SURGICAL METHODS EMPLOYING PURIFIED AMPHIPHILIC PEPTIDE
COMPOSITIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. provisional
patent application serial no. 61/773,359, filed March 6, 2013, which application is hereby
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] This application makes reference to a sequence listing submitted in electronic
form as an ascii .txt file named "2004837-0033_ST" on March 6, 2014. The .txt file was
generated on February 27, 2014 and is 13 kb in size.
BACKGROUND
[0003] Surgical procedures are performed to correct a variety of medical problems
encountered by patients. Typically, an incision is made to access a surgical site within the
body of a patient. Blood vessels may be clamped to prevent and/or minimize bleeding;
retractors may be employed to expose the surgical site or allow it to remain open thereby
permitting a surgeon to perform one or more tasks associated with t e procedure. Depending
on the work to be performed, several incisions and/or dissections may be necessary in order
to penetrate to the surgical site. For example, to gain access to a location in the abdomen, it
may be necessary to dissect skin, subcutaneous tissue, muscle layers and/or peritoneum. As
is the case in some surgical procedures, it may also be necessary for a surgeon to cut into
bone. For example, some surgical procedures may involve cutting the skull to gain access to
the brain, or cutting the chest to gain access to the heart. Bleeding can and typically does
occur at multiple points in the performance of any or all of these procedures.
[0004] Some bleeding during surgery is to be expected. However, extensive bleeding
(i.e., beyond what is typically encountered in a given surgical procedure) can be dangerous,
even life-threatening. In some cases, severe bleeding may cause a surgical procedure to be
terminated. In some cases, a transfusion may be necessary. Blood or blood expanders are
typically employed during a surgical procedure to compensate for blood loss. In some cases,
steps taken to address blood loss can add considerable time to a surgical procedure and/or
lead to longer recovery times for patients.
[0005] The standard of care for controlling bleeding during surgical procedures
includes the use of synthetic products, materials derived from animals, or human blood
components that are locally administered in an as needed manner or by established
methodology. Such products and materials are primarily composed of tissue-building
proteins are well suited for surgical application as they are biocompatible and demonstrate
effectiveness. However, they are not without limitation. For example, these products can
present a risk of infection through t e presence of infection substances, e.g., viruses. Further,
animal-derived products present their own risks in that they can trigger untoward immune
responses, potentially including anaphylactic shock, when the patient's body reacts to foreign
antigens in the products.
[0006] The present invention provides, among other things, improved surgical
procedures that, for example, employ materials that are safer and more effective in
controlling and arresting bleeding encountered by surgeons while performing surgical tasks.
The present invention also provides surgical procedures that are performed in shorter time
and/or involve less bleeding than typically occurs in standard procedures.
SUMMARY
[0007] The present invention provides, among other things, improved surgical
methods for treating and/or stopping bleeding (e.g., exudative bleeding) during surgery. It is
contemplated that peptide compositions provided by the present invention are particularly
useful for use in surgical methods employed to stop bleeding, such as coronary artery bypass
and liver resection in whole or in part. In some embodiments, use of peptide compositions as
described herein in surgical methods provides an improvement in stopping and/or controlling
bleeding during a surgical procedure (e.g., intrabody).
[0008] In some embodiments, the present invention provides in a method of
performing an intrabody surgical procedure on a patient or subject in which an incision is
made in a body so that a) access to a site including a damaged portion of an internal organ or
tissue is provided for a first period of time, b) removal, repair, or replacement of some or all
of the damaged portion is performed during the first period of time, and c) the incision is
closed at the first period of time's end, an improvement comprising within t e first period of
time, performing at least one application within the site of a composition comprising a of 0.1
- 10% peptide solution, wherein the peptide comprises an amino acid sequence of RADA
repeats; and wherein the solution is characterized by an ability to transition between two
states: an un-gelled state adopted when one or more particular ions is substantially absent,
and a gelled state adopted when t e one or more ions is present at or above a threshold level,
wherein the one or more ions is or becomes present in the location; and permitting the
composition to remain in the site for a second period of time, sufficient for the peptides in the
composition to transition to their gelled state.
