U.S. patent application number 13/363489 was filed with the patent office on 2012-06-14 for solid dressing for treating wounded tissue.
Invention is credited to Dawson Beall, Martin MacPhee.
Application Number | 20120148658 13/363489 |
Document ID | / |
Family ID | 39033515 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148658 |
Kind Code |
A1 |
MacPhee; Martin ; et
al. |
June 14, 2012 |
SOLID DRESSING FOR TREATING WOUNDED TISSUE
Abstract
Disclosed are solid dressings for treated wounded tissue in
mammalian patients, such as a human, comprising a haemostatic layer
consisting essentially of fibrinogen and a fibrinogen activator,
wherein the fibrinogen is present in an amount between 3.0
mg/cm.sup.2 of the wound facing surface of the dressing and 13.0
mg/cm.sup.2 of the wound facing surface of the dressing. Also
disclosed are methods for treating wounded tissue.
Inventors: |
MacPhee; Martin;
(Darnestown, MD) ; Beall; Dawson; (Gaithersburg,
MD) |
Family ID: |
39033515 |
Appl. No.: |
13/363489 |
Filed: |
February 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11882876 |
Aug 6, 2007 |
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13363489 |
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60835423 |
Aug 4, 2006 |
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Current U.S.
Class: |
424/446 ;
424/130.1; 424/184.1; 424/445; 424/450; 424/542; 424/94.64;
514/15.3; 514/7.6; 514/9.7; 602/43 |
Current CPC
Class: |
A61L 15/225 20130101;
A61F 13/0226 20130101; A61F 13/00068 20130101; A61L 26/0042
20130101; A61L 15/64 20130101; A61K 38/363 20130101; A61F
2013/00106 20130101; A61L 15/26 20130101; A61L 2300/418 20130101;
A61F 2013/00536 20130101; A61L 26/009 20130101; A61L 2400/04
20130101; A61F 13/00012 20130101; A61L 2300/604 20130101; A61L
15/325 20130101; A61F 13/0246 20130101; A61F 13/00017 20130101;
A61L 15/58 20130101; A61F 2013/00472 20130101; A61F 2013/0054
20130101; A61F 13/00029 20130101; A61K 38/4833 20130101; A61L
15/225 20130101; A61F 13/00991 20130101; A61F 13/00021 20130101;
A61F 13/02 20130101; A61F 2013/00174 20130101; A61F 2013/0091
20130101; A61L 2300/606 20130101; A61L 15/44 20130101; A61L 15/32
20130101; A61L 2300/252 20130101; C12Y 304/21005 20130101; A61L
2300/10 20130101; A61L 15/38 20130101; A61L 26/0052 20130101; A61F
2013/00931 20130101; C08L 89/00 20130101; C08L 89/00 20130101; A61F
13/00063 20130101; A61L 15/28 20130101; A61L 26/0052 20130101; A61L
26/0066 20130101; A61L 2300/608 20130101; A61P 7/04 20180101; A61L
15/18 20130101; A61F 13/00034 20130101; A61L 2300/254 20130101;
A61P 17/02 20180101 |
Class at
Publication: |
424/446 ;
514/15.3; 424/445; 514/7.6; 424/130.1; 514/9.7; 424/184.1; 424/450;
424/94.64; 424/542; 602/43 |
International
Class: |
A61L 15/44 20060101
A61L015/44; A61K 38/18 20060101 A61K038/18; A61K 39/395 20060101
A61K039/395; A61K 38/22 20060101 A61K038/22; A61F 13/02 20060101
A61F013/02; A61K 9/127 20060101 A61K009/127; A61K 38/48 20060101
A61K038/48; A61K 35/58 20060101 A61K035/58; A61P 17/02 20060101
A61P017/02; A61P 7/04 20060101 A61P007/04; A61K 38/36 20060101
A61K038/36; A61K 39/00 20060101 A61K039/00 |
Claims
1. A solid dressing for treating wounded tissue in a mammal
comprising at least one haemostatic layer consisting essentially of
fibrinogen and a fibrinogen activator, wherein said fibrinogen is
present in an amount between 3.0 mg/cm.sup.2 of the wound facing
surface of said dressing and 13.0 mg/cm.sup.2 of the wound facing
surface of said dressing.
2. The solid dressing of claim 1, further comprising at least one
support layer.
3. The solid dressing of claim 2, wherein said support layer
comprises a backing material.
4. The solid dressing of claim 2, wherein said support layer
comprises an internal support material.
5. The solid dressing of claim 2, wherein said support layer
comprises a resorbable material.
6. The solid dressing of claim 2, wherein said support layer
comprises a non-resorbable material.
7. The solid dressing of claim 6, wherein said non-resorbable
material is selected from the group consisting of silcone polymers,
gauze and latexes.
8. The solid dressing of claim 3, further comprising at least
physiologically acceptable adhesive between said haemostatic layer
and said backing layer.
9. The solid dressing of claim 5, wherein said resorbable material
is selected from the group consisting of proteinaceous materials
and carbohydrate substances.
10. The solid dressing of claim 9, wherein said proteinaceous
material is at least one substance selected from the group
consisting of keratin, silk, fibrin, collagen and gelatin.
11. The solid dressing of claim 9, wherein said carbohydrate
substance is selected from the group consisting of alginic acid and
salts thereof, chitin, chitosan, cellulose, n-acetyl glucosamine,
proteoglycans, glycolic acid polymers, lactic acid polymers,
glycolic acid/lactic acid co-polymers and mixtures of two or more
thereof.
12. The solid dressing of claim 1, wherein said haemostatic layer
also contains a fibrin cross-linker and/or a source of calcium
ions.
13. The solid dressing of claim 1, wherein said haemostatic layer
also contains one or more of the following: at least one filler, at
least one solubilizing agent, at least one foaming agent and at
least one release agent.
14. The solid dressing of claim 13, wherein said filler is selected
from the group consisting of sucrose, lactose, maltose, keratin,
silk, fibrin, collagen, gelatin, albumin, polysorbate, chitin,
chitosan, alginic acid and salts thereof, cellulose, proteoglycans,
glycolic acid polymers, lactic acid polymers, glycolic acid/lactic
acid co-polymers, and mixtures of two or more thereof.
15. The solid dressing of claim 13, wherein said solubilizing agent
is selected from the group consisting of sucrose, lactose, maltose,
dextrose, mannose, trehalose, mannitol, sorbitol, albumin, sorbate,
polysorbate, and mixtures of two or more thereof.
16. The solid dressing of claim 13, wherein said release agent is
selected from the group consisting of gelatin, mannitol, sorbitol,
polysorbate, sorbitan, lactose, maltose, trehalose, sorbate,
glucose and mixtures of two or more thereof.
17. The solid dressing of claim 13, wherein said foaming agent is
selected from the group consisting of mixtures of sodium
bicarbonate/citric acid, sodium bicarbonate/acetic acid, calcium
carbonate/citric acid and calcium carbonate/acetic acid.
18. The solid dressing of claim 1, wherein said haemostatic layer
also contains at least one therapeutic supplement selected from the
group consisting of antibiotics, anticoagulants, steroids,
cardiovascular drugs, growth factors, antibodies (poly and mono),
chemoattractants, anesthetics, antiproliferatives/antitumor agents,
antivirals, cytokines, colony stimulating factors, antifungals,
antiparasitics, antiinflammatories, antiseptics, hormones,
vitamins, glycoproteins, fibronectin, peptides, proteins,
carbohydrates, proteoglycans, antiangiogenins, antigens,
nucleotides, lipids, liposomes, fibrinolysis inhibitors and gene
therapy reagents.
19. The solid dressing of claim 1, wherein said haemostatic layer
is made from a single aqueous solution containing a mixture of said
fibrinogen and said fibrinogen activator.
20. The solid dressing of claim 1, wherein said haemostatic layer
is cast as a single piece.
21. The solid dressing of claim 1, wherein said haemostatic layer
is substantially homogeneous throughout.
22. The solid dressing of claim 1, wherein said haemostatic layer
is composed of a plurality of particles, each of said particles
consisting essentially of fibrinogen and thrombin.
23. The solid dressing of claim 22, wherein said haemostatic layer
further contains at least one binding agent in an amount effective
to improve the adherence of said particles to one another.
24. The solid dressing of claim 23, wherein said binding agent is
selected from the group consisting of sucrose, mannitol, sorbitol,
gelatin, maltose, povidone, chitosan and
carboxymethylcellulose.
25. The solid dressing of claim 1, wherein said haemostatic layer
is a monolith.
26. The solid dressing of claim 1, wherein said haemostatic layer
has been lyophilized.
27. The solid dressing of claim 1, wherein said haemostatic layer
has moisture content of at least 6%.
28. The solid dressing of claim 1, wherein said haemostatic layer
has moisture content of less than 6%.
29. The solid dressing of claim 1, wherein said fibrinogen is a
mammalian fibrinogen.
30. The solid dressing of claim 29, wherein said mammalian
fibrinogen is selected from the group consisting of bovine
fibrinogen, porcine fibrinogen, ovine fibrinogen, equine
fibrinogen, caprine fibrinogen, feline fibrinogen, canine
fibrinogen, murine fibrinogen and human fibrinogen.
31. The solid dressing of claim 1, wherein said fibrinogen is
selected from the group consisting of bird fibrinogen and fish
fibrinogen.
32. The solid dressing of claim 29 or 31, wherein said fibrinogen
is selected from the group consisting of recombinantly produced
fibrinogen and transgenic fibrinogen.
33. The solid dressing of claim 1, wherein said fibrinogen
activator is selected from the group consisting of thrombins,
prothrombins, snake venoms, and mixtures of any two or more
thereof.
34. The solid dressing of claim 33, wherein said thrombin is
mammalian thrombin.
35. The solid dressing of claim 34, wherein said mammalian thrombin
is selected from the group consisting of bovine thrombin, porcine
thrombin, ovine thrombin, equine thrombin, caprine thrombin, feline
thrombin, canine thrombin, murine thrombin and human thrombin.
36. The solid dressing of claim 34, wherein said thrombin is
selected from the group consisting of bird thrombin and fish
thrombin.
37. The solid dressing of claim 34 or 36, wherein said thrombin is
selected from the group consisting of recombinantly produced
thrombin and transgenic thrombin.
