U.S. patent application number 16/256857 was filed with the patent office on 2019-12-26 for methods and dressings for sealing internal injuries.
The applicant listed for this patent is RESOURCE TRANSITION CONSULTANTS, LLC. Invention is credited to Dawson BEALL, Jerry KANELLOS, Martin MACPHEE, Belinda WILMER.
Application Number | 20190388579 16/256857 |
Document ID | / |
Family ID | 40341589 |
Filed Date | 2019-12-26 |
![](/patent/app/20190388579/US20190388579A1-20191226-D00001.png)
United States Patent
Application |
20190388579 |
Kind Code |
A1 |
MACPHEE; Martin ; et
al. |
December 26, 2019 |
METHODS AND DRESSINGS FOR SEALING INTERNAL INJURIES
Abstract
Disclosed are solid and frozen haemostatic materials and
dressings consisting essentially of a fibrinogen component and a
fibrinogen activator. Also disclosed are methods of treating
internal wounded tissue in a mammal by applying one or more of
these haemostatic materials and dressings.
Inventors: |
MACPHEE; Martin;
(Darnestown, MD) ; KANELLOS; Jerry; (Victoria,
AU) ; WILMER; Belinda; (Martinsburg, WV) ;
BEALL; Dawson; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RESOURCE TRANSITION CONSULTANTS, LLC |
Edmonds |
WA |
US |
|
|
Family ID: |
40341589 |
Appl. No.: |
16/256857 |
Filed: |
January 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15269541 |
Sep 19, 2016 |
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16256857 |
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14990987 |
Jan 8, 2016 |
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15269541 |
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14829630 |
Aug 18, 2015 |
9259503 |
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14990987 |
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13080086 |
Apr 5, 2011 |
9131929 |
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14829630 |
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12222277 |
Aug 6, 2008 |
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13080086 |
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60935311 |
Aug 6, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 15/32 20130101;
A61L 15/64 20130101; A61P 33/00 20180101; A61L 26/009 20130101;
A61K 38/363 20130101; A61P 31/12 20180101; A61B 17/00491 20130101;
A61L 2300/254 20130101; A61P 31/00 20180101; A61P 31/10 20180101;
A61P 17/02 20180101; A61L 2300/252 20130101; C12Y 304/21005
20130101; A61B 2017/0065 20130101; A61B 17/0057 20130101; A61P 3/02
20180101; A61P 7/02 20180101; A61P 29/00 20180101; A61L 26/0042
20130101; A61K 38/4833 20130101; A61L 15/28 20130101; A61P 9/00
20180101; A61L 2400/04 20130101; A61P 35/00 20180101; A61F
2013/00472 20130101; A61L 2300/23 20130101; A61L 15/44
20130101 |
International
Class: |
A61L 15/64 20060101
A61L015/64; A61L 15/28 20060101 A61L015/28; A61B 17/00 20060101
A61B017/00; A61L 15/32 20060101 A61L015/32; A61K 38/48 20060101
A61K038/48; A61K 38/36 20060101 A61K038/36; A61L 15/44 20060101
A61L015/44 |
Claims
1. A method for treating wounded internal tissue in a mammal
comprising applying to wounded internal tissue at least one
haemostatic material consisting essentially of a fibrinogen
component and a fibrinogen activator for a time sufficient to join
or approximate said wounded tissue and/or to reduce the flow of
fluid from said wounded tissue, wherein said haemostatic material
has a plurality of particles, wherein said particles each have the
same composition, and wherein the moisture content of said
hemostatic material is from 6% to 44%.
2. A method for treating wounded internal tissue in a mammal
comprising applying to wounded internal tissue at least one
haemostatic material consisting essentially of a fibrinogen
component and a fibrinogen activator for a time sufficient to join
or approximate said wounded tissue and/or to reduce the flow of
fluid from said wounded tissue, wherein said haemostatic material
has a plurality of particles, wherein said particles each have the
same composition, and wherein the moisture content of said
hemostatic material is from 1% to 6%.
3. A method for treating wounded internal tissue in a mammal
comprising applying to wounded internal tissue at least one
haemostatic material consisting essentially of a fibrinogen
component and a fibrinogen activator for a time sufficient to join
or approximate said wounded tissue and/or to reduce the flow of
fluid from said wounded tissue, wherein said haemostatic material
is cast or formed as a single piece.
4. The method of claim 1, wherein said haemostatic material
includes at least one support layer.
5. The method of claim 4, wherein said support layer comprises a
backing material.
6. The method of claim 4, wherein said support layer comprises an
internal support material.
7. The method of claim 4, wherein said support layer comprises a
resorbable material.
8. The method of claim 4, wherein said support layer comprises a
non-resorbable material.
9. The method of claim 8, wherein said non-resorbable material is
selected from the group consisting of silicone polymers, paper,
gauze, plastics, non-resorbable suture materials, and latexes.
10. The method of claim 4, further comprising at least one
physiologically acceptable adhesive between said haemostatic
material and said backing layer.
11. The method of claim 7, wherein said resorbable material is
selected from the group consisting of proteinaceous materials,
carbohydrate substances and resorbable suture materials.
12. The method of claim 11, wherein said proteinaceous material is
at least one substance selected from the group consisting of
keratin, silk, fibrin, collagen, and gelatin.
13. The method of claim 11, 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.
14. The method of claim 1, wherein said haemostatic material also
contains a fibrin crosslinker and/or a source of calcium ions.
15. The method of claim 1, wherein said haemostatic material 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.
16. The method of claim 15, 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.
17. The method of claim 15, 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.
18. The method of claim 15, 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.
19. The method of claim 15, 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.
20-44. (canceled)
Description
[0001] This application is a continuation-in-part application, and
therefore claims benefit of the filing date, of prior U.S.
Provisional Patent Application No. 60/935,311, the disclosure of
which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to dressings for treating
internal injured tissue in a mammalian patient, such as a human and
methods of using the same.
BACKGROUND OF THE INVENTION
[0003] There are a large number of medical procedures that result
in injuries to blood vessels. Similarly, there are numerous
examples of bleeding caused by traumatic injuries, hematological
disorders, and from unknown causes. When the site of bleeding is
not readily accessible, such as an injured vessel located deep
within the flesh, or inside a body cavity, a simple and effective
method of hemorrhage control that can access the site within the
body and seal the injured vessel is needed. Similarly, tissue may
be divided by either traumatic injury or surgical procedure, and
require scaling to approximate the edges of the injury in order to
restore function. Current sealing products and devices have one or
mote deficiencies, usually due to their inadequate performance, or
their reliance upon non-natural components that interfere with
normal healing.
[0004] The need for improved technologies to address these injuries
is significant. Pot example, in the case of blood vessels that have
been deliberately punctured as part of a diagnostic and/or
therapeutic procedure (such as cardiac catheterization, balloon
angioplasty, vascular stenting and the like), over seven million
such procedures are currently performed every year, but with a 9%
overall complication rate and a 1-3% major complication rate (See
Millennium Research Group: Global Markets for Vascular Closure
Devices 2006). These complications can lead to significant
morbidity, increased expense, a requirement for additional
procedures and/or devices, extended time in the medical facility
and conversion of outpatients to inpatients. Commercially available
products now available only reduce the major complication rate by
one half of one percent (See Atora et al: Am Heart J. 2007 April;
153(4):606-11) to 2.4%. Nevertheless, despite this poor
performance, even these devices are currently used since the costs
and consequences of procedure-induced complications is so high (See
Resnic et al. Am J Cardiol. 2007 Mat 15; 99(6):766-70).
[0005] Not only are there the above described complications
associated with therapy itself, closure of the access hole(s)
created in the blood vessel is a significant source of additional
complications, including uncontrolled hemorrhage, pseudoaneurysm,
hematoma, arteriovenous fistula, arterial thrombosis, infection,
and retained devices (Set Meyerson et al. Angiographic Access Site
Complications in the Era of Artificial Closure Devices Vase
Endovasc Surg, 2002; 36 (2) 137-44). These additional complications
may lead to prolonged closure procedures, hospitalization, the
requirement for surgical repair, and even tissue loss or death.
[0006] Currently, the primary means of closing the access hole in
the vessel has been to allow a natural blood clot to form at the
puncture site. This has generally been accomplished by manual
compression, but various products have recently been developed in
an attempt to reduce the time required to achieve vascular closure.
Such devices automate the application of pressure over the injury
site, suture the hole in the vessel, clip the hole shut, or apply
some sort of patch or pad that allegedly increases the formation of
a natural dot at the site. These devices are convenient and gaining
in popularity, but their overall safety appears over estimated.
Indeed, far from being risk free, these devices may be associated
with unique levels of hemorrhagic and cardiac risks including
myocardial infarction, stroke and death (See Rao, S. Implications
of bleeding and blood transfusion in percutaneous coronary
intervention. Rev Cardiovasc Med. 2007, 8 Suppl 3:S18-26.).
[0007] Significant risks have been repotted to be associated with
all classes of vascular closure devices. Most seriously, the
severity and the difficulty in treating complications are generally
greater when vascular closure devices are used (See Nehler et al.
Iatrogenic vascular injuries from percutaneous vascular suturing
devices, J. Vase Surg 2001 May; 33(5):943-7; Castelli et al:
Incidence of vascular injuries after use of the Angio-Seal closure
device following endovascular procedures in a single center. World
J Surg. 2006 March; 30(3):280-4.). The use of such devices is even
associated with higher risks among patients having complications of
pseudoaneurysms, failure to successfully treat such
pseudoaneurysms, blood loss, transfusions, extensive operations to
correct the problems and arterial infections (See Sprouse et al.