[0009] In some embodiments, a first period of time is greater than five minutes; in
some embodiments, greater than 10, 20, 30, 40, 50, or 60 minutes. In some embodiments, a
first period of time is about one hour. In some embodiments, a first period of time is less
than five minutes; in some embodiments, less than 10, 20, 30, 40, 50 or 60 minutes. In some
embodiments, a first period of time is less than one hour.
[0010] In some embodiments, the present invention further comprises an
improvement of performing, within a second period of time, at least one other medical
procedure in a site. In some embodiments, a second period of time is less than five minutes;
in some embodiments, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In some embodiments,
a second period of time is about 5 minutes. In some embodiments, a second period of time is
greater than five minutes; in some embodiments, a second period of time is greater than 6, 7,
8, 9, 10, or more minutes. In some embodiments, a second period of time is about 10
minutes.
[0011] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0012] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human subjects include mammals. In some certain
embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs, cattle,
sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs, llamas,
mules, rabbits, reindeer, water buffalo and yaks.
[0013] In some embodiments, an intrabody surgical procedure of t e present
invention is a resection of or at least a portion of t e liver. In some embodiments, a resection
of the liver in whole or in part is performed. In some embodiments, of the present invention
further comprises an improvement of completing the liver resection within a first period of
time that is less than four hours (e.g., less than 3.75, 3.50, 3.00, 2.75, 2.00, 1.75, 1.50, or
1.00) and therefore reduced as compared with the standard first period of time absent such
improvement, which standard first period of time is within the range of five to six hours (e.g.,
within the range of about five to about six hours, inclusive; in some embodiments, about 5.0,
5.1, 5.2, 5.3. 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 hours).
[0014] In some embodiments, the present invention further comprises an
improvement of not applying fibrin glue or SURGICEL® or a combination thereof within the
site during the first period of time. In some embodiments, the present invention comprises an
improvement of applying the composition comprising a solution of peptides in addition to
fibrin glue or SURGICEL® or a combination thereof within the site during the first period of
time.
[0015] In some embodiments, at least one first application is completed prior to any
other surgical activity within the site.
[0016] In some embodiments, an intrabody surgical procedure of the present
invention is a coronary artery bypass.
[0017] In some embodiments, a patient or subject is dosed with an anti-coagulant
prior to surgery.
[0018] In some embodiments, an intrabody surgical procedure of the present
invention is a coronary artery bypass in which an improvement further comprises completing
the surgical procedure within a first period of time that is at least 20 minutes (e.g., at least 21,
22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes) per graft shorter as compared with the standard
first period of time absent such improvement; in some certain embodiments, about 20
minutes.
[0019] In some embodiments, a peptide solution of the present invention has a
concentration within the range of about 0 .1% to about 10% (e.g., about 0.1 - 10%; 0.2 -
9.9%, 0.3 - 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%,
1.0 - 9.1%, 1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 -
8.4%, 1.8 - 8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%,
2.5 - 7.6%, 2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 -
6.9%, 3.3 - 6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%,
4.0 - 6.1%, 4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 -
5.1% or 4.9 - 5.0%). In various embodiments, the concentration is within the range of 0.1 -
5%, 0.25 - 4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 -
3.25%, 2.0 - 3.0%, or 2.25 - 2.75%. In various embodiments, a peptide solution of the
present invention has a concentration within the range of 1 - 3%, inclusive; in some certain
embodiments, about 1.0%, in some embodiments, about 1.5%; in some embodiments, about
2.0%; in some embodiments, about 2.5%; in some embodiments, about 3%.
[0020] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0021] In some embodiments, the present invention provides a method of performing
an intrabody surgical procedure on a patient or subject comprising exposing a location within
the patient's body to access a damaged portion of an internal organ or tissue for a first period
of time in order to remove, repair, or replace at least some portion of the organ or tissue
during the first period of time, applying, to a site within the location, a composition
comprising a 0 .1 - 10% peptide solution, wherein the peptide comprises an amino acid
sequence of RADA repeats; and wherein the solution is characterized by an ability to
transition between two states: an un-gelled state adopted when one or more particular ions is
substantially absent, and a gelled state adopted when the one or more ions is present at or
above a threshold level, wherein the one or more ions is or becomes present in the location,
retaining the composition in the location for a second period of time, wherein the peptides in
the composition transitions to a gelled state.
