U.S. patent application number 10/446806 was filed with the patent office on 2003-11-06 for wound dressings with elastase-sequestering.
Invention is credited to Cohen, Kelman I., Diegelmann, Robert F., Edwards, Judson Vincent, Yager, Dorne.
Application Number | 20030206944 10/446806 |
Document ID | / |
Family ID | 24050242 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030206944 |
Kind Code |
A1 |
Cohen, Kelman I. ; et
al. |
November 6, 2003 |
Wound dressings with elastase-sequestering
Abstract
The invention provides wound dressings and methods of their use,
especially for the treatment of chronic, non-healing wounds. The
wound dressings are composed of a support matrix, such as cotton
cellulose, and an active agent associated with the support matrix.
The active agent may be a protease inhibitor or a protease
sequestrant, in particular an inhibitor or sequestrant of a
neutrophil-derived cationic protease such as elastase.
Inventors: |
Cohen, Kelman I.; (Richmond,
VA) ; Diegelmann, Robert F.; (Richmond, VA) ;
Yager, Dorne; (Chesterfield, VA) ; Edwards, Judson
Vincent; (Mandeville, LA) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON, P.C.
11491 SUNSET HILLS ROAD
SUITE 340
RESTON
VA
20190
US
|
Family ID: |
24050242 |
Appl. No.: |
10/446806 |
Filed: |
May 29, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10446806 |
May 29, 2003 |
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09794227 |
Feb 28, 2001 |
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6599523 |
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09794227 |
Feb 28, 2001 |
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09515172 |
Feb 29, 2000 |
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Current U.S.
Class: |
424/445 ;
442/123; 514/21.7; 514/23 |
Current CPC
Class: |
C08L 1/28 20130101; Y10T
442/2525 20150401; A61L 15/28 20130101; A61L 15/28 20130101; A61L
2300/434 20130101; A61L 15/44 20130101 |
Class at
Publication: |
424/445 ; 514/18;
514/17; 514/23; 442/123 |
International
Class: |
A61K 038/06; A61K
038/05; A61K 031/7024; A61K 031/70; A61K 009/70 |
Goverment Interests
[0002] This invention was made in part with grants from the
National Institutes of Health under grant numbers GM 20298 and NRSA
GM 19122. The government may have certain rights in this invention.
Claims
We claim:
1. A method for sequestering elastase at a wound site comprising
the step of contacting said wound site with a wound dressing
selected from the group consisting of carboxymethylcellulose,
dialdehyde gauze, sulfonated gauze, and phosphorylated gauze.
2. The method of claim 1 wherein said wound dressing is
carboxymethylcellulose.
3. The method of claim 1 wherein said wound dressing is dialdehyde
gauze.
4. The method of claim 1 wherein said wound dressing is sulfonated
gauze.
5. The method of claim 1 wherein said wound dressing is
phosphorylated gauze.
6. A wound dressing for treating a wound, comprising a support
matrix, and an active agent associated with said support matrix,
wherein said active agent is selected from the group consisting of
an inhibitor of a neutrophil-derived protease and a protease
sequestrant.
7. The wound dressing of claim 6 wherein said support matrix is
selected from the group consisting of cellulose and
carboxymethylcellulose.
8. The wound dressing of claim 6 wherein said neutrophil-derived
protease is elastase.
9. The wound dressing of claim 6 wherein said inhibitor is selected
from the group consisting of Val--Pro--Val, Val--Pro--Val-O
Methylester, Ala--Ala--Pro--Val-chloromehylketone,
Ala--Ala--Pro--Val-pentafluoroethyl- ketone, propyl-3-ketone,
glucose-6-citrate, and levulinate.
10. The wound dressing of claim 6 wherein said protease sequestrant
is selected from the group consisting of aldehyde, sulfate and
phosphate.
11. The wound dressing of claim 6 wherein said active agent is
associated with said support matrix by a means selected from the
group consisting of covalent bonding, non-covalent bonding and
ionic bonding.
12. A method for enhancing would healing, comprising, contacting
said wound with the wound dressing of claim 6.
13. The method of claim 12 wherein said wound is chronic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application 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 is
incorporated herein in entirety by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] 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.
[0005] 2. Background of the Invention
[0006] 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.
[0007] 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)
[0008] 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.
[0009] 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.
[0010] 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.
[0011] Further examples include:
[0012] U.S. Pat. No. 5,098,417 to Yamazaki etal. teaches the ionic
bonding of physiologically active agents to cellulosic wound
dressings.
[0013] U.S. Pat. No. 4,453,939 to Zimmerman et al. teaches the
inclusion of aprotonin in composition for "sealing and healing" of
wounds.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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).
[0019] 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.
[0020] 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.
SUMMARY OF THE INVENTION
[0021] 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 and an active agent
associated with the support matrix. In preferred embodiments of the
invention, the support matrix is cellulose or carboxymethylated
cellulose.
[0022] The active agents may be protease inhibitors. Protease
inhibitors especially suited to the practice of the instant
invention include those which inhibit neutrophil-derived proteases,
an overabundance of which are found in chronic wounds. In
particular, these are cationic proteases, such as elastase.
Examples of such inhibitors include peptide inhibitors such as di-
or tri-peptide sequence such as Val--Pro, Pro--Val, Ala--Pro--Val
or Val--Pro--Ala; or tetrapeptide sequences containing
Ala--Pro--Val or Val--Pro. These inhibitors may be associated with
the support matrix via covalent, non-covalent or ionic linkages.
Further, the inhibitors may be dissociable from the matrix. Upon
exposure to the wound fluid, the inhibitors may be released from
the matrix and migrate into the wound microenvironment.
[0023] The active agents may also be sequestrants. Substances
suitable as sequestrants may also be protease inhibitors (as listed
above). Alternatively, sequestrants may be of a more general
nature, for example, sulfonyl, phosphate, or aldehyde groups
associated with the support matrix. The sequestrants bind proteases
found in the wound fluid and remove them from the wound
microenvironment.
[0024] 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.