38. The solid dressing of claim 34, wherein said thrombin is
present in an amount between 2.50 Units/mg of fibrinogen component
and 0.025 Units/mg of the fibrinogen.
39. The solid dressing of claim 18, wherein said therapeutic
supplement is present in an amount equal to or greater than its
solubility limit in fibrin.
40. A method of treating wounded tissue in a mammal, comprising
placing a solid dressing of claim 1 to said wounded tissue and
applying sufficient pressure to said dressing for a sufficient time
for enough fibrin to form to reduce the loss of blood and/or other
fluid from said wounded tissue.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/882,876, entitled, "Solid Dressing for
Treating Wounded Tissue," filed Aug. 6, 2007, which is incorporated
herein by reference.
FIELD OF THE INVENTION
Background of the Invention
[0002] The present invention relates to a solid dressing for
treating wounded tissue in a mammalian patient, such as a human.
The materials and methods available to stop bleeding in
pre-hospital care (gauze dressings, direct pressure, and
tourniquets) have, unfortunately, not changed significantly in the
past 2000 years. See L. Zimmerman et al., Great Ideas in the
History of Surgery (San Francisco, Calif.: Norman Publishing;
1993), 31. Even in trained hands they are not uniformly effective,
and the occurrence of excessive bleeding or fatal hemorrhage from
an accessible site is not uncommon. See J. M. Rocko et al., J.
Trauma 22:635 (1982).
[0003] Mortality data from Vietnam indicates that 10% of combat
deaths were due to uncontrolled extremity hemorrhage. See SAS/STAT
Users Guide, 4th ed. (Cary, N.C.: SAS Institute Inc.; 1990). Up to
one third of the deaths from ex-sanguination during the Vietnam War
could have been prevented by the use of effective field hemorrhage
control methods. See SAS/STAT Users Guide, 4th ed. (Cary, N.C.: SAS
Institute Inc.; 1990).
[0004] Although civilian trauma mortality statistics do not provide
exact numbers for pre-hospital deaths from extremity hemorrhage,
case and anecdotal reports indicate similar occurrences. See J. M.
Rocko et al. These data suggest that a substantial increase in
survival can be affected by the pre-hospital use of a simple and
effective method of hemorrhage control.
[0005] There are now in use a number of newer haemostatic agents
that have been developed to overcome the deficiencies of
traditional gauze bandages. These haemostatic agents include the
following: [0006] Microporous polysaccharide particles
(TraumaDEX.RTM., Medafor Inc., Minneapolis, Minn.); [0007] Zeolite
(QuikClot.RTM., Z-Medica Corp, Wallington, Conn.); [0008]
Acetylated poly-N-acetyl glucosamine (Rapid Deployment Hemostat.TM.
(RDH), Marine Polymer Technologies, Danvers, Mass.); [0009]
Chitosan (HemCon.RTM. bandage, HemCon Medical Technologies Inc.,
Portland Oreg.); [0010] Liquid Fibrin Sealants (Tisseel VH, Baxter,
Deerfield, Ill.) [0011] Human fibrinogen and thrombin on equine
collagen (TachoComb-S, Hafslund Nycomed Pharma, Linz, Austria);
[0012] Microdispersed oxidized cellulose (m.cndot.doc.TM.,
Alltracel Group, Dublin, Ireland); [0013] Propyl gallate
(Hemostatin.TM., Analytical Control Systems Inc., Fishers, Ind.);
[0014] Epsilon aminocaproic acid and thrombin (Hemarrest.TM. patch,
Clarion Pharmaceuticals, Inc.); [0015] Purified bovine corium
collagen (Avitene.RTM. sheets (non-woven web or Avitene
Microfibrillar Collagen Hemostat (MCH), Davol, Inc., Cranston,
R.I.); [0016] Controlled oxidation of regenerated cellulose
(Surgicel.RTM., Ethicon Inc., Somerville, N.J.); [0017] Aluminum
sulfate with an ethyl cellulose coating (Sorbastace Microcaps,
Hemostace, LLC, New Orleans, La.); [0018] Microporous
hydrogel-forming polyacrylamide (BioHemostat, Hemodyne, Inc.,
Richmond Va.); and [0019] Recombinant activated factor VII
(NovoSeven.RTM., NovoNordisk Inc., Princeton, N.J.). These agents
have met with varying degrees of success when used in animal models
of traumatic injuries and/or in the field.
[0020] One such agent is a starch-based haemostatic agent sold
under the trade name TraumaDEX.TM.. This product comprises
macroporous polysaccharide particles that are poured directly into
or onto a wound. The particles appear to exert their haemostatic
effect by absorbing water from the blood and plasma in the wound,
resulting in the accumulation and concentration of clotting factors
and platelets. In two studies of a lethal groin wound model,
however, this agent showed no meaningful benefit over standard
gauze dressings. See McManus et al., Business Briefing: Emergency
Medical Review 2005, pp. 76-79 (presently available on-line at
www.touchbriefings.com/pdf/1334/Wedmore.pdf).
[0021] Another particle-based agent is QuickClot.TM. powder, a
zeolite granular haemostatic agent that is poured directly into or
onto a wound. The zeolite particles also appear to exert their
haemostatic effect through fluid absorption, which cause the
accumulation and concentration of clotting factors and platelets.
Although this agent has been used successfully in some animal
studies, there remains concern about the exothermic process of
fluid absorption by the particles. Some studies have shown this
reaction to produce temperatures in excess of 143.degree. C. in
vitro and in excess of 50.degree. C. in vivo, which is severe
enough to cause third-degree burns. See McManus et al., Business
Briefing: Emergency Medical Review 2005, at 77. The exothermic
reaction of QuikClot.TM. has also been observed to result in gross
and histological tissue changes of unknown clinical significance.
Acheson et al., Trauma 59:865-874 (2005).
[0022] Unlike these particle-based agents, the Rapid Deployment
Hemostat.TM. appears to exert its haemostatic effect through red
blood cell aggregation, platelet activation, clotting cascade
activation and local vasoconstriction. The Rapid Deployment
Hemostat.TM. is an algae-derived dressing composed of
poly-N-acetyl-glucosamine. While the original dressing design was
effective in reducing minor bleeding, it was necessary to add gauze
backing in order to reduce blood loss in swine models of aortic and
liver injury. See McManus et al., Business Briefing: Emergency
Medical Review 2005, at 78.
[0023] Another poly-N-acetyl-glucosamine-derived dressing is the
HemCon.TM. Chitosan Bandage, which is a freeze-dried chitosan
dressing purportedly designed to optimize the mucoadhesive surface
density and structural integrity of the chitosan at the site of the
wound. The HemCon.TM. Chitosan Bandage apparently exerts its
haemostatic effects primarily through adhesion to the wound,
although there is evidence suggesting it may also enhance platelet
function and incorporate red blood cells into the clot it forms on
the wound. This bandage has shown improved hemostasis and reduced
blood loss in several animal models of arterial hemorrhage, but a
marked variability was observed between bandages, including the
failure of some due to inadequate adherence to the wound. See
McManus et al., Business Briefing: Emergency Medical Review 2005,
at 79.
[0024] Liquid fibrin sealants, such as Tisseel VH, have been used
for years as an operating room adjunct for hemorrhage control. See
J. L. Garza et al., J. Trauma 30:512-513 (1990); H. B. Kram et al.,
J. Trauma 30:97-101(1990); M. G. Ochsner et al., J. Trauma
30:884-887 (1990); T. L. Matthew et al., Ann. Thorac. Surg.
50:40-44 (1990); H. Jakob et al., J. Vasc. Surg., 1:171-180 (1984).
The first mention of tissue glue used for hemostasis dates back to
1909. See Current Trends in Surgical Tissue Adhesives: Proceedings
of the First International Symposium on Surgical Adhesives, M. J.
MacPhee et al., eds. (Lancaster, Pa.: Technomic Publishing Co;
1995). Liquid fibrin sealants are typically composed of fibrinogen
and thrombin, but may also contain Factor XIII/XIIIa, either as a
by-product of fibrinogen purification or as an added ingredient (in
certain applications, it is therefore not necessary that Factor
XIII/Factor XIIIa be present in the fibrin sealant because there is
sufficient Factor XIII/XIIIa, or other transaminase, endogenously
present to induce fibrin formation). As liquids, however, these
fibrin sealants have not proved useful for treating traumatic
injuries in the field.
[0025] Dry fibrinogen-thrombin dressings having a collagen support
(e.g. TachoComb.TM., TachoComb.TM. H and TachoSil available from
Hafslund Nycomed Pharma, Linz, Austria) are also available for
operating room use in many European countries. See U. Schiele et
al., Clin. Materials 9:169-177 (1992). While these
fibrinogen-thrombin dressings do not require the pre-mixing needed
by liquid fibrin sealants, their utility for field applications is
limited by a requirement for storage at 4.degree. C. and the
necessity for pre-wetting with saline solution prior to application
to the wound. These dressings are also not effective against high
pressure, high volume bleeding. See Sondeen et al., J. Trauma
54:280-285 (2003).
[0026] A dry fibrinogen/thrombin dressing for treating wounded
tissue is also available from the American Red Cross (ARC). As
disclosed in U.S. Pat. No. 6,762,336, this particular dressing is
composed of a backing material and a plurality of layers, the outer
two of which contain fibrinogen (but no thrombin) while the inner
layer contains thrombin and calcium chloride (but no fibrinogen).
While this dressing has shown great success in several animal
models of hemorrhage, the bandage is fragile, inflexible, and has a
tendency to break apart when handled. See McManus et al., Business
Briefing: Emergency Medical Review 2005, at 78; Kheirabadi et al.,
J. Trauma 59:25-35 (2005). In addition, U.S. Pat. No. 6,762,336
teaches that this bandage should contain 15 mg/cm.sup.2 of
fibrinogen to successfully pass a porcine arteriotomy test that is
less robust than that disclosed in this application (see Example
XI). Moreover, although U.S. Pat. No. 6,762,336 discloses that
bandages comprising two layers of fibrinogen, each with a
concentration of 4 mg/cm.sup.2 to 15 mg/cm.sup.2 may provide
effective control of hemorrhage, it further teaches that
"fibrinogen dose is related to quality. The higher dose is
associated with more firm and tightly adhered clots. While lower
fibrinogen doses are effective for hemorrhage control during the
initial 60 minutes, longer term survival will likely depend on clot
quality."