[0022] In some embodiments, a first period of time is greater than five minutes; in
some embodiments, greater than 10, 20, 30, 40, 50, or 60 minutes. In some embodiments, a
first period of time is about one hour. In some embodiments, a first period of time is less
than five minutes; in some embodiments, less than 10, 20, 30, 40, 50 or 60 minutes. In some
embodiments, a first period of time is less than one hour.
[0023] In some embodiments, the present invention further comprises an
improvement of performing, during the second period of time, at least one other medical
procedure in the location. In some embodiments, a second period of time is less than five
minutes; in some embodiments, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In some
embodiments, a second period of time is about 5 minutes. In some embodiments, a second
period of time is greater than five minutes; in some embodiments, a second period of time is
greater than 6, 7, 8, 9, 10, or more minutes. In some embodiments, a second period of time is
about 10 minutes.
[0024] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0025] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human patients include mammals. In some certain
embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs, cattle,
sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs, llamas,
mules, rabbits, reindeer, water buffalo and yaks.
[0026] In some embodiments, an intrabody surgical procedure of the present
invention is a liver resection and is completed within a first period of time that is less than
four hours (e.g., less than 3.75, 3.50, 3.00, 2.75, 2.00, 1.75, 1.50, or 1.00) and therefore
reduced as compared with the standard first period of time absent the application, which
standard first period of time is within the range of five to six hours (e.g., within the range of
about five to about six hours, inclusive; in some embodiments, about 5.0, 5.1, 5.2, 5.3. 5.4,
5.5, 5.6, 5.7, 5.8, 5.9, 6.0 hours).
[0027] In some embodiments, the present invention further comprises an
improvement of a method that excludes application of fibrin glue or SURGICEL® or a
combination thereof within the site during the first period of time. In some embodiments, the
present invention further comprises an improvement of applying the composition comprising
a solution of peptides in addition to fibrin glue or SURGICEL® or a combination thereof
within the site during the first period of time.
[0028] In some embodiments, at least one first application is completed prior to any
other surgical activity within the site. In some embodiments, a patient is dosed with an anti
coagulant prior to surgery.
[0029] In some embodiments, a peptide solution of the present invention has a
concentration within t e range of about 0 .1% to about 10% (e.g., about 0.1 - 10%; 0.2 -
9.9%, 0.3 - 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%,
1.0 - 9.1%, 1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 -
8.4%, 1.8 - 8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%,
2.5 - 7.6%, 2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 -
6.9%, 3.3 - 6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%,
4.0 - 6.1%, 4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 -
5.1% or 4.9 - 5.0%). In various embodiments, the concentration is within the range of 0.1 -
5%, 0.25 - 4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 -
3.25%, 2.0 - 3.0%, or 2.25 - 2.75%.
[0030] In some embodiments, a peptide solution of the present invention has a
concentration within the range of 1 - 3%; inclusive. In some certain embodiments, about
1.0%, in some embodiments, about 1.5%; in some embodiments, about 2.0%; in some
embodiments, about 2.5%; in some embodiments, about 3%.
[0031] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0032] In some embodiments, the present invention provides a method of treating
exudative bleeding during liver surgery in a patient or subject, the method comprising the
steps of (a) applying to a location of exudative bleeding in a patient or subject a composition
comprising a 0 .1 - 10% peptide solution, wherein the peptide comprises an amino acid
sequence of RADA repeats; and wherein the solution is characterized by an ability to
transition between two states: an un-gelled state adopted when one or more particular ions is
substantially absent, and a gelled state adopted when the one or more ions is present at or
above a threshold level, wherein the one or more ions is or becomes present in the location;
(b) retaining the applied composition in the location, with t e one or more ions, for a period
of time sufficient for t e composition to gel; and (c) performing one or more liver surgery
tasks in the location without first removing the composition.