[0025] The dressings may be applied to wounds in order to enhance
would healing, especially the healing of chronic wounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A and 1B. Reaction progress curves for the inhibition
of HLE by fiber-inhibitor (1A) and known HLE inhibitor
MEOSuc--Val--Pro--Val-chl- oromethylketone (1B).
[0027] FIGS. 2A-C. Dose response relations for fiber-inhibitor in
elastase-containing would fluid. Residual elastase activity in
wound fluid after exposure to increasing quantities fiber-inhibitor
was measured. Measurements were carried out after 5 (FIG. 2A), 15
(FIG. 2B) and 60 (FIG. 2C) minutes of incubation of the wound fluid
with the indicated quantities of fiber-inhibitor. Data is
absorbance at 410 nm resulting from catalysis of substrate
N-methoxysuccinyl-Ala--Ala--Pro--Va- l-p-nitroanilide by residual
HLE in the samples.
[0028] FIG. 3. Percent levels of dicarbonyls in dialdehyde cotton
gauze (DAG I and II) and carboxylates on carboxymethylated
cellulose (CMC III and IV) as determined by titration of modified
cotton fibers. Data are mean .+-.S.D. of triplicate
determinations.
[0029] FIGS. 4A and 4B. 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.
[0030] FIGS. 5A-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).
[0031] FIG. 6. 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0032] 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.
[0033] The term active agent is meant to include (but not be
limited to) protease inhibitors and protease sequestrants. Those of
skill in the art will recognize that the two categories are not,
however, mutually exclusive. They may overlap in that a protease
inhibitor may also function as a sequestrant, and a sequestrant may
or may not also inhibit the protease. Further, the term "active
agent" is meant to encompass 1) substances that are associated with
the wound dressing as a result of having been added to the wound
dressing (either chemically attached or otherwise physically
compositioned onto the dressing), and 2) functional groups that are
inherent within the wound dressing material itself and derivatives
and chemical modifications of such functional groups. An example of
the latter is the hydroxyl groups of cellulose.
[0034] The term protease inhibitor is meant to include those
materials which effect a diminution in protease activity. Such
inhibitors may include, for example, inhibitors of the active site
of the protease, allosteric inhibitors, reversible and irreversible
inhibitors, substrate analogs of various types, peptides and
peptidemimetics, antibiotics, and the like. In a preferred
embodiment of the instant invention, the protease inhibitor
inhibits a neutrophil-derived protease. In yet another preferred
embodiment, the neutrophil-derived protease is neutrophil
elastase.
[0035] Examples of protease inhibitors which may be utilized in the
practice of the present invention include but are not limited to:
an alkyl amino acid such as Ala, Leu, Ile, Val, and Nle; a di- or
tri-peptide sequence such as Val--Pro, Pro--Val, Ala--Pro--Val or
Val--Pro--Ala; tetrapeptide sequences containing Ala--Pro--Val or
Val--Pro- and possessing as a terminal residue amino acids such as
Ala, Lys, Arg, Trp, Phe, Gln, His, and Tyr. Such inhibitors may be
linked through the amino- or carboxy- terminus to the wound
dressing material via, for example, a salt bridge. Alternatively,
the inhibitors may be embedded in or otherwise associated with the
wound dressing material. When the inhibitor is an amino acid or
peptide, it may also be derivatized at its amino- or COOH-terminus
as, for example, an acid, carboxamide, alcohol, ester, ketone,
aldehyde, ketomethylester, .alpha.-ketoesters, methyl
chloroformate, pentafluoroethylketone, trifluoromethylketones,
boronic acids or oleic acid. The inhibitor may also be
alpha-antitrypsin or any protein serine protease inhibitor.
[0036] In the case of protease inhibitors, they may either be
immobilized on the matrix, or they may be releasable into the wound
fluid. For example, covalently associated inhibitors may be
released via hydrolysis. Or inhibitors that are compositioned onto
the matrix may be released simply by hydration and dissolution into
the wound fluid. The released inhibitors are then free to migrate
into the wound fluid in order to exert their beneficial effect
(inhibiting deleterious proteases) throughout the wound
microenvironment.
[0037] The term sequestrant is meant to include active agents
capable of binding and retaining a protease in a manner which
removes the protease from the wound bed. The concentration of the
protease in the wound environment is thus decreased. The
sequestrant may be specific for the protease, e.g. designed to bind
to the protease active site (either reversibly or irreversibly), or
designed to bind to some other distinguishing feature of the
protease. For example, the sequestrant may be an antibody directed
to an epitope of a protease or a class of proteases. Alternatively,
binding may be of a more general nature. For example, binding may
be directed to a general class of proteases such as the cationic
proteases. In this case, the active agent may be an anionic group
such as phosphate, sulfate, carboxylate, and the like. The anionic
group may be attached directly to the wound dressing material (e.g.
to the hydroxyl functions of cellulose cotton) or may be attached
to the wound dressing material indirectly by means of a linking
group such as an alkyl chain. Further, the anionic group may be
part of another substituent that is associated with the wound
dressing material, e.g. the anion may be the carboxyl function of
an amino acid or peptide, or a phosphate group that is attached to
an amino acid or peptide. Any rationally designed inhibitor or
sequestrant that may be directly linked to the support matrix and
which possesses affinity for the protease may be utilized in the
practice of the present invention.
[0038] 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. The compounds of this invention may be applied to
wound dressings as agents that may be released into the wound and
thereby inhibit proteases such as human elastase and thus prevent
growth factor and tissue degradation. Alternatively, the inhibitors
of this invention are covalently bound 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.
[0039] The therapeutic administration of the modified wound
dressings containing inhibitors include a pharmacologically
effective dose of the inhibitor or sequestrant when used in 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, and specific
protease inhibitor/sequestrant utilized. The amount of active agent
required can be readily determined by those skilled in the art.
[0040] The term "patient" used herein is taken to mean mammals such
as sheep, horses, cattle, pigs, dogs, cats, rats, mice and
primates, including humans.