The management of peripheral vascular complications associated with
the use of percutaneous suture-mediated closure devices. J Vase
Surg. 2001 April; 33(4):688-693.). Moreover, some of these
complications can be deadly, particularly in patients with
diabetes, obesity and previously implanted devices (all conditions
commonly found in patients in whom such closure devices are
frequently used) (See Hollis and Retiring. Femoral endarteritis
associated with percutaneous suture closure: new technology,
challenging complications, J Vase Surg. 2003 July; 38(1):83-7.),
Accordingly, there, remains a great need to develop a vascular
closure system that avoids the problems associated with use of
known vascular closure devices.
[0008] Another medical situation involving treatment of injured
internal tissue is the repair of herniations. There are numerous
types and locations of hernia, and the surgical repair techniques
vary widely depending thereon. Both open and endoscopic procedures
are currently in use, and may involve the use of sutures alone or
sutures in combination with various kinds of meshes or supports for
the injured tissue. Major complications for most hernia repair
procedures include pain and the requirement to re-do the repair
(Set American College of Surgeons. When you need an operation . . .
. About Hernia Repair, available at:
http://www.facs.org/public_info/operation/herntep.pdf).
[0009] Similarly, there is also a need to improve the therapeutic
options for treatment of simple bleeding conditions such as
epistaxis, which requires professional medical treatment in 1 of 7
people in their lifetime (See Evans: Epistaxis, emedicine (2007)
available at www.emedicine.com/EMERG/topic806.htm). In fact,
epistaxis is frequently cited as the most common ENT emergency (See
Hussain et al: Evaluation of aerology and efficacy of management
protocols of epistaxis. Ayub Med Coll Abottabad, 2006 Oct.-Dec.;
18(4):63-6) The difficulty in treating these cases is evidenced by
the fact that 1.6 out of every 10,000 patients are hospitalized for
epistaxis that is refractory to normal treatment (See Viehweg et
al: Epistaxis: diagnosis and treatment, J. Oral Maxillofac Surg
2006 March; 64(3):5 11-8). Current treatment options include
packing, chemical cauterization, electrocautery, surgical ligation
and embolization (Ser. Ortiz & Bhattachatyya: Management
pitfalls in the use of embolization for the treatment of severe
epistaxis, Ear Nose Throat J. 2002 Mat; 82(3):178-83.) Frequently,
multiple treatments with different technologies are requited to
effectively treat this often life-threatening condition (See
Siniluoto et al: Embolization for the treatment of posterior
epistaxis. An analysis of 31 cases. Arch Otolaryngol Head Neck
Surg. 1993 August; 119(8):837-41; Gifford & Orlandi: Epistaxis.
Otoloaryngol Clin North Am. 2008 June; 41(3):525-36, vii).
[0010] 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: [0011] Microporous polysaccharide particles
(TtaumaDEX.RTM., Medafor Inc., Minneapolis, Minn.); [0012] Zeolite
(QuikClotC.RTM., Z-Medica Corp, Wallington, Conn.); [0013]
Acetylated poly-N-acetyl glucosamine (Rapid Deployment Hemostat.TM.
(RDH), Marine Polymer Technologies, Danvers, Mass.); [0014]
Chitosan (HemCon.RTM. bandage, HcmCon Medical Technologies inc.,
Portland Oreg.); [0015] liquid Fibrin Sealants (Tisscel V H,
Baxter, Deerfield, Ill.) [0016] Human fibrinogen and thrombin on
equine collagen (TachoComb-S, Hafslund Nycomed Pharma, Linz,
Austria); [0017] Microdispersed oxidized cellulose
(m.circle-solid.doc.TM., Alltracel Group, Dublin, Ireland); [0018]
Propyl gallate (Hemostatin.TM., Analytical Control Systems Inc.,
Fishers, Ind.); [0019] Epsilon aminocaptoic acid and thrombin
(Hemarrest.TM. patch, Clarion Pharmaceuticals, Inc); [0020]
Purified bovine corium collagen (Avitene.RTM. sheets (non-woven web
or Avitene Microfibrillar Collagen Hemostat (MCH), Davol, Inc.,
Cranston, R.I.); [0021] Controlled oxidation of regenerated
cellulose (Surgicel.RTM., Ethicon Inc., Somerville, N.J.); [0022]
Aluminum sulfate with an ethyl cellulose coating (Sorbastace
Microcaps, Hemostace, LLC, New Orleans, La.); [0023] Microporous
hydrogel-forming polyacrylamide (BioHemostat, Hemodyne, Inc.,
Richmond Va.); and [0024] Recombinant activated factor VII
(NovoSevcn.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, and with limited success
in the sealing of therapeutic vascular injuries.
[0025] Liquid fibrin sealants, such as Tisseel V H, 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. Vase. 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 dierc is
sufficient Factor XIII/XIIIa, or other transaminase, endogenously
present to induce fibrin formation). As liquids, however, these
fibrin sealants have not proved useful outside certain specific
procedures.
[0026] 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).
[0027] A dry fibrinogen/thrombin dressing for creating wounded
tissue is also 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 (sec 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."
[0028] 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 fillet 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 (in) 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.
[0029] A number of different techniques, including the use of
liquid fibrin sealant, have been proposed for scaling the punctures
in blood vessels made to secure vascular access. For example, U.S.
Pat. No. 7,357,794 discloses devices, systems and methods for acute
or chronic delivery of substances or apparatus to extravascular
treatment sites. U.S. Pat. No. 7,335,220 discloses apparatus and
methods for scaling a vascular puncture using an expanding
lyophilized hydrogel plug. U.S. Pat. No. 7,300,663 discloses
adhesion and scaling of tissue with compositions containing
polyfunctional crosslinking agents and protein polymers. U.S. Pat.
No. 7,399,483 discloses a carrier with solid fibrinogen and solid
thrombin. U.S. Pat. No. 7,335,220 discloses apparatus and methods
for scaling vascular punctures. U.S. Pat. No. 7,115,588 discloses
methods for treating a breach or puncture in a blood vessel. U.S.
Pat. No. 7,008,442 discloses vascular sealant delivery devices
using liquid formulations. U.S. Pat. No. 6,890,342 discloses to
methods and apparatus for closing vascular puncture using a
guidewire and/or other surgical implement extending from the wound
on which a haemostatic material is moved into contact with an area
of the blood vessel surrounding the wound U.S. Pat. No. 6,818,008
discloses percutaneous puncture scaling method using flowable
sealants. U.S. Pat. No. 6,699,262 discloses a percutaneous tissue
track closure assembly and method using flowable materials. U.S.
Pat. No. 6,613,070 discloses sealing vascular penetrations with
haemostatic gels. U.S. Pat. No. 6,500,152 discloses a device for
introducing a two-component liquid fibrin adhesive into a puncture
channel. U.S. Pat. No. 6,325,789 also discloses a device for
scaling puncture wounds using liquid or paste fibrin sealant. U.S.
Pat. No. 5,814,066 discloses methods of reducing femoral arterial
bleeding using percutaneous application of liquid fibrin sealant.
U.S. Pat. Nos. 5,725,551, 5,486,195 and 5,443,481 each disclose the
use of two component liquid fibrin sealant for artery closure U.S.
Pat. No. 5,649,959 discloses an assembly for sealing a puncture in
a vessel which maintains the fibrinogen and thrombin separately. To
date, however, all of these, remain little-used in therapy, most
likely due to the difficult and time consuming preparation
requirements for two-component liquid fibrin sealant
compositions.
[0030] Similarly, two component liquid fibrin sealants have been
used to attach surgical meshes in the treatment of abdominal
hernias. The surgical results have been excellent, typically as
good or better than the efficacy of suture and staple fixation,
with reduced complications and post-operative pain. (See Schwab et
al., Hernia. 2006 June; 10(3):272-7)
[0031] Liquid fibrin sealant has also be used to treat epistaxis,
endoscopic sinus surgery and endonasal surgery ((See Vaiman et al.
Fibrin glue treatment for epsitaxis, Rhinology. 2002 June;
40(2):99-91; Vaiman et al. Use of fibrin glue as a haemostatic in
edopscopic sinus surgery. Ann Otol Rhinol Laryngol, 2005 Mat;
114(3): 237-41; Vaiman et al. Fibrin sealant: alternative to nasal
packing in endonasal operations. A prospective randomized study.
Isr Med Assoc J. 2005 September; 7(9); 571-4.). All these reports
indicate that liquid fibrin sealant may be used with some success
at controlling hemorrhage from various locations just inside the
nose all the way into the sinuses. However, the time and efforts
associated with preparing such sealants make them less than ideal
for daily clinical use
[0032] Accordingly, there remains a need in the art for solid
dressings that can be used to achieve hemostasis and sealing of
internal wounded tissue, particularly highly vascularized tissue,
and single blood vessels. Additionally, treatment of tissues that
have been divided (e.g. due to accident, pathology or surgical
intervention) and require re-approximation to promote healing would
also benefit from a solid dressing capable of adequate tissue
sealing.
[0033] The assessment of such dressings requires new techniques
that go beyond those previously disclosed for testing haemostatic
dressings. The ability of dressings to seal an injured blood vessel
has been 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 teal 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 tighty 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.
[0034] In order to minimize the amount of time and the number of
animal studies required to develop dressings intended to treat
accessible traumatic injuries, an improved ex vivo testing
procedure has been 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). A new test has also been developed 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. These newer tests are described in U.S.
patent application Ser. No. 11/882,874, the disclosure of which is
herein incorporated by reference in its entirety.
[0035] The newer tests described in U.S. patent application Ser.