[0033] In some embodiments, exudative bleeding is caused by electrocauterization.
[0034] In some embodiments, a patient or subject is dosed with an anticoagulant prior
to the start of the liver surgery.
[0035] In some embodiments, a peptide composition of the present invention that
comprises a solution is applied endoscopically. In some embodiments, one or more liver
surgery tasks is performed endoscopically. In some embodiments, one or more liver surgery
tasks is performed laproscopically. In some certain embodiments, one or more liver surgery
tasks include liver separation. In some certain embodiments, one or more liver surgery tasks
include vascular exfoliation.
[0036] In some embodiments, a peptide solution of the present invention has a
concentration within the range of about 0.1% to about 10% (e.g., 0.1 - 10%; 0.2 - 9.9%, 0.3
- 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%, 1.0 - 9.1%,
1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 - 8.4%, 1.8 -
8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%, 2.5 - 7.6%,
2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 - 6.9%, 3.3 -
6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%, 4.0 - 6.1%,
4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 - 5.1% or 4.9 -
5.0%). In various embodiments, the concentration is within the range of 0.1 - 5%, 0.25 -
4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 - 3.25%, 2.0 -
3.0%, or 2.25 - 2.75%. In some embodiments, a peptide solution of the present invention has
a concentration within the range of 1 - 3%; inclusive. In some certain embodiments, about
1.0%, in some embodiments, about 1.5%; in some embodiments, about 2.0%; in some
embodiments, about 2.5%; in some embodiments, about 3%.
[0037] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0038] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0039] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human patients or subjects include mammals. In some
certain embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs,
cattle, sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs,
llamas, mules, rabbits, reindeer, water buffalo and yaks.
[0040] In some embodiments, the present invention provides a method of treating
bleeding during graft collection during coronary artery bypass surgery in a patient or subject
comprising (a) applying to a graft collection site a composition comprising a 0.1 - 10%
peptide solution, wherein the peptide comprises an amino acid sequence of RADA repeats;
and wherein the solution is characterized by an ability to transition between two states: an ungelled
state adopted when one or more particular ions is substantially absent, and a gelled
state adopted when the one or more ions is present at or above a threshold level, wherein the
one or more ions is or becomes present in the location; and (b) retaining the composition in
the location, with the one or more ions, for a period of time sufficient for the composition to
gel.
[0041] In some embodiments, bleeding is caused by electrocauterization.
[0042] In some embodiments, applying a peptide composition of the present
invention is performed prior to initiation of graft collection. In some certain embodiments,
graft collection is performed without removing an applied peptide composition which is
present in a gelled state.
[0043] In some embodiments, applying a peptide composition of the present
invention is performed after initiation but prior to completion of graft collection. In some
certain embodiments, graft collection is performed without removing an applied peptide
composition which is present in a gelled state.
[0044] In some embodiments, retaining a peptide composition of the present
invention in a location comprises retaining through performance of at least one step graft
collection step. In some embodiments, retaining a peptide composition of the present
invention in a location comprises retaining through completion of graft collection steps. In
various embodiments, graft collection is performed and/or completed without removing an
applied peptide composition of the present invention.
[0045] In some embodiments, a peptide solution of t e present invention has a
concentration within the range of about 0.1% to about 10% (e.g., 0.1 - 10%; 0.2 - 9.9%, 0.3
- 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%, 1.0 - 9.1%,
1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 - 8.4%, 1.8 -
8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%, 2.5 - 7.6%,
2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 - 6.9%, 3.3 -
6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%, 4.0 - 6.1%,
4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 - 5.1% or 4.9 -
5.0%). In various embodiments, the concentration is within the range of 0.1 - 5%, 0.25 -
4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 - 3.25%, 2.0 -
3.0%, or 2.25 - 2.75%. In some embodiments, a peptide solution of the present invention has
a concentration within the range of 1 - 3%; inclusive. In some certain embodiments, about
1.0%, in some embodiments, about 1.5%; in some embodiments, about 2.0%; in some
embodiments, about 2.5%; in some embodiments, about 3%.
