U.S. patent application number 17/535470 was filed with the patent office on 2022-06-02 for vascular embolic system.
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 Satoru Kobayashi, Kentaro Takamura.
Application Number | 20220168462 17/535470 |
Document ID | / |
Family ID | 1000006150010 |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220168462 |
Kind Code |
A1 |
Kobayashi; Satoru ; et
al. |
June 2, 2022 |
VASCULAR EMBOLIC SYSTEM
Abstract
Systems and methods of blocking a biological vessel 2 are
provided. The systems and methods may comprise introducing to the
vessel an amphiphilic peptide. The peptide may comprise at least
thirteen amino acids that may alternate between a hydrophobic amino
acid and a hydrophilic amino acid. The peptide may form a beta-
sheet spontaneously in an aqueous solution in the presence of a
cation.
Inventors: |
Kobayashi; Satoru;
(Chigasaki, JP) ; Takamura; Kentaro; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
3-D Matrix, Ltd. |
Tokyo |
|
JP |
|
|
Assignee: |
3-D Matrix, Ltd.
Tokyo
JP
|
Family ID: |
1000006150010 |
Appl. No.: |
17/535470 |
Filed: |
November 24, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14442677 |
May 13, 2015 |
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PCT/IB2013/060145 |
Nov 14, 2013 |
|
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17535470 |
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61726250 |
Nov 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 17/12113 20130101;
A61L 31/047 20130101; C07K 7/08 20130101; A61L 24/0031 20130101;
A61L 2400/06 20130101; A61B 17/12109 20130101; A61B 6/504 20130101;
A61B 6/481 20130101; A61L 2430/36 20130101; A61L 2400/04 20130101;
A61L 24/108 20130101; A61M 5/007 20130101; A61B 17/12186 20130101;
A61K 49/0438 20130101 |
International
Class: |
A61L 24/10 20060101
A61L024/10; A61L 31/04 20060101 A61L031/04; A61B 6/00 20060101
A61B006/00; A61B 17/12 20060101 A61B017/12; A61K 49/04 20060101
A61K049/04; A61L 24/00 20060101 A61L024/00; C07K 7/08 20060101
C07K007/08 |
Claims
1. A method of blocking one or more targeted biological vessels in
a subject comprising: introducing a catheter into a target
biological vessel; positioning an end of the catheter in, near,
upstream or downstream from a target area of a biological vessel in
which at least a partial obstruction of the vessel is desired;
administering through the catheter a solution comprising an
amphiphilic peptide comprising at least 12 amino acids that
alternate between a hydrophobic amino acid and a hydrophilic amino
acid in an effective amount and in an effective concentration to
form a hydrogel at the target site the hydrogel thereby forming at
least a partial blockage of the target biological vessel; removing
the catheter from the biological vessel with the at least partial
obstruction in place.
2. The method of claim 1, wherein the peptide solution comprises a
contrast agent.
3. The method of claim 2, further comprising visualizing a region
comprising at least a portion of the targeted biological vessel or
vessels.
4. The method of claim 3, wherein visualizing the region comprising
at least a portion of the biological vessel comprises visualizing
the region during at least one of: identifying the target area of
the biological vessel; introducing the catheter; positioning the
end of the catheter in the target area; administering the solution;
removing the catheter; and visualizing the biological vessel after
removing the catheter.
5. The method of claim 4, wherein visualizing the region comprises
imaging using X-ray radiography.
6. The method of claim 3, wherein visualizing the region provides
for selective administration of the solution to a targeted
biological vessel.
7. The method of claim 3, further comprising visualizing a
previously targeted region of a vessel in a time period about two
weeks subsequent to administration of a peptide solution.
8. The method of claim 1, wherein at least one of the effective
amount, the effective concentration and the identity of the peptide
is based in part on the diameter of the target area of the
biological vessel.
9. The method of claim 1, wherein at least one of the effective
amount, the effective concentration and the identity of the peptide
is based in part on the flow rate of the blood in the biological
vessel.
10. (canceled)
11. The method of claim 1, wherein the concentration effective to
form at least a partial blockage of the target biological vessel
comprises a concentration in a range of about 0.1 weight per volume
(w/v) percent to about 3 w/v percent peptide.
12. The method of claim 1, wherein the amount effective to cause at
least partial blockage of the biological vessel comprises a volume
in a range of about 0.1 mL to about 5 mL.
13. The method of claim 1, further comprising monitoring the area
surrounding the at least partial blockage comprising the
self-assembled peptide hydrogel to determine an effectiveness of
the at least partial obstruction in treating the subject's
pathology.
14. The method of claim 1, wherein the formed blockage is used in
the treatment of disorders, malformations, or congenital ailments
in one or more biological vessels.
15. The method of claim 14, wherein the formed blockage is used in
the treatment of one of patent ductus arteriosus (PDA) and major
aortopulmonary collateral artery (MAPCA).
16. The method of claim 14, wherein the formed blockage is used in
the treatment of a disorder, malformation, or congenital ailment
selected from the group consisting of recurrent hemolysis,
arteriovenous malformations, cerebral aneurysms, gastrointestinal
bleeding, epistaxis, post-partum hemorrhage, surgical hemorrhage,
and uterine fibroids.
17. The method of claim 1, wherein the formed blockage is used in
the reduction of the size and/or number of cancerous cells.
18-21. (canceled)
22. The method of claim 1, wherein administering the solution
comprises administering the solution in a single dose.
23. The method of claim 1, wherein treatment of the subject
comprises administering one or more doses of the same or a
different solution of peptide in separate applications.
24. The method of claim 1, wherein the peptide has an amino acid
sequence of one of RADARADARADARADA (SEQ ID NO: 7), IEIKIEIKIEIKI
(SEQ ID NO: 8), and IEIKIEIKIEIKIEIKI (SEQ ID NO: 9).
25-53. (canceled)
54. The method of claim 1 wherein the subject is heparinized, on
other anticoagulation therapy and/or in which normal coagulation is
impaired.
Description
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 14, 2013, is named T2071-7000WO_SL.txt and is 23,527 bytes
in size.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to macroscopic membranes that may be
used in medical, research, and industrial applications. More
particularly, this disclosure relates to membranes, hydrogels,
compositions and solutions that may be used in a vascular embolic
system and embolization procedures. The vascular embolic system may
provide an approach to at least partially block biological pathways
or channels including vessels, veins, portal veins, arteries, and
ducts that may transport blood and other fluids, such as lymph
fluids.
SUMMARY
[0003] A method of blocking a biological vessel in a subject is
provided. The method comprises introducing a catheter into a
biological vessel and positioning an end of the catheter in a
target area of the biological vessel in which at least a partial
obstruction is desired. The method further comprises administering
through the catheter a solution comprising an amphiphilic peptide
comprising at least 12 amino acids that alternate between a
hydrophobic amino acid and a hydrophilic amino acid in an effective
amount and in an effective concentration to form a hydrogel under
physiological conditions to allow at least partial blockage of the
biological vessel. The method further comprises removing the
catheter from the biological vessel with the at least partial
obstruction in place.