No. 11/882,874 were designed to simulate trauma-derived, accessible
wounds with high pressure and flow characteristics. Therefore, for
the evaluation of methods and compositions for treating; wounded
internal tissue, it was preferable to develop additional assays to
mote accurately simulate the peripheral vasculature and the effects
of surrounding tissue.
SUMMARY OF THE INVENTION
[0036] It is therefore an object of the present invention to
provide solid dressings that can treat wounded internal mammalian
tissue. It is further an object of the present invention to provide
a method of treating wounded internal 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.
[0037] In accordance with these and other objects, a first
embodiment of the present invention is directed to a method for
treating wounded internal tissue in a mammal comprising applying to
wounded internal tissue at least one haemostatic material
consisting essentially of a fibrinogen component and a fibrinogen
activator for a time sufficient to reduce the flow of fluid from
the wounded tissue, wherein the haemostatic material is
substantially homogeneous.
[0038] Another embodiment, is directed to a method for treating
wounded internal tissue in a mammal comprising applying to wounded
internal tissue at least one haemostatic material consisting
essentially of a fibrinogen component and a fibrinogen activator
for a time sufficient to reduce the flow of fluid from the wounded
tissue, wherein the haemostatic material is cast or formed from a
single aqueous solution containing the fibrinogen component and the
fibrinogen activator.
[0039] Another embodiment is directed to a method for treating
wounded internal tissue m a mammal comprising applying to wounded
internal tissue at least one haemostatic material consisting
essentially of a fibrinogen component and a fibrinogen activator
for a time sufficient to reduce the flow of fluid from the wounded
tissue, wherein the haemostatic material is cast or formed as a
single piece.
[0040] Still other embodiments of the invention are directed to the
various solid and frozen haemostatic materials useful in the
inventive methods.
[0041] 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
[0042] FIG. 1 is a diagram of the set-up for the ex vivo porcine
carotid arteriotomy assay described herein.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0043] 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.
[0044] 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 dearly and unambiguously dictates
otherwise.
[0045] "Patient" as used herein refers to human or animal
individuals in need of medical care and/or treatment.
[0046] "Wound" or "wounded tissue" as used herein refers to any
damage to any internal 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 any mammalian internal
tissue, such as an organ or blood vessel. A wound may be in a soft
internal tissue, such as an organ, or in hard internal tissue, such
as bone. The "damage" may have been caused by any agent or source,
including traumatic injury, infection or surgical intervention.
Thus, the "damage" being treated according to the methods of the
present invention may be the result of either an accident or an
intentional act.
[0047] "Resorbable material" as used herein refers to a substance
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.
[0048] "Stability" as used herein refers to the retention of those
characteristics of a substance that determine activity and/or
function.
[0049] "Suitable" as used herein is intended to mean that a
substance (or mixture of substances) docs not adversely affect the
stability of the dressings or any component thereof.
[0050] "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 material of the dressings.
[0051] "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.
[0052] "Filler" as used herein refers to a compound or mixture of
compounds that provide bulk and/or porosity to the haemostatic
material.
[0053] "Release agent" as used herein refers to a compound or
mixture of compounds that facilitates removal of a dressing from a
manufacturing mold.
[0054] "Foaming agent" as used herein refers to a compound or
mixture of compounds that produces gas when hydrated under suitable
conditions.
[0055] "Solid" as used herein is intended to mean that a
haemostatic material or dressing will not substantially change in
shape or form when placed on a rigid surface and then left to stand
at room temperature for 24 hours.
[0056] "Frozen" as used herein is intended to mean that a
haemostatic material or dressing will not substantially change in
shape or form when placed on a rigid surface and then left to stand
at 0.degree. C. for 24 hours, but will substantially change in
shape or form when placed on a rigid surface and then left at room
temperature for 24 hours. Thus, in the context of the present
invention, a "solid" dressing is not "frozen" and a "frozen"
composition is not "solid".
[0057] "Substantially homogeneous" as used herein is intended to
mean that the haemostatic material has a uniform composition
throughout, within the tolerances described herein. Thus, a
"substantially homogeneous" haemostatic material according to the
present invention may be composed of a plurality of particles,
provided that each of those particles has the same composition.
[0058] A first preferred embodiment of the present invention is
directed to a method for treating wounded internal tissue, in a
mammal comprising applying to wounded internal tissue at least one
haemostatic material consisting essentially of a fibrinogen
component and a fibrinogen activator for a time sufficient to
reduce the flow of fluid from the wounded tissue, wherein the
haemostatic material is substantially homogeneous.
[0059] Another embodiment is directed to a method for treating
wounded internal tissue in a mammal comprising applying to wounded
internal tissue at least one haemostatic material consisting
essentially of a fibrinogen component and a fibrinogen activator
for a time sufficient to reduce the flow of fluid from the wounded
tissue, wherein the haemostatic material is cast or formed from a
single aqueous solution containing the fibrinogen component and the
fibrinogen activator.
[0060] Another embodiment is directed to a method for treating
wounded internal tissue in a mammal comprising applying to wounded
internal tissue at least one haemostatic material consisting
essentially of a fibrinogen component and a fibrinogen activator
for a time sufficient to reduce the flow of fluid from the wounded
tissue, wherein the haemostatic material is cast or formed as a
single piece.
[0061] Another preferred embodiment is directed to a frozen
haemostatic material for treating wounded internal tissue in a
mammal consisting essentially of a fibrinogen component and a
fibrinogen activator.
[0062] As used herein, "consisting essentially of" is intended to
mean that the fibrinogen component and the fibrinogen activator are
the only necessary and essential ingredients of the haemostatic
material when it is used as intended to treat wounded internal
tissue. Accordingly, the haemostatic material may contain other
ingredients in addition to the fibrinogen component 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 component 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 material without deviating from the
spirit of the present invention.
[0063] According to certain embodiments of the present invention,
the haemostatic material is formed or cast as a single piece. Once
such is formed or cast, the haemostatic material may then be used
as is or it may be further processed, for example by grinding into
a powder of pre-determined particle size. Such particles may then
be used as is or may be combined with other substances for a
particular application, e.g. such particles of haemostatic material
may be mixed with a foaming agent or aerosol gas or may be combined
with one or mote binding agents and applied to a support
material.
[0064] The haemostatic materials of the present invention may be
formed or cast in any shape or form suitable for a given
application. For example, the haemostatic material may be formed or
cast, in the shape of a cone or cylinder or the like. Such a shape
is particularly suitable for use in applications where the damage
to the tissue being treated is a hole to be plugged or sealed, a
vein which has been intentionally punctured as part of a medical
procedure, such as angioplasty. In such applications, the
haemostatic material may alternatively be in the shape of a disk,
optionally with a hole for use in conjunction with a guide wire.
Additionally, each of these forms can also be prepared by combining
particles of the inventive haemostatic materials with at least one
suitable binding agent in an appropriate mold.
[0065] The haemostatic material may also be formed or cast in the
shape of a flat sheet. Such a form is particularly suitable for use
in applications where tissue needs to be scaled or approximated,
for example in connection with endoscopic surgery or, hernia
repair. Alternatively, a flat sheet may be prepared by combining
particles of the inventive haemostatic materials with one or more
suitable binding agents, optionally in a mold.
[0066] The haemostatic material may also optionally contain one or
more suitable fillets, such as sucrose, lactose, maltose, silk,
fibrin, collagen, albumin (natural or recombinantly produced),
polysorbate (Tween.TM.), chitin, chitosan and its derivatives (e.g.
NOCC-chitosan), alginic acid and salts thereof, cellulose and
derivatives thereof, proteoglycans, hyaluron and its derivatives,
such as hyaluronic acid, glycolic acid polymers, lactic add
polymers, glycolic acid/lactic acid co-polymers, and mixtures of
two or more thereof.
[0067] The haemostatic material may also optionally contain one or
more suitable solubilizing agents, including detergents and
tensities. Illustrative examples of suitable solubilizing agents
include, but are not limited to, the following: sucrose, dextrose,
mannose, trehalose, mannitol, sorbitol, albumin, hyaluron and its
derivatives, such as hyaluronic add, sorbate, polysorbate
(Tween.TM.), sorbitan (SPAN.TM.) and mixtures of two or mote
thereof.
[0068] ) The haemostatic material may also optionally contain one
or more suitable foaming agents, such as a mixture of a
physiologically acceptable add (e.g. citric add 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 parades 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. Such a
foaming agent may be introduced into the aqueous mixture of the
fibrinogen component and the fibrinogen activator, or may be
introduced into an aqueous solution of the fibrinogen component
and/or an aqueous solution of the fibrinogen activator prior to
mixing. Alternatively, the inventive haemostatic materials may be
ground to parades of a predetermined size and then combined with a
suitable foaming agent.
[0069] The haemostatic material 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.
[0070] The haemostatic materials of the present invention are most
preferably prepared by mixing aqueous solutions of the fibrinogen
component and the fibrinogen activator under conditions which
minimize the activation of the fibrinogen component by the
fibrinogen activator. This aqueous mixture of the fibrinogen
component and the fibrinogen activator may then be frozen until
used to treat wounded tissue. Alternatively, the mixture may then
subjected to a process, such as lyophilization or freeze-drying, to
reduce the moisture content to a predetermined effective level,
i.e. to a level where the dressing is solid and therefore will not
substantially change in shape or form upon standing 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, either alone or in
combination.
