U.S. patent application number 17/519638 was filed with the patent office on 2022-04-14 for preventing biological tissue adhesion.
This patent application is currently assigned to 3-D Matrix, Ltd.. The applicant listed for this patent is 3-D Matrix, Ltd.. Invention is credited to Eun Seok Gil, Satoru Kobayashi, Lisa Spirio.
Application Number | 20220111126 17/519638 |
Document ID | / |
Family ID | 1000006108934 |
Filed Date | 2022-04-14 |
United States Patent
Application |
20220111126 |
Kind Code |
A1 |
Kobayashi; Satoru ; et
al. |
April 14, 2022 |
PREVENTING BIOLOGICAL TISSUE ADHESION
Abstract
Methods and materials for mitigating biological tissue adhesion
are described herein. One method for mitigating adhesion to a
biological tissue includes administering an effective amount of a
self-assembling peptide solution to the biological tissue, wherein
the self-assembling peptide is between about 7 amino acids and 32
amino acids in length and the self-assembling peptide solution
forms a hydrogel under physiological conditions.
Inventors: |
Kobayashi; Satoru;
(Chigasaki, JP) ; Gil; Eun Seok; (Action, MA)
; Spirio; Lisa; (S. Weymouth, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
3-D Matrix, Ltd. |
Tokyo |
|
JP |
|
|
Assignee: |
3-D Matrix, Ltd.
Tokyo
JP
|
Family ID: |
1000006108934 |
Appl. No.: |
17/519638 |
Filed: |
November 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16085730 |
Sep 17, 2018 |
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PCT/IB2017/000312 |
Mar 17, 2017 |
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17519638 |
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62310121 |
Mar 18, 2016 |
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62310131 |
Mar 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 31/043 20130101;
A61L 31/145 20130101 |
International
Class: |
A61L 31/14 20060101
A61L031/14; A61L 31/04 20060101 A61L031/04 |
Claims
1. A method for mitigating adhesion to a biological tissue, the
method comprising administering an effective amount of a
self-assembling peptide solution to the biological tissue, wherein
the self-assembling peptide is between about 7 amino acids and 32
amino acids in length and the self-assembling peptide solution
forms a hydrogel under physiological conditions, and wherein the
hydrogel mitigates adhesion to the biological tissue.
2. A method for mitigating adhesion between biological tissue, the
method comprising administering an effective amount of a
self-assembling peptide solution to a biological tissue at a
surgical site, wherein the self-assembling peptide is between about
7 amino acids and 32 amino acids in length and the self-assembling
peptide solution forms a hydrogel under physiological conditions,
and wherein the hydrogel mitigates adhesion of another biological
tissue to the biological tissue at the surgical site.
3-40. (canceled)
Description
FIELD OF THE INVENTION
[0001] This disclosure relates to materials and methods that may be
used in medical, research, and industrial applications. More
particularly, this disclosure relates to materials and methods that
may be used to promote anti-adhesion, for example by preventing or
mitigating biological tissue adhesion.
BACKGROUND
[0002] Postoperative tissue adhesion occurs in connection with
various procedures such as those in neurosurgery, thoracic surgery,
urology, obstetrics, gynecology, digestive system surgery and
orthopedic surgery. Adhesion generally relates to a physiological
and biological repair reaction. It is considered difficult to
prevent such adhesion completely, though the nature of adhesion may
vary between different tissues.
[0003] Currently, sodium hyaluronate, carboxymethyl cellulose,
oxidized regenerated cellulose, and expanded
polytetrafluoroethylene (ePTFE) are used clinically as
anti-adhesion materials. However, these materials may have limited
efficacy and/or side effects and/or are difficult to use. For
example, such materials are commonly provided in sheet form, which
may be difficult to immobilize at the application site. They also
may be difficult to use during endoscopic surgeries and cannot
necessarily be effectively applied to bleeding sites.
[0004] Accordingly, there remains a need for improved treatments
for preventing or mitigating biological tissue adhesion.
SUMMARY
[0005] The invention is based, at least in part, upon the discovery
that certain amphiphilic peptide solution can be surprisingly and
advantageously used for preventing or mitigating biological tissue
adhesion.
[0006] In various aspects, the invention provides a method for
mitigating adhesion to a biological tissue, the method comprising
administering an effective amount of a self-assembling peptide
solution to the biological tissue, wherein the self-assembling
peptide is between about 7 amino acids and 32 amino acids in length
and the self-assembling peptide solution forms a hydrogel under
physiological conditions, and wherein the hydrogel mitigates
adhesion to the biological tissue.
[0007] In various aspects, the invention provides a method for
mitigating adhesion between biological tissue, the method
comprising administering an effective amount of a self-assembling
peptide solution to a biological tissue at a surgical site, wherein
the self-assembling peptide is between about 7 amino acids and 32
amino acids in length and the self-assembling peptide solution
forms a hydrogel under physiological conditions, and wherein the
hydrogel mitigates adhesion of another biological tissue to the
biological tissue at the surgical site.
[0008] In various aspects, the invention provides a use of an
effective amount of a self-assembling peptide solution for
mitigating adhesion to a biological tissue, wherein the
self-assembling peptide is between about 7 amino acids and 32 amino
acids in length and the self-assembling peptide solution forms a
hydrogel under physiological conditions, and wherein the hydrogel
mitigates adhesion to the biological tissue.
[0009] In various aspects, the invention provides a use of an
effective amount of a self-assembling peptide solution for
mitigating adhesion to a biological tissue, wherein the
self-assembling peptide is between about 7 amino acids and 32 amino
acids in length and the self-assembling peptide solution forms a
hydrogel under physiological conditions, and wherein the hydrogel
mitigates adhesion of another biological tissue to the biological
tissue at the surgical site.
[0010] In various aspects, the invention provides a method of
promoting anti-adhesion, comprising: introducing a delivery device
to a target area; positioning an end of the delivery device in the
target area at which anti-adhesion is desired; administering
through the delivery device a solution comprising a self-assembling
peptide comprising between about 7 amino acids and 32 amino acids
in an effective amount and in an effective concentration to the
target area to form a hydrogel under physiological conditions of
the target area to promote anti-adhesion; and removing the delivery
device from the target area.
[0011] In various aspects, the invention provides a composition
comprising a self-assembling peptide comprising between about 7
amino acids and 32 amino acids in an effective amount and in an
effective concentration for use in forming a hydrogel under
physiological conditions to promote anti-adhesion.
[0012] In various aspects, the invention provides a kit for
promoting anti-adhesion, comprising a self-assembling peptide
comprising between about 7 amino acids and about 32 amino acids in
an effective amount to form a hydrogel under physiological
conditions to promote anti-adhesion and instructions for
administering the self-assembling peptide to a target area.
[0013] In various aspects, the invention provides a method of
promoting anti-adhesion in epicardial ablation, comprising
introducing a delivery device to a target area associated with the
epicardial ablation, positioning an end of the delivery device in
the target area at which anti-adhesion is desired, administering
through the delivery device a solution comprising a self-assembling
peptide comprising between about 7 amino acids and 32 amino acids
in an effective amount and in an effective concentration to the
target area to form a hydrogel under physiological conditions of
the target area to promote anti-adhesion, and removing the delivery
device from the target area.
[0014] In various aspects, the invention provides a kit for
promoting anti-adhesion in epicardial ablation, comprising a
self-assembling peptide comprising between about 7 amino acids and
about 32 amino acids in an effective amount to form a hydrogel
under physiological conditions to promote anti-adhesion and
instructions for administering the self-assembling peptide to a
target area associated with epicardial ablation.
[0015] In various aspects, the invention provides a method of
facilitating promotion of anti-adhesion in epicardial ablation,
comprising: providing a solution comprising a self-assembling
peptide comprising between about 7 amino acids to about 32 amino
acids in an effective amount and in an effective concentration to
form a hydrogel in a target area associated with the epicardial
ablation under physiological conditions to promote anti-adhesion;
and providing instructions for administering the solution to the
target area through introduction of the solution through a delivery
device positioned in the target area.
[0016] In various aspects, the invention provides a macroscopic
scaffold consisting essentially of a plurality of self-assembling
peptides, each of the self-assembling peptides comprising between
about 7 amino acids and about 32 amino acids in an effective amount
that is capable of being positioned within a target area associated
with epicardial ablation to promote anti-adhesion.
[0017] As will be understood by those skilled in the art, any of
the aspects above can be combined with any one or more of the
features below.
[0018] In various embodiments, the biological tissue comprises an
epicardium.
[0019] In various embodiments, the biological tissue comprises an
epicardium subjected to ablation, preferably ventricular
tachycardia ablation.
[0020] In various embodiments, the self-assembling peptide
comprises about 12 to about 16 amino acids that alternate between
hydrophobic and a hydrophilic amino acids.
[0021] In various embodiments, the self-assembling peptide
comprises a sequence selected from RADA, IEIK, TTTT, ATAT, TVTV,
ASAS, SSSS, VVVTTTT, and a combination thereof.
[0022] In various embodiments, the self-assembling peptide
comprises a sequence selected from (RADA).sub.4, (IEIK).sub.3I, and
(KLDL).sub.3.
[0023] In various embodiments, the self-assembling peptide is about
0.1 to about 10 w/v % of the solution or about 0.1 to about 3.5 w/v
% of the solution.
[0024] In various embodiments, the self-assembling peptide is about
1, about 2.5, or about 3 w/v % of the solution.
[0025] In various embodiments, the effective amount is
approximately 0.1 mL per 1 cm.sup.2 to approximately 5 mL per 1
cm.sup.2 of target area.
[0026] In various embodiments, the effective amount is
approximately 1 mL per 1 cm.sup.2 of target area.
[0027] In various embodiments, the hydrogel is formed before
administering the self-assembling peptide solution to target
area.
[0028] In various embodiments, the hydrogel is formed after
administering the self-assembling peptide solution to target
area.
[0029] In various embodiments, the solution further comprises a
biologically active agent.
[0030] In various embodiments, the solution is substantially free
of cells and/or drugs.
[0031] In various embodiments, the self-assembling peptide solution
is administered in vivo.
[0032] In various embodiments, the biological tissue is a human
tissue.
[0033] In various embodiments, the solution is an aqueous solution
and wherein a concentration of the peptide in the aqueous solution
is about 0.1 weight per volume (w/v) percent to about 3 w/v
percent.
[0034] In various embodiments, the method further comprises
visualizing the target area prior to introducing and/or subsequent
to removing the delivery device from the target area.
[0035] In various embodiments, the method further comprises
monitoring the target area after removing the delivery device.
[0036] In various embodiments, the method further comprises
preparing the solution comprising the self-assembling peptide.
[0037] In various embodiments, the target site relates to a
catheter approach to epicardial ablation.
[0038] In various embodiments, the target site is a surgical
site.
[0039] In various embodiments, the hydrogel treats, prevents,
and/or mitigates intra-abdominal adhesion formation.
[0040] In various embodiments, biological tissue comprises
intraperitoneum, cecum, intestine, preferentially large intestine,
and/or colon.
[0041] In various embodiments, hydrogel treats, prevents, and/or
mitigates pelvic adhesion formation.