[0004] A kit for blocking a biological vessel in a subject is
provided. The kit comprises a solution comprising an amphiphilic
peptide comprising at least 12 amino acids that alternate between a
hydrophobic amino acid and a hydrophilic amino acid in an effective
amount and in an effective concentration to form a hydrogel under
physiological conditions to allow at least partial blockage of the
biological vessel. The kit further comprises instructions for
administering the solution to the biological vessel in the
subject.
[0005] A method of facilitating blocking a biological vessel in a
subject is provided. The method comprises providing a solution
comprising an amphiphilic peptide comprising at least 12 amino
acids that alternate between a hydrophobic amino acid and a
hydrophilic amino acid in an effective amount and in an effective
concentration to form a hydrogel under physiological conditions to
allow at least partial blockage of the biological vessel. The
method further comprises providing instructions for administering
the solution to a target area of the biological vessel through
introduction of the solution to a catheter positioned in the
biological vessel.
BRIEF DESCRIPTION OF THE DRAWING
[0006] The accompanying drawings are not intended to be drawn to
scale. For purposes of clarity, not every component may be
labeled.
[0007] In the drawings:
[0008] FIG. 1 is an image of a cross-section of a portal vein
embolism using a peptide solution of the present disclosure;
[0009] FIG. 2 is a contrast image of a normal hepatic artery;
[0010] FIG. 3 is a contrast image of an injection of the materials
of the present disclosure;
[0011] FIG. 4 is a contrast image of a hepatic artery after
injection with the materials of the present disclosure;
[0012] FIG. 5 is a contrast image of a hepatic artery two weeks
after injection with the materials of the present disclosure;
[0013] FIG. 6A is a histopathological image of a peptide hydrogel
located in a hepatic artery;
[0014] FIG. 6B is a histopathological image of peptide hydrogel
located in a hepatic artery;
[0015] FIG. 6C is a histopathological image of peptide hydrogel
located in a hepatic artery; and
[0016] FIG. 7 is a hepatic cell necrosis image of a peptide
hydrogel embolism location;
[0017] FIG. 8 is an image of an artery before embolization; and
[0018] FIG. 9 is an image of an artery after embolization.
DETAILED DESCRIPTION
[0019] Embolization is a procedure that creates a blockage,
lodging, occlusion, or embolism in one or more biological pathways
or channels. The biological pathways or channels may include
vessels, veins, portal veins, arteries, and ducts that may
transport blood and other fluids, such as lymph fluids.
Embolization is used to treat a wide variety of conditions
affecting different organs of a subject's body, including the human
body. The one or more vessels may be targeted to purposely prevent
or reduce the circulation of blood to a desired target. The
embolization procedure may be used to purposely create such a
blockage, lodging or occlusion in order to deprive tumors or other
pathological processes of their blood supply (perfusion).
Embolization may be used to treat disorders, malformations, or
congenital ailments in biological vessels. For example,
embolization may be used to treat patent ductus arteriosus (PDA).
The embolization treatment may be used to treat major
aortopulmonary collateral artery (MAPCA), recurrent hemotysis,
arteriovenous malformations, cerebral aneurysms, gastrointestinal
bleeding, epistaxis, post-partum hemorrhage, surgical hemorrhage,
and uterine fibroids.
[0020] Embolization may be accomplished by several different
techniques. It may be accomplished by administering a material,
such as a liquid to a desired or predetermined location, such as a
target area. Administering may include applying or injecting a
material, such as a liquid to a desired or predetermined location,
such as a target area.
[0021] Embolization may be used to shut down or block all or a
portion of a vessel which forms an aneurysm in order to prevent the
aneurysm from rupturing. Embolization may also be used to shut down
or block all or a portion of certain blood vessels that surround a
region of a subject that is being operated on, for example, during
surgery of cerebral arteriovenous malformation (AVM).
[0022] Obstructing materials may be used to create a blockage or
occlusion in a biological vessel to accomplish embolization.
Obstructing materials may include metal coils, collagens,
cyanoacrylates, and other materials. The materials may be inserted
or placed at the desired surgical sites with the use of a catheter.
Balloons may also be implanted in a target vessel and filled with
saline.
[0023] Metal coils may remain in vivo permanently, but the safety
of these coils in long-term applications is unknown. The metal
coils may also result in incompatibility with magnetic devices.
Collagens may have biological incompatabilities and cyanoacrylates
may become toxic in vivo.
[0024] Specific liquid embolization agents may include onyx,
n-butyl-2-cyanoacrylate (nbca) and ethiodol, made from iodine,
poppyseed oil. Schlerosing agents, which harden the endothelial
lining of vessels may also be used. Examples of such agents include
ethanol, ethanolamine oleate and sotradecol. Particulate
embolization agents include polyvinyl alcohol and acrylic gelatin
microspheres.
[0025] The present disclosure provides for methods of embolizing or
blocking biological vessels, methods of facilitating blocking a
biological vessel, and kits for use in blocking a biological
vessel. Biological vessels may include blood vessels and lymph
ducts. Blood vessels may include arteries, veins, portal veins and
capillaries. The term vascular may refer to biological vessels,
including arteries, veins, portal veins, capillaries, and
ducts.
[0026] The methods may comprise blocking or obstructing a
biological vessel in a subject or methods of facilitating blocking
or obstructing a biological vessel in a subject. 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.
[0027] The embolism, blockage or obstruction may be partial or
complete. By complete it is meant that the embolism, blockage or
obstruction prevents substantially all blood flow past the
embolism, blockage, or obstruction. The systems and methods may
include administration, application, or injection of a
self-assembling peptide, or a solution comprising a self-assembling
peptide, to a predetermined or desired target area. The
self-assembling peptide may be applied or introduced to a
biological vessel in the form of a self-assembling peptide
solution, hydrogel, membrane or other form.
[0028] The self-assembling peptide solution may be an aqueous
self-assembling peptide solution. The self-assembling peptide, also
referred to herein as "peptide" or "amphiphilic peptide" may be
administered, applied, or injected in a solution that is
substantially cell-free.
[0029] In certain embodiments, the self-assembling peptide may be
administered, applied, or injected in a solution that is
cell-free.
[0030] The self-assembling peptide may also be administered,
applied or injected in a solution that is substantially drug-free.
In certain embodiments, the self-assembling peptide may be
administered, applied, or injected in a solution that is drug-free.
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.
[0031] Administration of a solution may comprise, consist of, or
consist essentially of administration of a solution comprising,
consisting of, or consisting essentially of an amphiphilic peptide
comprising, consisting of, or consisting essentially of at least 12
amino acids that alternate between a hydrophobic amino acid and a
hydrophilic amino acid.
[0032] The systems and methods may comprise administering a
self-assembling peptide to a predetermined or desired target as a
hydrogel. A hydrogel is a term that may refer to a colloidal gel
that is dispersed in water. 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.
[0033] When using the term "administering," it 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 a solution,
such as an aqueous solution, or by way of a hydrogel, with or
without additional components.