[0071] As used herein, "moisture content" refers to levels
determined by procedures substantially similar to the FDA-approved,
modified Karl Fischer method (Centers for Biologies Evaluation and
Research, FDA, Docket No. 89D-0140, 83-93; 1990 and references
cited therein) or by near infrared spectroscopy. Suitable moisture
contends) for a particular inventive haemostatic material may be
determined empirically by one skilled in the art depending upon the
intended application(s) thereof.
[0072] For example, in certain embodiments of the present
invention, higher moisture contents are associated with mote
flexible solid dressings. Thus, in solid dressings intended to be
deformed in use, it may be preferred for the haemostatic material
to have a moisture content of at least 6% and even more preferably
in the range of 6% to 44%.
[0073] Similarly, in other embodiments of the present invention,
lower moisture contents are associated with mote rigid solid
dressings. Thus, in solid dressings intended to be used as formed
or cast, it may be preferred for the haemostatic material to have a
moisture content of less than 6% and even mote preferably in the
range of 1% to 6%.
[0074] Accordingly, illustrative examples of suitable moisture
contents for the inventive haemostatic materials 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%.
[0075] The fibrinogen component in the haemostatic material may be
any suitable fibrinogen known and available to those skilled in the
art. The fibrinogen component may also be a functional derivative
or metabolite of a fibrinogen, such the fibrinogen a, p and/or y
chains, soluble fibrin I or fibrin II, or a mixture of two or more
thereof. A specific fibrinogen (or functional derivative or
metabolite) 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 mounts 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.
[0076] Preferably, the fibrinogen employed as the fibrinogen
component 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 vitally 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.
[0077] The fibrinogen component should be present in the inventive
haemostatic materials in an amount effective to react with the
fibrinogen activator and form sufficient fibrin to reduce the flow
of fluid from wounded internal tissue. According to certain
preferred embodiments of the present invention, when the
haemostatic material is frozen, the fibrinogen component is present
in an amount of from 4.70 mg to 18.75 mg (.+-.0.009 mg) pet square
centimeter of the surface(s) of the haemostatic material intended
to contact the wounded internal tissue. *******
[0078] According to other preferred embodiments, when the
haemostatic material is a solid, regardless of form, the fibrinogen
component is present in an amount of from 5.00 mg to 450.00 mg
(.+-.0.009 mg) per square centimeter of the surface(s) intended to
contact the wounded internal tissue being treated. Greater or
lesser amounts, however, may be employed depending upon the
particular application intended for the solid dressing.
[0079] For example, when the haemostatic material is in the shape
of a rod or cylinder, the fibrinogen component is more preferably
present in an amount of from 25.00 mg to 75.00 mg (.+-.0.009 mg)
pet square centimeter of the surface(s) intended to contact the
wounded internal tissue being treated. Alternatively, when the
haemostatic material is in the shape of a flat sheet or disk, the
fibrinogen component is more preferably present in an amount of
from 5.00 to 56.00 mg (.+-.0.009 mg) per square centimeter of the
surface(s) intended to contact the wounded internal tissue being
treated. Still alternatively, when the haemostatic material is
powdered, either loose or compressed, the fibrinogen component is
more preferably present in an amount from 26.00 mg to 450.00 mg
(.+-.0.09 mg) per square centimeter of the surface(s) intended to
contact the wounded internal tissue being treated.
[0080] The fibrinogen activator employed in the haemostatic
materials of the present invention may be any of the substances or
mixtures of substances known by those skilled in the art to convert
fibrinogen (or a fibrinogen equivalent) 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 jaratacussu; and
mixtures of any two or mote of these. See, e.g., Dascombe et al.,
Thromb. Haemost. 78:947-51 (1997); Hahn et al., J. 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).
[0081] Preferably, the fibrinogen activator is a thrombin. Mote
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, the thrombin employed is preferably a lyophilized
mixture of human plasma proteins which has been sufficiently
purified and vitally 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 the inventive
haemostatic materials 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 pH in the range of 5.5-8.5
may be acceptable.
[0082] In addition to the inventive haemostatic materials), the
solid and frozen dressings of the present invention may optionally
further comprise one or more support materials. As used herein, a
"support material" refers to a material that sustains or improves
the structural integrity of the solid or frozen dressing and/or the
fibrin clot formed when such a dressing is applied to wounded
tissue. The support material may be an internal support material or
a surface support material. Moreover, in the case of the latter, if
the dressing is in a form that, has a wound facing side, the
support material may be on the wound facing side or it may be on
the non-wound facing side or both.
[0083] 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), 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 materials) may be selected empirically by those skilled
in the art depending upon the intended use of the solid
dressing.
[0084] 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).
[0085] Any suitable non-resorbable material known and available to
those skilled in the art may be employed as the support material.
Illustrative examples of suitable non-resorbable materials include,
but are not limited to, plastics, silicone polymers, paper and
paper products, latex, gauze plastics, non-resorbable suture
materials, latexes and suitable derivatives thereof.
[0086] According to other preferred embodiments, the support
material comprises an internal support material. Such an internal
support material is preferably fully contained within the
haemostatic materials) of a solid or frozen dressing. The internal
support material may take any form suitable for the intended
application of the haemostatic material. For example, according to
certain embodiments, the internal support material may be particles
of a predetermined suitable size which are dispersed throughout the
haemostatic material. Alternatively, a sheet or film or internal
support material may be included in the solid or frozen haemostatic
material.
[0087] According to still other preferred embodiments, the support
material may comprise a backing material on the surface(s) of the
dressing opposite the wound-facing surface. As with the internal
support material, the backing material may be a resorbable material
or a non-resorbable material, or a mixture thereof, such as a
mixture of two or mote resorbable materials or a mixture of two or
more non-resorbable materials or a mixture of resorbable materials)
and non-resorbable materials).
[0088] According to still other preferred embodiments, the dressing
comprises both a backing material and an internal support material
in addition to the haemostatic material(s). According to still
other preferred embodiments, the 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 dressing comprises a backing material, a front
support material and an internal support material in addition to
the haemostatic layer(s).
[0089] According to certain preferred embodiments, the haemostatic
materials) may also contain a binding agent to maintain the
physical integrity of the haemostatic material(s). Illustrative
examples of suitable binding agents include, but are not limited
to, sucrose, mannitol, sorbitol, gelatin, hyaluron and its
derivatives, such as hyaluronic add, maltose, povidone, starch,
chitosan and its derivatives, and cellulose derivatives, such as
carboxymethylcellulose, as well as mixtures of two or more
thereof.
[0090] According to certain embodiments of the present invention,
particularly where the solid or frozen dressing is manufactured
using a mold, the dressings may also optionally further comprise a
release layer in addition to the haemostatic materials) 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 or frozen dressing from a mold in
which it has been manufactured. A preferred such agent is sucrose,
but other suitable release agents include gelatin, hyaluron and its
derivatives, including hyaluronic acid, mannitol, sorbitol and
glucose. Alternatively, such one or mote release agents may be
contained in the haemostatic material.
[0091] The haemostatic material and any layer(s) 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 material subsequently affixed thereto.
[0092] 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 dot 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 dot after application of the bandage to wounded
tissue.
[0093] Suitable fibrinogen components and suitable fibrinogen
activators for the haemostatic materials 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.
[0094] According to certain preferred embodiments of the present
invention, the fibrinogen component 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 component is bird fibrinogen or
fish fibrinogen. According to any of these embodiments, the
fibrinogen component may be recombinantly produced fibrinogen or
transgenic fibrinogen.
[0095] 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.
[0096] As ft general proposition, the purity of the fibrinogen
component 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 component 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 component 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. The
fibrinogen component and/or fibrinogen activator and/or the
inventive haemostatic materials may also be subjected to suitable
sterilization treatments, including, but not limited to, treatment
with one or more of the following: heat, gamma radiation, c-beam
radiation, plasma radiation and ethylene oxide.
[0097] 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.
[0098] According to certain preferred embodiments of the present
invention, when the fibrinogen activator is human thrombin, the
amount of human thrombin employed is between 0.03 and 16.10 Units
(all values being.+-.0.009) per square centimeter of the surface(s)
of the haemostatic material intended to contact the wounded
internal tissue. Greater or lesser amounts, however, may be
employed depending upon the particular application intended for the
solid dressing.
[0099] For example, when the haemostatic material is a solid in the
shape of a rod or cylinder, the fibrinogen activator is mote
preferably present in an amount of from 2.50 Units to 7.50 Units
(.+-.0.009 Units) pet square centimeter of the surface(s) intended
to contact the wounded internal tissue being treated.
Alternatively, when the haemostatic material is a solid in the
shape of a flat sheet or disk, the fibrinogen activator is more
preferably present in an amount of from 0.03 Units to 16.10 Units
(.+-.0.009 Units) pet square centimeter of the surface(s) intended
to contact the wounded internal tissue being treated. Still
alternatively, when the haemostatic material is a powdered solid,
either loose or compressed, the fibrinogen activator is more
preferably present in an amount of about 1.3 Units (.+-.0.09 mg)
per square centimeter of the surfaced) intended to contact the
wounded internal tissue being treated. Still alternatively, when
the haemostatic material is frozen, the fibrinogen activator is
more preferably present in an amount of about 1.3 Units (i 0.09 mg)
pet square centimeter of the surface(s) intended to contact the
wounded internal tissue being treated.
[0100] According to still other preferred embodiments of the
present invention, when the fibrinogen activator is human thrombin,
the amount of human thrombin employed is between 0.0087 and 1.0000
Units (all values being.+-.0.00009) per milligram of the fibrinogen
component Greater or lesser amounts, however, may be employed
depending upon the particular application intended for the solid
dressing.