[0042] In various embodiments, hydrogel treats, prevents, and/or
mitigates adhesion formation in an obstetric or gynecologic
procedure.
[0043] In various embodiments, obstetric or gynecologic procedure
comprises cesarean delivery, abdominal hysterectomy, preferentially
myomectomy, ovarian cystectomy, or surgery for an invasive
gynecologic malignancy.
[0044] In various embodiments, treating, preventing, and/or
mitigating adhesion formation treats, prevents, and/or mitigates
small bowel obstruction, infertility, chronic pain, and
dyspareunia.
[0045] These and other advantages of the present technology will be
apparent when reference is made to the following description.
DETAILED DESCRIPTION
[0046] The invention is based, at least in part, upon the discovery
that certain amphiphilic peptide solution can be surprisingly and
advantageously used to mitigate biological tissue adhesion.
[0047] Broadly, the methods and materials may employ a hydrogel
barrier for preventing or mitigating biological tissue adhesion.
The mitigation may be partial or complete. The materials and
methods may include administration, application, or injection of a
self-assembling peptide, or a solution comprising a self-assembling
peptide, or a composition comprising a self-assembling peptide, to
a predetermined or desired target area, thereby preventing or
mitigating biological tissue adhesion.
[0048] For example, in various aspects and embodiments, the
invention provides methods and materials for mitigating adhesion to
a biological tissue. The method includes administering an effective
amount of a self-assembling peptide solution to the biological
tissue, where the self-assembling peptide is between about 7 amino
acids and 32 amino acids in length and the self-assembling peptide
solution forms a hydrogel under physiological conditions. In
certain embodiments, the adhesion is between two biological
tissues. In other certain embodiments, the biological tissue may
comprise an epicardium. In yet other certain embodiments, the
biological tissue may comprise an epicardium subjected to ablation.
In further other certain embodiments, the biological tissue may
comprise an epicardium subjected to ventricular tachycardia
ablation.
[0049] Through administration of the solution comprising the
self-assembling peptide, a hydrogel barrier may be formed. The
hydrogel barrier may be formed in the target area to promote
anti-adhesion. The self-organizing or self-assembling peptides of
the present disclosure may include application of the
self-organizing or self-assembling peptides to a predetermined or
desired target. The self-organizing or self-assembling peptide may
be applied or introduced to a target site in the form of a peptide
solution, hydrogel, membrane or other form. A target site may be a
predetermined area of a subject that requires a particular
treatment. In some embodiments, the target site may relate to a
surgical site, such as an endoscopic surgical site. In other
embodiments, the target site may be a bleeding site.
[0050] Various features of the invention are discussed, in turn,
below.
[0051] As used herein, the term "subject" is intended to include
human and non-human animals, for example, vertebrates, large
animals, and primates. In certain embodiments, the subject is a
mammalian subject, and in particular embodiments, the subject is a
human subject. Although applications with humans are clearly
foreseen, veterinary applications, for example, with non-human
animals, are also envisaged herein. The term "non-human animals" of
the invention includes all vertebrates, for example, non-mammals
(such as birds, for example, chickens; amphibians; reptiles) and
mammals, such as non-human primates, domesticated, and
agriculturally useful animals, for example, sheep, dog, cat, cow,
pig, rat, among others.
[0052] The term "self-assembling peptide" may refer to a peptide
comprising a self-assembling motif. Self-assembling peptides are
peptides that are capable of self-assembly into structures
including but not limited to, macroscopic membranes or
nanostructures. For example, the self-assembling peptide may
exhibit a beta-sheet structure in aqueous solution in the presence
of specific conditions to induce the beta-sheet structure. These
specific conditions may include adjusting the pH of a
self-assembling peptide solution. The adjustment may be an increase
or a decrease in the pH of the self-assembling peptide solution.
The increase in pH may be an increase in pH to a physiological pH.
The specific conditions may also include adding a cation, such as a
monovalent cation, to a self-assembling peptide solution. The
specific conditions may include conditions related to the pancreas.
The self-assembling peptides may be referred to as or be a part of
a composition, peptide solution, peptide powder, hydrogel, or
scaffold. The self-assembling peptide may be administered to a
target area in the form of a peptide solution, composition,
hydrogel, membrane, scaffold or other form.
[0053] During self-organization or self-assembly, the peptide may
form nanofibers. The self-organization or self-assembly may cause
gelling of the peptide in solution. The gelling may provide or form
a hydrogel. The peptide may form a beta-sheet spontaneously in the
solution under the physiological pH level. The peptide may form a
beta-sheet spontaneously in the solution under physiological
conditions and/or in the presence of a cation.
[0054] The term "hydrogel" may refer to a material that is
comprised of a polymer and a high percentage of water, for example,
at least 90% water.
[0055] The self-assembling peptide may be an amphiphilic
self-assembling peptide. By "amphiphilic" it is meant that the
peptide comprises hydrophobic portions and hydrophilic portions. In
some embodiments, an amphiphilic peptide may comprise, consist
essentially of, or consist of alternating hydrophobic amino acids
and hydrophilic amino acids. By alternating, it is meant to include
a series of three or more amino acids that alternate between a
hydrophobic amino acid and a hydrophilic amino acid, and it need
not include each and every amino acid in the peptide sequence
alternating between a hydrophobic and a hydrophilic amino acid. In
certain embodiments, the peptide may comprise a first portion that
is amphiphilic and a second portion that is not amphiphilic.
[0056] The self-assembling peptide, also referred to herein as
"peptide" or "self-assembling oligopeptides," may be administered
to the pre-determined or desired target area in the form of a
self-assembling peptide solution, composition, hydrogel, membrane,
scaffold or other form. The hydrogel may also be referred to as a
membrane or scaffold throughout this disclosure.
[0057] The pre-determined or desired target area may be biological
tissue. The pre-determined or desired target area may be at or near
the location of a surgery. The pre-determined or desired target
area may be established based on the site of or other area that may
have undergone a surgical procedure, or an unintentional or
intentional trauma. In some embodiments, the target site may be an
epicardium. In some other embodiments, the target site may be an
epicardium subjected to ablation. In further some other
embodiments, the target site may be an epicardium subjected to
ventricular tachycardia ablation. In various embodiments, the
target site comprises intraperitoneum, cecum, intestine,
preferentially large intestine, and/or colon. In various
embodiments, the target site comprises a biological tissue subject
to cesarean delivery, abdominal hysterectomy, preferentially
myomectomy, ovarian cystectomy, or surgery for an invasive
gynecologic malignancy.
[0058] The self-assembling peptide solution may be an aqueous
self-assembling peptide solution. The self-assembling peptide may
be administered, applied, or injected in a solution that is
substantially cell-free, or free of cells. In certain embodiments,
the self-assembling peptide may be administered, applied, or
injected in a solution that is cell-free or free of cells.
[0059] The self-assembling peptide may also be administered,
applied, or injected in a solution that is substantially drug-free
or free of drugs. In certain embodiments, the self-assembling
peptide may be administered, applied, or injected in a solution
that is drug-free or free of drugs. In certain other embodiments,
the self-assembling peptide may be administered, applied, or
injected in a solution that is substantially cell-free and
substantially drug-free. In still further certain other
embodiments, the self-assembling peptide may be administered,
applied, or injected in a solution that is cell-free and drug
free.
[0060] The self-assembling peptide solution may comprise, consist
of, or consist essentially of the self-assembling peptide. The
self-assembling peptide may be in a modified or unmodified form. By
modified, it is meant that the self-assembling peptide may have one
or more domains that comprise one or more amino acids that, when
provided in solution by itself, would not self-assemble. By
unmodified, it is meant that the self-assembling peptide may not
have any other domains other than those that provide for
self-assembly of the peptide. That is, an unmodified peptide
consists of alternating hydrophobic and hydrophilic amino acids
that may self-assemble into a beta-sheet, or a macroscopic
structure, such as a hydrogel.
[0061] The self-assembling peptide can be at least about 7 amino
acids, between about 7 and 32 amino acids, or between about 12 and
16 amino acids. Other peptides that do not comprise, consist of, or
consist essentially of at least about 7 amino acids may be
contemplated by this disclosure. The self-assembling peptides may
be composed of about 6 to about 200 amino acid residues. In certain
embodiments, about 8 to about 32 residues may be used in the
self-assembling peptides, while in other embodiments
self-assembling peptides may have about 7 to about 17 residues. In
certain other examples, the self-assembling peptides may be
peptides of at least 8 amino acids, at least about 12 amino acids,
or at least about 16 amino acids.
[0062] The materials and methods may comprise administering a
self-assembling peptide to a predetermined or desired target. The
peptide may be administered as a hydrogel or form a hydrogel upon
administration. A hydrogel is a term that may refer to a colloidal
gel that is dispersed in water. The hydrogel may also be referred
to as a membrane or scaffold throughout this disclosure. The
systems and methods may also comprise applying a self-assembling
peptide to a predetermined or desired target as a solution such as
an aqueous peptide solution.
[0063] The term "administering," is intended to include, but is not
limited to, applying, introducing or injecting the self-assembling
peptide, in one or more of various forms including, but not limited
to, by itself, by way of solution, such as an aqueous solution, or
by way of a composition, hydrogel, or scaffold, with or without
additional components.
[0064] The method may comprise introducing a delivery device at or
near a predetermined or desired target area of a subject. The
method may comprise introducing a delivery device comprising at
least one of a syringe, tube, pipette, catheter, catheter syringe,
or other needle-based device to the predetermined or desired target
area of a subject. The self-assembling peptide may be administered
by way of a syringe, tube, pipette, catheter, catheter syringe, or
other needle-based device to the predetermined or desired target
area of a subject. The gauge of the syringe needle may be selected
to provide an adequate flow of a composition, a solution, a
hydrogel, or a liquid from the syringe to the target area. This may
be based in some embodiments on at least one of the amount of
self-assembling peptide in a composition, peptide solution, or a
hydrogel being administered, the concentration of the peptide
solution, in the composition, or the hydrogel, and the viscosity of
the peptide solution, composition, or hydrogel. The delivery device
may be a conventional device or designed to accomplish at least one
of to reach a specific target area, achieve a specific dosing
regime, deliver a specific target volume, amount, or concentration,
and deliver accurately to a target area.
[0065] The method of mitigating biological tissue adhesion may
comprise introducing a delivery device into the subject and
positioning an end of the delivery device in a predetermined or
target area, such as a portion of a surgical site. The
self-assembling peptide may be administered by way of a delivery
device to the target area in which at least is desired. The use of
a delivery device may provide a more selective administration of
the peptide to provide for a more accurate delivery to the target
area. Selective administration of the peptide may allow for
enhanced and more targeted delivery of the peptide solution,
composition, or hydrogel such that is successful and positioned in
the desired location in an accurate manner. The selective
administration may provide enhanced, targeted delivery that
markedly improves the positioning and effectiveness of the
treatment over use of another delivery device. Delivery devices
that may be used in the systems, methods, and kits of the
disclosure may include a syringe, tube, needle, pipette, syringe
catheter, other needle-based device, or catheter.