[0027] Other fibrinogen/thrombin-based dressings have also been
proposed. For example, U.S. Pat. No. 4,683,142 discloses a
resorptive sheet material for closing and healing wounds which
consists of a glycoprotein matrix, such as collagen, containing
coagulation proteins, such as fibrinogen and thrombin. U.S. Pat.
No. 5,702,715 discloses a reinforced biological sealant composed of
separate layers of fibrinogen and thrombin, at least one of which
also contains a reinforcement filler such as PEG, PVP, BSA,
mannitol, FICOLL, dextran, myo-inositol or sodium chlorate. U.S.
Pat. No. 6,056,970 discloses dressings composed of a bioabsorbable
polymer, such as hyaluronic acid or carboxymethylcellulose, and a
haemostatic composition composed of powdered thrombin and/or
powdered fibrinogen. U.S. Pat. No. 7,189,410 discloses a bandage
composed of a backing material having thereon: (i) particles of
fibrinogen; (ii) particles of thrombin; and (iii) calcium chloride.
U.S. Patent Application Publication No. US 2006/0155234 A1
discloses a dressing composed of a backing material and a plurality
of fibrinogen layers which have discrete areas of thrombin between
them. To date, none of these dressings have been approved for use
or are available commercially.
[0028] Accordingly, there remains a need in the art for a solid
dressing that can be used to treat wounded tissue, particularly
wounded tissue resulting from traumatic injury in the field.
SUMMARY OF THE INVENTION
[0029] It is therefore an object of the present invention to
provide a solid dressing that can treat wounded mammalian tissue,
particularly wounded tissue resulting from a traumatic injury. It
is further an object of the present invention to provide a method
of treating wounded mammalian tissue, particularly human tissue.
Other objects, features and advantages of the present invention
will be set forth in the detailed description of preferred
embodiments that follows, and will in part be apparent from that
description and/or may be learned by practice of the present
invention. These objects and advantages will be realized and
attained by the compositions and methods described in this
specification and particularly pointed out in the claims that
follow.
[0030] In accordance with these and other objects, a first
embodiment of the present invention is direct to a solid dressing
for treating wounded tissue in a mammal comprising at least one
haemostatic layer consisting essentially of fibrinogen and a
fibrinogen activator, wherein the fibrinogen is present in an
amount between about 3.0 mg/cm.sup.2 of the surface area of the
wound facing side of the dressing and 13.0 mg/cm.sup.2 of the
surface area of the wound facing side of the dressing.
[0031] Another embodiment is directed to a method of treating
wounded tissue using a solid dressing comprising at least one
haemostatic layer consisting essentially of fibrinogen and a
fibrinogen activator, wherein the fibrinogen is present in an
amount between about 11.0 mg/cm.sup.2 of the surface area of the
wound facing side of the dressing and 13.0 mg/cm.sup.2 of the
surface area of the wound facing side of the dressing.
[0032] It is to be understood that the foregoing general
description and the following detailed description of preferred
embodiments are exemplary and explanatory only and are intended to
provide further explanation, but not limitation, of the invention
as claimed herein.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIGS. 1A-1C are graphs showing the results achieved in
Example 1.
[0034] FIG. 2 is a diagram of the set-up for the ex vivo porcine
arteriotomoy assay described herein.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications mentioned herein are incorporated by
reference.
[0036] As used herein, use of a singular article such as "a," "an,"
and "the" is not intended to excluded pluralities of the article's
object unless the context clearly and unambiguously dictates
otherwise.
[0037] "Patient" as used herein refers to human or animal
individuals in need of medical care and/or treatment.
[0038] "Wound" as used herein refers to any damage to any tissue of
a patient which results in the loss of blood from the circulatory
system and/or any other fluid from the patient's body. The tissue
may be an internal tissue, such as an organ or blood vessel, or an
external tissue, such as the skin. The loss of blood may be
internal, such as from a ruptured organ, or external, such as from
a laceration. A wound may be in a soft tissue, such as an organ, or
in hard tissue, such as bone. The damage may have been caused by
any agent or source, including traumatic injury, infection or
surgical intervention.
[0039] "Resorbable material" as used herein refers to a material
that is broken down spontaneously and/or by the mammalian body into
components which are consumed or eliminated in such a manner as not
to interfere significantly with wound healing and/or tissue
regeneration, and without causing any significant metabolic
disturbance.
[0040] "Stability" as used herein refers to the retention of those
characteristics of a material that determine activity and/or
function.
[0041] "Suitable" as used herein is intended to mean that a
material does not adversely affect the stability of the dressings
or any component thereof.
[0042] "Binding agent" as used herein refers to a compound or
mixture of compounds that improves the adherence and/or cohesion of
the components of the haemostatic layer(s) of the dressings.
[0043] "Solubilizing agent" as used herein refers to a compound or
mixture of compounds that improves the dissolution of a protein or
proteins in aqueous solvent.
[0044] "Filler" as used herein refers to a compound or mixture of
compounds that provide bulk and/or porosity to the haemostatic
layer(s) of a dressing.
[0045] "Release agent" as used herein refers to a compound or
mixture of compounds that facilitates removal of a dressing from a
manufacturing mold.
[0046] "Foaming agent" as used herein refers to a compound or
mixture of compounds that produces gas when hydrated under suitable
conditions.
[0047] "Solid" as used herein is intended to mean that the dressing
will not substantially change in shape or form when placed on a
rigid surface, wound-facing side down, and then left to stand at
room temperature for 24 hours.
[0048] A first preferred embodiment of the present invention is
directed to a solid dressing for treating wounded tissue in a
patient which comprises a haemostatic layer consisting of
fibrinogen and a fibrinogen activator, wherein the fibrinogen is
present in an amount between 3.0 mg/cm.sup.2 of the surface area of
the wound facing side of the dressing and 13.0 mg/cm.sup.2 of the
surface area of the wound facing side of the dressing, all values
being .+-.0.09 mg/cm.sup.2.
[0049] As used herein, "consisting essentially of" is intended to
mean that the fibrinogen and the fibrinogen activator are the only
necessary and essential ingredients of the haemostatic layer(s) of
the solid dressing when it is used as intended to treat wounded
tissue. Accordingly, the haemostatic layer may contain other
ingredients in addition to the fibrinogen and the fibrinogen
activator as desired for a particular application, but these other
ingredients are not required for the solid dressing to function as
intended under normal conditions, i.e. these other ingredients are
not necessary for the fibrinogen and fibrinogen activator to react
and form enough fibrin to reduce the flow of blood and/or fluid
from normal wounded tissue when that dressing is applied to that
tissue under the intended conditions of use. If, however, the
conditions of use in a particular situation are not normal, for
example the patient is a hemophiliac suffering from Factor XIII
deficiency, then the appropriate additional components, such as
Factor XIII/XIIIa or some other transaminase, may be added to the
haemostatic layer(s) without deviating from the spirit of the
present invention. Similarly, the solid dressing of the present
invention may contain one or more of these haemostatic layers as
well as one or more other layers, such as one or more support
layers (e.g. a backing material or an internal support material)
and release layers.
[0050] Other preferred embodiments of the present invention include
similar solid dressings wherein the fibrinogen is present in an
amount between 11.0 mg/cm.sup.2 of the surface area of the wound
facing side of the dressing and 13.0 mg/cm.sup.2 of the surface
area of the wound facing side of the dressing, all values being
.+-.0.09 mg/cm.sup.2. Other preferred embodiments include similar
solid dressings wherein the fibrinogen is present in an amount
between 3.0 mg/cm.sup.2 and 9.0 mg/cm.sup.2 Still other preferred
embodiments are directed to similar solid dressings wherein the
amount of fibrinogen is: 3.0 mg/cm.sup.2 of the surface area of the
wound facing side of the dressing; 5.0 mg/cm.sup.2; 7.0
mg/cm.sup.2; 9.0 mg/cm.sup.2; 11.0 mg/cm.sup.2; or 13.0 mg/cm.sup.2
(all values being .+-.0.09 mg/cm.sup.2).
[0051] Another preferred embodiment of the present invention is
directed to a method for treating wounded tissue in a mammal,
comprising placing a solid dressing of the present invention to
wounded tissue and applying sufficient pressure to the dressing for
a sufficient time for enough fibrin to form to reduce the loss of
blood and/or other fluid from the wound.
[0052] According to certain embodiments of the present invention,
the haemostatic layer(s) of the solid dressing is formed or cast as
a single piece. According to certain other embodiments of the
present invention, the haemostatic layer is made or formed into or
from a single source, e.g. an aqueous solution containing a mixture
of the fibrinogen and the fibrinogen activator. With each of these
embodiments of the present invention, the haemostatic layer(s) is
preferably substantially homogeneous throughout.
[0053] According to other preferred embodiments, the haemostatic
layer(s) of the solid dressing are composed of a plurality of
particles, each of which consists essentially of fibrinogen and a
fibrinogen activator. According to such embodiments, the
haemostatic layer may also contain a binding agent to facilitate or
improve the adherence of the particles to one another and/or to any
support layer(s). Illustrative examples of suitable binding agents
include, but are not limited to, sucrose, mannitol, sorbitol,
gelatin, hyaluron and its derivatives, such as hyaluronic acid,
maltose, povidone, starch, chitosan and its derivatives, and
cellulose derivatives, such as carboxymethylcellulose, as well as
mixtures of two or more thereof.
[0054] The haemostatic layer(s) of the solid dressing may also
optionally contain one or more suitable fillers, such as sucrose,
lactose, maltose, silk, fibrin, collagen, albumin, hyaluron and its
derivatives, such as hyaluronic acid, polysorbate (Tween.TM.),
chitin, chitosan and its derivatives, such as NOCC-chitosan,
alginic acid and salts thereof, cellulose and derivatives thereof,
proteoglycans, glycolic acid polymers, lactic acid polymers,
glycolic acid/lactic acid co-polymers, and mixtures of two or more
thereof.
[0055] The haemostatic layer of the solid dressing may also
optionally contain one or more suitable solubilizing agents, such
as sucrose, dextrose, mannose, trehalose, mannitol, sorbitol,
albumin, hyaluron and its derivatives, such as hyaluronic acid,
sorbate, polysorbate (Tween.TM.), sorbitan (SPAN.TM.) and mixtures
of two or more thereof.