[0101] For example, when the haemostatic material is a solid in the
shape of a rod or cylinder, the fibrinogen activator is more
preferably present in an amount of about 0.1 Units (.+-.0.09 Units)
per milligram of the fibrinogen component. Alternatively, when the
haemostatic material is a solid in the shape of a flat sheet or
disk, the fibrinogen activator is mote preferably present in an
amount of from 0.1 Units to 1.00 Units (.+-.0.009 Units) per
milligram of the fibrinogen component. Still alternatively, when
the haemostatic material is a powdered solid, either loose or
compressed, the fibrinogen activator is more preferably present in
an amount of about 0.0087 Units to 0.0500 Units (.+-.0.00009 Units)
per milligram of the fibrinogen component. Still alternatively,
when the haemostatic material is frozen, the fibrinogen activator
is more preferably present in an amount of about 0.07 Units to 0.10
Units (.+-.0.009 Units) per milligram of the fibrinogen
component.
[0102] During use of the inventive haemostatic materials, the
fibrinogen component 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 fibrinogen activator 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.
[0103] In some embodiments of the present invention, the inventive
haemostatic materials 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 aptotonin, 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, such
as epinephrine; chemoattractant; 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.
[0104] The inventive haemostatic materials, and the solid and
frozen dressings containing them, may be applied to any internal
wounded tissue in a mammal using any of the suitable techniques
and/or devices known and available to one skilled in the medical
arts. For example, when used to treat vascular punctures, the
haemostatic material(s) may be applied via a catheter, either with
or without a guide wire. The inventive materials and dressings may
also be applied in conjunction with endoscopic techniques,
including endoscopic surgery, laparascopic surgery and
telc-robotic/tele-prescesne surgery. According to such embodiments,
it is preferable to use a "plunger" or "tamper" to facilitate
passage of the inventive materials through surrounding tissue to
the wounded internal tissue being treated. The inventive materials
and dressings may also be applied manually.
[0105] 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
[0106] The following is a list of acronyms used in the Examples
below: [0107] CFB: Complete Fibrinogen Buffer (100 mM Sodium
Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mM Sodium
Citrate, 1.5% Sucrose, Human Scrum Albumin (80 mg/g of total
protein) and Tween.TM. 80 (non-animal source) 15 mg/g total
protein) [0108] 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) [0109] ERL: Enzyme Research
laboratories [0110] EVPA: Ex Vivo Porcine Arteriotomy [0111] EVPCA:
Ex Vivo Porcine Carotid Arteriotomy [0112] FD: Inventive
haemostatic dressing [0113] HSA: Human Serum Albumin [0114] HD: A
"sandwich" fibrin sealant haemostatic dressing as disclosed in U.S.
Pat. No. 6,762,336 [0115] IFB; Incomplete Fibrinogen Buffer; CFB
without HSA and Tween [0116] Fibrinogen Dose: In a solid mass, the
amount of fibrinogen within the mass divided by the surface area to
be treated. Usually expressed in mg of Fibrinogen pet cm.sup.2,
where the mass of fibrinogen is determined via a clottable protein
assay [0117] PETG: Glycol-modified Polyethlyleneteterapthalate
[0118] PPG: Polypropylene [0119] PVC: Poly vinyl chloride [0120]
T:F Thrombin to Fibrinogen ratio. In a test article, the amount of
thrombin activity per unit of fibrinogen. Usually expressed in
thrombin NIH Units pet mg of fibrinogen (measured via a clottable
protein assay) [0121] Thrombin Dose: In a solid mass, the amount of
thrombin within the mass divided by the surface area to be treated.
Usually expressed in NIH Units of thrombin per cm.sup.2 [0122]
TRIS: trishydroxymethylaminomethane
(2-amino-2-hydroxymethyl-1,3-propanediol)
[0123] The ability of the dressings to seal an injured blood vessel
was determined by modifications of an ex viva 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. Mote
specifically, the procedure disclosed in U.S. Pat. No. 6,762,336
requited 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. Set, 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.
[0124] 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 teal
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).
[0125] In addition, a new test was derived to directly measure
adherence of the dressing to the injured tissue.
Example 1
[0126] In order to apply the haemostatic test articles to the
surface of an injured artery surrounded by a tissue stimulant, the
test articles were housed in cylindrical molds made of 10 or 3 ml,
polypropylene syringes (Becton Dickinson) with the luer-lock end
removed. The plungers were withdrawn to the 6 mL and 2 mL mark
respectively. Pot 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 a pre-cooled Genesis.TM. lyophylizer
(Virus, Gardiner, N.Y.). The chamber was scaled and the temperature
equilibrated. The chamber was then evacuated and the dressings
lyophilized via a primary and secondary drying cycle.
[0127] They were subsequently performance tested in a modified EVPA
assay (Deep Tissue EVPA). Briefly, in one version, 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 (FIG. 1). In another variant, the foam was replaced with a
piece of tissue, specifically, bovine muscle, in which a hole had
been prepared as with the foam. The foam was maintained at
37.degree. C. by placement in a 37.degree. C. water bath, while the
muscle tissue was maintained at 3.degree. C. by placement on a
37.degree. C. block heater. 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 described hereafter.
Deep Tissue EVPA Testing
[0128] Equipment and Supplies: [0129] In-line high pressure
transducer (Ashcroft Duralife.TM. or equivalent) [0130] Peristaltic
pump (Pharmacia Biotech.TM., Model P-1 or equivalent) [0131]
Voltmeter (Craftsman.TM. Professional Model 82324 or equivalent)
[0132] Computer equipped with software for recording pressure or
voltage information [0133] Tygon.TM. tubing (assorted sizes) with
attachments [0134] Water bath (Baxter Durabath.TM. or equivalent),
preset to 37.degree. C. [0135] Incubation chamber (VWR.TM., Model
1400G or equivalent), preset to 37.degree. C. [0136] Thermometer to
monitor temperatures of both water bath and oven [0137] Assorted
forceps, hemostats, and scissors [0138] 10 cc. and 20 cc. syringes
with an approximately 0.6 cm bole 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. [0139] O-rings (size 10 and 13) [0140] Plastic
Shields to fit the 10 cc and 20 cc syringes (approximately 3.5 cm
in length) [0141] P-1000 Pipetman.TM. with tips [0142] Programmable
Logic Controller (PLC) to control the pumps to maintain the desired
pressure profile (Optional. Manual control may be used if
desired.)
[0143] 1. Materials and Chemicals [0144] Porcine descending aortas
(Pel-Freez Biologicals.TM., Catalog #59402-2 or equivalent) [0145]
Cyanoacrylate glue (Vetbond.TM., 3M or equivalent) [0146] 18-gauge
needle(s) [0147] 0.9% Saline, maintained at 37.degree. C. [0148]
Red food coloring [0149] Vascular Punch(es), 2.8 mm or other [0150]
Plastic Wrap
[0151] 2. Artery Cleaning and Storage [0152] 1. Store arteries at
-20.degree. C. until used. [0153] 2. Thaw arteries at 37.degree. C.
in H.sub.2O bath. [0154] 3. Clean fat and connective tissue from
exterior surface of artery. [0155] 4. Cut the arteries into 5 cm
segments. [0156] 5. The arteries may be refrozen to -20.degree. C.
and stored until use.
[0157] 3. Artery Preparation for Assay [0158] 1. Turn the artery
inside-out so that the smooth, interior wall is facing outwards.
[0159] 2. Stretch a size 13 O-ting over a 20 cc syringe or a size
10 O-ting over a 10 cc syringe with an approximately 0.6 cm (0.25
in) hole drilled into one side. [0160] 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 [0161] 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 [0162] 5.
Using the blade of some surgical scissors, gently scrape the
surface of the artery in order to roughen the surface of the
artery. [0163] 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) [0164] 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. [0165] 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.
[0166] Place the artery in the warmed, moistened container and
place in the incubation chamber. Allow the arteries to warm for at
least 30 minutes.
[0167] 4. Solution and Equipment Preparation [0168] 1. Check to see
that the water bath, block heater and incubation chamber are
maintained at 37.degree. C. [0169] 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. [0170] 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. [0171] 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. [0172] 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.
[0173] 5. Application of the Dressing [0174] 1. Slip either the
warmed (at 37.degree. C.) plastic foam form or the warmed tissue
over the artery. Align the hole in it to correspond to the hole in
the artery and the surrounding tissue (FIG. 1). [0175] 2. Open the
haemostatic dressing (Test Article) pouch and remove haemostatic
dressing & Applicator. [0176] 3. Slowly 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 the data collection
forms. [0177] NOTE: By way of example, a representative (13-15
mg/cm.sup.2 of fibrinogen) 2.4.times.2.4 cm haemostatic dressing
should generally 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.
[0178] 4. immediately pass the dressing in the applicator down thru
the hole in the foam to the artery surface. Depress the plunger by
hand and hold by hand for 3 minutes, after which the applicator is
withdrawn as the plunger was depressed further. [0179] 5. After
polymerization, note the condition of the haemostatic dressing. Any
variation from the positive control should be noted on the data
collection form.
[0180] EXCLUSION CRITERION: The mesh support material must remain
over the hole in the artery. If it has shifted during the
polymerization and does not completely covet the hole the
haemostatic dressing must be excluded.
Testing Procedure
[0181] 1. Diagram of Testing Equipment Set-Up
[0182] The set-up of the testing equipment is shown in FIG. 1. Some
additional, unshown components may be utilized to read out
(pressure gauge) or control the pressure within the system
[0183] 2. Equipment and Artery Assembly
[0184] Fill the artery and syringe with red 0.9% saline warmed to
3.degree. C., taking care to minimize the amount of air bubbles
within the syringe and 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 mote
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
[0185] 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
step 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.