[0066] Use of a delivery device, such as a catheter, may include
use of accompanying devices, such as a guidewire used to guide the
catheter into position, or an endoscope that may allow proper
placement of the catheter and visualization of the target area,
and/or the path to the target area. The endoscope may be a tube
that may comprise at least one of a light and a camera or other
visualization device to allow images of the subject's body to be
viewed. The guidewire or endoscope may be introduced into the
subject, for example, by way of an incision in the skin. The
endoscope may be introduced to the target area prior to introducing
the delivery device to the target area.
[0067] The use of the delivery device, such as a syringe, tube,
needle, pipette, syringe catheter, other needle-based device,
catheter, or endoscope may require determining the diameter or size
of the opening in which there is a target area, such that at least
a portion of the syringe, tube, needle, pipette, syringe catheter,
other needle-type device, catheter, or endoscope may enter the
opening to administer the peptide, peptide solution, composition,
or hydrogel to the target area.
[0068] In certain embodiments, the hydrogel may be formed in vitro
and administered to the desired location in vivo. In certain
examples, this location may be the target area. In other examples,
this location may be upstream, downstream of the area, or
substantially near the area. It may be desired to allow a migration
of the hydrogel to the area in which it is desired to.
Alternatively, another procedure may position the hydrogel in the
area in which it is desired. The desired location or target area
may be at least a portion of an area in which it is desired to
provide or promote anti-adhesion, e.g., prevent or mitigate
biological tissue adhesion, in a subject.
[0069] In certain aspects of the disclosure, the hydrogel may be
formed in vivo. A solution comprising the self-assembling peptide,
such as an aqueous solution, may be inserted to an in vivo location
or area of a subject to provide or promote anti-adhesion, e.g.,
prevent or mitigate biological tissue adhesion, in a subject. In
certain examples, the hydrogel may be formed in vivo at one
location, and allowed to migrate to the area in which it is desired
to provide or promote anti-adhesion, e.g., prevent or mitigate
biological tissue adhesion, in a subject. Alternatively, another
procedure may place the hydrogel in the area in which it is desired
to provide or promote anti-adhesion, e.g., prevent or mitigate
biological tissue adhesion, in a subject. The peptides of the
present disclosure may be in the form of a powder, a solution, a
gel, or the like. Since the self-assembling peptide gels in
response to changes in solution pH and salt concentration, it can
be distributed as a liquid that gels upon contact with a subject
during application or administration.
[0070] In certain environments, the peptide solution may be a weak
hydrogel and, as a result, it may be administered by way of a
delivery device as described herein.
[0071] In accordance with some embodiments, the self-assembling
peptides may be amphiphilic, alternating between hydrophobic amino
acids and hydrophilic amino acids.
[0072] In accordance with one or more embodiments, a subject may be
evaluated to determine a need to provide or promote anti-adhesion,
e.g., prevent or mitigate biological tissue adhesion, in a subject.
Once the evaluation has been completed, a peptide solution to
administer to the subject may be prepared.
[0073] In some embodiments, a biologically active agent may be used
with the materials and methods of the present disclosure. A
biologically active agent may comprise a compound, including a
peptide, DNA sequence, chemical compound, or inorganic or organic
compound that may impart some activity, regulation, modulation, or
adjustment of a condition or other activity in a subject or in a
laboratory setting. The biologically active agent may interact with
another component to provide such activity. The biologically active
agent may be referred to as a drug in accordance with some
embodiments herein. In certain embodiments, one or more
biologically active agents may be gradually released to the outside
of the peptide system. For example, the one or more biologically
active agents may be gradually released from the hydrogel. Both in
vitro and in vivo testing has demonstrated this gradual release of
a biologically active agent. The biologically active agent may be
added to the peptide solution prior to administering to a subject,
or may be administered separately from the solution to the subject.
The one or more biologically active agents may be encapsulated
within the system, for example, they may be encapsulated in the
hydrogel, solution, or nanofibers.
[0074] The self-assembling peptides may exhibit a beta-sheet
structure in aqueous solution in the presence of physiological pH
and/or a cation, such as a monovalent cation, or other conditions
applicable to a surgical site.
[0075] The peptides may be generally stable in aqueous solutions
and self-assemble into large, macroscopic structures, scaffolds, or
matrices when exposed to physiological conditions, physiological
pH, or physiological levels of salt. Once the hydrogel is formed it
may not decompose, or may decompose or biodegrade after a period of
time. The rate of decomposition may be based at least in part on at
least one of the amino acid sequence and conditions of its
surroundings.
[0076] By "macroscopic" it is meant as having dimensions large
enough to be visible under magnification of 10-fold or less. In
preferred embodiments, a macroscopic structure is visible to the
naked eye. A macroscopic structure may be transparent and may be
two-dimensional, or three-dimensional. Typically each dimension is
at least 10 .mu.m, in size. In certain embodiments, at least two
dimensions are at least 100 .mu.m, or at least 1000 .mu.m in size.
Frequently at least two dimensions are at least 1-10 mm in size,
10-100 mm in size, or more. In certain embodiments, the size of the
filaments may be about 10 nanometers (nm) to about 20 nm. The
interfilament distance may be about 50 nm to about 80 nm. The
self-assembling peptides of the present disclosure may have a
length of about 5 nm. The self-assembling peptides of the present
disclosure may have a nanofiber diameter in a range of about 10 nm
to about 20 nm and an average pore size is in a range of about 5 nm
to about 200 nm. In certain embodiments, the nanofiber diameter,
the pore size, and the nanofiber density may be controlled by at
least one of the concentration of peptide solution used and the
amount of peptide solution used, such as the volume of peptide
solution. As such, at least one of a specific concentration of
peptide in solution and a specific amount of peptide solution to
provide at least one of a desired nanofiber diameter, pore size,
and density to adequately provide for anti-adhesion may be
selected.
[0077] "Physiological conditions" may occur in nature for a
particular organism, cell system, or subject which may be in
contrast to artificial laboratory conditions. The conditions may
comprise one or more properties such as one or more particular
properties or one or more ranges of properties. For example, the
physiological conditions may include a temperature or range of
temperatures, a pH or range of pH's, a pressure or range of
pressures, and one or more concentrations of particular compounds,
salts, and other components. For example, in some examples, the
physiological conditions may include a temperature in a range of
about 20 to about 40 degrees Celsius. In some examples, the
atmospheric pressure may be about 1 atm. The pH may be in the range
of a physiological pH. For example, the pH may be in a range of
about 6 to about 8. The physiological conditions may include
cations such as monovalent metal cations that may induce membrane
or hydrogel formation. These may include sodium chloride (NaCl).
The physiological conditions may also include a glucose
concentration, sucrose concentration, or other sugar concentration,
of between about 1 mM and about 20 mM.
[0078] The peptides may also be complementary and structurally
compatible. Complementary refers to the ability of the peptides to
interact through ionized pairs and/or hydrogen bonds which form
between their hydrophilic side-chains, and structurally compatible
refers to the ability of complementary peptides to maintain a
constant distance between their peptide backbones. Peptides having
these properties participate in intermolecular interactions which
result in the formation and stabilization of beta-sheets at the
secondary structure level and interwoven filaments at the tertiary
structure level.
[0079] Both homogeneous and heterogeneous mixtures of peptides
characterized by the above-mentioned properties may form stable
macroscopic membranes, filaments, and hydrogels. Peptides which are
self-complementary and self-compatible may form membranes,
filaments, and hydrogels in a homogeneous mixture. Heterogeneous
peptides, including those which cannot form membranes, filaments,
and hydrogels in homogeneous solutions, which are complementary
and/or structurally compatible with each other may also
self-assemble into macroscopic membranes, filaments, and
hydrogels.
[0080] The membranes, filaments, and hydrogels may be
non-cytotoxic. The hydrogels of the present disclosure may be
digested and metabolized in a subject. The hydrogels may be
biodegraded in 30 days or less. They have a simple composition, are
permeable, and are easy and relatively inexpensive to produce in
large quantities. The membranes and filaments, hydrogels or
scaffolds may also be produced and stored in a sterile condition.
The optimal lengths for membrane formation may vary with at least
one of the amino acid composition, solution conditions, and
conditions at the target site.
[0081] The amino acids of the self-assembling or amphiphilic
peptides may be selected from d-amino acids, 1-amino acids, or
combinations thereof. The hydrophobic amino acids may include Ala,
Val, Ile, Met, Phe, Tyr, Trp, Ser, Thr and Gly. The hydrophilic
amino acids may be basic amino acids, for example, Lys, Arg, His,
Orn; acidic amino acids, for example, Glu, Asp, or amino acids
which form hydrogen bonds, for example, Asn, Gln. Acidic and basic
amino acids may be clustered on a peptide. The carboxyl and amino
groups of the terminal residues may be protected or not protected.
Membranes or hydrogels may be formed in a homogeneous mixture of
self-complementary and self-compatible peptides or in a
heterogeneous mixture of peptides which are complementary and
structurally compatible to each other. Peptides fitting the above
criteria may self-assemble into macroscopic membranes under
suitable conditions, described herein.
[0082] The peptide may comprise or consist essentially of a
sequence selected from the group consisting of: RADA, IEIK, TTTT,
ATAT, TVTV, ASAS, SSSS, VVVTTTT, and combinations thereof. Other
peptide sequences are contemplated and are within the scope of this
disclosure. In certain embodiments, the peptide may comprise or
consist essentially of a repeated sequence of arginine, alanine,
and aspartic acid.
[0083] The peptides of the present disclosure may include peptides
having the repeating sequence of arginine, alanine, aspartic acid
and alanine (Arg-Ala-Asp-Ala (RADA)), and such peptide sequences
may be represented by (RADA).sub.p, wherein p=2-50.
[0084] Other peptide sequences may be represented by
self-assembling peptides having the repeating sequence of
isoleucine, glutamic acid, isoleucine and lysine (Ile-Glu-Ile-Lys
(IEIK)), and such peptide sequences are represented by
(IEIK).sub.p, wherein p=2-50. Other peptide sequences may be
represented by self-assembling peptides having the repeating
sequence of isoleucine, glutamic acid, isoleucine and lysine
(Ile-Glu-Ile-Lys (IEIK)), and such peptide sequences are
represented by (IEIK).sub.pI, wherein p=2-50.
[0085] Other peptide sequences may be represented by
self-assembling peptides having the repeating sequence of lysine,
leucine, aspartic acid, and leucine (Lys-Leu-Asp-Leu (KLDL)), and
such peptide sequences are represented by (KLDL).sub.p, wherein
p=2-50. Other peptide sequences may be represented by
self-assembling peptides having the repeating sequence of lysine,
leucine, and aspartic acid (Lys-Leu-Asp (KLD)), and such peptide
sequences are represented by (KLD).sub.p, wherein p=2-50. As
specific examples of self-assembling peptides according to the
invention there may be a self-assembling peptide RADA16 having the
sequence
Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala
(RADA)4, a self-assembling peptide IEIK13 having the sequence
Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (IEIK).sub.3I,
a self-assembling peptide IEIK17 having the sequence
Ile-Glu-Ile-Lys-Ile-Glu-Ile
-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (IEIK).sub.4I or a
self-assembling peptide KLDL12 having the sequence
Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (KLDL).sub.3.