[0056] The haemostatic layer of the solid dressing may also
optionally contain one or more suitable foaming agents, such as a
mixture of a physiologically acceptable acid (e.g. citric acid or
acetic acid) and a physiologically suitable base (e.g. sodium
bicarbonate or calcium carbonate). Other suitable foaming agents
include, but are not limited to, dry particles containing
pressurized gas, such as sugar particles containing carbon dioxide
(see, e.g., U.S. Pat. No. 3,012,893) or other physiologically
acceptable gases (e.g. Nitrogen or Argon), and pharmacologically
acceptable peroxides.
[0057] The haemostatic layer(s) of the solid dressing may also
optionally contain a suitable source of calcium ions, such as
calcium chloride, and/or a fibrin cross-linker, such as a
transaminase (e.g. Factor XIII/XIIIa) or glutaraldehyde.
[0058] The haemostatic layer of the solid dressing is preferably
prepared by mixing aqueous solutions of the fibrinogen and the
fibrinogen activator under conditions which minimize the activation
of the fibrinogen by the fibrinogen activator. The mixture of
aqueous solutions is then subjected to a process such as
lyophilization or free-drying to reduce the moisture content to the
desired level, i.e. to a level where the dressing is solid and
therefore will not substantially change in shape or form upon
standing, wound-facing surface down, at room temperature for 24
hours. Similar processes that achieve the same result, such as
drying, spray-drying, vacuum drying and vitrification, may also be
employed.
[0059] As used herein, "moisture content" refers to the amount
freely-available water in the dressing. "Freely-available" means
the water is not bound to or complexed with one or more of the
non-liquid components of the dressing. The moisture content
referenced herein refers to levels determined by procedures
substantially similar to the FDA-approved, modified Karl Fischer
method (Meyer and Boyd, Analytical Chem., 31:215-219, 1959; May et
al., J. Biol. Standardization, 10:249-259, 1982; Centers for
Biologics Evaluation and Research, FDA, Docket No. 89D-0140, 83-93;
1990) or by near infrared spectroscopy. Suitable moisture
content(s) for a particular solid dressing may be determined
empirically by one skilled in the art depending upon the intended
application(s) thereof.
[0060] For example, in certain embodiments of the present
invention, higher moisture contents are associated with more
flexible solid dressings. Thus, in solid dressings intended for
extremity wounds, it may be preferred to have a moisture content of
at least 6% and even more preferably in the range of 6% to 44%.
[0061] Similarly, in other embodiments of the present invention,
lower moisture contents are associated with more rigid solid
dressings. Thus, in solid dressings intended for flat wounds, such
as wounds to the abdomen or chest, it may be preferred to have a
moisture content of less than 6% and even more preferably in the
range of 1% to 6%.
[0062] Accordingly, illustrative examples of suitable moisture
contents for solid dressings include, but are not limited to, the
following (each value being .+-.0.9%): less than 53%; less than
44%; less than 28%; less than 24%; less than 16%; less than 12%;
less than 6%; less than 5%; less than 4%; less than 3%; less than
2.5%; less than 2%; less than 1.4%; between 0 and 12%,
non-inclusive; between 0 and 6%; between 0 and 4%; between 0 and
3%; between 0 and 2%; between 0 and 1%; between 1 and 16%; between
1 and 11%; between 1 and 8%; between 1 and 6%; between 1 and 4%;
between 1 and 3%; between 1 and 2%; and between 2 and 4%.
[0063] The fibrinogen in the haemostatic layer(s) of the solid
dressings may be any suitable fibrinogen known and available to
those skilled in the art. A specific fibrinogen for a particular
application may be selected empirically by one skilled in the art.
As used herein, the term "fibrinogen" is intended to include
mixtures of fibrinogen and small amounts of Factor XIII/Factor
XIIIa, or some other such transaminase Such small amounts are
generally recognized by those skilled in the art as usually being
found in mammalian fibrinogen after it has been purified according
to the methods and techniques presently known and available in the
art, and typically range from 0.1 to 20 Units/mL.
[0064] Preferably, the fibrinogen employed as the fibrinogen
component of the solid dressing is a purified fibrinogen suitable
for introduction into a mammal Typically, such fibrinogen is a part
of a mixture of human plasma proteins which include Factor
XIII/XIIIa and have been purified to an appropriate level and
virally inactivated. A preferred aqueous solution of fibrinogen for
preparation of a solid dressing contains around 37.5 mg/mL
fibrinogen at a pH of around 7.4.+-.0.1. Suitable fibrinogen for
use as the fibrinogen component has been described in the art, e.g.
U.S. Pat. No. 5,716,645, and similar materials are commercially
available, e.g. from sources such as Sigma-Aldrich, Enzyme Research
Laboratories, Haematologic Technologies and Aniara.
[0065] The fibrinogen activator employed in the haemostatic
layer(s) of the solid dressing may be any of the substances or
mixtures of substances known by those skilled in the art to convert
fibrinogen into fibrin. Illustrative examples of suitable
fibrinogen activators include, but are not limited to, the
following: thrombins, such as human thrombin or bovine thrombin,
and prothrombins, such as human prothrombin or prothrombin complex
concentrate (a mixture of Factors II, VII, IX and X); snake venoms,
such as batroxobin, reptilase (a mixture of batroxobin and Factor
XIIIa), bothrombin, calobin, fibrozyme, and enzymes isolated from
the venom of Bothrops jararacussu; and mixtures of any two or more
of these. See, e.g., Dascombe et al., Thromb. Haemost. 78:947-51
(1997); Hahn et al., Biochem. (Tokyo) 119:835-43 (1996); Fortova et
al., J. Chromatogr. S. Biomed. Appl. 694:49-53 (1997); and
Andriao-Escarso et al., Toxicon. 35: 1043-52 (1997).
[0066] Preferably, the fibrinogen activator is a thrombin. More
preferably, the fibrinogen activator is a mammalian thrombin,
although bird and/or fish thrombin may also be employed in
appropriate circumstances. While any suitable mammalian thrombin
may be used in the solid dressing, the thrombin employed in the
haemostatic layer is preferably a lyophilized mixture of human
plasma proteins which has been sufficiently purified and virally
inactivated for the intended use of the solid dressing. Suitable
thrombin is available commercially from sources such as
Sigma-Aldrich, Enzyme Research Laboratories, Haematologic
Technologies and Biomol International. A particularly preferred
aqueous solution of thrombin for preparing a solid dressing
contains thrombin at a potency of between 10 and 2000.+-.50
International Units/mL, and more preferred at a potency of
25.+-.2.5 International Units/mL. Other constituents may include
albumin (generally about 0.1 mg/mL) and glycine (generally about
100 mM.+-.0.1 mM). The pH of this particularly preferred aqueous
solution of thrombin is generally in the range of 6.5-7.8, and
preferably 7.4.+-.0.1, although a in the range of 5.5-8.5 may be
acceptable.
[0067] In addition to the haemostatic layer(s), the solid dressing
may optionally further comprise one or more support layers. As used
herein, a "support layer" refers to a material that sustains or
improves the structural integrity of the solid dressing and/or the
fibrin clot formed when such a dressing is applied to wounded
tissue.
[0068] According to certain preferred embodiments of the present
invention the support layer comprises a backing material on the
side of the dressing opposite the side to be applied to wounded
tissue. Such a backing material may be affixed with a
physiologically-acceptable adhesive or may be self-adhering (e.g.
by having a sufficient surface static charge). The backing material
may comprise one or more resorbable materials or one or more
non-resorbable materials or mixtures thereof. Preferably, the
backing material is a single resorbable material.
[0069] Any suitable resorbable material known and available to
those skilled in the art may be employed in the present invention.
For example, the resorbable material may be a proteinaceous
substance, such as silk, fibrin, keratin, collagen and/or gelatin.
Alternatively, the resorbable material may be a carbohydrate
substance, such as alginates, chitin, cellulose, proteoglycans
(e.g. poly-N-acetyl glucosamine), hyaluron and its derivatives,
such as hyaluronic acid, glycolic acid polymers, lactic acid
polymers, or glycolic acid/lactic acid co-polymers. The resorbable
material may also comprise a mixture of proteinaceous substances or
a mixture of carbohydrate substances or a mixture of both
proteinaceous substances and carbohydrate substances. Specific
resorbable material(s) may be selected empirically by those skilled
in the art depending upon the intended use of the solid
dressing.
[0070] According to certain preferred embodiments of the present
invention, the resorbable material is a carbohydrate substance.
Illustrative examples of particularly preferred resorbable
materials include, but are not limited to, the materials sold under
the trade names VICRYL.TM. (a glycolic acid/lactic acid copolymer)
and DEXON.TM. (a glycolic acid polymer).
[0071] Any suitable non-resorbable material known and available to
those skilled in the art may be employed as the backing material.
Illustrative examples of suitable non-resorbable materials include,
but are not limited to, plastics, silicone polymers, paper and
paper products, latex, gauze and the like.
[0072] According to other preferred embodiments, the support layer
comprises an internal support material. Such an internal support
material is preferably fully contained within a haemostatic layer
of the solid dressing., although it may be placed between two
adjacent haemostatic layers in certain embodiments. As with the
backing material, the internal support material may be a resorbable
material or a non-resorbable material, or a mixture thereof, such
as a mixture of two or more resorbable materials or a mixture of
two or more non-resorbable materials or a mixture of resorbable
material(s) and non-resorbable material(s).
[0073] According to still other preferred embodiments, the support
layer may comprise a front support material on the wound-facing
side of the dressing, i.e. the side to be applied to wounded
tissue. As with the backing material and the internal support
material, the front support material may be a resorbable material
or a non-resorbable material, or a mixture thereof, such as a
mixture of two or more resorbable materials or a mixture of two or
more non-resorbable materials or a mixture of resorbable
material(s) and non-resorbable material(s).
[0074] According to still other preferred embodiments, the solid
dressing comprises both a backing material and an internal support
material in addition to the haemostatic layer(s), i.e. the solid
dressing comprises two support layers in addition to the
haemostatic layer(s). According to still other preferred
embodiments, the solid dressing comprises both a front support
material and an internal support material in addition to the
haemostatic layer(s). According to still other preferred
embodiments, the solid dressing comprises a backing material, a
front support material and an internal support material in addition
to the haemostatic layer(s).
[0075] According to certain embodiments of the present invention,
particularly where the solid dressing is manufactured using a mold,
the solid dressings may also optionally further comprise a release
layer in addition to the haemostatic layer(s) and support layer(s).