[0046] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0047] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0048] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human patients or subjects include mammals. In some
certain embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs,
cattle, sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs,
llamas, mules, rabbits, reindeer, water buffalo and yaks.
[0049] In some embodiments, t e present invention provides a method of performing
a coronary artery bypass graft procedure in a patient or subject comprising (a) applying to a
cardiac location in t e patient a composition comprising a 0.1 - 10% peptide solution,
wherein the peptide an amino acid sequence of RADA repeats; and wherein the solution is
characterized by an ability to transition between two states: an un-gelled state adopted when
one or more particular ions is substantially absent, and a gelled state adopted when the one or
more ions is present at or above a threshold level, wherein the one or more ions is or becomes
present in the location.
[0050] In some embodiments, a cardiac location is an anastomy site on a coronary
artery. In some embodiments, a cardiac location is an anastomy site on a graft vessel. In
some embodiments, a cardiac location is an annula connection site for an oxygenator.
[0051] In various embodiments, a peptide composition of the present invention is
applied to a cardiac location without additional pressure.
[0052] In some embodiments, applying a peptide composition of the present
invention is performed after initiation but prior to completion of graft collection. In some
certain embodiments, graft collection is performed without removing an applied peptide
composition which is present in a gelled state.
[0053] In some embodiments, applying a peptide composition of the present
invention is performed after initiation but prior to completion of graft collection. In some
certain embodiments, graft collection is performed without removing an applied peptide
composition which is present in a gelled state.
[0001] In some embodiments, an applied peptide composition of the present invention
is retained at the site through performance of at least one step graft collection step. In some
embodiments, an applied peptide composition of the present invention is retained at the site
through the completion of graft collection steps. In various embodiments, graft collection is
performed and/or completed without removing an applied peptide composition of the present
invention.
[0054] In some embodiments, a method of performing a coronary artery bypass graft
procedure in a patient or subject is provided, an improvement comprising excluding
application of fibrin glue or SURGICEL® within t e site.
[0055] In some embodiments, a method of performing a coronary artery bypass graft
procedure in a patient or subject is provided, an improvement comprising applying a peptide
composition of t e present invention in addition to application of fibrin glue or SURGICEL®
within the site.
[0056] In some embodiments, a peptide solution of the present invention has a
concentration within the range of about 0.1% to about 10% (e.g., 0.1 - 10%; 0.2 - 9.9%, 0.3
- 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%, 1.0 - 9.1%,
1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 - 8.4%, 1.8 -
8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%, 2.5 - 7.6%,
2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 - 6.9%, 3.3 -
6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%, 4.0 - 6.1%,
4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 - 5.1% or 4.9 -
5.0%). In various embodiments, the concentration is within the range of 0.1 - 5%, 0.25 -
4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 - 3.25%, 2.0 -
3.0%, or 2.25 - 2.75%. In some embodiments, a peptide solution of the present invention has
a concentration within the range of 1 - 3%; inclusive. In some certain embodiments, about
1.0%, in some embodiments, about 1.5%; in some embodiments, about 2.0%; in some
embodiments, about 2.5%; in some embodiments, about 3%.
[0057] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0058] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0059] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human patients or subjects include mammals. In some
certain embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs,
cattle, sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs,
llamas, mules, rabbits, reindeer, water buffalo and yaks.
[0060] In some embodiments, the present invention provides a pre-filled syringe for
use in a surgical procedure comprising a peptide composition of the present invention as
described herein.
[0061] In some embodiments, a pre-filled syringe for use in a surgical procedure is
provided, comprising a barrel comprising a 0 .1 - 10% peptide solution, wherein the peptide
comprises an amino acid sequence of RADA repeats; and wherein t e peptide solution is
characterized by an ability to transition between two states: an ungelled state adopted when
one or more particular ions is substantially absent, and a gelled state adopted when t e one or
more ions is present at or above a threshold level, wherein the one or more ions is or becomes
present in the location; and, a non-metal nozzle; wherein said barrel and non-metal nozzle are
capable of forming a secure connection in a liquid-tight manner.