[0034] The method of blocking the biological vessel in a subject
may comprise introducing a syringe, pipette, catheter, or other
needle-based device into the biological vessel. The self-
assembling peptide may be administered by way of a syringe,
pipette, catheter, or other needle-based device into the biological
vessel. The gauge of the syringe needle may be selected to provide
an adequate flow of 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 or peptide solution being administered,
the concentration of the peptide in solution, and the viscosity of
the peptide solution.
[0035] The method of blocking the biological vessel in the subject
may comprise introducing a catheter into the biological vessel and
positioning an end of the catheter in a target area of the
biological vessel in which at least a partial obstruction is
desired. The self-assembling peptide may be administered by way of
a catheter to the target area of a biological vessel in which at
least a partial obstruction is desired. The use of a catheter 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 such that blockage of the
biological vessel 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 blockage in the biological
vessel over use of a syringe or other means.
[0036] Use of the catheter may include use of accompanying devices,
such as a guidewire used to guide the catheter into position. The
guidewire may be introduced into the biological vessel prior to
introducing the catheter. Once the administration of the peptide
solution is complete, or once the at least partial obstruction or
blockage is in place, the catheter may be removed from the
biological vessel.
[0037] The use of a syringe, needle, pipette, other needle-based
device, or catheter may require determining the diameter of the
biological vessel which is targeted, such that at least a portion
of the syringe, needle, pipette, other needle-type device, or
catheter may enter the biological vessel to administer the peptide,
peptide solution, or hydrogel to the target area.
[0038] 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 area in which it is desired to
create an embolism. In other examples, this location maybe upstream
or downstream of the area in which it is desired to form an
embolism. In this case, it may be desired to allow an unassisted
movement or migration of the hydrogel to the area in which it is
desired to form an embolism. Alternatively, another procedure may
position the hydrogel in the area in which it is desired to form an
embolism. The desired location or target area may be a portion of a
biological vessel. The desired location or target area may be a
portion within a biological vessel.
[0039] 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 allow an embolism to be created at that
location. In certain examples, the hydrogel may be formed in vivo
at one location, and allowed to move the hydrogel unassisted to the
area in which it is desired to form an embolism. Alternatively,
another procedure may place the hydrogel in the area in which it is
desired to form an embolism. 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.
[0040] The particular self-assembling peptides of the present
disclosure may provide for improved adhesion to tissue over other
agents that may be used in biological vessel embolization. The
improved adhesion may be due to the composition of the peptide (for
example, the particular amino acids of the peptide), the structure
of the peptide once self- assembled, or due to the self-assembly
process itself. In certain embodiments, it may benefit the
procedure to remove excess body fluid, such as blood or bile, from
the target site or area in which it is desired to provide a
hydrogel for occlusion.
[0041] In some embodiments, the peptide or hydrogel may not adhere
to the tissue or biological vessel. As the peptide or peptide
solution is administered, it comes in contact with the blood or
other fluid in the biological vessel, which causes gelation inside
the vessel. The peptide solution can move within the vessel, but as
it begins to gel it loses its fluidity and will remain in position
at a position or target area in the biological vessel. The peptides
in the form of a hydrogel may remain in place without adhesion to
the tissue or biological vessel.
[0042] This disclosure relates to aqueous solutions, hydrogels, and
membranes comprising self-assembling peptides, sometimes referred
to as self-assembling oligopeptides, or amphiphilic peptides. The
peptides may be comprised of an amphiphilic peptide having about 6
to about 200 amino acid residues with the hydrophilic amino acids
and hydrophobic amino acids alternately bonded. The self-assembling
peptides may exhibit a beta-structure in aqueous solution in the
presence of physiological pH and/or a cation, such as a monovalent
cation.
[0043] The order of effectiveness of the monovalent cations appears
to be Li.sup.+>Na.sup.+>K.sup.+>Cs.sup.+. Cs.sup.+ may
produce the least amount of membranes and in addition, yields
nonmembranous precipitates. The effectiveness of the monovalent
cations may correlate inversely with the crystal radii of the ions:
Li.sup.+ (0.6 Angstroms), Na.sup.+ (0.95 Angstroms), K.sup.+ (1.33
Angstroms), and Cs.sup.+ (1.69 Angstroms) (Pauling, 1960). A
correlation may also be seen with the hydrated radii of the ions:
Li.sup.+ (3.4 Angstroms), Na.sup.+ (2.76 Angstroms), K.sup.+ (2.32
Angstroms), and Cs.sup.+ (2.28 Angstroms), and with the order of
enthalpies of the monovalent cations (Pauling, 1960). The presence
of the the monovalent metal cations may act as a catalyst or may be
incorporated into the membrane. The size of the filaments (10-20
nm) and interfilament distance (50-80 nm) in some membranes formed
may suggest that hydrated ions may stabilize the intermolecular
interaction. Some anions, including divalent anions, acetate,
Cl.sup.-, SO.sub.4.sup.-2, and PO.sub.4 .sup.-2, and organic ions,
NH.sub.4.sup.+ and Tris-Cl, may not induce membrane formation.
[0044] Concentrations of monovalent metal cations (NaCl) as low as
5 mM and as high as 5 M have been found to induce membrane
formation within a few minutes in certain embodiments. Thus,
membrane formation may be independent of salt concentration over
this wide range. Salt concentrations of less than 5 mM may also
induce membrane formation, but at a slower rate.
[0045] The peptides may be generally stable in aqueous solutions
and self-assemble into large, extremely stable macroscopic
structures or matrices when exposed to physiological conditions or
levels of salt. The presence of a monovalent alkali metal ion such
as sodium ions and potassium ions present at physiological levels
promote formation of a hydrogel from the peptide solution. Once the
hydrogel is formed it may not decompose even under common protein
denaturing conditions such as high temperature or with denaturing
agents such as acids, alkalis, proteases, urea, guanidine
hydrochloride or the like. The self-assembled peptides may be
visible to the naked eye when stained with a dye, Congo Red, and
can form sheet-like or fibril structures which have high tensile
strength. These materials are substantially resistant to change in
pH, heat, and enzymatic proteolysis. The self-assembled peptides
have a fibrous microstructure with small pores as revealed by
electron microscopy.
[0046] Physiological conditions may occur in nature for a
particular organism or cell system, 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 a range of about 6 to
about 8. The physiological conditions may include cations such as
monovalent metal cations that may induce membrane 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.
[0047] The self-assembling peptides of the present disclosure may
have at least 8 amino acids, at least 12 amino acids, or at least
16 amino acids. 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.
[0048] 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 in a
homogeneous mixture. Heterogeneous peptides, including those which
cannot form membranes in homogeneous solutions, which are
complementary and/or structurally compatible with each other may
also self-assemble into macroscopic membranes, filaments, and
hydrogels.
[0049] Macroscopic membranes, filaments, and hydrogels formed of
the self-assembling peptides may be stable in aqueous solution, in
serum, and in ethanol, and may be highly resistant to degradation
by heat, alkaline and acidic pH (stable at pH 1.5-11), chemical
denaturants (for example, guanidine-HCl, urea and sodium dodecyl
sulfate), and proteases in vitro (for example, trypsin,
alpha-chymotrypsin, papain, protease K, and pronase). They may be
non-cytotoxic.