As used herein, a "release layer" refers to a layer containing one
or more agents ("release agents") which promote or facilitate
removal of the solid dressing from a mold in which it has been
manufactured. A preferred such agent is sucrose, but other suitable
release agents include gelatin, mannitol, sorbitol, hyaluron and
its derivatives, such as hyaluronic acid, and glucose.
Alternatively, such one or more release agents may be contained in
the haemostatic layer.
[0076] The various layers of the inventive dressings may be affixed
to one another by any suitable means known and available to those
skilled in the art. For example, a physiologically-acceptable
adhesive may be applied to a backing material (when present), and
the haemostatic layer(s) subsequently affixed thereto.
[0077] In certain embodiments of the present invention, the
physiologically-acceptable adhesive has a shear strength and/or
structure such that the backing material can be separated from the
fibrin clot formed by the haemostatic layer after application of
the dressing to wounded tissue. In other embodiments, the
physiologically-acceptable adhesive has a shear strength and/or
structure such that the backing material cannot be separated from
the fibrin clot after application of the bandage to wounded
tissue.
[0078] Suitable fibrinogens and suitable fibrinogen activators for
the haemostatic layer(s) of the solid dressing may be obtained from
any appropriate source known and available to those skilled in the
art, including, but not limited to, the following: from commercial
vendors, such as Sigma-Aldrich and Enzyme Research Laboratories; by
extraction and purification from human or mammalian plasma by any
of the methods known and available to those skilled in the art;
from supernatants or pastes derived from plasma or recombinant
tissue culture, viruses, yeast, bacteria, or the like that contain
a gene that expresses a human or mammalian plasma protein which has
been introduced according to standard recombinant DNA techniques;
and/or from the fluids (e.g. blood, milk, lymph, urine or the like)
of transgenic mammals (e.g. goats, sheep, cows) that contain a gene
which has been introduced according to standard transgenic
techniques and that expresses the desired fibrinogen and/or desired
fibrinogen activator.
[0079] According to certain preferred embodiments of the present
invention, the fibrinogen is a mammalian fibrinogen such as bovine
fibrinogen, porcine fibrinogen, ovine fibrinogen, equine
fibrinogen, caprine fibrinogen, feline fibrinogen, canine
fibrinogen, murine fibrinogen or human fibrinogen. According to
other embodiments, the fibrinogen is bird fibrinogen or fish
fibrinogen. According to any of these embodiments, the fibrinogen
may be recombinantly produced fibrinogen or transgenic
fibrinogen.
[0080] According to certain preferred embodiments of the present
invention, the fibrinogen activator is a mammalian thrombin, such
as bovine thrombin, porcine thrombin, ovine thrombin, equine
thrombin, caprine thrombin, feline thrombin, canine thrombin,
murine thrombin and human thrombin. According to other embodiments,
the thrombin is bird thrombin or fish thrombin. According to any of
these embodiments, the thrombin may be recombinantly produced
thrombin or transgenic thrombin.
[0081] As a general proposition, the purity of the fibrinogen
and/or the fibrinogen activator for use in the solid dressing will
be a purity known to one of ordinary skill in the relevant art to
lead to the optimal efficacy and stability of the protein(s).
Preferably, the fibrinogen and/or the fibrinogen activator has been
subjected to multiple purification steps, such as precipitation,
concentration, diafiltration and affinity chromatography
(preferably immunoaffinity chromatography), to remove substances
which cause fragmentation, activation and/or degradation of the
fibrinogen and/or the fibrinogen activator during manufacture,
storage and/or use of the solid dressing. Illustrative examples of
such substances that are preferably removed by purification
include: protein contaminants, such as inter-alpha trypsin
inhibitor and pre-alpha trypsin inhibitor; non-protein
contaminants, such as lipids; and mixtures of protein and
non-protein contaminants, such as lipoproteins.
[0082] The amount of the fibrinogen activator employed in the solid
dressing is preferably selected to optimize both the efficacy and
stability thereof. As such, a suitable concentration for a
particular application of the solid dressing may be determined
empirically by one skilled in the relevant art. According to
certain preferred embodiments of the present invention, when the
fibrinogen activator is human thrombin, the amount of human
thrombin employed is between 2.50 Units/mg of fibrinogen component
and 0.025 Units/mg of the fibrinogen (all values being .+-.0.0009).
Other preferred embodiments are directed to similar solid dressings
wherein the amount of thrombin is between 0.250 Units/mg of
fibrinogen and 0.062 Units/mg of fibrinogen and solid dressings
wherein the amount of thrombin is between 0.125 Units/mg of
fibrinogen and 0.080 Units/mg of fibrinogen.
[0083] During use of the solid dressing, the fibrinogen and the
fibrinogen activator are preferably activated at the time the
dressing is applied to the wounded tissue by the endogenous fluids
of the patient escaping from the hemorrhaging wound. Alternatively,
in situations where fluid loss from the wounded tissue is
insufficient to provide adequate hydration of the protein layers,
the fibrinogen component and/or the thrombin may be activated by a
suitable, physiologically-acceptable liquid, optionally containing
any necessary co-factors and/or enzymes, prior to or during
application of the dressing to the wounded tissue.
[0084] In some embodiments of the present invention, the
haemostatic layer(s) may also contain one or more supplements, such
as growth factors, drugs, polyclonal and monoclonal antibodies and
other compounds. Illustrative examples of such supplements include,
but are not limited to, the following: fibrinolysis inhibitors,
such as aprotonin, tranexamic acid and epsilon-amino-caproic acid;
antibiotics, such as tetracycline and ciprofloxacin, amoxicillin,
and metronidazole; anticoagulants, such as activated protein C,
heparin, prostacyclins, prostaglandins (particularly (PGI.sub.2),
leukotrienes, antithrombin III, ADPase, and plasminogen activator;
steroids, such as dexamethasone, inhibitors of prostacyclin,
prostaglandins, leukotrienes and/or kinins to inhibit inflammation;
cardiovascular drugs, such as calcium channel blockers,
vasodilators and vasoconstrictors; chemoattractants; local
anesthetics such as bupivacaine; and antiproliferative/antitumor
drugs such as 5-fluorouracil (5-FU), taxol and/or taxotere;
antivirals, such as gangcyclovir, zidovudine, amantidine,
vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine and
antibodies to viral components or gene products; cytokines, such as
alpha- or beta- or gamma-Interferon, alpha- or beta-tumor necrosis
factor, and interleukins; colony stimulating factors;
erythropoietin; antifungals, such as diflucan, ketaconizole and
nystatin; antiparasitic gents, such as pentamidine;
anti-inflammatory agents, such as alpha-1-anti-trypsin and
alpha-1-antichymotrypsin; anesthetics, such as bupivacaine;
analgesics; antiseptics; hormones; vitamins and other nutritional
supplements; glycoproteins; fibronectin; peptides and proteins;
carbohydrates (both simple and/or complex); proteoglycans;
antiangiogenins; antigens; lipids or liposomes; oligonucleotides
(sense and/or antisense DNA and/or RNA); and gene therapy reagents.
In other embodiments of the present invention, the backing layer
and/or the internal support layer, if present, may contain one or
more supplements. According to certain preferred embodiments of the
present invention, the therapeutic supplement is present in an
amount greater than its solubility limit in fibrin.
[0085] The following examples are illustrative only and are not
intended to limit the scope of the invention as defined by the
appended claims. It will be apparent to those skilled in the art
that various modifications and variations can be made in the
methods of the present invention without departing from the spirit
and scope of the invention. Thus, it is intended that the present
invention cover the modifications and variations of this invention
provided they come within the scope of the appended claims and
their equivalents.
EXAMPLES
[0086] The ability of the dressings to seal an injured blood vessel
was determined by an ex vivo porcine arteriotomy (EVPA) performance
test, which was first described in U.S. Pat. No. 6,762,336. The
EVPA performance test evaluates the ability of a dressing to stop
fluid flow through a hole in a porcine artery. While the procedure
described in U.S. Pat. No. 6,762,336 has been shown to be useful
for evaluating haemostatic dressings, it failed to replicate
faithfully the requirements for success in vivo. More specifically,
the procedure disclosed in U.S. Pat. No. 6,762,336 required testing
at 37.degree. C., whereas, in the real world, wounds are typically
cooler than that. This decreased temperature can significantly
reduce the rate of fibrin formation and its haemostatic efficacy in
trauma victims. See, e.g., Acheson et al., J. Trauma 59:865-874
(2005). The test in U.S. Pat. No. 6,762,336 also failed to require
a high degree of adherence of the dressing to the injured tissue. A
failure mode in which fibrin forms but the dressing fails to attach
tightly to the tissue would, therefore, not be detected by this
test. Additionally, the pressure utilized in the procedure (200
mHg) may be exceeded during therapy for some trauma patients. The
overall result of this is that numerous animal tests, typically
involving small animals (such as rats and rabbits), must be
conducted to accurately predict dressing performance in large
animal, realistic trauma studies and in the clinical
environment.
[0087] In order to minimize the amount of time and the number of
animal studies required to develop the present invention, an
improved ex vivo testing procedure was developed. To accomplish
this, the basic conditions under which the dressing test was
conducted were changed, and the severity of the test parameters was
increased to include testing at lower temperatures (i.e.
29-33.degree. C. vs. 37.degree. C., representing the real
physiologic challenge at realistic wound temperatures (Acheson et
al., J. Trauma 59:865-874 (2005)), higher pressures (i.e. 250 mmHg
vs. 200 mmHg), a longer test period (3 minutes vs. 2 minutes) and
larger sized arterial injuries (U.S. Pat. No. 6,762,336 used an 18
gauge needle puncture, whereas the revised procedure used puncture
holes ranging from 2.8 mm to 4 mm.times.6 mm).
[0088] In addition, a new test was derived to directly measure
adherence of the dressing to the injured tissue. Both these tests
showed greatly improved stringency and are thus capable of
surpassing the previous ex vivo test and replacing many in vivo
tests for efficacy.