[0062] In some embodiments, a peptide solution of the present invention has a
concentration within the range of about 0.1% to about 10% (e.g., 0.1 - 10%; 0.2 - 9.9%, 0.3
- 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 - 9.3%, 0.9 - 9.2%, 1.0 - 9.1%,
1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%, 1.6 - 8.5%, 1.7 - 8.4%, 1.8 -
8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 - 7.8%, 2.4 - 7.7%, 2.5 - 7.6%,
2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%, 3.1 - 7.0%, 3.2 - 6.9%, 3.3 -
6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 - 6.3%, 3.9 - 6.2%, 4.0 - 6.1%,
4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%, 4.7 - 5.2%, 4.8 - 5.1% or 4.9 -
5.0%). In various embodiments, the concentration is within the range of 0.1 - 5%, 0.25 -
4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 - 3.25%, 2.0 -
3.0%, or 2.25 - 2.75%. In some embodiments, a peptide solution of the present invention has
a concentration within the range of 1 - 3%; inclusive. In some certain embodiments, about
1.0%, in some embodiments, about 1.5%; in some embodiments, about 2.0%; in some
embodiments, about 2.5%; in some embodiments, about 3%.
[0063] In various embodiments, a peptide of the present invention comprises an
amino acid sequence that comprises two, three or four repeats of RADA (SEQ ID NO: 1); in
some embodiments, an amino acids sequence of two RADA repeats (e.g., RADARADA;
SEQ ID NO:2); in some embodiments, an amino acids sequence of three repeats (e.g.,
RADARADARADA; SEQ ID NO:3); in some embodiments, an amino acids sequence of
four RADA repeats (e.g., RADARADARADARADA; SEQ ID NO:4).
[0064] In some embodiments, one or more ions are selected from potassium (K+) and
sodium (Na+) . In some embodiments, one or more ions are potassium (K+) and sodium
(Na+) . In various embodiments, a threshold level is characterized by physiological conditions
present within a surgical site of a patient or subject. In some embodiments, a threshold level
is provided by contact with bodily fluids, blood, tissues and/or a combination thereof within
the surgical site of a patient or subject.
[0065] In various embodiments, patients or subjects are human or non-human. In
some certain embodiments, non-human patients or subjects include mammals. In some
certain embodiments, mammals include rodents (e.g., mice or rats), dogs, cats, horses, pigs,
cattle, sheep, goats, alpacas, bantengs, bison, camels, deer, donkeys, gayals, guinea pigs,
llamas, mules, rabbits, reindeer, water buffalo and yaks.
[0066] In some embodiments, a pre-filled syringe of the present invention is used in a
surgical procedure selected from the group consisting of coronary artery bypass graft
(CABG), hepatectomy, pure laparoscopic hepatectomy (PLH), endoscopic mucosal resection
(EMR), endoscopic sub mucosal dissection (ESD), thoracoscopic partial lung resection,
lymph node dissection, open partial nephrectomy, laparoscopic partial nephrectomy, aorta
replacement and orthopedic bone surgery.
[0067] In some embodiments, a pre-filled syringe of the present invention comprises
a non-metal nozzle that is rigid. In some embodiments, a pre-filled syringe of the present
invention comprises a non-metal nozzle that is flexible. In some certain embodiments, a nonmetal
nozzle is flexible such that it is capable for use in an endoscopic surgical procedure. In
some certain embodiments, a non-metal nozzle is flexible such that it is capable for use in a
laparoscopic surgical procedure.
[0068] In some embodiments, a pre-filled syringe of the present invention comprises
a peptide solution as described herein in a volume within the range of about 1- 50 mL (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
mL). In some embodiments, a volume within the range of about 1to about 10 mL, inclusive;
in some certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mL. In some embodiments,
about 1mL. In some embodiments, about 3 mL. In some embodiments, about 5 mL; in
some embodiments, about 10 mL. In some embodiments, a volume within the range of about
20 mL to about 30 mL. In some embodiments, a volume within a range of about 30 mL to
about 40 mL. In some embodiments, a volume within a range of about 40 mL to about 50
mL; in some embodiments about 30 mL.