[0086] The criteria of amphiphilic sequence, length,
complementarity and structural compatibility apply to heterogeneous
mixtures of peptides. For example, two different peptides may be
used to form the membranes: peptide A,
Val-Arg-Val-Arg-Val-Asp-Val-Asp-Val-Arg-Val -Arg-Val-Asp-Val-Asp
(VRVRVDVDVRVRVDVD) has Arg and Asp as the hydrophilic residues and
peptide B,
Ala-Asp-Ala-Asp-Ala-Lys-Ala-Lys-Ala-Asp-Ala-Asp-Ala-Lys-Ala-Lys
ADADAKAKADADAKAK, has Lys and Asp. Peptides A and B are
complementary; the Arg on A can form an ionized pair with the Asp
on B and the Asp on A can form an ionized pair with the Lys on B.
Thus, in a heterogeneous mixture of peptides A and B, membranes
would likely form, but they would be homogeneously composed of
either peptide A or B.
[0087] Membranes and hydrogels can also be formed of heterogeneous
mixtures of peptides, each of which alone would not form membranes,
if they are complementary and structurally compatible to each
other. For example, mixtures of (Lys-Ala-Lys-Ala).sub.4
(KAKA).sub.4 and (Glu-Ala-Glu-Ala)4 (EAEA).sub.4 or of
(Lys-Ala-Lys-Ala).sub.4 (KAKA).sub.4 and (Ala-Asp-Ala-Asp).sub.4
(ADAD).sub.4 would be expected to form membranes, but not any of
these peptides alone due to lack of complementarity.
[0088] Peptides, which are not perfectly complementary or
structurally compatible, can be thought of as containing mismatches
analogous to mismatched base pairs in the hybridization of nucleic
acids. Peptides containing mismatches can form membranes if the
disruptive force of the mismatched pair is dominated by the overall
stability of the interpeptide interaction. Functionally, such
peptides can also be considered as complementary or structurally
compatible. For example, a mismatched amino acid pair may be
tolerated if it is surrounded by several perfectly matched pairs on
each side.
[0089] Each of the peptide sequences disclosed herein may provide
for peptides comprising, consisting essentially of, and consisting
of the amino acid sequences recited.
[0090] The present disclosure provides materials, methods, and kits
for solutions, hydrogels, and scaffolds comprising, consisting
essentially of, or consisting of the peptides recited herein.
[0091] A 1 weight per volume (w/v) percent aqueous (water) solution
and a 2.5 w/v percent of (RADA).sub.4 is available as the product
PuraMatrix.TM. peptide hydrogel by 3-D Matrix Co., Ltd.
[0092] Certain peptides may contain sequences which are similar to
the cell attachment ligand RGD (Arginine-Glycine-Aspartic acid).
The suitability of these peptides for supporting in vitro cell
growth was tested by introducing a variety of cultured primary and
transformed cells to homopolymer sheets of
Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys
(AEAEAKAKAEAEAKAK (EAK16)), RAD16, RADA16, and heteropolymers of
RAD16 and EAK16. The RAD-based peptides may be of particular
interest because the similarity of this sequence to RGD. The RAD
sequence is a high affinity ligand present in the extracellular
matrix protein tenascin and is recognized by integrin receptors.
The EAK16 peptide and other peptides disclosed herein were derived
from a region of a yeast protein, zuotin.
[0093] A list of peptides that may form membranes, hydrogels or
scaffolds in homogeneous or heterogeneous mixtures are listed in
Table 1.
TABLE-US-00001 TABLE 1 Potential hydrogel-forming peptides SEQUENCE
SEQ ID NAME (N .fwdarw. C) NO RADA RADA SEQ ID NO: 1 IEIK IEIK SEQ
ID NO: 2 TTTT TTTT SEQ ID NO: 3 ATAT ATAT SEQ ID NO: 4 TVTV TVTV
SEQ ID NO: 5 ASAS ASAS SEQ ID NO: 6 SSSS SSSS SEQ ID NO: 7 VVVTTTT
VVVTTTT SEQ ID NO: 8 KLDL KLDL SEQ ID NO: 9 KLD KLD SEQ ID NO: 10
(RADA).sub.4 RADARADARADARADA SEQ ID NO: 11 (IEIK).sub.3I
IEIKIEIKIEIKI SEQ ID NO: 12 (IEIK).sub.4I IEIKIEIKIEIKIEIKI SEQ ID
NO: 13 (KLDL).sub.3 KLDLKLDLKLDL SEQ ID NO: 14 Peptide A
VRVRVDVDVRVRVDVD SEQ ID NO: 15 Peptide B ADADAKAKADADAKAK SEQ ID
NO: 16 (KAKA).sub.4 KAKAKAKAKAKAKAKA SEQ ID NO: 17 (EAEA).sub.4
EAEAEAEAEAEAEAEA SEQ ID NO: 18 (ADAD).sub.4 ADADADADADADADAD SEQ ID
NO: 19 EAK16 AEAEAKAKAEAEAKAK SEQ ID NO: 20 RAD16 ARADARADARADARAD
SEQ ID NO: 21 KAKA16 KAKAKAKAKAKAKAKA SEQ ID NO: 22 KAKA5 KAKAK SEQ
ID NO: 23 KAE16 AKAKAEAEAKAKAEAE SEQ ID NO: 24 AKE16
AKAEAKAEAKAEAKAE SEQ ID NO: 25 EKA16 EAKAEAKAEAKAEAKA SEQ ID NO: 26
EAK8 AEAEAKAK SEQ ID NO: 27 EAK12 AEAKAEAEAKAK SEQ ID NO: 28 KEA16
KAEAKAEAKAEAKAEA SEQ ID NO: 29 AEK16 AEAKAEAKAEAKAEAK SEQ ID NO: 30
ARD8 ARARADAD SEQ ID NO: 31 DAR16 ADADARARADADARAR SEQ ID NO: 32
RAD16 ARADARADARADARAD SEQ ID NO: 33 DRA16 DARADARADARADARA SEQ ID
NO: 34 ADR16 ADARADARADARADAR SEQ ID NO: 35 ARA16 ARARADADARARADAD
SEQ ID NO: 36 ARDAKE16 ARADAKAEARADAKAE SEQ ID NO: 37 AKEW16
AKAEARADAKAEARAD SEQ ID NO: 38 ARKADE16 ARAKADAEARAKADAE SEQ ID NO:
39 AKRAED16 AKARAEADAKARADAE SEQ ID NO: 40 AQ16 AQAQAQAQAQAQAQAQ
SEQ ID NO: 41 VQ16 VQVQVQVQVQVQVQVQ SEQ ID NO: 42 YQ16
YQYQYQYQYQYQYQYQ SEQ ID NO: 43 HQ16 HQHQHQHQHQHQHQHQ SEQ ID NO: 44
AN16 ANANANANANANANAN SEQ ID NO: 45 VN16 VNVNVNVNVNVNVNVN SEQ ID
NO: 46 YN16 YNYNYNYNYNYNYNYN SEQ ID NO: 47 HN16 HNHNHNHNHNHNHNHN
SEQ ID NO: 48 ANQ16 ANAQANAQANAQANAQ SEQ ID NO: 49 AQN16
AQANAQANAQANAQAN SEQ ID NO: 50 VNQ16 VNVQVNVQVNVQVNVQ SEQ ID NO: 51
VQK16 VQVNVQVNVQVNVQVN SEQ ID NO: 52 YNQ16 YNYQYNYQYNYQYNYQ SEQ ID
NO: 53 YQN16 YQYNYQYNYQYNYQYN SEQ ID NO: 54 HNQ16 HNHQHNHQHNHQHNHQ
SEQ ID NO: 55 HQN16 HQHNHQHNHQHNHQHN SEQ ID NO: 56 AKQD18
AKAQADAKAQADAKAQAD SEQ ID NO: 57 VKQ18 VKVQVDVKVQVDVKVQVD SEQ ID
NO: 58 YKQ18 YKYQYDYKYQYDYKYQYD SEQ ID NO: 59 HKQ18
HKHQHDHKHQHDHKHQHD SEQ ID NO: 60 RAD RAD SEQ ID NO: 61 AAAAAAK
AAAAAAK SEQ ID NO: 62 AAAAAAD AAAAAAD SEQ ID NO: 63 TTTTTTT TTTTTTT
SEQ ID NO: 64 ATATATAT ATATATAT SEQ ID NO: 65 TVTVTVTV TVTVTVTV SEQ
ID NO: 66 ASASASAS ASASASAS SEQ ID NO: 67 SSSSSSS SSSSSSS SEQ ID
NO: 68 (RADA).sub.50 RADARADARADARADARA SEQ ID DARADARADARADARADA
NO: 69 RADARADARADARADARA DARADARADARADARADA RADARADARADARADARA
DARADARADARADARADA RADARADARADARADARA DARADARADARADARADA
RADARADARADARADARA DARADARADARADARADA RADARADARADARADARA DA
(IEIK).sub.50 IEIKIEIKIEIKIEIKIE SEQ ID IKIEIKIEIKIEIKIEIK NO: 70
IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE
IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK
IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE IK
(IEIK).sub.50I IEIKIEIKIEIKIEIKIE SEQ ID IKIEIKIEIKIEIKIEIK NO: 71
IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE
IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK
IEIKIEIKIEIKIEIKIE IKIEIKIEIKIEIKIEIK IEIKIEIKIEIKIEIKIE IKI
(KLDL).sub.50I KLDLKLDLKLDLKLDLKL SEQ ID
DLKLDLKLDLKLDLKLDL NO: 72 KLDLKLDLKLDLKLDLKL DLKLDLKLDLKLDLKLDL
KLDLKLDLKLDLKLDLKL DLKLDLKLDLKLDLKLDL KLDLKLDLKLDLKLDLKL
DLKLDLKLDLKLDLKLDL KLDLKLDLKLDLKLDLKL DLKLDLKLDLKLDLKLDL
KLDLKLDLKLDLKLDLKL DL (KLD).sub.50 KLDKLDKLDKLDKLDKLD SEQ ID
KLDKLDKLDKLDKLDKLD NO: 73 KLDKLDKLDKLDKLDKLD KLDKLDKLDKLDKLDKLD
KLDKLDKLDKLDKLDKLD KLDKLDKLDKLDKLDKLD KLDKLDKLDKLDKLDKLD
KLDKLDKLDKLDKLDKLD KLDKLD (KLDL).sub.2 KLDLKLDL SEQ ID NO: 74
(KLDL).sub.3 KLDLKLDLKLDL SEQ ID NO: 75 (AGAG).sub.4
AGAGAGAGAGAGAGAG SEQ ID NO: 76 (LALA).sub.4 LALALALALALALALA SEQ ID
NO: 77 LALAL LALAL SEQ ID NO: 78 (ALALAGAG).sub.2 ALALAGAGALALAGAG
SEQ ID NO: 79 (ALAG).sub.4 ALAGALAGALAGALAG SEQ ID NO: 80
(GALA).sub.4 GALAGALAGALAGALA SEQ ID NO: 81 AGAGALAL AGAGALAL SEQ
ID NO: 82 AGALAGAGA AGALAGAGALAL SEQ ID LAL NO: 83 (LAGA).sub.4
LAGALAGALAGALAGA SEQ ID NO: 84 (AGAL).sub.4 AGALAGALAGALAGAL SEQ ID
NO: 85
[0094] Without wishing to be bound by any particular theory, it is
believed that the self-assembly of the peptides may be attributable
to hydrogen bonding and hydrophobic bonding between the peptide
molecules by the amino acids composing the peptides.