[0089] The following is a list of acronyms used in the Examples
below:
CFB: Complete Fibrinogen Buffer (100 mM Sodium Chloride, 1.1 mM
Calcium Chloride, 10 Mm Tris, 10 mM Sodium Citrate, 1.5% Sucrose,
Human Serum Albumin (80 mg/g of total protein) and Tween.TM. 80
(animal source) 15 mg/g total protein) CTB: Complete Thrombin
Buffer (150 mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Tris
and 100 mM L-Lysine with the addition of HSA at 100 ug/ml)
ERL: Enzyme Research Laboratories
EVPA: Ex Vivo Porcine Arteriotomy
[0090] FD: Inventive haemostatic dressing
HSA: Human Serum Albumin
[0091] HD: A "sandwich" fibrin sealant haemostatic dressing as
disclosed in U.S. Pat. No. 6,762,336 IFB: Incomplete Fibrinogen
Buffer; CFB without HSA and Tween
PETG: Glycol-modified Polyethlylenetetrapthalate
PPG: Polypropylene
[0092] PVC: Poly vinyl chloride TRIS: trishydroxymethylaminomethane
(2-amino-2-hydroxymethyl-1,3-propanediol)
Example 1
[0093] Backing material (DEXON.TM.) was cut and placed into each
PETG 2.4.times.2.4 cm mold. Twenty-five microliters of 2% sucrose
was pipetted on top of each of the four corners of the backing
material. Once completed the molds were placed in a -80.degree. C.
freezer for at least 60 minutes. Fibrinogen (Enzyme Research
Laboratories.TM.) was formulated in CFB. The final pH of the
fibrinogen was 7.4.+-.0.1. The fibrinogen concentrations were
adjusted to 37.5, 31.7, 25.9, 20.16, 14.4, 8.64, and 4.3 mg/ml.
When 2 ml of fibrinogen was delivered into the molds, this would
result in a fibrinogen dose of 13, 11, 9, 7, 5, 3 or 1.5
mg/cm.sup.2. Once prepared the fibrinogen was placed on ice until
use. Thrombin was formulated in CTB. The final pH of the thrombin
was 7.4.+-.0.1. The concentrations of thrombin were adjusted so
that when mixed with the fibrinogen solutions as described below,
the combination would produce a solution that contained 0.1
units/mg of Fibrinogen. Once prepared the thrombin was placed on
ice until use. The temperature of the fibrinogen and thrombin prior
to dispensing was 4.degree. C..+-.2.degree. C. Molds were removed
from the -80.degree. C. freezer and placed on a copper plate that
was placed on top of dry ice. A repeat pipettor was filled with
fibrinogen and second repeat pipettor was filled with thrombin. Two
ml of fibrinogen and 300 micro liters of thrombin were dispensed
simultaneously into each mold. Once the molds were filled they were
allowed to freeze and then returned to the -80.degree. C. freezer
for at least two hours. The frozen dressings were then placed into
a pre-cooled Genesis.TM. lyophylizer (Virtis, Gardiner, N.Y.). The
chamber was sealed and the temperature equilibrated. The chamber
was then evacuated and the dressings lyophilized via a primary and
secondary drying cycle.
[0094] The dressings were removed from the lyophylizer, sealed in
foil pouches and stored at room temperature until testing.
Subsequently, the dressings were evaluated in the EVPA, Adherence
and Weight Assays.
[0095] The results are given in the following Table and depicted
graphically in FIGS. 1A-1C.
TABLE-US-00001 Weight Weight EVPA Peel Test Adherence Held Held
Group Pass/Total Adherence Std Dev (mean) (g) Std Dev 13
mg/cm.sup.2 6/6 4.0 0.0 198.0 12.6 11 mg/cm.sup.2 6/6 3.8 0.4 163
48.5 9 mg/cm.sup.2 5/6 3.0 0.0 88 20.0 7 mg/cm.sup.2 6/6 3.2 0.4 93
17.6 7 mg/cm.sup.2 5/6 3.0 0.0 94.7 8.2 5 mg/cm.sup.2 5/5 2.8 0.4
76 34.2 3 mg/cm.sup.2 5/5 2.4 0.5 48 27.4 1.5 mg/cm.sup.2 0/6 0.1
0.2 4.7 11.4
Example 2
[0096] Monolithic dressings were manufactured as follows: backing
material was cut and placed into each PETG 2.4.times.2.4cm mold.
Twenty-five microliters of 2% sucrose was pipetted on top of each
of the four corners of the backing material Once completed the
molds were placed in a -80.degree. C. freezer for at least 60
minutes.
[0097] For all dressings, ERL fibrinogen lot 3114 was formulated in
CFB. The final pH of the fibrinogen was 7.4.+-.0.1. The fibrinogen
concentration was adjusted to 37.5 mg/ml. Once prepared the
fibrinogen was placed on ice until use. Thrombin was formulated in
CTB. The final pH of the thrombin was 7.4.+-.0.1. The thrombin was
adjusted to deliver 0.1 units/mg of Fibrinogen or 25 Units/ml
thrombin. Once prepared the thrombin was placed on ice until use.
The temperature of the fibrinogen and thrombin prior to dispensing
was 4.degree. C..+-.2.degree. C. Molds were removed from the
-80.degree. C. freezer and placed on a copper plate that was placed
on top of dry ice. A repeat pipettor was filled with fibrinogen and
second repeat pipettor was filled with thrombin. Simultaneously 2
ml of fibrinogen and 300 micro liters of thrombin were dispensed
into each mold. Once the molds were filled they were returned to
the -80.degree. C. freezer for at least two hours before being
placed into the freeze dryer. Dressings were then lyophilized as
described above. Once complete the dressings were stored in low
moisture transmission foil bags containing 5 grams of
desiccant.
[0098] Trilayer dressings were manufactured as described
previously.sup.1, using the same materials as described above.
Subsequently, the dressings were placed under conditions of 100%
relative humidity at 37.degree. C. for various times in order to
increase their relative moisture content to desired levels. The
dressings were evaluated visually and for their handling and other
physical characteristics. Following this evaluation, a sample of
each of the dressings was tested to determine their moisture
content. The remaining dressings were performance tested in the
EVPA, Adherence and Weight Held assays.
Results
[0099] The results of the assays are given in the Tables below:
TABLE-US-00002 TABLE 1 Performance Data of Inventive Solid
Dressings Exposure Time Weight to 100% Humidity % EVPA Peel Test
Held (g) @ 37.degree. C. Mois- # Adherence (mean .+-. Std.
(minutes) ture Pass/Total (.+-.Std. Dev.) Dev.) 0 2.5 2/2 4.0 .+-.
0 148 .+-. 28.3 1 5.8 2/2 3.5 .+-. 0.71 123 .+-. 7.1 15 16 2/2
.sup. 2.5 .+-. .71 108 .+-. 14.1 45 24 2/2 4.0 .+-. 0 168 .+-. 0 60
28 2/2 4.0 .+-. 0 273 .+-. 7.1 225 44 2/2 .sup. 2 .+-. 0 58 .+-.
14.1 1200 52 ND ND ND
TABLE-US-00003 TABLE 2 Performance Data for Tri-layer Dressings
Exposure Time to 100% Humidity EVPA Weight @ 37.degree. C. % # Peel
Test Held (g) (minutes) Moisture Pass/Total Adherence (mean) 0 3
1/1 2.0 78 15 22 1/1 2.0 78 60 33.7 0/1 0.5 28
TABLE-US-00004 TABLE 3 Integrity and Handling Characteristics of
Inventive Solid Dressings Exposure Time During Hydration to 100%
Force Humidity Prior To Hydration Required After @ 37.degree. C.
Surface Speed of for Hydration (minutes) Appearance Curling
Integrity Flexible Hydration Hydration Appearance 0 Normal No
Excellent No Normal No Normal (Smooth, No (No cracks or "skin")
flaking off) 1 Normal '' Excellent Yes '' '' '' (Smooth, No (No
cracks or "skin") flaking off) 15 Normal '' Excellent '' '' '' ''
(Smooth, No (No cracks or "skin") flaking off) 45 Normal ''
Excellent '' '' '' '' (Smooth, No (No cracks or "skin") flaking
off) 60 Normal Slight Excellent '' '' '' '' (Smooth, No (No cracks
or "skin") flaking off) 225 Normal Yes Excellent '' '' '' ''
(Smooth, No (No cracks or "skin") flaking off) 1200 Normal Curling
Excellent '' n/d n/d Mottled & (Smooth, No up on (No cracks or
lumpy "skin") itself flaking off)
TABLE-US-00005 TABLE 4 Integrity and Handling Characteristics of
Trilayer Dressings Exposure Time During Hydration to 100% Force
Humidity Prior To Hydration Required After @ 37.degree. C. Surface
Speed of for Hydration (minutes) Appearance Curling Integrity
Flexibility Hydration Hydration Appearance 0 Normal No Good; some
No Normal No Normal delamination 15 Irregular No Good; some Yes
Slow No Mottled delamination 60 Skinned Yes Good; some Yes Very
Slow Yes Very delamination Mottled and lumpy
Conclusions:
[0100] The monolithic dressings were fully functional at very high
levels of moisture. As much as 28% moisture was found to retain
complete functionality. When the moisture levels rose to 44%, the
dressings were still functional, however some of their activity was
reduced Higher levels of moisture may also retain some function.
The original dressings, at 2.5% moisture content, were not
flexible, but had all the other desired properties including
appearance, a flat surface, integrity, rapid and uncomplicated
hydration and a smooth appearance post hydration. Once the moisture
content was increased to 5.8%, the monolithic dressings became
flexible, while retaining their functionality and desirable
characteristics. They retained their flexibility, without curling
or losing their integrity or appearing to form excessive amounts of
fibrin prior to hydration.
[0101] This contrasted with the tri-layer dressings, which began to
lose their desirable characteristics upon the addition of moisture,
and lost them entirely by the time moisture had increased to 33%.
At no time did these dressings become flexible.
Example 3
[0102] For dressings utilizing a backing, the backing material was
cut and placed into each PETG 2.4.times.2.4 cm mold. Twenty-five
microliters of 2% sucrose was pipetted on top of each of the four
corners of the backing material. Once completed the molds were
placed in a -80.degree. C. freezer for at least 60 minutes. For
dressings without backing material, PETG 2.4.times.2.4 cm molds
were placed in a -80.degree. C. freezer for at least 60
minutes.