Failure Criteria
[0186] Haemostatic dressings that start leaking saline at any point
during testing are considered to have reached the end of the assay.
[0187] 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).
[0188] 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, dearly labeled,
and entered by hand into the data tables.
Exclusion Criteria
[0189] If the total testing period exceeds the maximum allowed for
that procedure, regardless of cause, results most 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 support material does not completely cover
the hole in the artery, the results must be excluded.
Adherence Performance Tearing
[0190] Equipment and Supplies
[0191] Hemostat(s), Porcine artery and haemostatic dressing,
optionally after performance of EVPA assay.
[0192] Preparation of the Artery+Dressing
[0193] 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
docs 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.
[0194] Performance of the Adherence Assay
[0195] 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. [0196] NOTE: If the
FD developed a channel leak during the performance of the EVPA
Assay, test die adherence on the opposite of the haemostatic
dressing to obtain a more accurate assessment of the overall
adherence.
[0197] 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-00001 TABLE 1.1 Dressing Amount Performance Score of
Adherence 4 90+% 3 75-90% 2 50-75% 1 ~50% 0.5 Only the plug holds
the hemostat 0 No adherence
[0198] Exclusion Criteria
[0199] The mesh support material 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.
[0200] Success Criteria
[0201] Dressings that are given an adherence score of 3 are
considered to have passed the assay.
[0202] Failure Criteria
[0203] 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.
[0204] Weight Held Performance Assay
[0205] After the initial scoring of the "Adherence Test", weights
may then be added to the hemostat in an incremental manner until
the mesh support material 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.
Example 2
[0206] Similar to the need to evaluate a test article in the
context of sealing and injury deep within surrounding tissue, there
was also a need to test products that can seal injured tissue where
the injured vessels are smaller and thinner-walled than an aorta.
The following assay accomplishes this goal.
[0207] According to this modification, the porcine carotid artery
is attached to a barbed female connector using cotton thread with
the connective tissue side exposed. This is in contrast to the
standard EVPA where the internal side is exposed. As the carotid
arteries used in the VA model are more elastic and friable than the
aorta, it is mote, difficult to treat or abrade the surface without
damaging and compromising the artery. To ensure that no teats have
occurred during the removal of the bulk of the connective tissue,
the artery is connected to the barbed connector and solution is
pumped into it. If the artery is intact, a 1.5 mm hole is punched
into the artery using a biopsy punch.
[0208] After the artery is prepped, it is connected to the pump
system and placed on top of a piece of foam with a concave "hollow"
cut: into the surface. This serves as a support for the artery
during application of the FD and "compression" of the artery. The
test article is applied to the top of the hole and wet with
37.degree. C. 0.9% NaCl. The artery is covered with plastic wrap,
and a weight warmed to .about.38-40.degree. C. is then placed on
top of the artery. The artery is partially compressed instead of
being pressed flat because of the support of the foam.
[0209] After the weight has been applied for 5 min., it is then
removed, and the pump is fumed on. When the solution is coming out
of the end of the artery, it is then clamped and allowed to
pressurize until 250 mmHg or a leak occurs, whichever comes
first.
[0210] In development of the assay, the following variables were
considered and tested:
[0211] Tissue Selection: In order to mimic a vascular access
procedure, a tissue substrate that was elastic yet strong was
needed. Contact, with rendering companies such as PelFreeze and
Animal Technologies revealed 2 types of arteries collected that
could be potentially used to mimic the vascular access procedure:
porcine renal arteries and porcine carotid arteries. These arteries
were comparable in size to a human femoral artery. Both types were
purchased to examine their usefulness. The porcine renal artery was
too short in useable length (less than 2''), to small an internal
diameter, and not as elastic as desired. The porcine carotid
artery, however, was highly elastic and offered useable segments of
3-5'' without branching or collateral arteries.
[0212] Artery Hole Size: To determine a size to use for the assay,
the actual surgical procedure was mimicked insofar as possible. A
hole was put into the artery using an 18-gauge needle. A 200 uL
pipette tip was then pushed into the hole to the point where the
diameter was .about.3.5 mm, just larger than a 10 F catheter. The
rip was left: in place for 2 hrs. and was dien removed. The
resulting hole was larger than the initial 18-gauge needle punch
and, when compared to 2.8, 2.0, and 1.5 mm holes, was very similar
to the 1.5 mm hole produced by the biopsy punch.
[0213] Surface Preparation: In the EVPA assay, the interior surface
of a porcine aorta is gently abraded using the edge of a pair of
scissors to provide a "damaged" surface to which the FD would
adhere, mimicking large trauma. For the vascular access procedure,
obtaining a uniform, reproducible surface on which to test the FD
was important. Starting with the familiar, the carotid artery was
turned inside-out and abraded. However, this did not work as the
carotid artery is highly elastic, and the scraping of the surface
created tears that tendered the artery unusable. Using the exterior
surface, the arteries that had the connective tissue carefully
removed down to the level of the artery provided a surface that was
uniform and best mimicked the vascular access procedure.
[0214] Integration of the Artery into the Pump System: To best
mimic the vascular access procedure, the use of the artery without
any internal support to interfere with compression was desired. In
order to incorporate the artery into the pump system, it was
necessary to attach the artery at one end to the tubing and still
have an open end to allow solution flow prior to pressurization.
After examining different types of tubing and connectors, a barbed
low-pressure female connector was chosen. The barb could be either
1/8'' or 1/4'', depending upon the inner diameter of the carotid
artery. To attach the artery to the barbed end, cable ties,
o-rings, and thread were tested. Only the thread prevented leakage
during pressurization.
[0215] Arterial Support: In trying to partially-compress the artery
on a flat surface, it became clear that some form of support was
needed to prevent the artery from shifting during application of
the FD and to prevent total compression of the artery. A variety of
materials were tested, including gel packs, Styrofoam packaging
material, and foam pieces. Foam pieces that had a concave trough
cut into the top surface offered the best support: the trough held
the artery in place, and it was cut just deep enough to allow
partial compression of the artery.
[0216] Compression Method: In the actual surgical procedure,
hemostasis is more commonly achieved by manual compression of the
artery for a period of .about.20 min. During this time, arterial
flow is maintained. Application of a weight to the artery was
tested in order to mimic this at the lab bench. Various weights in
beakers just large enough to contain the weight were tested on
arteries in the foam arterial support. With this set-up, both 200 g
and 500 g weight inside a glass beaker (to provide a uniform
surface for compression) just large enough to accommodate the
weight proved to be ideal for compression. Weights lower or higher
provided insufficient or too much compression, respectively.
[0217] ) Temperature maintenance: FXIII, a component of the FD that
is responsible for cross-linking of fibrin monomers, is thermally
labile, and the assay needs maintained around normal body and wound
temperatures of 34-36.degree. C. As this set-up cannot be easily
transferred to an incubator as in the EVPA, another method had to
be devised. Various methods were considered such as warmed gel
packs, heating pads, and warming lamps. While these methods would
produce a warmer-than-ambient temperature, they were difficult to
control to the level that this assay requites. The most practical
method was the use of a heat block set to 37.degree. C. While a
heat block can maintain a constant temperature for very long
periods of time, they were not sufficient to warm the artery and
IT) to 34-36.degree. in the 5 minute time frame of the assay. As
the weight that is applied could be a potential heat source, it was
warmed in the incubator prior to application, and this addition to
the 37.degree. C. heat block was sufficient to maintain the
34-36.degree. C. temperature range.
[0218] Data Collection: For this assay, the following pieces of
data are collected: amount of saline required to wet the dressing,
ease of wetting, artery temperature after the incubation period,
maximum pressure obtained, failure mode (channel leak, leak through
plug), qualitative assessment of the adherence of the dressing to
the artery, and overall comments on dressing appearance (mottled,
pre-formed fibrin, thin, etc.)
1 Test Protocol for Ex Vivo Porcine Carotid Artery Assay
(EVPCA)
[0219] Equipment and Supplies [0220] In line high pressure
transducer(Ashcroft Duralife or equivalent) [0221] Peristaltic pump
(Pharmacia Biotech, Model P-1 or equivalent) [0222] Voltmeter
(Craftsman Professional Model 82324 or equivalent) [0223] Computer
equipped with software for recording pressure or voltage
information [0224] Tygon tubing (asst. sizes) with attachments
[0225] Water bath (Baxter Durabath or equivalent), preset to
37.degree. C. [0226] Heat Block (Thermolyne Type 16500 Dri-Bath or
equivalent) [0227] Incubation chamber (VWR, Model 1400G or
equivalent), preset to .about.40.degree. C. [0228] Thermometer to
monitor temperatures of water bath, heat block, and oven [0229]
Calibration weights: 200 g and 500 g [0230] Beakers to hold
calibration weights [0231] Biopsy punch(es), 1.5 mm or other
required sizes [0232] Assorted forceps, hemostats, and scissors
[0233] P-200 and P-100 Pipetman with tips [0234] Plastic 1/8'' and
1/4'' low pressure fittings, female connector with a barbed tubing
connection [0235] Plastic strips 1/8'' and 1/4'' wide
[0236] Materials and Chemicals [0237] Porcine carotid arteries (xxx
or equivalent) [0238] 0.9% Saline, maintained at 37.degree. C.
[0239] Red food coloring [0240] Quilting thread or other
heavy-gauge thread [0241] Plastic Wrap [0242] Foam pieces with a
concave area cut into the surface
[0243] Preliminary Procedures [0244] Artery Cleaning and Storage
[0245] 1. Store arteries at -20.degree. C. until used. [0246] 2.