[0041] The term "wound dressing" used herein is taken to include
any pharmaceutically acceptable wound covering or support matrix
such as:
[0042] a) films, including those of a semipermeable or a
semi-occlusive nature such as polyurethane copolymers, acrylamides,
acrylates, paraffin, polysaccharides, cellophane and lanolin.
[0043] 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.
[0044] 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.
[0045] d) foams such as polysaccharide which consist of a
hydrophilic open-celled contact surface and hydrophobic closed-cell
polyurethane.
[0046] e) impregnates including pine mesh gauze, paraffin and
lanolin-coated gauze, polyethylene glycol-coated gauze, knitted
viscose, rayon, and polyester.
[0047] 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.
[0048] Preferred wound dressings are polysaccharide containing
support matrices capable of ionically or covalently bonding the
active agents thereto, or having the active agent compositioned
with or upon, and is envisioned to include chitosans, alginates and
cotton or carboxymethylated cotton in the form of gauze, films,
hydrocolloide, hydrogels, hydroactives, foams, impregnates,
absorptive powders and pastes, as known in the art and described in
USP 24:NP 19; The United States Pharmacopeia: The National
Formulary, USP 24:NF 19, United States Pharmacopeial Convention,
INC., Rockville, Md., Jan. 1, 2000, incorporated by reference
herein.
[0049] 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.
[0050] The active agents may be applied as a reactively bound
constituent of a wound dressing or may be compositioned for
application to a treatment site via moistened fibers in the
dressing. Dressing systems may be either single or multi-phase;
with the one-phase system consisting of the wound dressing with the
active agent. An exemplary multi-phase system would employ the
wound dressing and a suspension of a physiologically acceptable
diluent. Exemplary pharmaceutical carriers which may function as
the diluent can be a sterile physiologically acceptable liquids
such as water and oils and may optionally further contain
surfactants and other pharmaceutically acceptable adjuvants.
[0051] An exemplary but non-exhaustive list of oils which can be
employed in these preparations are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, and mineral oil. in general water, saline, and glycols, such
as polyethylene glycols are preferred liquid carriers.
[0052] 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.
[0053] Further, the wound dressings of the present invention may be
comprised of a single active agent, or of a plurality of active
agents on the same dressing. For example, a sequestrant and an
inhibitor may both be associated with the wound dressing. Or a
releasable inhibitor and a sequestrant may both be associated with
the wound dressing. The wound dressings may also include other
therapeutically beneficial substances such as antibiotics,
vitamins, and the like.
[0054] 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.
[0055] The invention is illustrated by the following Examples which
are intended to be illustrative but should in no way be construed
as limiting.
EXAMPLES
[0056] Abbreviations:
[0057] CMC: carboxymethyl cellulose
[0058] DIC: diisopropyl carbodiimide
[0059] DIPEA: diisopropylethylamine
[0060] DMAP:dimethylaminopyridine
[0061] DMF: dimethyl formamide
[0062] DMSO: dimethyl sulfoxide
[0063] DS: degree of substitution
[0064] EDCI: N'-ethyl-N'-(3-dimethylaminopropyl)carbodiimide
hydrochloride
[0065] FAB MS: fast atom bombardment mass spectrometry
[0066] Fmoc: 4-fluoronylmethyloxycarbonyl
[0067] HLE: human neutrophil elastase
[0068] HOBT:hydroxybenzotriazole
[0069] NMI: N-methylimidazole
[0070] NMM: N-methylmorpholine
[0071] TFA: trifluoroacetic acid
Example 1
Assessment of Elastase Sequences as Sequestrants
[0072] Methods
[0073] General Synthesis and Formulation of Conjugates of Cotton
Cellulose and Inhibitor Sequences
[0074] Desized, scoured, bleached and mercerized cotton gauze was
used for the synthesis. The cotton twill fabric was cut as circular
discs (8.5 cm. in diameter) for the synthesis.
[0075] Carboxymethylated cotton cellulose was prepared by refluxing
100% cotton twill (290 grams) for one hour with 25%
monochloroacetic acid in a sodium hydroxide solution of
methanol:isopropanol (13:87, v :v) and 0.5% TX-100. The degree of
substitution of carboxymethylation or carboxyl content was
determined by measuring the carboxyl content of the cotton with an
acid base titration. The carboxyl content was calculated from the
following equation:
DS=[(162)(% COOH)]4500-(R)(%COOH)
[0076] where R is the molecular weight of the ether substituent
minus one i.e., 58 for carboxymethylcellulose.
[0077] Esterification of cotton cellulose was accomplished through
base-catalyzed carbodiimide/HOBT acetylation. Cotton samples used
in the synthesis were pre-treated with 20 ml 25% TFA/DCM (10 min),
washed with 5.times.20 ml DCM, 2.times.20 ml, 10% DIPEA, 5.times.20
ml (5 min), and 2.times.20 ml DCM. The cotton discs were vacuum
dried on a Buchner funnel, and esterified in a beaker placed in an
ultrasonic bath. Finoc-glycine esterification was accomplished by
reacting the cotton discs in a 20 ml DMF solution with 0.3 M
Fmoc-Glycine/DIC/HOBT and 0.03 DMAP. The cotton discs were washed
with DMF and water and glycine estimated from amino acid analysis
to be 200 micromoles/gram of cotton. Cotton samples of this type
prepared with glycine linkers may then be used to assemble peptide
sequences or may be used to form the counterion of a peptide or
amino acid carboxy salt. Thus the amino salts of glycine cotton
cellulose conjugates may be formed with elastase peptide inhibitors
illustrative of the claims.
[0078] Val--Pro--Val--Gly Peptide Synthesis on Cotton Cellulose
[0079] The Val--Pro--Val recognition sequence was synthesized with
glycine as a COOH-terminal linker on 8.5 cm discs of cotton twill.