[0103] For all dressings, ERL fibrinogen lot 3114 was formulated in
CFB. The final pH of the fibrinogen was 7.4.+-.0.1. The fibrinogen
concentration was adjusted to 37.5 mg/ml. Once prepared the
fibrinogen was placed on ice until use. Thrombin was formulated in
CTB. The final pH of the thrombin was 7.4.+-.0.1. The thrombin was
adjusted to deliver 0.1 units/mg of Fibrinogen or 25 Units/ml
thrombin. Once prepared the thrombin was placed on ice until use.
The temperature of the fibrinogen and thrombin prior to dispensing
was 4.degree. C..+-.2.degree. C. Molds were removed from the
-80.degree. C. freezer and placed on a copper plate that was placed
on top of dry ice. A repeat pipettor was filled with fibrinogen and
second repeat pipettor was filled with thrombin. Simultaneously 2
ml of fibrinogen and 300 micro liters of thrombin were dispensed
into each mold. Once the molds were filled they were returned to
the -80.degree. C. freezer for at least two hours before being
placed into the freeze dryer. Dressings were then lyophylized as
described below.
[0104] Both groups were performance tested in the EVPA assay. In
addition, the group which had a backing was also tested in the
Adherence and Weight Held assays.
Results:
TABLE-US-00006 [0105] Weight Weight EVPA Peel Test Adherence Held
Held Group # Pass/Total Adherence Std Dev (mean) (g) Std Dev
Backing 6/6 3.7 0.5 153 37.3 No Backing 9/12
Conclusions:
[0106] Dressings formulated with backing material performed well,
with all dressings passing the EVPA test, and high values for
adherence and weight held. Dressings without backing material were
not quite as effective in the EVPA assay, however, surprisingly 75%
of them passed the EVPA test. Without the backing the other tests
could not be performed. The ability of the dressings made without a
backing to succeed in the EVPA assay indicates that these dressings
would be effective in treating arterial injuries and even more
effective in treating venous and small vessel injuries.
Example 4
[0107] For all dressings, ERL fibrinogen lot 3130 was formulated in
CFB. The final pH of the fibrinogen was 7.4.+-.0.1. The fibrinogen
concentration was adjusted to 37.5 mg/ml. Once prepared the
fibrinogen was placed on ice until use. Thrombin was formulated in
CTB. The final pH of the thrombin was 7.4.+-.0.1. The thrombin was
adjusted to deliver 0.1 units/mg of Fibrinogen or 25 Units/ml
thrombin. For the group with shredded VICRYL.TM. mesh dispersed
within, this support material was cut into approximately 1
mm.times.1 mm pieces and dispersed within the thrombin solution
prior to filling the molds. Once prepared the thrombin was placed
on ice until use. The temperature of the fibrinogen and thrombin
prior to dispensing was 4.degree. C..+-.2.degree. C. Cylindrical
molds made of 10 or 3 mL polypropylene syringes (Becton Dickinson)
with the luer-lock end removed were used. The plungers were
withdrawn to the 6 mL and 2 mL mark respectively. For dressings
utilizing a backing, the support material was cut and placed into
each mold and pushed down until it was adjacent to the plunger.
Once prepared the molds were placed upright and surrounded by dry
ice, leaving the opening exposed at the top. 1 ml of fibrinogen and
0.15 mL of thrombin (with or without backing material dispersed
within) were dispensed into the 10 mL molds and 1 ml of fibrinogen
and 0.15 mL of thrombin (with or without support material dispersed
within) were dispensed into the 3 mL molds, which were allowed to
freeze for 5 minutes. The molds were then placed into the
-80.degree. C. freezer for at least two hours before being placed
into the freeze dryer and lyophylized as described above.
[0108] Upon removal from the lyophylizer, both groups were
performance tested in a modified EVPA assay. Briefly, a plastic
foam form was slipped over the artery. This covering had a hole in
it that corresponded to the hole in the artery and the surrounding
tissue. Warm saline was added to the surface of the dressing and
the mold was immediately passed down thru the hole in the foam to
the artery surface. The plunger was then depressed and held by hand
for 3 minutes, after which the mold was withdrawn as the plunger
was depressed further. At this point the artery was pressurized and
the assay continued as before.
Results
TABLE-US-00007 [0109] Mold EVPA Result Maximum Support Material
Size (@250 mmHg) Pressure None 10 ml Pass >250 mmHg Dexon Mesh
Backing 10 ml Pass '' '' 3 ml Pass '' Shredded Dexon Mesh
(Dispersed) 10 ml Pass '' '' 3 ml Fail 150 mmHg
Conclusions:
[0110] Dressings that included no backing or a DEXON.TM. mesh
backing performed well, with all passing the EVPA test at 250 mmHg
When the support material was dispersed throughout the composition,
the dressings also performed well, with the large size (10 mL mold)
dressings holding the full 250 mmHg of pressure, while the smaller
held up to 150 mmHg of pressure. This indicates that the use of a
support material may be optional, and it's location may be on the
`back` of the dressing, or dispersed thou the composition, as
desired.
Example 5
[0111] Dressings made with a support material on the "back" (i.e.
the non-wound-facing side) of the dressing were manufactured by
first cutting the mesh support material and placing it into each
PETG 10.times.10 cm mold. Twenty-five microliters of 2% sucrose was
pipetted on top of each of the four corners of the backing
material. Once completed the molds were placed in a -80.degree. C.
freezer for at least 60 minutes.
[0112] For dressings made with a support material on the "front"
(i.e. the wound-facing side) of the dressing, these were
manufactured without any support material in the mold. The support
mesh was placed atop the dressing immediately after dispensing of
the fibrinogen and thrombin into the mold (see below), and lightly
pressing it into the surface prior to its freezing. In all other
ways the manufacture of the dressings was similar as described
below.
[0113] For all dressings, ERL fibrinogen lot 3114 was formulated in
CFB. The final pH of the fibrinogen was 7.4.+-.0.1. The fibrinogen
concentration was adjusted to 37.5 mg/ml. Once prepared the
fibrinogen was placed on ice until use. Thrombin was formulated in
CTB. The final pH of the thrombin was 7.4.+-.0.1. The thrombin was
adjusted to deliver 0.1 units/mg of Fibrinogen or 25 Units/ml
thrombin. Once prepared the thrombin was placed on ice until use.
The thrombin was adjusted to deliver 0.1 units/mg of Fibrinogen or
25 Units/ml thrombin. Once prepared the thrombin was placed on ice
until use. The temperature of the fibrinogen and thrombin prior to
dispensing was 4.degree. C..+-.2.degree. C. The mold was removed
from the -80.degree. C. freezer and placed on an aluminum plate
that was placed on top of dry ice. The aluminum plate had a 0.25
inch hole drilled in the center and a fitting attached so that a
piece of tubing could be attached to a vacuum source. The mold was
centered over the hole in the aluminum plate and vacuum was turned
on. The vacuum served two purposes it prevented the mold from
moving and it held it flat against the aluminum plate. Thirty-five
milliliters of fibrinogen and 5.25 milliliters of Thrombin were
placed in 50 ml test tube, inverted three times and poured into the
mold. Once the molds were filled and the support material applied
as described above, they were returned to the -80.degree. C.
freezer for at least two hours before being placed into the freeze
dryer. Dressings were then lyophylized as described previously.
[0114] Both groups were performance tested in the EVPA assay. In
addition, the group which had a backing was also tested in the
Adherence and Weight Held assays.
Results:
TABLE-US-00008 [0115] EVPA Adher- Weight Weight # ence Adher- Held
Held Support Material Pass/ Test ence (mean) Std (Mesh) Orientation
Total Score Std Dev (g) Dev Back (away from injury 6/6 3.5 0.5 136
49 site) Front (immediately 6/6 3.8 0.4 163 32 adjacent to injury
site)
Conclusions:
[0116] Dressings formulated with backing material in either
orientation well, with all dressings passing the EVPA test, and
high values for adherence and weight held. This indicates that the
location of a support material may be on the `back` of the
dressing, or the `front`, of the composition as desired.
EVPA Performance Testing
[0117] Equipment and Supplies: [0118] In-line high pressure
transducer (Ashcroft Duralife.TM. or equivalent) [0119] Peristaltic
pump (Pharmacia Biotech.TM., Model P-1 or equivalent) [0120]
Voltmeter (Craftsman.TM. Professional Model 82324 or equivalent)
[0121] Computer equipped with software for recording pressure or
voltage information [0122] Tygon.TM. tubing (assorted sizes) with
attachments [0123] Water bath (Baxter Durabath.TM. or equivalent),
preset to 37.degree. C. [0124] Incubation chamber (VWR.TM., Model
1400G or equivalent), preset to 37.degree. C. [0125] Thermometer to
monitor temperatures of both water bath and oven [0126] Assorted
forceps, hemostats, and scissors [0127] 10 cc. and 20 cc. syringes
with an approximately 0.6 cm hole drilled in center and smaller
hole drilled through both syringe and plunger. This hole, drilled
into the end of the syringe, will be used to keep the plunger drawn
back and stationary. [0128] O-rings (size 10 and 13) [0129] Plastic
Shields to fit the 10 cc and 20 cc syringes (approximately 3.5 cm
in length) [0130] P-1000 Pipetman.TM. with tips [0131]
Sphygmomanometer with neonatal size cuff and bladder [0132]
Programmable Logic Controller (PLC) to control the pumps to
maintain the desired pressure profile (Optional. Manual control may
be used if desired.)
[0133] 1. Materials and Chemicals [0134] Porcine descending aortas
(Pel-Freez Biologicals.TM., Catalog # 59402-2 or equivalent) [0135]
Cyanoacrylate glue (Vetbond.TM., 3M or equivalent) [0136] 18-gauge
needle(s) [0137] 0.9% Saline, maintained at 37.degree. C. [0138]
Red food coloring [0139] Vascular Punch(es), 2.8 mm or other [0140]
Plastic Wrap
[0141] 2. Artery Cleaning and Storage [0142] 1. Store arteries at
-20.degree. C. until used. [0143] 2. Thaw arteries at 37.degree. C.
in H.sub.2O bath. [0144] 3. Clean fat and connective tissue from
exterior surface of artery. [0145] 4. Cut the arteries into
.about.5 cm segments. [0146] 5. The arteries may be refrozen to
-20.degree. C. and stored until use.
[0147] 3. Artery Preparation for Assay [0148] 1. Turn the artery
inside-out so that the smooth, interior wall is facing outwards.