Thaw arteries at 37.degree. C. in H.sub.2O bath. [0247] 3. Clean
fat and connective tissue from exterior surface of artery. [0248]
4. The arteries may be refrozen to -20.degree. C. and stored until
use.
[0249] Artery Preparation for Assay [0250] 1. Make sure that all
connective tissue is removed from the artery. [0251] 2. If any
collateral arteries or other large holes are visible, cut the
artery at the hole. If a small section of artery is produced from
the cut, discard if. If 2 pieces are produced that are at least
11/2'' long, both pieces may be used for assays. [0252] 3. Insert
the barbed end of a low pressure connector, either 1/8'' or 1/4''
depending upon the internal diameter of the artery, into the larger
end of the artery. [0253] 4. Cut a piece of quilting or other heavy
thread .about.6'' long. Using a square knot, tie the artery to the
connector so that it docs not come off of the connector. [0254]
Note: As it is possible to tear the artery during the cleaning
process, it is important to "leak test" the artery prior to
performing the assay. [0255] 5. Connect the artery to the male
connector at the end of the tubing attached to the pump system.
[0256] 6. Turn on the pump with the open end of the artery pointed
upwards and allow the artery to fill with the red 0.9% NaCl. When
the artery is full, clamp the artery closed using a hemostat.
[0257] 7. Watch the artery as it pressurizes to sec if any holes or
teats are present. If a hole is present, turn off the pump, unclamp
the artery over a beaker to catch the saline solution, and
disconnect it from the pump system.
[0258] For arteries with holes [0259] If the hole is near the open
end of the artery, cut off the artery at the hole, leaving the
artery attached to the connector. [0260] If the hole is neat the
connector, remove the artery from the connector, cut the artery at
the hole, and re-attach it to the connector as outlined above.
[0261] For arteries that have pieces cut off, the remaining piece
should be at least 11/2'' long. If not, it should be discarded.
[0262] If the hole is near the middle of the artery, check the size
of the hole. If it is less than 1.5 mm, it may be used for the
assay as a hole may be punched around it. If the hole is larger
than 1.5 mm, the artery should be discarded.
[0263] For arteries without holes [0264] If no holes are seen,
allow the artery to pressurize to .about.100 mmHg (a reading of 2.0
on the pressure gauge). Turn off the pump and unclamp the artery
over a beaker, and disconnect it from the pump system. [0265] If
any holes become visible during this period, unclamp the artery
over a beaker, disconnect form the pump system, and fix the artery
according to the procedures outlined above. Note: After the artery
has been inspected and any unwanted holes addressed, the test hole
may then be punched in the artery [0266] 8. Insert a plastic strip
into the open end of the artery so that it goes most of the way
into the artery. [0267] 9. Using the biopsy punch, carefully punch
a hole in the artery. Make sure that the punch connects with the
plastic strip so that no additional holes are punched in the
artery. [0268] 10. The punch should totally remove the center
portion. If it does not, gently remove it with forceps or by
re-cutting it using the biopsy punch. [0269] 11. Place the artery
in the warmed, moistened container and place in the
.about.40.degree. C. incubation chamber to keep the artery moist
prior to assay
[0270] Solution and Equipment Preparation [0271] 1. Turn on the
heat block and check to see that it is maintained at 37.degree. C.
[0272] 2. Check to see that the water bath is maintained at
37.degree. C. and incubation chamber is maintained at
.about.40.degree. C. [0273] 3. 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. [0274] 4. Place 0.9% saline into
containers in a 37.degree. C. water bath so that the solutions will
be warmed prior to performing the assay. [0275] 5. The peristaltic
pump should be calibrated so that it delivers approximately 3
ml/min. If not, adjust the settings at this point. [0276] 6. 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.
[0277] Application of the FD or HD [0278] 1. Place a piece of foam
with the concave surface on top of the heating block and cover with
a piece of plastic wrap. [0279] 2. Remove an artery from the
warming box and attach it to the pump system. [0280] 3. Allow the
artery to rest in the concave hollow of the foam piece. [0281] 4.
Open the haemostatic dressing pouch and remove haemostatic
dressing(s). Place any extras in the vacuum dessicator. [0282] 5.
Place the dressing, mesh support material side UP (or the side
closest to the bottom of the mold if no support material is
present), over the hole in the artery [0283] 6. Slowly wet the
haemostatic dressing with an amount of saline appropriate for the
article being tested [0284] 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. A dressing of
1.5.times.1.5 cm would require 300 .mu.l of saline or other blood
substitute, and a 0.7.times.0.7 cm dressing would requite 70 .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. [0285] NOTE: Wet the haemostatic
dressing drop wise with 0.9% saline warmed to 37.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.
[0286] 7. Covet the artery with plastic wrap, taking care that the
dressing doesn't slide around on the surface of the artery. [0287]
8. Place a warmed weight carefully on top of the dressing so that
it does not shift off of the hole in the artery. [0288] 9. Allow
the weight to remain on the artery on top of the 37.degree. C. heat
block for the duration of the polymerization time. [0289] NOTE:
Time, pressure, and hole size can be altered according to the
requirements of the experiment; changes from the standard
conditions should be noted on the data collection forms. [0290] 10.
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.
[0291] EXCLUSION CRITERION: The mesh support material must remain
over die hole in the artery. If it has shifted during the
polymerization and does not completely cover the hole the
haemostatic dressing must be excluded.
[0292] Testing Procedure
[0293] A diagram of testing equipment set-up is shown in FIG.
1.
TABLE-US-00002 TABLE 2.1 Conversion Table for Pressure (PSI) to
mmHg and Voltage Pressure Gauge mm Hg Voltage Reading (PSI)
Equivalent Equivalent 1.0 50 1.25 2.0 100 1.50 3.0 150 l.75 4.0 200
2.00 5.0 250 2.25
[0294] Equipment and Artery Assembly [0295] 1. After the
polymerization period is complete, carefully remove the plastic
wrap so that the dressing is not disturbed. [0296] 2. Turn on the
pump and gently lift the open end of the artery with a hemostat.
Allow the artery to fill to the top with 0.9% NaCl. This is done to
minimize air bubbles in the system. [0297] 3. The system should be
operated according to a pre-determined range of pressures and hold
times as appropriate for the article being tested. Should the
pressure drop below the desired maximum during due hold period, the
pump should be turned on again until the maximum pressure is
achieved. [0298] 4. Should a leak in the system develop other than
failure of the FD or HD (i.e. leaking from a hole in the artery,
etc.), attempts to correct the problem should be taken. This might
involve clamping the leak for the remainder of the assay. Should
the attempts to fix the problem be ineffective, the test article
will be excluded from analysis and called a "system failure" (See
Exclusion Criteria below). [0299] 5. 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.
[0300] Success/Fail and Exclusion Criteria
[0301] Success criteria [0302] 1. Haemostatic dressings that are
able to withstand various pressures for 3 minutes are considered to
have passed the assay. [0303] 2. 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. [0304] 3. The entire testing period from application of
the haemostatic dressing to completion must fall within
pre-established criteria. [0305] NOTE: For a single-step increase
to maximum pressure the entire testing period should not exceed 15
minutes. Other time limits may be established for other test
procedures, and should be noted on the data collection forms.
[0306] 4. The maximum pressure reached should be recorded on the
data collection form. NOTE: Typical challenge is 250 mmHg for three
minutes in one step, but that may be altered based on the article
being tested. Tire pressure, for example, may be increased in
"steps" with holds at various pressures until the 250 mmHg is
achieved. One example is increasing the pressure in 50 mmHg
increments with a 1 minute hold at each step to ensure that the FD
or HD can hold these pressures.
[0307] Failure criteria [0308] 1. Haemostatic dressings that start
leaking saline at the point of FD or HD attachment at any point
during testing are considered to have failed the assay. [0309]
NOTE; Build failures that are earned 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). [0310] 2.
When leakage from the FD or HD docs 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. [0311] 3. The
pressures at which leakage occurred should be recorded on the data
collection form. [0312] 4. 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.
[0313] Exclusion Criteria [0314] 1. If the total testing period
exceeds the maximum allowed for that procedure, regardless of
cause, results must be excluded. [0315] 2. If there are leaks from
holes that can't be fixed by clamping or finger pressure the
results must be excluded. [0316] 3. If the mesh support material
docs not completely covet the hole in the artery, the results must
be excluded
Example 3
[0317] 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 support material dispersed
within, it 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 support material, 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 support 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. The compositions are shown in Table
3.1 below.
TABLE-US-00003 TABLE 3.1 Mold Fibrinogen Dose Thrombin Dose T:F
Size (mg/cm.sup.2) (U/cm.sup.2) Ratio 3 ml 75 7.5 0.1 10 ml 25 2.5
0.1
[0318] Upon removal from the lyophylizer, both groups were
performance tested in a modified EVPA assay as described in Example
1 above. 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 described in
Example 1 above.
TABLE-US-00004 TABLE 3.1 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 mm Hg
[0319] Conclusions: Dressings that included no support material or
a DEXON.TM. mesh support material 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
throughout the composition, as desired.
[0320] The results demonstrate that the dressings were effective at
the highest pressure tested regardless of size, and that they
functioned effectively regardless of the presence or absence of the
support material. Higher performance was associated with the
presence of support material, and a larger applicator.