The synthetic protocol for the synthesis of Val--Pro--Val--Gly on
cotton consisted of the following steps as described by Eichler et
al, 1991. Acetylation of Fmoc-Gly-bound cellulose cotton was
accomplished with acetic anhydride/NMI/DMF 1:2:3 (v/v/v) for 60
min. The cotton discs were washed with DMF(3.times.10 mL)and DCM
(2.times.10 mL).
[0080] Deprotection of Fmoc was accomplished in 20% piperidine/DMF,
15 min; wash (3 .times.DMF, 2.times.DCM); coupling (0.3M Fmoc-amino
acid/HOBT/DIC in DMF, 90 min); wash (3 .times.DMF; 2.times.DCM).
Ten microliters of a bromophenol blue/DMF solution was added.
during the coupling step. Two hundred milligram samples were
subjected to amino acid analysis. The resulting ratio of amino
acids from the analysis was 1:2 (Pro:Val) and the resulting yield
was 1.1 micromoles/gram cotton.
[0081] Synthesis Of Val--Pro--Val--O-Methylester
[0082] A solution of carbobenzoxy-Val--Pro--OH (1 g, 2.8 mmol) in
30 ml of dry tetrahydrofuran was cooled to -5.degree. C. and
N-methylmorpholine (0.29 g, 2.8 mmol) and isobutyl chloroformate
(0.391 g, 2.8 mmol) were added and stirred for 1 h. A solution of
Val-OMe (0.49 g, 2.8 mmol) in dioxane/water (7:3) was adjusted to
pH 7 with diisopropylethylamine. The solutions were combined and
the mixture stirred for 3 h, water added and the tetrahydrofuran
evaporated. The resulting oil was extracted with ethyl acetate and
subjected to a work-up of 1N HCl, saturated NaCl, and drying over
sodium sulfate yielding a clear oil. The product was confirmed by
FAB MS ([M+1]=463) and the N-protecting group was removed by
catalytic hydrogenolysis using ammonium formate. (Anwer, M. K.,
& Spatola, A. F.(1980) Synthesis 11, 929-932). The product
Val--Pro--Val--OMe may be used in the formation of
carboxymethylcellulose-Val--Pro--Val--OMe conjugate.
[0083] Synthesis of Carboxymethylcellulose- Val--Pro--Val--Ome
Conjugate
[0084] Two carboxymethylated cotton discs (circular 8.5 cm discs
weighing 2.6 g each with a degree of substitiution of .about.25%)
were reacted with 0.15 M Val--Pro--Val--OMe/HOBT/DIC in 10 ml DMF
mixed in a beaker and placed in an ultrasonic bath. The reaction
was monitored with bromophenol blue (20 uL, 0.01 M bromophenol),
and was allowed to proceed overnight. Conversion of blue to yellow
signals completion of the reaction. Three hundred milligram samples
of the cotton cellulose conjugates were subject to amino acid
analysis. The resulting ratio of amino acid from the analysis was
1:2 (Pro:Val) and the resulting yield was 8.5 micromoles /gram
cotton.
[0085] Preparation of Carboxymethylcellulose-Ala--Ala--Pro-
Valine-chloromethylketone and its Cotton Conjugate
[0086] A stirred suspension of N-tosyl-L-valine acid chloride (0.95
g, 3 mmoles) in anhydrous ether (30 mL) was treated in an ice bath
with ethereal diazomethane (6 mmoles) in anhydrous ether. The
reaction mixture was left overnight, then treated with dry hydrogen
chloride for 2 h. The chloroketone is obtained on removal of the
solvent. A solution of carbobenzoxy-Ala--Ala--Pro--OH (2.8 mmol) in
30 ml of dry tetrahydrofuran was cooled to -5.degree. C. and
N-methylmorpholine (0.29 g, 2.8 mmol) and isobutyl chloroformate
(0.391 g, 2.8 mmol) were added and stirred for 1 h. A solution of
N-tosyl-L-valine chloromethylketone (2.8 mmol) in dioxane/water
(7:3) was adjusted to pH 7 with diisopropylethylamine. The
solutions were combined and the mixture stirred for 3 h, water
added and the tetrahydrofuran evaporated. The resulting oil was
extracted with ethyl acetate and subjected to a work-up of 1 N HCl,
saturated NaCl, and drying over sodium sulfate yielding a clear
oil. The N-protecting group was removed by catalytic hydrogenolysis
using ammonium formate. The resulting product was filtered and
lyophilized to give the peptide
Ala--Ala--Pro--Val-chloromethylketone. Two carboxymethylated cotton
discs (circular 8.5 cm discs weighing 2.6 g each with a degree of
substitution, 25%) were reacted with
Ala--Ala--Pro--Val-pentafluoroethylketone/HOBT/DIC in 10 ml DMF
mixed in a beaker and placed in an ultrasonic bath. The reaction
was monitored-with bromophenol blue (20 uL, 0.01 M bromophenol),
and was allowed to proceed overnight. Conversion of blue to yellow
signals completion of the reaction. Three hundred milligram samples
of the cotton cellulose conjugates were subject to amino acid
analysis The resulting ratio of amino acid from the analysis was
2:1:1 (Ala:Pro:Val) and the resulting yield was 0.484
micromoles/gram cotton.
[0087] Stepwise Preparation of
Carboxymethylcellulose-Ala--Ala--Pro--Val -pentafluoroethylketone
and its Cotton Conjugate
[0088] Step 1. Preparation of
Boc-Valyl-N-methyl-o-methylcarboxamide
[0089] To a solution of N-(tert-butoxycarbonyl)-L-valine in
ethylene chloride was added dimethylaminanopyridine,
N,O,-dimethylhydroxylamine hydrochloride, NMM and EDCl and the
solution was stirred at room temperature for 20 h. The solution was
washed with 10% HCl, saturated NaHC0.sub.3 and brine, and the
solvent was removed in vacuo to give a colorless oil.
[0090] Step 2. Preparation of Boc-Valyl-pentafluoroethylketone.