[0149] 2. Stretch a size 13 O-ring over a 20 cc syringe or a size
10 O-ring over a 10 cc syringe with an approximately 0.6 cm (0.25
in) hole drilled into one side. [0150] 3. Pull the artery onto the
syringe, taking care not to tear the artery or have a too loose
fit. The artery should fit snugly to the syringe. Slide another
O-ring of the same size onto the bottom of the syringe [0151] 4.
Carefully pull both O-rings over the ends of the artery. The
distance between the O-rings should be at least 3.5 cm [0152] 5.
Using the blade of some surgical scissors, gently scrape the
surface of the artery in order to roughen the surface of the
artery. [0153] 6. Use a 18-gauge needle to poke a hole through the
artery over the site of the hole in the syringe barrel (see note
above) [0154] 7. The tip of the biopsy punch is inserted through
the hole in the artery. Depress the punch's plunger to make an open
hole in the artery. Repeat a couple of times to ensure that the
hole is open and free of connective tissue. [0155] 8. Patch holes
left by collateral arteries. Generally this is done by cutting a
patch from a latex glove and gluing it over the hole with
cyanoacrylate glue. Allow the glue to cure for at least 10 minutes.
[0156] 9. Place the artery in the warmed, moistened container and
place in the incubation chamber. Allow the arteries to warm for at
least 30 minutes.
[0157] 4. Solution and Equipment Preparation [0158] 1. Check to see
that the water bath and incubation chamber are maintained at
29-33.degree. C. [0159] 2. Make sure that there is sufficient 0.9%
saline in the pump's reservoir for completion of the day's assays.
Add more if needed. [0160] 3. Place 0.9% saline and 0.9% saline
with a few drops of red food coloring added into containers in a
water bath so that the solutions will be warmed prior to performing
the assay. [0161] 4. Prepare the container for warming the arteries
in the incubation chamber by lining with KimWipes.TM. and adding a
small amount of water to keep the arteries moist. [0162] 5. Check
the tubing for air bubbles. If bubbles exist, turn on the pump and
allow the 0.9% saline to flow until all bubbles are removed.
[0163] 5. Application of the Dressing [0164] 1. Open the
haemostatic dressing pouch and remove haemostatic dressing [0165]
2. Place the haemostatic dressing, mesh backing side UP, over the
hole in the artery [0166] 3. Slowly wet the haemostatic dressing
with an amount of saline appropriate for the article being
tested
[0167] NOTE: A standard (13-15 mg/cm.sup.2 of fibrinogen)
2.4.times.2.4 cm haemostatic dressing should be wet with 800 .mu.l
of saline or other blood substitute. The amount of saline used can
be adjusted depending on the requirements of the particular
experiment being performed; however, any changes should be noted on
the data collection forms.
[0168] NOTE: Wet the haemostatic dressing drop wise with 0.9%
saline warmed to 29-33.degree. C. or other blood substitute, taking
care to keep the saline from running off the edges. Any obvious
differences in wetting characteristics from the positive control
should be noted on data collection forms. [0169] 4. Place the
shield gently onto the haemostatic dressing, taking care that it
lies flat between the O-rings. Press lightly to secure in place
[0170] 5. Wrap the artery and haemostatic dressing with plastic
wrap [0171] 6. Wrap with blood pressure cuff, taking care that the
bladder is adjacent to the haemostatic dressing. [0172] 7. Pump up
the bladder to 100-120 mmHg, and monitor the pressure and pump
again if it falls below 100 mmHg. Maintain pressure for 5
minutes.
[0173] NOTE: Time and pressure can be altered according to the
requirements of the experiment; changes from the standard
conditions should be noted on the data collection forms. [0174] 8.
After polymerization, carefully unwrap the artery and note the
condition of the haemostatic dressing. Any variation from the
positive control should be noted on the data collection form.
[0175] EXCLUSION CRITERION: The mesh backing must remain over the
hole in the artery. If it has shifted during the polymerization and
does not completely cover the hole the haemostatic dressing must be
excluded.
Testing Procedure
[0176] 6. Diagram of Testing Equipment Set-Up
[0177] The set-up of the testing equipment is shown in FIG. 2. Some
additional, unshown components may be utilized to read out
(pressure gauge) or control the pressure within the system.
[0178] 7. Equipment and Artery Assembly
[0179] Fill the artery and syringe with red 0.9% saline warmed to
37.degree. C., taking care to minimize the amount of air bubbles
within the syringe & artery. Filling the artery with the
opening uppermost can assist with this. Attach the artery and
syringe to the testing apparatus, making sure that there are as few
air bubbles in the tubing as possible. The peristaltic pump should
be calibrated so that it delivers approximately 3 ml/min. If
available, the PLC should be operated according to a pre-determined
range of pressures and hold times as appropriate for the article
being tested. If under manual control, the pressure/time profile to
be followed is attained by manually turning the pump on and off
while referencing the system pressure as read out by one or more
pressure-reading components of the system. Following the conclusion
of testing, the haemostatic dressing is subjectively assessed with
regard to adhesion to the artery and formation of a plug in the
artery hole. Any variations from the positive control should be
noted on the data collection form.
Success Criteria
[0180] Haemostatic dressings that are able to withstand pressures
for 3 minutes are considered to have passed the assay. When a
haemostatic dressing has successfully passed the assay the data
collection should be stopped immediately so that the natural
decrease in pressure that occurs in the artery once the test is
ended isn't included on the graphs. Should the operator fail to
stop data collection, these points can be deleted from the data
file to avoid confusing the natural pressure decay that occurs
post-test with an actual dressing failure. The entire testing
period from application of the haemostatic dressing to completion
must fall within pre-established criteria. The maximum pressure
reached should be recorded on the data collection form.
[0181] NOTE: Typical challenge is 250 mmHg for three minutes in one
step, but that may be altered based on the article being tested.
Changes from the standard procedure should be noted on the data
collection forms.
Failure Criteria
[0182] Haemostatic dressings that start leaking saline at any point
during testing are considered to have failed the assay.
[0183] NOTE: Build failures that are caused by artery swelling can
be ignored and the test continued or re-started (as long as the
total testing time doesn't fall beyond the established limit)
[0184] When leakage does occur, the pressure should be allowed to
fall .about.20 mmHg before data collection is stopped so that the
failure is easily observed on the graphs. The pressures at which
leakage occurred should be recorded on the data collection form.
Should the data collection stop in the middle of the experiment due
to equipment failure the data can be collected by hand at 5 second
intervals until the end of the test or haemostatic dressing
failure, whichever happens first. The data points should be
recorded on the back of the data collection form, clearly labeled,
and entered by hand into the data tables.
Exclusion Criteria
[0185] If the total testing period exceeds the maximum allowed for
that procedure, regardless of cause, results must be excluded. If
there are leaks from collaterals that can't be fixed either by
patching or finger pressure the results must be excluded. If the
test fails because of leaks at the O-rings, the results must be
excluded. If the mesh backing does not completely cover the hole in
the artery, the results must be excluded.
Adherence Performance Testing
[0186] 8. Equipment and Supplies
[0187] Hemostat(s), Porcine artery and haemostatic dressing
(usually after completion of the EVPA Assay although it does not
need to be performed to do the Adherence Assay).
[0188] 9. Preparation of the Artery+Dressing
[0189] After application of the dressing without completion of the
EVPA Assay, the dressing is ready for the Adherence Assay and
Weight Limit Test (if applicable). After application of the
dressing and subsequent EVPA Analysis, the artery and syringe
system is then disconnected slowly from the pump so that solution
does not spray everywhere. The warmed, red saline solution from the
EVPA Assay remains in the syringe until the Adherence Assay and
Weight Limit Test (if applicable) is completed.
Performance of the Adherence Assay
[0190] 1. After preparation of the artery and dressing (with or
without EVPA analysis), gently lift the corner of the mesh and
attach a hemostat of known mass to the corner.
[0191] NOTE: If the FD developed a channel leak during the
performance of the EVPA Assay, test the adherence on the opposite
of the haemostatic dressing to obtain a more accurate assessment of
the overall adherence. [0192] 2. Gently let go of the hemostat,
taking care not to allow the hemostat to drop or twist. Turn the
syringe so that the hemostat is near the top and allow the hemostat
to peel back the dressing as far as the dressing will permit. This
usually occurs within 10 seconds. After the hemostat has stopped
peeling back the dressing, rate the adherence of the bandage
according to the following scale:
TABLE-US-00009 [0192] Dressing Performance Score Amount of
Adherence 4 .sup. 90+% 3 75-90% 2 50-75% 1 .sup. ~50% 0.5 Only the
plug holds the hemostat 0 No adherence
Exclusion Criteria
[0193] The mesh backing must remain over the hole in the artery. If
it has shifted during the polymerization and does not completely
cover the hole the haemostatic dressing must be excluded.
Success Criteria
[0194] Dressings that are given an adherence score of 3 are
considered to have passed the assay.
Failure Criteria
[0195] If a dressing does not adhere to the artery after
application and/or prior to performing the EVPA assay, it is given
a score of 0 and fails the adherence test. If a dressing receives a
score .ltoreq.2, the dressing is considered to have failed the
Adherence Assay.
Weight Held Performance Assay
[0196] After the initial scoring of the "Adherence Test", weights
may then be added to the hemostat in an incremental manner until
the mesh backing is pulled entirely off of the artery. The maximum
weight that the dressing holds is then recorded as a measure of the
amount of weight the dressing could hold attached to the
artery.
Moisture Assay
[0197] Moisture determinations were carried out using a Brinkman
Metrohm Moisture Analyzer System. The system contains the following
individual components, 774 Oven Sample Processor, 774SC Controller,
836 Titrando, 5 ml and 50 ml 800 Dosino Units and a 801 Stirrer.
The system was connected to a computer using the Brinkman Tiamo
software for data collection, analysis and storage. The moisture
system is set-up and run according to the manufactures
recommendations and specifications to measure the moisture content
of lyophilized samples using the Karl Fischer method.
[0198] All components were turned on and allowed to reach operating
temperature prior to use. Lactose and water were run as standards
and to calibrate the instrument. Once the machine was successfully
calibrated, samples were prepared as follows. Dressing pieces
weighing at least 30 mg were placed into vials and capped. The
vials were placed in the 774 Oven Sample Processor in numerical
order, and one empty capped vial is placed in the conditioning
space. The machine was then run to determine the moisture content
(residual moisture) in the controls and samples.
* * * * *
References