[0050] The methods and methods of facilitating of the present
disclosure may comprise administering or providing instructions for
administering through a catheter a solution comprising an
amphiphilic peptide comprising at least 12 amino acids that
alternate between a hydrophobic amino acid and a hydrophilic amino
acid in an effective amount and in an effective concentration to
form a hydrogel under physiological conditions to allow at least
partial blockage of a biological vessel.
[0051] The methods of facilitating may comprise providing the
solution comprising an amphiphilic peptide comprising the at least
12 amino acids that alternate between a hydrophobic amino acid and
a hydrophilic amino acid in an effective amount and in an effective
concentration to form a hydrogel under physiological conditions to
allow at least partial blockage of a biological vessel.
[0052] The methods and methods of facilitating may comprise adding
a contrast agent to the peptide solution or providing instructions
to add a contrast agent to the solution. Alternatively, the peptide
solution may be manufactured with a contrast agent. The contrast
agent may provide a visual image during use of X-ray techniques,
such as fluoroscopy or angiography. A nonionic radiopaque contrast
media may be included, such as, for example, a water-soluble iodine
based solution. The water-soluble iodine based solution may be
iopamidol.
[0053] The methods and methods of facilitating of the present
disclosure may comprise visualizing a region comprising at least a
portion of the biological vessel or providing instructions to
visualize a region comprising at least a portion of the biological
vessel. The visualization may occur during at least one of
identifying the target area, introducing the catheter, positioning
the end of the catheter in the target area, administering of the
solution, and observing the biological vessel after removing the
catheter.
[0054] The visualizing may be accomplished through imaging using
X-ray radiography. Methods and methods of facilitating may comprise
visualizing or providing instructions to visualize the region using
X-ray radiography. Visualizing may occur for a period of time after
administering the peptide or removing the catheter. For example, it
may occur for up to 5 minutes or an hour after administering the
peptide or removing the catheter. Visualization may also occur
after one or more pre-determined intervals. For example,
visualization may occur about 24 hours after administering the
peptide or removing the catheter, after about one week, after about
two weeks, or after about four weeks. Visualization may occur after
about 3 months. Visualization may also occur after about 6 months.
Instructions may be provided to visualize the region at any one or
more of the times disclosed herein and for any period of time. For
example, at one week, the visualization may occur for 1 minute or 5
minutes. At four weeks, the visualization may occur for 10 minutes
or 3 minutes.
[0055] Visualizing or monitoring the area surrounding the formed
blockage may also occur for a period of time or at one or more
pre-determined intervals after administering the peptide or
removing the catheter. This may occur to determine any one or more
of the effectiveness of the blockage, any degradation of the
blockage, and any cell or tissue necrosis.
[0056] The methods of the present disclosure may further comprise
evaluating the subject to determine a need for blocking a
biological vessel and preparing the peptide solution. Preparing the
peptide solution may comprise adding a contrast agent to a
preliminary solution comprising peptides.
[0057] The method of facilitating may comprise providing
instructions to add a contrast agent to the solution. The method of
facilitating may comprising providing instructions to combine a
sufficient quantity or volume of the contrast agent in order to
adequately do at least one of: identify the target area, introduce
a catheter or other administration device, position an end of the
catheter in the target area, administer the peptide solution,
remove the catheter or other administration device, and observe the
biological vessel after removing the catheter. The use of the
contrast agent may allow visualization of the area to which the
peptide, peptide solution or hydrogel is administered.
[0058] 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 include Ala, Val,
Ile, Met, Phe, Tyr, Trp, Ser, Thr and Gly. The hydrophilic amino
acids can 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 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.
[0059] 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) (SEQ ID NO: 1)), and such
peptide sequences may be represented by (RADA).sub.p, wherein
p=2-50 (SEQ ID NO: 2). 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
(TEIK) (SEQ TD NO: 3)), and such peptide sequences are represented
by (IEIK).sub.p, wherein p=2-50 (SEQ ID NO: 4). 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) (SEQ ID NO: 5)), and such peptide sequences
are represented by (KLDL).sub.p, wherein p=2-50 (SEQ ID NO: 6). The
self-assembling peptides may be composed of about 8 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 12 to
about 17 residues. The peptides may have a length of about 5
nm.
[0060] 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).sub.4 (SEQ ID NO: 7), a self-assembling peptide IEIK13
having the sequence Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-
Ile-Glu-Ile-Lys-Ile (IEIK).sub.3I (SEQ ID NO: 8), 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 (SEQ ID NO: 9) or a self-assembling peptide
KLDL12 having the sequence
Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (KLDL).sub.3 (SEQ
ID NO: 10). A 1% aqueous solution of (RADA).sub.4 (SEQ ID NO: 7) is
available as the product PuraMatrix.TM. by 3D-Matrix Co., Ltd.
PuraMatrix.TM. contains 1% peptide having the sequence (RADA).sub.4
(SEQ ID NO: 7), in water.
[0061] 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) (SEQ ID NO: 11), RAD 16 (SEQ ID NO: 22),
RADA16 (SEQ ID NO: 7), and heteropolymers of RAD16 (SEQ ID NO:
[0062] 22) and EAK16 (SEQ ID NO: 11). 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 EAK 16 peptide (SEQ ID NO: 11) and other peptides
disclosed herein were derived from a region of a yeast protein,
zuotin.
[0063] 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.
[0064] 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
deliver and form an embolism upon administration to a biological
vessel may be selected.
[0065] As used herein, an amount of a peptide, peptide solution or
hydrogel effective to provide at least a partial obstruction,
blockage, or occlusion or treat a disorder, an "effective amount"
or a "therapeutically effective amount" refers to an amount of the
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.
[0066] 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 biological vessel that is
being treated or occluded, the desired thickness of the resulting
hydrogel that may be positioned in the desired target area, and the
length of time of treatment.
[0067] 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.
[0068] An effective amount and an effective concentration of the
peptide solution may be selected to at least partially obstruct or
block a biological vessel. In some embodiments, at least one of the
effective amount and the effective concentration may be based in
part on a diameter of the target area of the biological vessel. In
other embodiments, at least one of the effective amount and the
effective concentration is based in part on the flow rate of the
blood in the biological vessel. In other embodiments, at least one
of the effective amount and the effective concentration may be
based in part on a blood pressure of the blood in the biological
vessel. In still other embodiments, at least one of the effective
amount and the effective concentration may be based in part on an
average diameter of a red blood cell of the subject.
[0069] In yet other embodiments, at least one of the effective
amount and the effective concentration may be based in part on at
least one of the diameter of the target area of the biological
vessel, the flow rate of blood in the biological vessel, the blood
pressure of the blood in the biological vessel, and the average
diameter of a red blood cell of the subject.
[0070] The at least one of the effective amount and the effective
concentration may be based in part on providing nanofibers of a
hydrogel having an average pore size that is less than an average
diameter of a red blood cell of the subject. This may comprise
collecting a sample of blood from the subject to determine the
average red blood cell diameter to provide for the at least one of
the effective amount and the effective concentration.