[0091] To a -78.degree. C. solution of
Boc-Valyl-N-methylmethylcarboxamide was added condensed
pentafluoroethyliodide. To the mixture was added
methyllithium-lithium bromide complex while maintaining an internal
reaction temperature below -65.degree. C. The reaction mixture is
stirred at -65.degree. C. to -78.degree. C. for 1.5 h. The mixture
was poured into water and the aqueous phase was acidified with
potassium hydrogen sulfate. The aqueous phase was extracted with
additional Et.sub.2O (500 ml), and the combined organic extracts
were washed with saturated NaHCO.sub.3 and dried over
Na.sub.2SO.sub.4.
[0092] Step 3. Preparation of
Boc--Ala--Ala--Pro--Val-pentafluoroethylketo- ne.
[0093] A solution of Boc-Valyl-pentafluoroethylketone in
trifluoroacetic acid; methylene chloride (1:1, v:v) was prepared
and allowed to react for 30 min. The solvent was removed in vacuo
and the resulting deprotected peptide reacted with
Boc--Ala--Ala--Pro--OH through diisopropycarbodiimide/HOBT
coupling.
[0094] Further preparation of carboxymethyl
cellulose-O--Ala--Ala--Pro--Va- l-pentafluoroethylketone is as
follows:
[0095] Two carboxymethylated cotton discs (circular 8.5 cm discs
weighing 2.6 g each with a degree of substitution, 25%) were
reacted with 0.15M
Ala--Ala--Pro--Val-pentafluoroethylketone/HOBT/DIC in 10 ml DMF
mixed in a beaker and placed in an ultrasonic bath. The reaction
was monitored with bromophenol blue (20 uL, 0.01 M bromophenol),
and was allowed to proceed overnight. Conversion of blue to yellow
signals completion of the reaction. The cotton is then washed with
20 mL of DMF three times followed by three washes with methylene
chloride. The resulting peptido-cellulose conjugates on cotton were
subjected to amino analysis and found to contain 30 micromoles of
peptide per gram of cotton.
[0096] Preparation of Propyl-3-keto-(2.3,6)-O-Cellulose ether.
[0097] Four grams of cotton cellulose was suspended in a 300 ml
solution of dioxane and water (2:1) whereupon Dabco
(1,4-diazabicyclo[2.2.2.]octan- e) was added to pH 8, and 0.0246
moles of vinylpropylketone was added. The suspension is allowed to
stir overnight. Alternatively, the treated gauze soaked with the
solution of base and vinylpropyketone may be cured at 100.degree.
C. for one hour and the product rinsed with cold water for 30
minutes followed by drying at 85.degree. C.
[0098] Preparation of Levulinate-(2,3,6)-O-Cellulose ester
[0099] Esterification of cotton cellulose gauze with levulinic acid
was accomplished by reacting the cotton discs in a 20 ml DMF
solution with 0.3 M levulinic acid/DlC/1-HOBT and 0.03 M DMAP. The
esterification may also be performed under aqueous conditions with
a water soluble carbodiimide at the same molar concentrations via
convention pad and cure techniques employing citric acid and sodium
hypophosphite crosslinking of the levulinic acid.
[0100] Preparation of glucose-6-citrate-(2,3,6,)-O-Cellulose
ester.
[0101] Two gram samples of cotton gauze were padded with two dips
and two nips in a four percent solution of sodium hypophosphite, a
0.62M citric acid and a 0.12 M glucose solution on a laboratory
mangle. The padded gauze were dried and cured in ovens with
mechanical circulated air. Curing temperatures were set at
180.degree. C., and drying at 85.degree. C. The resulting add-on
weight of product was found to be 11% or an 11% increase in weight
based on the difference before and after the wet finishing
modification.
[0102] Ten milligrams of Ala--Pro--Val-Chloromethylketone acetate
salt was dissolved in a 0.05 M saline solution and applied to 2
grams of carboxymethylated cotton gauze to saturation. The gauze
was then lyophlized to dryness and a cotton cellulose sample taken
for amino acid analysis revealing 10 micromoles of peptide per gram
of cotton gauze.
[0103] Results
[0104] Chromatography was performed to measure the affinity of the
cotton cellulose-bound recognition sequences for elastase, and the
ability of the cotton fiber conjugates to sequester the elastase
from an aqueous environment. Since the synthesis was performed on
mercerized cotton, mercerized cotton was compared with unmercerized
cotton as a chromatographic stationary phase for elastase elution.
Less elastase was retained (4%) in the untreated mercerized cotton
column compared to untreated unmercerized (12%). This might be
expected since the crystallinity of the cotton fiber undergoes a
change upon mercerization. Table 1 outlines the comparative levels
of elastase retained, expressed as percent of retained elastase on
the cotton columns. The comparative levels of elastase retained on
the columns under physiological saline conditions suggests the
ability to sequester elastase from wound fluid. Two series of
elastase retention measurements were made based on the first
injection of elastase to the freshly prepared column and subsequent
percent elastase retained. The percent of retained elastase
following the first injection was higher for all samples when
compared with the repetitive injections.
[0105] Conjugate I gave the highest retention of elastase. Fifty
eight percent of elastase was retained on conjugate I as compared
with the CMC control of thirty percent on the first pass of
elastase solution over the column. Conjugate I is a COOH-terminal
methyl ester of Val--Pro--Val attached to carboxymethylated
cellulose at the amino-terminal valine. This results in the
COOH-terminus being more accessible for enzyme binding. The cotton
cellulose conjugate Val--Pro--Val--Gly sequence attached through
the COOH-terminal glycine to cotton cellulose retained less
elastase (26%) from the first injection. The percent elastase
retained with repetitive injections followed a similar trend to the
first-injected samples among the analogs tested. Conjugate I
demonstrated the highest average retention of elastase (37%).
1TABLE 1 Elastase Retention on Peptido-Cellulose Columns.sup.1
Cotton Description of Cotton Conjugates % Elastase Retained .+-.
SD.sup.2 I Carboxymethylated Cellulose-Val-Pro-Val-OMe 37 .+-. 0.71
II Val-Pro-Val-Gly-Cellulos- e 26 .+-. 0.71 III Carboxymethylated
Cellulose Cotton 32 .+-. 2.12 IV Unmercerized Cotton Twill 12 .+-.