[0095] As used herein, an "effective amount" or a "therapeutically
effective amount" refers to an amount of a peptide, peptide
solution or hydrogel effective to prevent or mitigate biological
tissue adhesion in a subject. In certain embodiments, such an
"effective amount" or "therapeutically effective amount" may refer
to an amount of a peptide, peptide solution or hydrogel which is
effective, upon single or multiple administration (application or
injection) to a subject, in treating, or in curing, alleviating,
relieving or improving a subject with a disorder beyond that
expected in the absence of such treatment. This may include a
particular concentration or range of concentrations of peptide in
the peptide solution or hydrogel and additionally, or in the
alternative, a particular volume or range of volumes of the peptide
solution or hydrogel. The method of facilitating may comprise
providing instructions to prepare at least one of the effective
amount and the effective concentration.
[0096] The self-assembling peptides of the present disclosure, such
as RADA16, may be peptide sequences that lack a distinct
physiologically or biologically active motif or sequence, and
therefore may not impair intrinsic cell function. Physiologically
active motifs may control numerous intracellular phenomena such as
transcription, and the presence of physiologically active motifs
may lead to phosphorylation of intracytoplasmic or cell surface
proteins by enzymes that recognize the motifs. When a
physiologically active motif is present in a peptide tissue
anti-adhesion agent, transcription of proteins with various
functions may be activated or suppressed. The self-assembling
peptides, of the present disclosure may lack such physiologically
active motifs and therefore do not carry this risk.
[0097] The optimal lengths for membrane formation may vary with the
amino acid composition. A stabilization factor contemplated by the
peptides of the present disclosure is that complementary peptides
maintain a constant distance between the peptide backbones.
Peptides which can maintain a constant distance upon pairing are
referred to herein as structurally compatible. The interpeptide
distance can be calculated for each ionized or hydrogen bonding
pair by taking the sum of the number of unbranched atoms on the
side-chains of each amino acid in the pair. For example, lysine has
5 and glutamic acid has 4 unbranched atoms on its side-chains,
respectively.
[0098] The dosage, for example, volume or concentration,
administered (for example, applied or injected) may vary depending
upon the form of the peptide (for example, in a peptide solution,
hydrogel, or in a dried form, such as a lyophilized form) and the
route of administration utilized. The exact formulation, route of
administration, volume, and concentration can be chosen in view of
the subject's condition and in view of the particular target area
or location that the peptide solution, hydrogel, or other form of
peptide will be administered. Lower or higher doses than those
recited herein may be used or required. Specific dosage and
treatment regimens for any particular subject may depend upon a
variety of factors, which may include the specific peptide or
peptides employed, the dimension of the area that is being treated,
the desired thickness of the resulting hydrogel that may be
positioned in the desired target area, and the length of time of
treatment. Other factors that may affect the specific dosage and
treatment regimens include age, body weight, general health status,
sex, time of administration, rate of degradation, the severity and
course of the disease, condition or symptoms, and the judgment of
the treating physician. In certain embodiments, the peptide
solution may be administered in a single dose. In other
embodiments, the peptide solution may be administered in more than
one dose, or multiple doses. The peptide solution may be
administered in at least two doses.
[0099] An effective amount and an effective concentration of the
peptide solution may be selected to at least partially provide or
promote anti-adhesion, e.g., prevent or mitigate biological tissue
adhesion. In some embodiments, at least one of the effective amount
and the effective concentration may be based in part on a dimension
or diameter of the target area. In other embodiments, at least one
of the effective amount and the effective concentration is based in
part on the flow rate of one or more fluids at or near the target
area.
[0100] The effective amount may include volumes of from about 0.1
milliliters (mL) to about 100 mL of a peptide solution. The
effective amount may include volumes of from about 0.1 mL to about
10 mL of a peptide solution. In certain embodiments, the effective
amount may be about 0.5 mL. In other embodiments, the effective
amount may be about 1.0 mL. In yet other embodiments, the effective
amount may be about 1.5 mL. In still yet other embodiments, the
effective amount may be about 2.0 mL. In some other embodiments,
the effective amount may be about 3.0 mL.
[0101] In certain embodiments, the effective amount may be
approximately 0.1 mL per 1 cm.sup.2 to approximately 5 mL per 1
cm.sup.2 of target area. In certain embodiments, the effective
amount may be approximately 1 mL per 1 cm.sup.2 of target area.
This effective amount may be used related to a concentration, such
as a 2.5 weight per volume percent of a peptide solution of the
present disclosure.
[0102] The effective concentration may be, as described herein, an
amount that may provide for or promote anti-adhesion, e.g., prevent
or mitigate biological tissue adhesion,. Various properties at or
near the target site may contribute to the selection or
determination of the effective concentration including at least one
of a dimension or diameter of the target area, and the flow rate of
one or more fluids at or near the target area.
[0103] The effective concentration may include peptide
concentrations in the solution in a range of about 0.1 weight per
volume (w/v) percent to about 10 w/v percent. The effective
concentration may include peptide concentrations in the solution in
a range of about 0.1 w/v percent to about 3.5 w/v percent. In
certain embodiments, the effective concentration may be about 1 w/v
percent. In other embodiments, the effective concentration may be
about 2.5 w/v percent. In yet other embodiments, the effective
concentration may be about 3.0 w/v percent.
[0104] In certain embodiments, a peptide solution having a higher
concentration of peptide may provide for a more effective hydrogel
that has the ability to stay in place and provide effective, or
promote anti-adhesion, e.g., prevent or mitigate biological tissue
adhesion,. For purposes of delivering the peptide solution, higher
concentrations of peptide solutions may become too viscous to allow
for effective and selective administration of the solution. It is
possible that if a high enough concentration is not selected, the
hydrogel may not be effective in the target area for the desired
period of time.
[0105] The effective concentration may be selected to provide for a
solution that may be administered by injection or other means using
a particular diameter or gauge catheter or needle.
[0106] Methods of the disclosure contemplate single as well as
multiple administrations of a therapeutically effective amount of
the peptides, compositions, peptide solutions, membranes,
filaments, and hydrogels as described herein. Peptides as described
herein may be administered at regular intervals, depending on the
nature, severity and extent of the subject's condition. In some
embodiments, a peptide, composition, peptide solution, membrane,
filament, or hydrogel may be administered in a single
administration. In some embodiments, a peptide, composition,
peptide solution, or hydrogel described herein is administered in
multiple administrations. In some embodiments, a therapeutically
effective amount of a peptide, composition, peptide solution,
membrane, filament, or hydrogel may be administered periodically at
regular intervals. The regular intervals selected may be based on
any one or more of the initial peptide concentration of the
solution administered, the amount administered, and the degradation
rate of the hydrogel formed. For example, after an initial
administration, a follow-on administration may occur after, for
example, one week, two weeks, four weeks, six weeks, or eight
weeks. The follow-on administration may comprise administration of
a solution having the same concentration of peptide and volume as
the initial administration, or may comprise administration of a
solution of lesser or great concentration of peptide and volume.
The selection of the appropriate follow-on administration of
peptide solution may be based on imaging the target area and the
area surrounding the target area and ascertaining the needs based
on the condition of the subject. The pre-determined intervals may
be the same for each follow-on administration, or they may be
different. In some embodiments, a peptide, peptide solution, or
hydrogel may be administered chronically at pre-determined
intervals to maintain at least a partial anti-adhesion, e.g.,
prevention or mitigation of biological tissue adhesion, in a
subject over the life of the subject. The pre-determined intervals
may be the same for each follow-on administration, or they may be
different. This may be dependent on whether the hydrogel formed
from the previous administration is partially or totally disrupted
or degraded. The follow-on administration may comprise
administration of a solution having the same concentration of
peptide and volume as the initial administration, or may comprise
administration of a solution of lesser or great concentration of
peptide and volume. The selection of the appropriate follow-on
administration of peptide solution may be based on imaging the
target area and the area surrounding the target area and
ascertaining the needs based on the condition of the subject.
[0107] The peptides can be chemically synthesized or they can be
purified from natural and recombinant sources. Using chemically
synthesized peptides may allow the peptide solutions to be
deficient in unidentified components such as unidentified
components derived from the extracellular matrix of another animal.
This property therefore may eliminate concerns of infection,
including risk of viral infection compared to conventional
tissue-derived biomaterials. This may eliminate concerns of
infection including infections such as bovine spongiform
encephalopathy (BSE), making the peptide highly safe for medical
use.
[0108] The initial concentration of the peptide may be a factor in
the size and thickness of the membrane, hydrogel, or scaffold
formed. In general, the higher the peptide concentration, the
higher the extent of membrane or hydrogel formation. Hydrogels, or
scaffolds formed at higher initial peptide concentrations (about 10
mg/ml) (about 1.0 w/v percent) may be thicker and thus, likely to
be stronger.
[0109] Formation of the, membranes, hydrogels, or scaffolds may be
very fast, on the order of a few minutes. The formation of the
membranes or hydrogels may be irreversible. In certain embodiments,
the formation may be reversible, and in other embodiments, the
formation may be irreversible. The hydrogel may form
instantaneously upon administration to a target area. The formation
of the hydrogel may occur within about one to two minutes of
administration. In other examples, the formation of the hydrogel
may occur within about three to four minutes of administration. In
certain embodiments the time it takes to form the hydrogel may be
based at least in part on one or more of the concentration of the
peptide solution, the volume of peptide solution applied, and the
conditions at the area of application or injection (for example,
the concentration of monovalent metal cations at the area of
application, the pH of the area, and the presence of one or more
fluids at or near the area). The process may be unaffected by pH of
less than or equal to 12, and by temperature. The membranes or
hydrogels may form at temperatures in the range of 1 to 99 degrees
Celsius.
[0110] The hydrogels may remain in position at the target area for
a period of time sufficient to provide a desired effect using the
methods and kits of the present disclosure. The desired effect
using the methods and kits of the present disclosure may be to
treat areas or to assist in healing of areas in which a surgical
procedure at or near the site of a surgery was performed. For
example, the desired effect using the methods and kits of the
present disclosure may be to treat areas or to assist in healing of
areas in which an endoscopic surgery is performed.
[0111] The period of time that the membranes or hydrogels may
remain at the desired area may be for about 10 minutes. In certain
examples, it may remain at the desired area for about 35 minutes.
In certain further examples, it may remain at the desired area for
one or more days, up to one or more weeks. In other examples, it
may remain at the desired area for up to 30 days, or more. It may
remain at the desired area indefinitely. In other examples, it may
remain at the desired area for a longer period of time, until it is
naturally degraded or intentionally removed. If the hydrogel
naturally degrades over a period of time, subsequent application or
injection of the hydrogel to the same or different location may be
performed.