[0071] The effective amount may be, as described herein, an amount
that may provide for a desired blockage in a biological vessel.
Various properties of the biological vessel may contribute to the
selection or determination of the effective amount including at
least one of the diameter of the target area of the biological
vessel, the flow rate of blood in the biological vessel, the blood
pressure of the blood in the biological vessel, and the average
diameter of a red blood cell of the subject.
[0072] 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 he about 2.0 mL. In some other embodiments,
the effective amount may be about 3.0 mL.
[0073] In some embodiments, a more effective blockage may be
achieved with a greater volume of peptide solution administered.
This may allow a longer or thicker hydrogel to form within the
biological vessel, allowing a more secure position of the hydrogel
in the target area. It is possible that if a high enough volume is
not selected, the hydrogel may not be effective in maintaining a
blockage in the target area for the desired period of time. This
may also be influenced based on the blood flow rate or blood
pressure in the vessel.
[0074] The effective concentration may be, as described herein, an
amount that may provide for a desired blockage in a biological
vessel. Various properties of the biological vessel may contribute
to the selection or determination of the effective concentration
including at least one of the diameter of the target area of the
biological vessel, the flow rate of blood in the biological vessel,
the blood pressure of the blood in the biological vessel, and the
average diameter of a red blood cell of the subject.
[0075] 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.
[0076] 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
blockage of the biological vessel. 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 maintaining a blockage in the target area for the
desired period of time. This may also be influenced based on the
blood flow rate or blood pressure in the vessel.
[0077] 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.
Methods of the disclosure contemplate single as well as multiple
administrations of a therapeutically effective amount of the
peptides, peptide solutions, and hydrogels as described herein.
Peptides as described herein may he administered at regular
intervals, depending on the nature, severity and extent of the
subject's condition. In some embodiments, a peptide, peptide
solution, or hydrogel may be administered in a single
administration. In some embodiments, a peptide, peptide solution,
or hydrogel described herein is administered in multiple
administrations. In some embodiments, a therapeutically effective
amount of a peptide, peptide solution, 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, 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 blockage of a biological vessel 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.
[0078] The self-assembling peptides of the present disclosure, such
as RADA16 (SEQ ID NO: 7), 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 occluding 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.
[0079] A 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 occluding effect,
thereby allowing the biological safety to be increased. In certain
examples, the sugar may be sucrose or glucose.
[0080] In certain embodiments, the peptide length may be more than
12 amino acids and preferably at least 16 residues. Very long
peptides, for example, of about 200 amino acids, may encounter
problems due to insolubility and intramolecular interactions which
destabilize membrane formation, but may also be contemplated
herein. Furthermore, peptides with a large amount of hydrophobic
residues may have insolubility problems. The optimal lengths for
membrane formation may vary with the amino acid composition.
[0081] An additional stabilization factor 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.
[0082] Examples of peptides that may form membranes in homogeneous
mixtures are shown in Table 1. These examples illustrate some of
the variety of amino acid arrangement and composition of
membrane-forming peptides.
TABLE-US-00001 TABLE 1 Potential membrane-forming peptides Name
Sequence (N.fwdarw.C) IEIK13 IEIKIEIKIEIKI (SEQ ID NO: 8) IEIK17
IEIKIEIKIEIKIEIKI (SEQ ID NO: 9) KAKA16 KAKAKAKAKAKAKAKA (SEQ ID
NO: 12) KAKAS KAKAK (SEQ ID NO: 13) KAE16 AKAKAEAEAKAKAEAE (SEQ ID
NO: 14) AKE16 AKAEAKAEAKAEAKAE (SEQ ID NO: 15) EKA16
EAKAEAKAEAKAEAKA (SEQ 11) NO: 11) EAK8 AEAEAKAK (SEQ ID NO: 16)
EAK12 AEAKAEAEAKAK (SEQ ID NO: 17) KEA16 KAEAKAEAKAEAKAEA (SEQ ID
NO: 18) AEK16 AEAKAEAKAEAKAEAK (SEQ ID NO: 19) ARD8 ARARADAD (SEQ
ID NO: 20) DAR16 ADADARARADADARAR (SEQ ID NO: 21) RAD16
ARADARADARADARAD (SEQ Ill NO: 22) DRA16 DARADARADARADARA (SEQ ID
NO: 23) RADA16 RADARADARADARADA (SEQ ID NO: 7) ADR16
ADARADARADARADAR (SEQ ID NO: 24) ARA16 ARARADADARARADAD (SEQ ID NO:
25) ARDAKE16 ARADAKAEARADAKAE (SEQ ID NO: 26) AKEW16
AKAEARADAKAEARAD (SEQ ID NO: 27) ARKADE16 ARAKADAEARAKADAE (SEQ ID
NO: 28) AKRAED16 AKARAEADAKARADAE (SEQ ID NO: 29) AQ16
AQAQAQAQAQAQAQAQ (SEQ ID NO: 30) VQ16 VQVQVQVQVQVQVQVQ (SEQ ID NO:
31) YQ16 YQYQYQYQYQYQYQYQ (SEQ ID NO: 32) IIQ16
IIQIIQIIQIIQIIQIIQIIQIIQ (SEQ ID NO: 33) AN16 ANANANANANANANAN (SEQ
ID NO: 34) VN16 VNVNVNVNVNVNVNVN (SEQ ID NO: 35) YN16
YNYNYNYNYNYNYNYN (SEQ ID NO: 36) HN16 HNHNHNHNHNHNHNHN (SEQ ID NO:
37) ANQ16 ANAQANAQANAQANAQ (SEQ ID NO: 38) AQN16 AQANAQANAQANAQAN
(SEQ Ill NO: 39) VNQ16 VNVQVNVQVNVQVNVQ (SEQ ID NO: 40) VQK16
VQVNVQVNVQVNVQVN (SEQ ID NO: 41) YNQ16 YNYQYNYQYNYQYNYQ (SEQ ID NO:
42) YQN16 YQYNYQYNYQYNYQYN (SEQ ID NO: 43) HNQ16 HNHQHNHQHNHQHNHQ
(SEQ ID NO: 44) HQN16 HQHNHQHNHQHNHQHN (SEQ ID NO: 45) AKQD18
AKAQADAKAQADAKAQAD (SEQ ID NO: 46) VKQ18 VKVQVDVKVQVDVKVQVD (SEQ
Ill NO: 47) YKQ18 YKYQYDYKYQYDYKYQYD (SEQ ID NO: 48) HKQ18
HKHQHDHKHQHDHKHQHD (SEQ ID NO: 49) .beta.-Amy1oid
DAEFRHDSGYEVHHQKLVFFAEDVGSNK (1-28) (SEQ ID NO: 50) .beta.-Amyloid
GSNKGAIIGLM (SEQ ID NO: 51) (25-35) Substance P RPKQQFGLM (SEQ ID
NO: 52) Spantide (D)RPKPQQ(D)WF(D)WLL* (SEQ ID NO: 53) *(D) in
Spantide is a D amino acid incorporated into the peptide
[0083] 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) (SEQ ID NO: 54), as shown in the appended
sequence listing), 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) (SEQ ID NO: 55), has Lys and Asp. Peptides A and
B arc 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. A calculation of the interpeptide distances in such pairs,
however, shows that the two peptides are not structurally
compatible. Using a conversion factor of 3 Angstroms per atom, the
difference in interpeptide distance between the two pairs would be
3 Angstroms. It is estimated that a variation in interpeptide
distance of more than 3-4 Angstroms would destabilize
intermolecular interactions leading to membrane formation. 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.