1.63 V Cellulase-treated Cotton 15 .+-. 0.35 VI
Val-Pro-Pro-Gly-Cotton (Cellulase treated) 12 .+-. 2.47 VII
Mercerized Cotton Twill 4 .+-. 1.41 .sup.1Elastase was injected
onto the cotton conjugate columns as described. .sup.2Percent
elastase retained represents the average of triplicate injections
on the same columns.
[0106] These results demonstrate that peptide sequences that are
covalently attached to a cellulose support can effect the
sequestration of proteases for which they are inhibitory.
Example 2
Inhibition of Elastase by a Synthetic Cotton-Bound Protease
Inhibitor
[0107] Materials and Methods
[0108] The peptide substrate and inhibitor, including
MeO--Suc--Ala--Ala--Pro--Val-p-nitroanalide and
MeO--Suc--Ala--Ala--Pro--- Val chloromethyketone, respectively were
obtained from Sigma (St. Louis, Mo.) and their purity confirmed by
Reversed Phase High Performance Liquid Chromatography (RPHPLC)
prior to experimental use. Leukocyte elastase (Sigma, St. Louis,
Mo.) obtained from human leukocytes (HLE) was solubilized from 1
unit vials (one unit of HLE will release one nanomole of
p-nitrophenol per second from N-t-Boc-Alanine p-nitrophenylester at
pH 6.5) and 0.2 unit aliquots employed per reaction. Cotton fibers
were taken from woven cotton twill, which was desized, scoured,
bleached and washed. The woven cotton was pretreated with the
cellulase enzyme, Cellusoft, and a 10% solution of trifluoroacetic
acid in methylene chloride followed by three washes with methylene
chloride. The cotton fabric was pre-treated with cellulase to
remove the non-cellulose constituents of the primary cell wall of
he cellulose cotton and improve binding of the peptide CMK.
[0109] Preparation of Fiber-Inhibitor Formulation
[0110] As a model to demonstrate the optimal conditions for
formulations, enzyme inhibition, and in vitro release, a low
molecular weight COOH-terminally modified tetrapeptide ketone was
impregnated into cotton fibers. Acetonitrile solutions (0.5 ml) of
the MeO--Suc--Ala--Ala--Pro--V- al chloromethyketone (1.2 mg/ml)
were applied to separate 300 mg samples of cotton twill fabric. The
use of acetonitrile in the application provides for rapid diffusion
of the inhibitor solution into the fabric. The inhibitor is thought
to bond non-covalently to the polysaccharide chain of the cellulose
fibers through hydrogen bonding. The fabric was made slightly
acidic through pre-treatment with trifluoroacetic acid solution to
promote acid catalyzed formation of a hemiketal between the peptide
ketone and accessible hydroxyls of the glucan rings in cellulose.
This would form a more durable affinity of the inhibitor for the
cotton cellulose, which is hydrolyzed under aqueous conditions.
Hemiketals are released to their corresponding ketones when
hydrated. Samples were allowed to air dry and pulverized in a Wiley
Mill of 80 mesh screen (150 micron size fibers). The pulverized
samples were lyophilized to remove trace amounts of
acetonitrile.
[0111] Enzyme Assays
[0112] Enzyme assays of HLE 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-nitoraniline at 410 nm from the enzymatic hydrolysis of
MeO--Suc--Ala--Ala--Pro--Val-pNA. In a typical experiment 250 .mu.l
of enzyme solution (0.52 units, or 2.08 units.ml) of elastase was
combined in a total volume of 1.5 ml buffer with 60 .mu.M
substrate. In a typical experiment fiber-inhibitor formulations
were assessed for elastase inhibition by mixing milligram
quantities of the pulverized cotton samples with enzyme solutions
in 5 ml Reacti-Vials (Pierce Chemical Company). The cotton fiber
suspension was filtered on 0.45 micron filters attached to a 5 ml
syringe. The filtrate was mixed with substrate and the enzyme
hydrolysis of substrate was measured spectrophotometrically.
[0113] Reaction progress curves were recorded on a Shimadzu UV-265
equipped with a recorder, and time points were obtained by applying
the program Un-Scan It.TM. (Silk Scientific, Ogden Utah) to the
recorded curves. A digitized scan produced between 280 and 450
poirs of absorbance-time data points. velocities were determined as
described (Williams and Morrison) at 10-40 points along the
progress curve.
[0114] Amino acid analysis was completed on an Applied Biosystems
amino acid analyzer. This consisted of the Model 420A
Derivatizer/Hydrolyzer where peptide samples were hydrolyzed in 6N
HCl, converted to the PTC-derivatives, and chromatographically
analyzed on the Model 130A, and the Model 920! Data Module.
[0115] High performance liquid chromatography studies were
completed with a Beckman Systems Gold 508 autosampler, programmable
solvent module 126, and diode array detector module 168 (214 nm).
Data were acquired and analyzed by computer automated Gold.TM.
Noveau software. Chromatographic analysis and separation of the
elastase inhibitor was performed on a Vydac 5 micron C18 peptide
reverse phase column (4.6.times.150 mm) with a linear gradient
mobile phase of 15 to 40% acetonitrile/aqueous 0.1% TFA and a flow
rate of 1 ml/min.
[0116] Patients and Wound Fluids
[0117] Fluids were harvested from seven-grade III sacral, ischial,
or trochanteric pressure ulcers of five patients with spinal cord
injuries. Three patients had two distinct wounds which were sampled
and which were considered separate data points. Patients ranged in
age from 50-65 years and had no significant comorbidities. All
wounds were present for a minimum of 2 months. There was no
evidence of gross infection in any of the wounds used in the study.