[0112] In certain embodiments, the self-assembling peptide may be
prepared with one or more components that may provide for enhanced
effectiveness of the self-assembling peptide or may provide another
action, treatment, therapy, or otherwise interact with one or more
components of the subject. For example, additional peptides
comprising one or more biologically or physiologically active amino
acid sequences or motifs may be included as one of the components
along with the self-assembling peptide. Other components may
include biologically active compounds such as a drug or other
treatment that may provide some benefit to the subject. For
example, a cancer treating drug or anticancer drug may be
administered with the self-assembling peptide, or may be
administered separately.
[0113] The peptide, peptide solution, or hydrogel may comprise
small molecular drugs to treat the subject or to prevent hemolysis,
inflammation, and infection. The small molecular drugs may be
selected from the group consisting of glucose, saccharose, purified
saccharose, lactose, maltose, trehalose, destran, iodine, lysozyme
chloride, dimethylisoprpylazulene, tretinoin tocoferil, povidone
iodine, alprostadil alfadex, anise alcohol, isoamyl salicylate,
.alpha.,.alpha.-dimethylphenylethyl alcohol, bacdanol, helional,
sulfazin silver, bucladesine sodium, alprostadil alfadex,
gentamycin sulfate, tetracycline hydrochloride, sodium fusidate,
mupirocin calcium hydrate and isoamyl benzoate. Other small
molecular drugs may be contemplated. Protein-based drugs may be
included as a component to be administered, and may include
erythropoietin, tissue type plasminogen activator, synthetic
hemoglobin and insulin.
[0114] A component may be included to protect the peptide solution
against rapid or immediate formation into a hydrogel. This may
include an encapsulated delivery system that may degrade over time
to allow a controlled time release of the peptide solution into the
target area to form the hydrogel over a desired, predetermined
period of time. Biodegradable, biocompatible polymers may be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid.
[0115] In some embodiments, sugar may be added to the
self-assembling peptide solution to improve the osmotic pressure of
the solution from hypotonicity to isotonicity without reducing the
tissue anti-adhesion effect, thereby allowing the biological safety
to be increased. In certain examples, the sugar may be sucrose or
glucose.
[0116] Any of the components described herein may be included in
the peptide solution or may be administered separate from the
peptide solution. Additionally, any of the methods and methods of
facilitating provided herein may be performed by one or more
parties.
[0117] A peptide, peptide solution, or hydrogel of the disclosure
may be provided in a kit. Instructions for administering the
solution to a target area of a subject may also be provided in the
kit. The peptide solution may comprise a self-assembling peptide
comprising at least about 7 amino acids in an effective amount and
in an effective concentration to form a hydrogel to at least
partially mitigate adhesion to a biological tissue. The peptide
solution may comprise a self-assembling peptide comprising between
about 7 amino acids to about 32 amino acids in an effective amount
and in an effective concentration to form a hydrogel to at least
partially mitigate adhesion to a biological tissue. The
instructions for administering the solution may comprise methods
for administering the peptide, peptide solution, or hydrogel
provided herein, for example, by a route of administration
described herein, at a dose, volume or concentration, or
administration schedule. The peptide may be amphiphilic and at
least a portion of the peptide may alternate between a hydrophobic
amino acid and a hydrophilic amino acid.
[0118] The kit may also comprise informational material. The
informational material may be descriptive, instructional, marketing
or other material that relates to the methods described herein. In
one embodiment, the informational material may include information
about production of the peptide, peptide solution, or hydrogel
disclosed herein, physical properties of the peptide, composition,
peptide solution or hydrogel, concentration, volume, size,
dimensions, date of expiration, and batch or production site.
[0119] The kit may also optionally include a device or materials to
allow for administration of the peptide or peptide solution to the
desired area. For example, a syringe, pipette, catheter, or other
needle-based device may be included in the kit. Additionally, or
alternatively, the kit may include a guidewire, endoscope, or other
accompanying equipment to provide selective administration of the
peptide solution to the target area.
[0120] The kit may comprise in addition to or in the alternative,
other components or ingredients, such as components that may aid in
positioning of the peptide solution, hydrogel or scaffold.
Instructions may be provided in the kit to combine a sufficient
quantity or volume of the peptide solution with a sucrose solution,
that may or may not be provided with the kit. Instructions may be
provided for diluting the peptide solution to administer an
effective concentration of the solution to the target area. The
instruction may describe diluting the peptide solution with a
diluent or solvent. The diluent or solvent may be water.
Instructions may further be provided for determining at least one
of the effective concentration of the solution and the effective
amount of the solution to the target area. This may be based on
various parameters discussed herein, and may include the diameter
of the lesion or wound at the target area.
[0121] Other components or ingredients may be included in the kit,
in the same or different compositions or containers than the
peptide, peptide solutions, or hydrogel. The one or more components
that may include components that may provide for enhanced
effectiveness of the self-assembling peptide or may provide another
action, treatment, therapy, or otherwise interact with one or more
components of the subject. For example, additional peptides
comprising one or more biologically or physiologically active
sequences or motifs may be included as one of the components along
with the self-assembling peptide. Other components may include
biologically active compounds such as a drug or other treatment
that may provide some benefit to the subject. For example, a cancer
treating drug or anticancer drug may be administered with the
self-assembling peptide, or may be administered separately. The
peptide, peptide solution, or hydrogel may comprise small molecular
drugs to treat the subject or to prevent hemolysis, inflammation,
and infection, as disclosed herein. A sugar solution such as a
sucrose solution may be provided with the kit. The sucrose solution
may be a 20% sucrose solution.
[0122] Other components which are disclosed herein may also be
included in the kit.
[0123] In some embodiments, a component of the kit is stored in a
sealed vial, for example, with a rubber or silicone closure (for
example, a polybutadiene or polyisoprene closure). In some
embodiments, a component of the kit is stored under inert
conditions (for example, under nitrogen or another inert gas such
as argon). In some embodiments, a component of the kit is stored
under anhydrous conditions (for example, with a desiccant). In some
embodiments, a component of the kit is stored in a light blocking
container such as an amber vial.
[0124] As part of the kit or separate from a kit, syringes or
pipettes may be pre-filled with a peptide, peptide solution, or
hydrogel as disclosed herein. Methods to instruct a user to supply
a self-assembling peptide solution to a syringe or pipette, with or
without the use of other devices, and administering it to the
target area through the syringe or pipette, with or without the use
of other devices, is provided. Other devices may include, for
example, a catheter with or without a guidewire.
[0125] In some embodiments of the disclosure, the self-assembling
peptides may be used as a coating on a device or an instrument such
as a stent or catheter, to suppress body fluid leakage. The
self-assembling peptides may also be incorporated or secured to a
support, such as gauze or a bandage, or a lining, that may provide
a therapeutic effect to a subject, or that may be applied within a
target area. The self-assembling peptides may also be soaked into a
sponge for use.
[0126] The membranes may also be useful for culturing cell
monolayers. Cells prefer to adhere to non-uniform, charged
surfaces. The charged residues and conformation of the
proteinaceous membranes promote cell adhesion and migration. The
addition of growth factors, such as fibroblast growth factor, to
the peptide membrane may further improve attachment, cell growth
and neurite outgrowth.
[0127] Although the peptide solution may be liquid at acidic pH,
the peptide may undergo self-organization or self-assembly upon
adjustment of a pH level of the solution to a neutral or
physiological pH level. The solution may be aqueous or
non-aqueous.
[0128] The following examples are illustrative and not restrictive.
Many variations of the technology will become apparent to those of
skill in the art upon review of this disclosure. The scope of the
technology should, therefore, be determined not with reference to
the examples, but instead should be determined with reference to
the appended claims along with their full scope of equivalents.
EXAMPLE
[0129] Anti-adhesion methods utilizing self-assembling peptide
solutions according to the present invention were assessed in a
rabbit cecal-sidewall model, which is used to assess
intra-abdominal adhesion formation and, more generally,
anti-adhesion capacity.
[0130] The self-assembling peptide solution used in this example
was PURASTAT.RTM. (3D Matrix, Inc.), which is a commercial 2.5%
solution of the RADA16 peptide. The test system was the rabbit, as
follows:
[0131] Species: Rabbit (Oryctolagus cuniculus)
[0132] Strain: New Zealand White
[0133] Source: USDA licensed supplier
[0134] Sex: Female, nulliparous and nonpregnant
[0135] Body Weight Range: 4.0 to 6.0 kg at selection
[0136] Age: No particular age is prescribed for this test
[0137] Acclimation Period: Minimum 5 days
[0138] Number of Animals: Twenty One (+4 reserves)
[0139] Identification Method: Ear tag
[0140] The rabbit is an appropriate model in numerous literature
references for evaluating reduction of post-surgical adhesions. The
rabbit represents the lowest sentient species that is physically
large enough to accommodate the sidewall defects and the size of
the implanted articles. The number of animals represented the
smallest number that will yield interpretable results. Husbandry,
Housing, and Environment Conditions conformed to NAMSA Standard
Operating Procedures based on the "Guide for the Care and Use of
Laboratory Animals." There are no available validated in vitro
assays or computer-simulated models that can mimic the complexity
of the rabbit model for post-surgical adhesion formation.
[0141] Methods. Pre-Operative Procedure: Within 2 days prior to the
first surgical day, animals were weighed and randomly assigned by
weight to treatment groups as indicated in Table 2.
TABLE-US-00002 TABLE 2 Cecal-Sidewall Model Animal Assignment
Treatment Group Number of Animals Terminal Interval (.+-.1 day)
Surgical Control* 5 14 days Test Article Treated 8 *Surgical
control group had the abrasion procedure, but no article was
applied.
[0142] On the day of surgery, each animal was injected
subcutaneously with 0.05 mg/kg of the analgesic buprenorphine and a
fentanyl patch (analgesic; 25 .mu.g/hr) was applied to an ear. Each
animal was intramuscularly injected with a combination ketamine
hydrochloride and xylazine (34 mg/kg+5 mg/kg) general anesthetic
dosed at 0.6 mL/kg. A veterinary ophthalmic ointment was applied to
both eyes of the animal to protect the corneas from drying. Each
animal received a prophylactic dose of enrofloxacin (antibiotic)
intramuscularly at 5.0 mg/kg. Each animal was clipped free of fur
over the abdomen. The abdomen was scrubbed with a germicidal soap
and wiped with 70% isopropyl alcohol. The surgical site was painted
with an antiseptic such as povidone iodine and the animal was
draped. Each animal was placed on isoflurane inhalant anesthetic
for continued general anesthesia. The vital signs (temperature,
heart rate, SPO2) of each animal was monitored during the
procedure.