[0084] Membranes and hydrogels may also he 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 (SEQ ID
[0085] NO: 12) and (Glu-Ala-Glu-Ala).sub.4 (EAEA).sub.4 (SEQ ID NO:
56) or of (Lys-Ala-Lys-Ala).sub.4 (KAKA).sub.4 (SEQ ID NO: 12) and
(Ala-Asp-Ala-Asp).sub.4 (ADAD).sub.4 (SEQ ID NO: 57) would be
expected to form membranes, but not any of these peptides alone due
to lack of complementarity.
[0086] 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 he 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. Mismatched peptides can be tested for ability to
self-assemble into macroscopic membranes using the methods
described herein.
[0087] 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.
[0088] The initial concentration of the peptide may be a factor in
the size and thickness of the membrane or hydrogel formed. In
general, it may be the case that the higher the peptide
concentration, the higher the extent of membrane formation.
Membranes or hydrogels may form from initial peptide concentrations
as low as about 0.5 mM or about 1 mg/ml (about 0.1 w/v percent).
However, membranes or hydrogels formed at higher initial peptide
concentrations (about 10 mg/ml (about 1 w/v percent)) may be
thicker and thus, likely to be stronger. It may be preferable when
producing the membranes or hydrogels to add peptide to a salt
solution or a physiological condition, rather than to add salt to a
peptide solution.
[0089] Formation of the membranes or hydrogels may be very fast, on
the order of a few minutes. The formation of the membranes or
hydrogels may form instantaneously upon application or injection to
a desired area. The formation of the membranes or hydrogels may
occur within one to two minutes of application or injection. In
other examples, the formation of the membranes or hydrogels may
occur within four minutes of application or injection. In certain
embodiments the time it takes to form the membranes or hydrogels
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 blood flow rate, the blood pressure, and the
diameter of the biological vessel).
[0090] The formation of the membranes or hydrogels may be
irreversible. The process may be unaffected by pH of less than or
equal to 12 (the peptides tend to precipitate out at pH above 12),
and by temperature. The membranes or hydrogels may form at
temperatures in the range of 4 to 90 degrees Celsius.
[0091] The membranes or 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 may be to reduce or prevent flow of a fluid through
a biological pathway or channel. The desired effect may be a
blockage, lodging, occlusion, or embolism in one or more biological
pathways or channels. The desired effect may be to purposely create
such a blockage, lodging or occlusion in order to deprive tumors or
other pathological processes of their blood supply (perfusion).
[0092] The desired effect using the methods and kits of the present
disclosure may be to treat disorders, malformations, or congenital
ailments in biological vessels. The desired effect may be to treat
one or more of patent ductus arteriosus (PDA), major aortopulmonary
collateral artery (MAPCA), recurrent hemotysis, arteriovenous
malformations, cerebral aneurysms, gastrointestinal bleeding,
epistaxis, post-partum hemorrhage, surgical hemorrhage, and uterine
fibroids. The desired effect may include providing at least a
partial blockage to produce cell necrosis or to reduce or eliminate
cancerous cells.
[0093] 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
several days, up to two weeks. In other examples, 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 in the biological
vessel, or another biological vessel may be performed.
[0094] 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.
[0095] 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.
[0096] A component may be included to protect the peptide solution
against rapid or immediate formation into a hydrogel. This may
include an encapsulated delivery systems that may degrade over time
to allow a controlled time release of the peptide solution into the
target area to form the hydrogel over time 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.
[0097] 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.
[0098] A peptide, peptide solution, or hydrogel of the disclosure
may be provided in a kit. Instructions for administering the
solution to a biological vessel in a subject may also be provided
in the kit. The peptide solution may comprise an amphiphilic
peptide comprising at least 12 amino acids that alternate between a
hydrophobic amino acid and a hydrophilic amino acid in an effective
amount and in an effective concentration to form a hydrogel under
physiological conditions to allow at least partial blockage of a
biological vessel. 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.
[0099] 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, peptide
solution or hydrogel, concentration, volume, size, dimensions, date
of expiration, and batch or production site.
[0100] 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 or other
accompanying equipment to provide selective administration of the
peptide solution to the target area.
[0101] The kit may comprise in addition to or in the alternative,
other components or ingredients, such as components that may aid in
contrast imaging. For example, the kit may comprise a contrast
agent. The contrast agent may provide a visual image during use of
X-ray techniques, such as fluoroscopy or angiography. A nonionic
radiopaque contrast media may be included, such as, for example, a
water-soluble iodine based solution. The water-soluble iodine based
solution may be iopamidol. Instructions may be provided in the kit
to combine a sufficient quantity or volume of the contrast agent in
order to adequately do at least one of: identify the target area,
introduce a catheter or other administration device, position an
end of the catheter in the target area, administer the peptide
solution, remove the catheter or other administration device, and
observe the biological vessel after removing the catheter. The use
of the contrast agent may allow visualization of the area to which
the peptide, peptide solution or hydrogel is administered.
Instructions may be provided for diluting the peptide solution to
administer an effective concentration of the solution to the
biological vessel. 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 biological
vessel. This may be based on various parameters discussed herein,
and may include the diameter of the biological vessel at the target
area.
[0102] 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.
[0103] Other components which are disclosed herein may also be
included in the kit.
[0104] 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.
[0105] 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.
[0106] 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
biological vessel. The self-assembling peptides may also be soaked
into a sponge for use.
[0107] In alternative embodiments, an atomizing sprayer filled with
a powder or solution of the self-assembling peptides may be
prepared. When such a spray is used for spraying onto an affected
area, the pH and salt concentration increase upon contact with the
body causing gelling.
[0108] Modification of the membranes may give them additional
properties. For example, the membranes may be further strengthened
by cross-linking the peptides after membrane formation by standard
methods. Collagen may be combined with the peptides to produce
membranes more suitable for use as artificial skin; the collagen
may be stabilized from proteolytic digestion within the membrane.
Furthermore, combining phospholipids with the peptides may produce
vesicles.
[0109] 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 can further improve attachment, cell growth
and neurite outgrowth.
[0110] The function and advantage of these and other embodiments of
the methods and kits disclosed herein will be more fully understood
from the example below. The following example is intended to
illustrate the benefits of the disclosed treatment approach, but do
not exemplify the full scope thereof.