Wound care in all but one ulcer consisted of normal saline-soaked
wet to dry dressings. A small margin of one wound was receiving
topical collagenase (Santyl) for enzymatic debridement. This wound
was irrigated copiously with normal saline prior to collection of
ulcer fluid. An occlusive dressing (Tegaderm: 3M, St Paul, Minn.)
was placed over the ulcers for 2-4 hours, and fluid was collected
by aspiration with a sterile tuberculin syringe. Fluids were
clarified by centrifugation at 14,000 g for 15 min at 4 C. The
protein concentration was determined with the Bio-Rad Protein assay
(Richmond, Calif.) with bovine serum albumin as a quantitative
standard.
[0118] Determination of elastase activity in wound fluid.
[0119] Elastase activity was determined by methods described
previously. (Nakajima et al.) One hundred microgram amounts of
protein were incubated in 1.0 ml of Hepes-NaOH buffer 100 mmole/L,
pH 7.5, NaCl 500 mmole/L, 10% DMSO, containing 0 to 5 mg of
cotton-bound fiber inhibitor. The heterogeneous reaction was
incubated at room temperature while shaking vigorously. The
inhibitor-protein mixture was then filtered through a 0.22 micron
filter into a cuvette. The reaction substrate was added to each of
the filtered samples to a final concentration of 100 .mu.mol/L.
Substrate hydrolysis was assessed by measuring A.sub.410 at 5 min,
15 min, and 60 min after substrate addition. Purified neutrophil
elastase was used to generate a standard curve.
[0120] Results
[0121] Elastase inhibition kinetics
[0122] Reaction progress curves for inhibition of human neutrophil
elastase (HLE) in the presence of fiber-inhibitor samples were
generated (FIG. 1A) and compared to reaction progress curves for
inhibition of HLE by the known inhibitor
MeOSuc--Val--Pro--Val-chloromethylketone (FIG. 1B). HLE
concentrations in the reaction mixtures were 0.5 and 0.2 units/ml
for the fiber-inhibitor and the MeOSuc--Val--Pro--Val-chlorometh-
ylketone studies, respectively. The weights of the cotton-bound
inhibitor samples employed in the inhibition study were in the low
milligram range (0.5 to 3.0 mg). The results show that the cotton
samples effected a 0.01-0.7 .mu.M inhibitor concentration, as
determined by a comparison to inhibition by
MeOSuc--Val--Pro-Valchloromethylketone. Further, a dose response
relation of enzyme inhibition was demonstrated in the reaction
progress curve for the cotton fiber-inhibitor samples.
[0123] The dose response of inhibition for HLE was apparent from
the linear relation of a plot of reciprocal initial velocities
(1/v.sub.0) versus weight of fiber- inhibitor. It was likewise
apparent that the dose response of inhibition for HLE using freely
dissolved inhibitor is within a similar concentration range to that
expected for release of inhibitor from the fiber into solution.
Thus, the initial velocities (v.sub.o) for the weighed
fiber-inhibitor samples were within a comparable range to those
observed for freely dissolved inhibitor concentrations assayed
separately.
[0124] Biphasic reaction progress curves were observed for HLE by
the free peptide chloromethyl ketone (CMK) and with peptide bound
to fiber. This is also indicative of a slow- binding inhibitor. The
reaction progress curves for slow-binding inhibitors may be
described by the expression of equation 1:
P=v.sub.st+(v.sub.o-v.sub.s)[1-exp(-k.sub.obs)/k.sub.obs+d
[0125] Values for k.sub.obs were derived from this equation by
applying it to the reaction progress curves of HLE. The k.sub.obs
values for the pre-incubation experiments of fiber-bound and freely
dissolved inhibitor with enzyme were generated. The k.sub.obs for
fiber- bound inhibition (Table 2) of HLE demonstrate the same range
and rate decrease as freely dissolved inhibitor.
2TABLE 2 Comparison of the k.sub.obs for Free and Bound HLE
Inhibitors Free Inhibitor Fiber Bound Inhibitor [Inhibitor] .mu.M
k.sub.obs (min.sup.-1) Fiber mass (mg) k.sub.obs (min.sup.-1)
Control 0.087 0.020 0.037 0.5 0.0042 0.040 0.057 1.0 0.0046 0.050
0.013 2.0 0.042 0.2 0.003 3.0 0.638
[0126] Measurement of enzyme inhibition and wound fluid
activities
[0127] Inhibitory activities were measured by comparing I.sub.50
values for the inhibitor bound and freely dissolved CMK inhibitor
from each of the reaction progress curves. I.sub.50 reflects the
inhibitor concentration or fiber-inhibitor weight in suspension at
50% inhibition using the control inhibitor-free reaction as a
benchmark of 100% activity. 150 values were assigned for the
inhibition of HLE based on a plot of initial rate versus freely
dissolved inhibitor and fiber-inhibitor concentration. For HLE the
plot of initial rate versus free inhibitor concentration revealed
an I.sub.50 of approximately 11 nM free inhibitor and 0.6 mg of
fiber-inhibitor as compared with 29 nM of free inhibitor (based on
a semi-quantitative RPHPLC determination) released by 0.6 mg of
fiber-inhibitor.
[0128] Assessment of the fiber-inhibitor on elastase activity in
wound fluid was performed by measuring substrate hydrolysis at
fixed time points following incubation of fiber-inhibitor with
HLE-containing wound fluid. A dose response of inhibition was
evident when fiber-inhibitor samples ranging from 1 mg to 5 mg were
incubated in the presence of wound fluid. Elastase activity levels
decrease from 40-60 mU in the absence of inhibitor to 0 to 10 mU in
the presence of 1 to 5 mg of fiber inhibitor (FIGS. 2A-C).
[0129] This decrease in elastase activity with increasing fiber
weight demonstrates the inhibitory activity of the serine protease
inhibitor as it is released into wound fluid.
[0130] The results shown in this Example demonstrate that protease
inhibitors which are attached to a cellulose support via a
hydrolyzable linkage are capable of effecting the inhibition of a
protease in wound fluid.
Example 3
Carboxymethylated and Dialdehyde Cotton Gauze
[0131] Methods
[0132] Preparation of Dialdehyde Cotton Gauze
[0133] 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.
