels) and
from stand point of process control this type of product formation
is not as desirable as process described below. TABLE-US-00002
TABLE 2 % Elastase Sequestering Fabric Process (SWFL) Example 4
Cotton W/4% Alginate 61.2 Then Silver Nitrate Example 5
Phosphorylated cotton with 4% Alginate, 59.4 then Silver
Nitrate
[0083] In this more preferred embodiment, the phosphorylated cotton
gauze (Comparative 2) is reacted with soluble silver compounds such
as silver nitrate or acetate to form silver complex with
exceptional elastase sequestering capacity (Comparative 4). However
the % CWR capacity of this product is not much different than that
of cotton. When such a product is then coated with alginates,
products with exceptional elastase sequestering and % CWR are
obtained (Examples 6 and 7). TABLE-US-00003 TABLE 3 % Elastase Seq.
Examples Description (SWFL) % CWR Comparative 2 PhosCot 44.2 100
Comparative 4 PhosCot, Silver derivative 85.0 100 (1000 ppm AgNO3)
Example 6 PhosCot, Silver derivative 79.0 275 (1000 ppm AgNO3),
then 4% Alginate Example 7 PhosCot, Silver derivative 72.5 383
(1000 ppm AgNO3), 6% Alginate
[0084] While phosphorylated cotton is preferred in the present
invention, other polysaccharide derivatives may complex with silver
such as phosphorylated starch, carboxymethyl cellulose, dialdehyde
gauze, and sulfonated gauze, and those of skill in the art will
recognize that the invention can be practiced with polysaccharides
which have the capacity to form complexes with transition metals
such as silver or copper.
[0085] It is also understood by those of skill in the art that
these alginate containing products could be cross-linked by
exposure to multivalent cations such as Ca2+, Cu2+, or Al3+ without
affecting their elastase sequestering properties or their % CWR
capacities as shown by examples that follow. In these examples,
after coating the fabric with alginate or silver alginate, the
products were brought in contact with 200 ppm calcium chloride (Ca)
or copper sulfate (Cu) and then the performances were measured. As
seen these products have great performances. Additionally these
products having large supply of silver or copper will have
antibacterial and antifungal properties. TABLE-US-00004 TABLE 4 %
Elastase Seq. Examples Description (SWFL) % CWR Example 8 PhosCot
w/3.7% Alg, then Ca 50.1 363 Example 9 PhosCot w/3.7% Alg (10%
Silv), 56.2 453 then Ca Example 10 PhosCot w/3.7% Alg, then Cu 53.1
364 Example 11 PhosCot w/3.7% Alg (10% Silv), 67.1 385 then Cu
These products have exceptional performance as dressings for
chronic wounds where management of exudates fluids is critical in
treatment of this class of wounds.
[0086] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
* * * * *