EXAMPLES
Example 1
[0111] Tests were performed on a rat using a 3% (weight per volume
(w/v)) PuraMatrix.TM. solution, a peptide solution comprising
Ac-RADARADARADARADA-NH.sub.2 (Ac-Arg-Ala-
Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH.sub.2)
(SEQ ID NO: 58) in water. An 18 gauge needle was used to inject 1
milliliter (mL) into a rat portal vein. Hematoxylin-Eosin (HE) dye
was used to implement a histopathological evaluation. It was
confirmed that an embolism developed in the portal vein using the
peptide solution. As shown in FIG. 1, the peptide solution appears
to have developed into a hydrogel 2 and resides in the rat portal
vein. A red blood cell 4 is also shown.
Example 2
[0112] Tests were performed in two beagles using a 2.5%
PuraMatrix.TM. solution, a peptide solution comprising
Ac-RADARADARADARADA-NH.sub.2
(Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH.su-
b.2) (SEQ ID NO: 58) in water. These tests were performed to
confirm the effectiveness of beagle hepatic artery embolism and
hepatic cell necrosis using the 2.5% peptide solution. The peptide
solution included iopamidol at a concentration of 612.4 mg/mL.
Iopamidol is a nonionic radiopaque contrast agent.
[0113] Under X-ray imaging of a beagle under full anesthesia, a
microcatheter (Terumo, minimum inner diameter of 0.50 mm) was
inserted by way of the carotid artery into the hepatic artery.
Hepatic artery contrast imaging was used to confirm that the
hepatic artery was operational. A 2 mL volume of the 2.5% peptide
solution (with Iopamidol) was injected.
[0114] Hepatic artery contrast imaging was used to confirm the
hepatic artery peptide solution embolism effect during surgery.
FIG. 2 displays a contrast image of a normal hepatic artery of the
beagle, while FIG. 3 shows the peptide solution injection. FIG. 4
shows a hepatic artery contrast image after a peptide solution
injection in which back flow of the contrast image was confirmed.
The presence of the peptide solution is evidenced by the darker
regions of the image.
[0115] After two weeks of monitoring elapsed, hepatic artery
contrast imaging was used to confirm the embolism effect. FIG. 5
shows a hepatic artery contrast image two weeks after a peptide
solution injection.
[0116] Subsequently, the liver was extracted. Hematoxylin-Eosin
(HE) dye was used to histopathologically confirm the peptide
solution embolism and hepatic impairment. As shown in FIGS. 6A-6C,
the peptide solution can be seen in each of these images of the
hepatic artery as the darkened areas of the images. FIG. 7 shows a
hepatic cell necrosis image at an embolism location, where all
cells appear to have at least some level of necrosis.
[0117] The results show that injection of the peptide solution
using a microcatheter may be accomplished. The peptide gel may be
visible using X-ray imaging. The hepatic artery embolism effect can
be seen during surgery and two weeks after surgery. Additionally,
the hepatic artery embolism effect and hepatic cell necrosis effect
using the peptide solution occurred and was confirmed
histopathologically.
Example 3
[0118] PuraMatrix.TM., a peptide solution comprising
Ac-RADARADARADARADA-NII.sub.2
(Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH.su-
b.2) (SEQ ID NO: 58) in water was used as an embolic agent in a
porcine model through angiography, gross necropsy assessment, and
histopathology assessment. One female Yorkshire cross swine was
tested. The weight of the swine at the time of testing was 46.5 kg.
Feed and water were provided per standard operating procedures.
There were no contaminants in the food or water that were expected
to interfere with the conduct or results of the study. The swine
was acquired from a test facility approved animal supplier. The
Swine participated in an incoming physical exam, and after a period
of acclimation was again examined. The swine was fasted a minimum
of 12 hours prior to the procedures. The animals were sedated and
anesthetized by an intramuscular or subcutaneous injection of
Telazol (2-10 mg/kg) and Xylazine (0.5=5.0 mg/kg). Propofol (to
effect) was given to aid in sedation. An endotracheal tube was used
to ensure proper ventilation and the animals were maintained under
general anesthesia with inhalant isofluorante (0.1 to 5.0%).
Heparin (50-300 units/kg, IV) was administered throughout the
procedure.
[0119] A 2.5% test solution of the peptide solution was used.
Approximately 800 microliters of the peptide solution was placed in
an eppendorf tube. Approximately 200 microliters of Isovue-370
(Iopamidol) contrast agent was added. The liquids were mixed slowly
so as to not create air bubbles.
[0120] On the day of testing, the swine was 2 months, 25 days old.
The swine was sedated and prepared for surgery. The femoral artery
was accessed and an introducer was placed. A guidewire was advanced
to the selected renal artery. A catheter was advanced to the
selected renal artery. Angiography was used to visualize the
location within the artery. The peptide solution was injected to
the desired location until the artery was occluded. This procedure
was repeated in the hepatic and splenic arteries. Angiography was
used throughout the procedure to visualize the vessels and devices
throughout testing. FIGS. 8 and 9 are representative examples of a
vessel before (FIG. 6) and after (FIG. 9) embolization. There were
no adverse events reported throughout the testing.
[0121] A summary of the data can be found in Table 2 below.
TABLE-US-00002 TABLE 2 Test Approximate Num- Embolization Volume
ber Site Time Placed Comments 1 Left Start 13:55 1.5 mL Successful
Kidney embolization Renal immediately Artery following injection of
peptide solution/Isovue 2 Right Start 14:12 1.5 mL Successful
Kidney embolization Renal immediately Artery following injection.
Slight flow reestablished at 14:18. Additional PuraMatrix.sup.Tm/
Iopamidol placed at 14:27. Angiogram showed full occlusion. 3
Hepatic Start 14:58 2.0 mL Successful Artery embolization
immediately following injection. At 15:08, the artery remained
occluded. At 15:32, the artery remained occluded. 4 Splenic Start
15:17 3.0 mL Vessel was Artery completed occluded at 15:21.
[0122] As shown in Table 2, injection into the left kidney renal
artery was successful, immediately following injection. Successful
embolization of the right kidney renal artery was successful
immediately following injection, however, a slight flow was
reestablished 6 minutes after the initial injection. An additional
injection was made 15 minutes after the initial injection, and a
full occlusion was obtained.
[0123] Successful embolization of the hepatic artery was also
obtained immediately following injection. The artery remained
occluded after 10 minutes and 34 minutes. Successful embolization
of the splenic artery was also obtained after four minutes.
[0124] The description and figures provided are for example only
and are not intended to be limiting. While exemplary embodiments of
the disclosure have been disclosed many modifications, additions,
and deletions may be made therein without departing from the spirit
and scope of the disclosure and its equivalents, as set forth in
the following claims.
[0125] Those skilled in the art would readily appreciate that the
various configurations described herein are meant to be exemplary
and that actual configurations will depend upon the specific
application for which the system and methods of the present
disclosure are used. Those skilled in the art will recognize, or be
able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments described herein.
[0126] Further, it is to be appreciated various alterations,
modifications, and improvements will readily occur to those skilled
in the art. Such alterations, modifications, and improvements are
intended to be part of this disclosure, and are intended to be
within the spirit and scope of the disclosure. Accordingly, the
foregoing description and drawings are by way of example only.