[0134] Preparation of Carboxymethylated Cotton Gauze
[0135] 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.
[0136] Determination of Dialdehyde Content and Degree of
Substitution of Carboxymethylcellulose
[0137] 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.)
[0138] Assay of Treated Gauze for Elastase Activity
[0139] 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.
[0140] Enzyme Assays
[0141] 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.
[0142] Results
[0143] 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.
[0144] FIG. 3 shows that percent levels of dicarbonyls in two
samples of dialdehyde cotton gauze (DAG I and DAG II) on periodate
finished cotton ranged from about 12 to 16%. As can be seen, the
percent levels of carboxylates on carboxymethylated cellulose
samples CMC III and IV were relatively low (approximately
1-2%).
[0145] As shown in FIG. 4A, 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 (see "Preparation of Dialdehyde
Cotton Gauze" under Methods above) 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.
[0146] Two different degree of substitution (DS) levels of carboxy
methylated cotton cellulose were also compared. As shown in FIG.
4B, higher substitution levels of carboxylate on cotton resulted in
an increased reduction in elastase activity in solution.
[0147] 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.
[0148] 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.
Example 4
Oxidized, Sulfonated, and Phosphorylated Cotton Gauze Dressings
Selectively Absorb Neutrophil Elasase Activity in Solution
[0149] Methods
[0150] Preparaton of Periodate-Oxidized, Sulfonated, and
Phosphorylated Cotton
[0151] 2,3 dialdehyde-anhydroglucos-cellulose (i.e. periodate
oxidized) cotton. Cotton gauze (12 ply- 4 in..times.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.
[0152] Sulfonated cotton. The cotton gauze may be sulfonated by
washing the dialdehyde oxycellulose with 5% sodium bisulfite
(NaHS0.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.
[0153] 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.
[0154] The phosphorylated and sulfonated cotton cellulose D.S.
levels were 0.035 and 0.011 respectively, as measured by elemental
analysis.
[0155] Carboxymethylated Cotton Gauze
[0156] 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.
[0157] Free-Swell Absorbency and Wicking Test
[0158] 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.
[0159] Patients and Wound Fluid
[0160] 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..RTM., 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.
[0161] Assay of Wound Fluid
[0162] 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.
[0163] Sequestration and Inhibition of Elastase Activity by
Finished Cotton Gauze
[0164] 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.
[0165] 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.
[0166] Enzyme Assays
[0167] 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.
[0168] Inhibition of Elastase Activity with Dialdehyde Starch
[0169] 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 x g for five minutes and the
supernatant was assayed for elastase activity as described
above.
[0170] Results
[0171] 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.
[0172] Effect of Modfied Gauzes on Elastase Activity
[0173] 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%.
[0174] 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. 5. 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. 6 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] Elastase-Lowering Activity in Wound Fluid
[0179] 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
[0180] 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 guaze tested. This
suggests that more elastase has been sequestered by DAG than by UT
and is reflected in the initial velocity (v.sub.o) values given in
Table 3. As can be seen, increasing the quantity of DAG resulted in
a dose dependent decrease in the amount of retained elastase
activity.
3TABLE 3 Gauze per Volume DAG Fluid UT Gauze Specific mg
gauze/.mu.l Specific Adsorption. UT Gauze Adsorption. DAG Wound
(.mu.g protein/mg V.sub.oe-03(s.sup.-1) .+-. (.mu.g protein/mg
V.sub.oe -03(s.sup.-1) .+-. Fluid* gauze) .+-. S.D.** S.D.***
gauze) .+-. S.D.** S.D.*** 2.5 8.74 .+-. 0.06 2.81 .+-. 0.068 7.42
.+-. 1.2 2.46 .+-. 0.038 7.5 1.10 .+-. 0.62 1.18 .+-. 0.047 3.49
.+-. 0.28 0.64 .+-. 0.028 10.8 1.69 .+-. 0.69 0.62 .+-. 0.129 2.82
.+-. 0.44 0.23 .+-. 0.14 14.2 1.60 .+-. 0.33 0.22 .+-. 0.057 2.69
.+-. 0.39 0.08 .+-. 0.03 17.5 1.40 .+-. 0.24 1.09 .+-. 0.137 1.83
.+-. 0.29 NA**** *mg gauze/.mu.l wound fluid (w.f.) was calculated
by dividing the gauze mass by the volume of the wound fluid (w.f.)
used in the experiment. For example (75 mg gauze/3 ml diluted w.f.)
.times. (1 ml diluted w.f./10 .mu.L (0.01 ml) w.f.) = 2.5 mg
gauze/.mu.L w.f. The elastase activity (0.25-0.27 units) of the
wound fluid used in these experiments was the same as shown in FIG.
5B. **Specific adsorption of protein on gauze (.mu.g protein/mg
gauze) was determined by dividing the residual protein mass by the
initial gauze mass. Residual protein mass remaining on the gauze
after exposure to wound fluid was calculated by subtracting the
protein mass remaining in solution from the initial protein mass of
the solution diluted 1:100 (wound fluid:buffer, v:v). [Protein]
1:100 solution .mu.g/mL .times. 3 mL = Initial protein mass; #
[Protein] after exposure .mu.g/mL .times. (3.0 mL .times. 0.9) =
Protein in solution after incubation. Initial protein mass -
protein mass after incubation = Gauze-bound protein (residual
protein). ***NA (No measurable rate or elastase activity).
****Reaction rates are reported as initial velocities (v.sub.o)
which were taken from the slope of the linear least squares fit of
absorbance-time data of the reaction progress profiles as described
in the Materials and Methods section under Enzyme Assays.
[0181] Data are mean .+-.SD of triplicate determinations. All are
significant when compared within the five groups of protein and
reaction rate data such that p<0.05 and were determined by
one-way ANOVA and analysis of variance.
[0182] 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.
[0183] 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.
[0184] The results obtained in this Example demonstrate that
dialdehyde cotton effects the sequestration of the protease
elasetase from wound fluid.
[0185] 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.
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