[0143] Operative Procedure. An approximate 12 cm in length skin
incision was made along the midline of the ventral abdomen,
beginning approximately 6 cm caudal to the xiphoid process. The
abdominal wall was opened by incising along the linea alba. The
entire cecum was exteriorized and abraded by wiping the entire
serosal surface with a sterile dry gauze sponge until punctate
bleeding was achieved. If the integrity of the cecal wall was
compromised, the animal was euthanized and replaced. The cecum was
repositioned in the abdomen. Bilateral defects measuring
approximately 2.times.4.5 cm were made to the peritoneum over the
abdominal sidewall. The defects were made approximately 4 cm
lateral to the midline incision and approximately 7-9 cm caudal to
the xiphoid process. An approximate 2.times.4.5 cm window of
peritoneum was excised using sharp dissection and the muscle was
disrupted by scraping the area with a scalpel blade to produce
bleeding. If less than desirable bleeding is noted, additional
bleeding will be induced by incising small blood vessels that are
traversing through the defect area. Representative photographs were
taken of the sidewall defect sites. For the test animals, the test
article was applied to the peritoneal wall to cover and coat the
sidewall defect sites. The defect sites remained untreated for the
negative control group. The sidewall and cecum were returned to
normal positioning and the abdominal wall was closed with a simple
continuous suture pattern using appropriate absorbable suture. The
subcutaneous tissue was closed with a simple continuous suture
pattern using the same suture type. The skin was closed with 4-0
nonabsorbable suture, stainless steel wound clips, or a combination
of these materials. The day of surgery was Day 0.
[0144] Post-Operative Observations. The animals were moved to a
recovery area and placed on a heat source. Animals were monitored
for recovery from the anesthetic. Once sternal recumbency was
achieved, each animal was fitted with an Elizabethan collar and
returned to its cage. Buprenorphine (SQ, 0.05 mg/kg) was
administered approximately 6 hours after the initial dose.
Enrofloxacin (IM, 5.0 mg/kg) was administered at the end of the day
of surgery and then twice a day for the first 2 days after
surgery.
[0145] At 14 (.+-.1) days after surgery, the animals were weighed
and euthanized by an intravenous injection of sodium pentobarbital
based euthanasia solution. The peritoneal cavity was opened and the
viscera were examined by a staff veterinarian. To maintain
consistency in grading, the same veterinarian conducted all of the
evaluations. The defect sites of each animal were photographed.
Each site was examined for adhesion formation. Adhesions were
graded for extent and strength as per the criteria shown in Tables
3 and 4:
TABLE-US-00003 TABLE 3 Adhesion Extent Scoring Score Description 0
0% 1 1-25% 2 26-50% 3 51-75% 4 76-100%
TABLE-US-00004 TABLE 4 Adhesion Strength Scoring Score Description
0 No adhesions present 1 Friable 2 Immature, easy to break 3
Mature, hard to break
[0146] The general location of the adhesions and the tissue or
organ involved in the adhesion was noted. Adhesions to the
abdominal incision were noted and described as well as any other
adhesions present within the abdominal cavity, however they were
not scored.
[0147] Following scoring, the wounds and cecum were excised and
placed in 10% neutral buffered formalin (NBF). Tissues was labeled
with the animal number, wound identification, day of termination.
No microscopic evaluation was conducted unless indicated by the
Study Director.
[0148] Evaluation and Statistical Analysis. The mean scores for
adhesion extent and strength and the incidence of adhesions were
calculated. A total adhesion score (extent+strength of each defect)
was calculated for each animal in the cecal-sidewall model. The
scores for adhesion extent, adhesion strength, and total adhesion
score were statistically compared. The mean and standard deviation
was calculated for each parameter. Descriptive statistics and group
comparisons data were accomplished using a validated statistical
program. After screening the data for normality and equal variance,
the appropriate parametric or nonparametric tests were performed.
Normally distributed data with equal variance was considered
parametric and compared using an unpaired t-test. If data was
nonparametric, two sample t-test of unequal variance (Welch test)
were conducted. Calculations resulting in probability (p) values
less than 0.05 were considered statistically significant and
appropriate post-hoc tests were run. For body weights, the mean and
standard deviation was calculated for each treatment group.
[0149] Conclusions. Results are presented in Table 5A and 5B below.
In the cecal side-wall model, 9/10 sites (5 animals total) formed
adhesions in the surgical controls (See Table 5A) and only 4/16
sites (8 animals total) formed adhesions with self-assembling
peptide solution treatment (See Table 5B). Preliminary statistical
calculations demonstrate a significant difference (p-value=0.00126;
result is significant at p<0.05) between the controls and
PuraStat treated animals. In conclusion, the self-assembling
peptide solution demonstrated anti-adhesive properties in the
rabbit cecal side wall model.
TABLE-US-00005 TABLE 5A Adhesions in Cecal Side Wall Model, Control
Group Volume (mL) Adhesion Adhesion Control/ of Test Adhesions
Extent Strength Additional Test Test Article at Defect Score Score
Adhesions Article Incision Additional Animal # Article (L/R) Site
(L/R) (L/R) (L/R) (Cecum to . . . ) Present Healing Comments 17490
Control cecum and 1 & 1/4 2 & 1/2 incision, omentum, no yes
at time of small cecum comment explant, intestine/ adhesion of
cecum cecum became adhered to left defect site 17486 Control cecum/
4/2 2/2 incision, cecum, no yes cecum omentum, large comment
intestine 17476 Control non- 2 & 2/ 1 & 2/ incision,
omentum, no yes abraded 2 & 1 2 & 2 cecum, small comment
cecum & intestine, abraded abdominal fat cecum/ cecum &
large intestine 17477 Control none/ 0/3 0/2 sidewall (without no
yes small defect), incision, comment intestine small intestine,
omentum, cecum, large intestine, (uterus to incision) 17472 Control
N/A cecum/ 4/1 & 1 2/2 & 2 incision, cecum, no yes
non-abraded cecum large intestine comment cecum to right (X2)
sidewall defect
TABLE-US-00006 TABLE 5B Adhesions in Cecal Side-Wall Model,
Self-Assembling Peptide Solution Treatment Group Volume (mL)
Adhesion Adhesion Control/ of Test Adhesions Extent Strength
Additional Test Test Article at Defect Score Score Adhesions
Article Incision Additional Animal # Article (L/R) Site (L/R) (L/R)
(L/R) (Cecum to . . . ) Present Healing Comments 17484 Test 6/9
none/ 0/0 0/0 incision, omentum, yes yes tan film adhered Article
none cecum, large to surface of liver intestine 17485 Test 6/6
none/ 0/0 0/0 incision, large yes yes tan film adhered Article none
intestine, small to surface of the intestine, cecum liver and
spleen 17482 Test 3/3 cecum/ 4/0 2/0 cecum, small no yes small
intestine Article none intestine, adhered to abdominal fat,
incision omentum 17479 Test 3/3 none/ 0/0 0/0 omentum, cecum, yes
yes tan film (minimal) Article none large intestine adhered to
surface of the liver 17470 Test 3/3 none/ 0/0 0/0 incision,
omentum, no yes Article none cecum, large intestine, small
intestine 17471 Test 8.5/5 none/ 0/0 0/0 incision, small yes yes
Article none intestine, omentum, large intestine 17467 Test 6/6
none/ 0/1 0/2 Incision, cecum yes yes small intestine Article cecum
adhere to cecum; tan material adhered to liver surface 17474 Test
6/6 cecum/ 1/2 2/2 incision, cecum, yes yes extensive cecum Article
cecum small intestine, to cecum large intestine, adhesions
messentery
Sequence CWU 1
1
8514PRTArtificial SequenceSynthetic peptide 1Arg Ala Asp
Ala124PRTArtificial SequenceSynthetic peptide 2Ile Glu Ile
Lys134PRTArtificial SequenceSynthetic peptide 3Thr Thr Thr
Thr144PRTArtificial SequenceSynthetic peptide 4Ala Thr Ala
Thr154PRTArtificial SequenceSynthetic peptide 5Thr Val Thr
Val164PRTArtificial SequenceSynthetic peptide 6Ala Ser Ala
Ser174PRTArtificial SequenceSynthetic Peptide 7Ser Ser Ser
Ser187PRTArtificial SequenceSynthetic peptide 8Val Val Val Thr Thr
Thr Thr1 594PRTArtificial SequenceSynthetic peptide 9Lys Leu Asp
Leu1103PRTArtificial SequenceSynthetic peptide 10Lys Leu
Asp11116PRTArtificial SequenceSynthetic peptide 11Arg Ala Asp Ala
Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala1 5 10
151213PRTArtificial SequenceSynthetic peptide 12Ile Glu Ile Lys Ile
Glu Ile Lys Ile Glu Ile Lys Ile1 5 101317PRTArtificial
SequenceSynthetic peptide 13Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys Ile Glu Ile Lys1 5 10 15Ile1412PRTArtificial
SequenceSynthetic peptide 14Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu
Asp Leu1 5 101516PRTArtificial SequenceSynthetic peptide 15Val Arg
Val Arg Val Asp Val Asp Val Arg Val Arg Val Asp Val Asp1 5 10
151616PRTArtificial SequenceSynthetic peptide 16Ala Asp Ala Asp Ala
Lys Ala Lys Ala Asp Ala Asp Ala Lys Ala Lys1 5 10
151716PRTArtificial SequenceSynthetic peptide 17Lys Ala Lys Ala Lys
Ala Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala1 5 10
151816PRTArtificial SequenceSynthetic peptide 18Glu Ala Glu Ala Glu
Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala1 5 10
151916PRTArtificial SequenceSynthetic peptide 19Ala Asp Ala Asp Ala
Asp Ala Asp Ala Asp Ala Asp Ala Asp Ala Asp1 5 10
152016PRTArtificial SequenceSynthetic peptide 20Ala Glu Ala Glu Ala
Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys1 5 10
152116PRTArtificial SequenceSynthetic peptide 21Ala Arg Ala Asp Ala
Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp1 5 10
152216PRTArtificial SequenceSynthetic peptide 22Lys Ala Lys Ala Lys
Ala Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala1 5 10
15235PRTArtificial SequenceSynthetic peptide 23Lys Ala Lys Ala Lys1
52416PRTArtificial SequenceSynthetic peptide 24Ala Lys Ala Lys Ala
Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu1 5 10
152516PRTArtificial SequenceSynthetic peptide 25Ala Lys Ala Glu Ala
Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu1 5 10
152616PRTArtificial SequenceSynthetic peptide 26Glu Ala Lys Ala Glu
Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala1 5 10
15278PRTArtificial SequenceSynthetic peptide 27Ala Glu Ala Glu Ala
Lys Ala Lys1 52812PRTArtificial SequenceSynthetic peptide 28Ala Glu
Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys1 5 102916PRTArtificial
SequenceSynthetic peptide 29Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala
Glu Ala Lys Ala Glu Ala1 5 10 153016PRTArtificial SequenceSynthetic
peptide 30Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu
Ala Lys1 5 10 15318PRTArtificial SequenceSynthetic peptide 31Ala
Arg Ala Arg Ala Asp Ala Asp1 53216PRTArtificial SequenceSynthetic
peptide 32Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala Arg
Ala Arg1 5 10 153316PRTArtificial SequenceSynthetic peptide 33Ala
Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp1 5 10
153416PRTArtificial SequenceSynthetic peptide 34Asp Ala Arg Ala Asp
Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala1 5 10
153516PRTArtificial SequenceSynthetic peptide 35Ala Asp Ala Arg Ala
Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg1 5 10
153616PRTArtificial SequenceSynthetic peptide 36Ala Arg Ala Arg Ala
Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp1 5 10
153716PRTArtificial SequenceSynthetic peptide 37Ala Arg Ala Asp Ala
Lys Ala Glu Ala Arg Ala Asp Ala Lys Ala Glu1 5 10
153816PRTArtificial SequenceSynthetic peptide 38Ala Lys Ala Glu Ala
Arg Ala Asp Ala Lys Ala Glu Ala Arg Ala Asp1 5 10
153916PRTArtificial SequenceSynthetic peptide 39Ala Arg Ala Lys Ala
Asp Ala Glu Ala Arg Ala Lys Ala Asp Ala Glu1 5 10
154016PRTArtificial SequenceSynthetic peptide 40Ala Lys Ala Arg Ala
Glu Ala Asp Ala Lys Ala Arg Ala Asp Ala Glu1 5 10
154116PRTArtificial SequenceSynthetic peptide 41Ala Gln Ala Gln Ala
Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln1 5 10
154216PRTArtificial SequenceSynthetic peptide 42Val Gln Val Gln Val
Gln Val Gln Val Gln Val Gln Val Gln Val Gln1 5 10
154316PRTArtificial SequenceSynthetic peptide 43Tyr Gln Tyr Gln Tyr
Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln1 5 10
154416PRTArtificial SequenceSynthetic peptide 44His Gln His Gln His
Gln His Gln His Gln His Gln His Gln His Gln1 5 10
154516PRTArtificial SequenceSynthetic peptide 45Ala Asn Ala Asn Ala
Asn Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn1 5 10
154616PRTArtificial SequenceSynthetic peptide 46Val Asn Val Asn Val
Asn Val Asn Val Asn Val Asn Val Asn Val Asn1 5 10
154716PRTArtificial SequenceSynthetic peptide 47Tyr Asn Tyr Asn Tyr
Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn1 5 10
154816PRTArtificial SequenceSynthetic peptide 48His Asn His Asn His
Asn His Asn His Asn His Asn His Asn His Asn1 5 10
154916PRTArtificial SequenceSynthetic peptide 49Ala Asn Ala Gln Ala
Asn Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln1 5 10
155016PRTArtificial SequenceSynthetic peptide 50Ala Gln Ala Asn Ala
Gln Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn1 5 10
155116PRTArtificial SequenceSynthetic peptide 51Val Asn Val Gln Val
Asn Val Gln Val Asn Val Gln Val Asn Val Gln1 5 10
155216PRTArtificial SequenceSynthetic peptide 52Val Gln Val Asn Val
Gln Val Asn Val Gln Val Asn Val Gln Val Asn1 5 10
155316PRTArtificial SequenceSynthetic peptide 53Tyr Asn Tyr Gln Tyr
Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln1 5 10
155416PRTArtificial SequenceSynthetic peptide 54Tyr Gln Tyr Asn Tyr
Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn1 5 10
155516PRTArtificial SequenceSynthetic peptide 55His Asn His Gln His
Asn His Gln His Asn His Gln His Asn His Gln1 5 10
155616PRTArtificial SequenceSynthetic peptide 56His Gln His Asn His
Gln His Asn His Gln His Asn His Gln His Asn1 5 10
155718PRTArtificial SequenceSynthetic peptide 57Ala Lys Ala Gln Ala
Asp Ala Lys Ala Gln Ala Asp Ala Lys Ala Gln1 5 10 15Ala
Asp5818PRTArtificial SequenceSynthetic peptide 58Val Lys Val Gln
Val Asp Val Lys Val Gln Val Asp Val Lys Val Gln1 5 10 15Val
Asp5918PRTArtificial SequenceSynthetic peptide 59Tyr Lys Tyr Gln
Tyr Asp Tyr Lys Tyr Gln Tyr Asp Tyr Lys Tyr Gln1 5 10 15Tyr
Asp6018PRTArtificial SequenceSynthetic peptide 60His Lys His Gln
His Asp His Lys His Gln His Asp His Lys His Gln1 5 10 15His
Asp613PRTArtificial SequenceSynthetic peptide 61Arg Ala
Asp1627PRTArtificial SequenceSynthetic peptide 62Ala Ala Ala Ala
Ala Ala Lys1 5637PRTArtificial SequenceSynthetic peptide 63Ala Ala
Ala Ala Ala Ala Asp1 5647PRTArtificial SequenceSynthetic peptide
64Thr Thr Thr Thr Thr Thr Thr1 5658PRTArtificial SequenceSynthetic
peptide 65Ala Thr Ala Thr Ala Thr Ala Thr1 5668PRTArtificial
SequenceSynthetic peptide 66Thr Val Thr Val Thr Val Thr Val1
5678PRTArtificial SequenceSynthetic peptide 67Ala Ser Ala Ser Ala
Ser Ala Ser1 5687PRTArtificial SequenceSynthetic peptide 68Ser Ser
Ser Ser Ser Ser Ser1 569200PRTArtificial SequenceSynthetic
peptideMISC_FEATURE(1)..(200)/note="This sequence may encompass
2-50 'Arg Ala Asp Ala' repeating
units"MISC_FEATURE(1)..(200)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions"VARIANT(9)..(200)/replace=" "
69Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala1
5 10 15Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp
Ala 20 25 30Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala
Asp Ala 35 40 45Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg
Ala Asp Ala 50 55 60Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala
Arg Ala Asp Ala65 70 75 80Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala
Asp Ala Arg Ala Asp Ala 85 90 95Arg Ala Asp Ala Arg Ala Asp Ala Arg
Ala Asp Ala Arg Ala Asp Ala 100 105 110Arg Ala Asp Ala Arg Ala Asp
Ala Arg Ala Asp Ala Arg Ala Asp Ala 115 120 125Arg Ala Asp Ala Arg
Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 130 135 140Arg Ala Asp
Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala145 150 155
160Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala
165 170 175Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala
Asp Ala 180 185 190Arg Ala Asp Ala Arg Ala Asp Ala 195
20070200PRTArtificial SequenceSynthetic
peptideMISC_FEATURE(1)..(200)/note="This sequence may encompass
2-50 'Ile Glu Ile Lys' repeating
units"MISC_FEATURE(1)..(200)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions"VARIANT(9)..(200)/replace=" "
70Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys1
5 10 15Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile
Lys 20 25 30Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys 35 40 45Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile
Glu Ile Lys 50 55 60Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys
Ile Glu Ile Lys65 70 75 80Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys Ile Glu Ile Lys 85 90 95Ile Glu Ile Lys Ile Glu Ile Lys Ile
Glu Ile Lys Ile Glu Ile Lys 100 105 110Ile Glu Ile Lys Ile Glu Ile
Lys Ile Glu Ile Lys Ile Glu Ile Lys 115 120 125Ile Glu Ile Lys Ile
Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys 130 135 140Ile Glu Ile
Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys145 150 155
160Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys
165 170 175Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys 180 185 190Ile Glu Ile Lys Ile Glu Ile Lys 195
20071201PRTArtificial SequenceSynthetic
peptideMISC_FEATURE(1)..(201)/note="This sequence may encompass
2-50 'Ile Glu Ile Lys' repeating
units"MISC_FEATURE(1)..(201)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions"VARIANT(9)..(201)/replace=" "
71Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys1
5 10 15Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile
Lys 20 25 30Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys 35 40 45Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile
Glu Ile Lys 50 55 60Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys
Ile Glu Ile Lys65 70 75 80Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys Ile Glu Ile Lys 85 90 95Ile Glu Ile Lys Ile Glu Ile Lys Ile
Glu Ile Lys Ile Glu Ile Lys 100 105 110Ile Glu Ile Lys Ile Glu Ile
Lys Ile Glu Ile Lys Ile Glu Ile Lys 115 120 125Ile Glu Ile Lys Ile
Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys 130 135 140Ile Glu Ile
Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys145 150 155
160Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys
165 170 175Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys 180 185 190Ile Glu Ile Lys Ile Glu Ile Lys Ile 195
20072200PRTArtificial SequenceSynthetic
peptideMISC_FEATURE(1)..(200)=note="This sequence may encompass
2-50 'Lys Leu Asp Leu' repeating
units"MISC_FEATURE(1)..(200)=note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions"VARIANT(9)..(200)/replace=" "
72Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu1
5 10 15Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp
Leu 20 25 30Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu
Asp Leu 35 40 45Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys
Leu Asp Leu 50 55 60Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu
Lys Leu Asp Leu65 70 75 80Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu
Asp Leu Lys Leu Asp Leu 85 90 95Lys Leu Asp Leu Lys Leu Asp Leu Lys
Leu Asp Leu Lys Leu Asp Leu 100 105 110Lys Leu Asp Leu Lys Leu Asp
Leu Lys Leu Asp Leu Lys Leu Asp Leu 115 120 125Lys Leu Asp Leu Lys
Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu 130 135 140Lys Leu Asp
Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu145 150 155
160Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu
165 170 175Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu
Asp Leu 180 185 190Lys Leu Asp Leu Lys Leu Asp Leu 195
20073150PRTArtificial SequenceSynthetic
peptideMISC_FEATURE(1)..(150)/note="This sequence may encompass
2-50 'Lys Leu Asp' repeating
units"MISC_FEATURE(1)..(150)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions"VARIANT(7)..(150)/replace=" "
73Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys1
5 10 15Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys
Leu 20 25 30Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys
Leu Asp 35 40 45Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys
Leu Asp Lys 50 55 60Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys
Leu Asp Lys Leu65 70 75 80Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp
Lys Leu Asp Lys Leu Asp 85 90
95Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys
100 105 110Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp
Lys Leu 115 120 125Asp Lys Leu Asp Lys Leu Asp Lys Leu Asp Lys Leu
Asp Lys Leu Asp 130 135 140Lys Leu Asp Lys Leu Asp145
150748PRTArtificial SequenceSynthetic peptide 74Lys Leu Asp Leu Lys
Leu Asp Leu1 57512PRTArtificial SequenceSynthetic peptide 75Lys Leu
Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu1 5 107616PRTArtificial
SequenceSynthetic peptide 76Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly
Ala Gly Ala Gly Ala Gly1 5 10 157716PRTArtificial SequenceSynthetic
peptide 77Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala
Leu Ala1 5 10 15785PRTArtificial SequenceSynthetic peptide 78Leu
Ala Leu Ala Leu1 57916PRTArtificial SequenceSynthetic peptide 79Ala
Leu Ala Leu Ala Gly Ala Gly Ala Leu Ala Leu Ala Gly Ala Gly1 5 10
158016PRTArtificial SequenceSynthetic peptide 80Ala Leu Ala Gly Ala
Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala Gly1 5 10
158116PRTArtificial SequenceSynthetic peptide 81Gly Ala Leu Ala Gly
Ala Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala1 5 10
15828PRTArtificial SequenceSynthetic peptide 82Ala Gly Ala Gly Ala
Leu Ala Leu1 58312PRTArtificial SequenceSynthetic peptide 83Ala Gly
Ala Leu Ala Gly Ala Gly Ala Leu Ala Leu1 5 108416PRTArtificial
SequenceSynthetic peptide 84Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala
Gly Ala Leu Ala Gly Ala1 5 10 158516PRTArtificial SequenceSynthetic
peptide 85Ala Gly Ala Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala Gly
Ala Leu1 5 10 15
* * * * *