Further, the depictions in the drawings do not limit the
disclosures to the particularly illustrated representations.
[0127] As used herein, the terms "comprising," "including,"
"carrying," "having," "containing," and "involving," whether in the
written description or the claims and the like, are open-ended
terms, i.e., to mean "including but not limited to." Thus, the use
of such terms is meant to encompass the items listed thereafter,
and equivalents thereof, as well as additional items. Only the
transitional phrases "consisting of" and "consisting essentially
of," are closed or semi-closed transitional phrases, respectively,
with respect to the claims Use of ordinal terms such as "first,"
"second," "third," and the like in the claims to modify a claim
element does not by itself connote any priority, precedence, or
order of one claim element over another or the temporal order in
which acts of a method are performed, but are used merely as labels
to distinguish one claim element having a certain name from another
element having a same name (but for use of the ordinal term) to
distinguish the claim elements.
Sequence CWU 1
1
5814PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Arg Ala Asp Ala12200PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(9)..(200)/replace="
"misc_feature(1)..(200)/note="This sequence many 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" 2Arg 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 20034PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 3Ile Glu Ile
Lys14200PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polypeptide"VARIANT(9)..(200)/replace="
"misc_feature(1)..(200)/note="This sequence many 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" 4Ile 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 20054PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 5Lys Leu Asp
Leu16200PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polypeptide"VARIANT(9)..(200)/replace="
"misc_feature(1)..(200)/note="This sequence many 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" 6Lys 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 200716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala
Asp Ala1 5 10 15813PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 8Ile Glu Ile Lys Ile Glu
Ile Lys Ile Glu Ile Lys Ile1 5 10917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 9Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu
Ile Lys1 5 10 15Ile1012PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 10Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu1 5
101116PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 11Ala Glu Ala Glu Ala Lys Ala Lys Ala
Glu Ala Glu Ala Lys Ala Lys1 5 10 151216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala
Lys Ala1 5 10 15135PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 13Lys Ala Lys Ala Lys1
51416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 14Ala Lys Ala Lys Ala Glu Ala Glu Ala
Lys Ala Lys Ala Glu Ala Glu1 5 10 151516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 15Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys
Ala Glu1 5 10 15168PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 16Ala Glu Ala Glu Ala Lys
Ala Lys1 51712PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 17Ala Glu Ala Lys Ala Glu
Ala Glu Ala Lys Ala Lys1 5 101816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala
Glu Ala1 5 10 151916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 19Ala Glu Ala Lys Ala Glu
Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys1 5 10 15208PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Ala Arg Ala Arg Ala Asp Ala Asp1 52116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala Arg
Ala Arg1 5 10 152216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 22Ala Arg Ala Asp Ala Arg
Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp1 5 10 152316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 23Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala
Arg Ala1 5 10 152416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 24Ala Asp Ala Arg Ala Asp
Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg1 5 10 152516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Ala Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp
Ala Asp1 5 10 152616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 26Ala Arg Ala Asp Ala Lys
Ala Glu Ala Arg Ala Asp Ala Lys Ala Glu1 5 10 152716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 27Ala Lys Ala Glu Ala Arg Ala Asp Ala Lys Ala Glu Ala Arg
Ala Asp1 5 10 152816PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 28Ala Arg Ala Lys Ala Asp
Ala Glu Ala Arg Ala Lys Ala Asp Ala Glu1 5 10 152916PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 29Ala Lys Ala Arg Ala Glu Ala Asp Ala Lys Ala Arg Ala Asp
Ala Glu1 5 10 153016PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 30Ala Gln Ala Gln Ala Gln
Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln1 5 10 153116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln
Val Gln1 5 10 153216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 32Tyr Gln Tyr Gln Tyr Gln
Tyr Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln1 5 10 153316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 33His Gln His Gln His Gln His Gln His Gln His Gln His Gln
His Gln1 5 10 153416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 34Ala Asn Ala Asn Ala Asn
Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn1 5 10 153516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 35Val Asn Val Asn Val Asn Val Asn Val Asn Val Asn Val Asn
Val Asn1 5 10 153616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 36Tyr Asn Tyr Asn Tyr Asn
Tyr Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn1 5 10 153716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 37His Asn His Asn His Asn His Asn His Asn His Asn His Asn
His Asn1 5 10 153816PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 38Ala Asn Ala Gln Ala Asn
Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln1 5 10 153916PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 39Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln
Ala Asn1 5 10 154016PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 40Val Asn Val Gln Val Asn
Val Gln Val Asn Val Gln Val Asn Val Gln1 5 10 154116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 41Val Gln Val Asn Val Gln Val Asn Val Gln Val Asn Val Gln
Val Asn1 5 10 154216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 42Tyr Asn Tyr Gln Tyr Asn
Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln1 5 10 154316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 43Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln
Tyr Asn1 5 10 154416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 44His Asn His Gln His Asn
His Gln His Asn His Gln His Asn His Gln1 5 10 154516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 45His Gln His Asn His Gln His Asn His Gln His Asn His Gln
His Asn1 5 10 154618PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 46Ala Lys Ala Gln Ala Asp
Ala Lys Ala Gln Ala Asp Ala Lys Ala Gln1 5 10 15Ala
Asp4718PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 47Val Lys Val Gln Val Asp Val Lys Val
Gln Val Asp Val Lys Val Gln1 5 10 15Val Asp4818PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 48Tyr Lys Tyr Gln Tyr Asp Tyr Lys Tyr Gln Tyr Asp Tyr Lys
Tyr Gln1 5 10 15Tyr Asp4918PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49His Lys His Gln His Asp His Lys His Gln His Asp His Lys
His Gln1 5 10 15His Asp5028PRTUnknownsource/note="Description of
Unknown Beta-amyloid peptide" 50Asp Ala Glu Phe Arg His Asp Ser Gly
Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys 20 255111PRTUnknownsource/note="Description of
Unknown Beta-amyloid peptide" 51Gly Ser Asn Lys Gly Ala Ile Ile Gly
Leu Met1 5 10529PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 52Arg Pro Lys Gln Gln Phe
Gly Leu Met1 55311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)D-ArgMOD_RES(7)..(7)D-TrpMOD_RES(9)..(9)D-Trp
53Arg Pro Lys Pro Gln Gln Trp Phe Trp Leu Leu1 5
105416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 54Val Arg Val Arg Val Asp Val Asp Val
Arg Val Arg Val Asp Val Asp1 5 10 155516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 55Ala Asp Ala Asp Ala Lys Ala Lys Ala Asp Ala Asp Ala Lys
Ala Lys1 5 10 155616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 56Glu Ala Glu Ala Glu Ala
Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala1 5 10 155716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57Ala Asp Ala Asp Ala Asp Ala Asp Ala Asp Ala Asp Ala Asp
Ala Asp1 5 10 155816PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide"source/note="N-term
acetylated"source/note="C-term amidated" 58Arg Ala Asp Ala Arg Ala
Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala1 5 10 15
* * * * *