Example 4
[0321] Dcxon.TM. Mesh support material was cut to fit into and
placed into each PETG 1.5.times.1.5 cm mold. Fifteen microliters of
2% sucrose was pipeted on top of each of the four comets of the
support material and the molds were placed inside a -80.degree. C.
freezer. Once completed the molds were placed in a -80.degree. C.
freezer. All molds remained m the -80.degree. C. freezer for at
least 60 minutes. Enzyme Research Laboratories (ERL) Fibrinogen lot
3150 was formulated in 100 mM Sodium Chloride, 1.1 mM Calcium
Chloride, 10 mM Tris, 10 mM Sodium Citrate, and 1.5% Sucrose
(Fibrinogen complete buffer). In addition, Human Serum Albumin was
added to 80 mg/g of total protein and Tween 80 (non-animal source)
was added to 15 mg/g total protein. 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 150 mM Sodium Chloride, 40 mM Calcium
Chloride, 10 mM Tris and 100 mM L-Lysine. The final pH of the
thrombin was 7.4+/-0.1. The thrombin was adjusted to 25 Units/ml
thrombin, resulting in 0.1 units/mg of Fibrinogen or 1.3
U/cm.sup.2. 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 pipetot was filled with fibrinogen and
second repeat pipetor was filled with thrombin. Simultaneously 0.8
ml of fibrinogen and 133 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 a pre-cooled Genesis.TM. lyophylizer (Virtis, Gardiner,
N.Y.). The chamber was scaled and the temperature equilibrated. The
chamber was then evacuated and the dressings lyophilized as
described in Example 3,
[0322] Test articles of a different size were also prepared as
follows. Support material was cut and placed into each PETG
0.7.times.0.7 cm mold. Five microliters of 2% sucrose was pipeted
on top of each of the four comets of the support material and the
molds were placed inside a -80.degree. C. freezer. Once completed
the molds were placed in a -80.degree. C. freezer. All molds
remained in the -80.degree. C. freezer for at least 60 minutes.
Enzyme Research Laboratories (ERL) Fibrinogen lot 3150 was
formulated in 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10
mM Tris, 10 mM Sodium Citrate, and 1.5% Sucrose (Fibrinogen
complete buffer). In addition, Human Scrum Albumin was added to 80
mg/g of total protein and Tween 80 (non-animal source) was added to
15 mg/g total protein. The final pH of the fibrinogen was
7.4+/-0.1. The fibrinogen concentration was adjusted to 39.2 mg/mL
Once prepared the fibrinogen was placed on ice until use. Thrombin
was formulated in 150 mM Sodium Chloride, 40 mM Calcium Chloride,
10 mM Tris and 100 mM L-Lysine. The final pH of the thrombin was
7.4+/-0.1. The thrombin was adjusted to 25 Units/ml thrombin, which
resulted in a final composition of 0.1 units/mg of Fibrinogen or
1.3 U thrombin/cm.sup.2. 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 pipetor was filled with
fibrinogen and second repeat pipetor was filled with thrombin.
Simultaneously 0.17 ml of fibrinogen and 26 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 and lyophylized as
described above.
[0323] The performance of the test articles was determined using
the EVPCA assay as described in Example 2 above.
TABLE-US-00005 TABLE 4.1 Test Fibrinogen Thrombin % % % % Article
Dose Dose T:F Reaching Reaching Reaching Reaching Size (cm.sup.2)
(mg/cm.sup.2) (U/cm.sup.2) (U/mg) 100 mmHg 150 mmHg 200 mmHg 250
mmHg 0.7 13 1.3 0.1 100 80 40 40 1.5 13 1.3 0.1 100 80 80 60
Example 5
[0324] Dcxon.TM. Mesh support material was cut to fit into and
placed into each PETG 1.5.times.1.5 cm mold. Fifteen microliters of
2% sucrose was pipeted on top of each of the four corners of the
support material and the molds were placed inside a -80.degree. C.
freezer. PETG 1.5.times.1.5 cm molds that did not contain support
material were also placed inside the -80.degree. C. freezer. In a
third group, the same amount of support material was cut into small
pieces (approximately less than 2 mm.times.2 mm) and placed into
PETG 1.5.times.1.5 cm molds (these dressings are referred to as
having their support material `dispersed`). Once completed the
molds were placed in a -80.degree. C. freezer. AD molds remained in
the -80.degree. C. freezer for at least 60 minutes. Enzyme Research
laboratories (ERL) Fibrinogen lot 3130 was formulated in 100 mM
Sodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mM Sodium
Citrate, and 1.5% Sucrose (Fibrinogen complete buffer). In
addition, Human Serum Albumin was added to 80 mg/g of total protein
and Tween 80 (non-animal source) was added to 15 mg/g total
protein. The final pH of the fibrinogen was 7.4+/-0.1. The
fibrinogen concentration was adjusted to 36.56 mg/ml and 14.06
mg/ml. Once prepared the fibrinogen was placed on ice until use.
Thrombin was formulated in 150 mM Sodium Chloride, 40 mM Calcium
Chloride, 10 mM Tris and 100 mM L-Lysine. The final pH of the
thrombin was 7.4+/-0.1. The thrombin was adjusted to deliver 0.01,
0.1 or 1 units/mg of Fibrinogen or 2.5, 25 or 250 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 pipetor was filled with fibrinogen and
second repeat pipetor was filled with thrombin. Simultaneously 0.8
ml of fibrinogen and 133 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. Table 5.1 shows the experimental
design.
TABLE-US-00006 TABLE 5.1 Experimental Design Fibrinogen Dose
Thrombin Dose T:F Support (mg/cm.sup.2) (U/cm.sup.2) (U/mg)
material 5 0.05 0.01 Yes 5 0.05 0.01 No 5 0.05 0.01 Dispersed 5 0.5
0.1 Yes 5 0.5 0.1 No 5 0.5 0.1 Dispersed 5 5 1 Yes 5 5 1 No 5 5 1
Disperscd 13 0.13 0.01 Yes 13 0.13 0.01 No 13 0.13 0.01 Disperscd
13 1.3 0.1 Yes 13 1.3 0.1 No 13 1.3 0.1 Disperscd 13 13 1 Yes 13 13
1 No 13 13 1 Dispersed
[0325] The performance of the test articles was determined using
the EVPCA assay as described in Example 2 above.
[0326] Results:
TABLE-US-00007 TABLE 5.2 Fibrinogen Thrombin Thrombin % % % Dose
Dose (U/mg Support Reaching Reaching Reaching (mg/cm.sup.2)
(U/cm.sup.2) fibrinogen) material 150 mmHg 200 mmHg 250 mmHg 5 0.05
0.01 Yes 66.6 50 50 5 0.05 0.01 No 0 16 0 5 0.05 0.01 Dispersed
13.3 0 0 5 0.5 0.1 Yes 66.6 66 50 5 0.5 0.1 No 60 20 0 5 0.5 0.1
Dispersed 40 0 0 5 5 1 Yes 33.3 0 0 5 5 1 No 0 0 0 5 5 1 Dispersed
0 0 0 13 0.13 0.01 Yes 100 50 33.3 13 0.13 0.01 No 33.3 0 0 13 0.13
0.01 Dispersed 20 20 0 13 1.3 0.1 Yes 66.6 50 16.6 13 1.3 0.1 No 0
0 0 13 1.3 0.1 Dispersed 100 80 40 13 13 1 Yes 33.3 33.3 33.3 13 13
1 No 33.3 0 0 13 13 1 Dispersed 33.3 16.6 16.6
Example 6
[0327] Dexon.TM. Mesh support material was cut to fit into and
placed into each PETG 0.7.times.0.7 cm mold. Five microliters of 2%
sucrose was pipeted on top of each of the four corners of the
support material and the molds were placed inside a -80.degree. C.
freezer. Once completed the molds were placed in a -80.degree. C.
freezer. All molds remained in the -80.degree. C. freezer for at
least 60 minutes. Enzyme Research Laboratories (ERL) Fibrinogen lot
3130 was formulated in 100 mM Sodium Chloride, 1.1 mM Calcium
Chloride, 10 mM Tris, 10 mM Sodium Citrate, and 1.5% Sucrose
(Fibrinogen complete buffer). In addition, Human Serum Albumin was
added to 80 mg/g of total protein and Tween 80 (non-animal source)
was added to 15 mg/g total protein. The final pH of the fibrinogen
was 7.4+/-0.1. The fibrinogen concentration was adjusted to 39.2
mg/ml and 32.06 mg/ml. Once prepared the fibrinogen was placed on
ice until use. Thrombin was formulated in 150 mM Sodium Chloride,
40 mM Calcium Chloride, 10 mM Tris and 100 mM L-Lysine. The final
pH of the thrombin was 7.4+/-0.1. The thrombin was adjusted to
deliver 1 unit/mg of Fibrinogen or 250 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 pipetor was filled with fibrinogen and second repeat
pipetor was filled with thrombin. Simultaneously 0.2 ml of
fibrinogen and 33 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. Table 6.1 shows the experimental design.
TABLE-US-00008 TABLE 6.1 Experimental Design Fibrinogen Dose
Thrombin Dose T:F Support (mg/cm.sup.2) (U/cm.sup.2) (U/mg)
material 16 16.1 1 Yes 13 13.0 1 Yes
[0328] The performance of the test articles was determined using
the EVPCA assay as described in Example 2 above.
[0329] Results:
TABLE-US-00009 TABLE 6.2 Fibrinogen % Pass % Pass % Pass Dose EVCPA
at EVCPA at EVCPA at (mg/cm.sup.2) 150 mm Hg 200 mm Hg 250 mm Hg 16
66.6 33.3 16.6 13 0 0 0
Example 7
[0330] Dexon.TM. Mesh support material was cut to fit into and
placed into each PETG 1.5.times.1.5 cm mold. Fifteen microliters of
2% sucrose was pipetted on top of each
References