[0069] In some embodiments, a kit comprising one or more pre-filled syringes as
described herein is provided. In some certain embodiments, a kit comprises one, two, three,
four, five, or more pre-filled syringes.
[0070] In some embodiments, a pharmaceutical package is provided comprising a
pre-filled syringe as described herein and a blister pack specifically formed to accept such
pre-filled syringe.
BRIEF DESCRIPTION OF THE DRAWING
[0071] The Drawing included herein, which is comprised of the following Figures, is
for illustration purposes only not for limitation.
[0072] FIG. 1 is a schematic illustration of the interactions between peptides in the
peptide scaffold. Various peptides with amino acid sequences of alternating hydrophobic and
hydrophilic residues self-assemble to form a stable scaffold of beta-sheets when exposed to
physiologically-equivalent electrolyte solutions (US 5,955,343 and US 5,670,483). The
peptide scaffolds are stabilized by numerous interactions between the peptides. For example,
the positively charged and negatively charged amino acid side chains from adjacent peptides
form complementary ionic pairs, and other hydrophilic residues such as asparagine and
glutamine participate in hydrogen-bonding interactions. The hydrophobic groups on adjacent
peptides participate in van der Waals interactions. The amino and carbonyl groups on the
peptide backbone also participate in intermolecular hydrogen-bonding interactions.
[0073] FIG. 2 shows an illustration of the constituents of a peptide solution and
conditions under which the peptide solution forms a fibrous network causing the solution to
gel. The peptide chain of RADA repeats is shown (top left) and the resulting formation of a
fibrous network after exposure to physiological conditions (top right). An electron
micrograph of the fibrous network is shown (bottom right) in addition to the adopted gelled
state (bottom left).
[0074] FIG. 3 shows a schematic illustration, not to scale, of t e locations of grafts
surgically performed on a heart in a typical coronary artery bypass graft (CABG) surgery.
The typical steps performed in a CABG surgery are detailed on the right.
[0075] FIG. 4 shows an schematic illustration, not to scale, of the placement of a
metal nail plate (left), a gamma nail (middle), and a ender pin (right) in a surgical procedure
to repair an intertrochanteric fracture.
[0076] FIG. 5 shows a schematic illustration, not to scale, of t e surgical site of a
thorascoscopic partial lung resection using a laparoscopy.
[0077] FIG. 6 shows a picture of a syringe that can be employed for the delivery of a
peptide solution to a surgical and/or bleeding site. A plunger, finger grip, gasket, barrel and
head cap are labeled.
[0078] FIG. 7 shows a pre-filled syringe with and without a specialized
nozzle/cannula attached for delivery of the peptide solution to a surgical and/or bleeding site.
[0079] FIG. 8 shows a pre-filled syringe with and without a specialized connector
attached for delivery of the peptide solution to a surgical and/or bleeding site administered
through a catheter.
[0080] FIG. 9 shows a pharmaceutical package containing a pre-filled syringe and
specialized nozzle/cannula individually supplied in a sterilized blister pack.
[0081] FIG. 10 shows a bar graph of the time during operation to stop bleeding in
minutes (x-axis) and the number of application sites (y-axis) according to three exemplary
surgical procedures (hepatectomy, angiostomy, endoscopy).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0082] The present invention is not limited to particular methods, and experimental
conditions described, as such methods and conditions may vary. It is also to be understood
that the terminology used herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting unless indicated, since the scope of the present
invention will be limited only by the appended claims.
[0083] Unless stated otherwise, all technical and scientific terms and phrases used
herein have the same meaning as commonly understood by one of ordinary skill in the art.
Although any methods and materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, t e preferred methods and materials
are now described. All publications mentioned herein are incorporated herein by reference.
DEFINITIONS
[0084] As used in this specification and the appended claims, the singular forms "a",
"an", and "the" include plural references unless the context clearly dictates otherwise. Thus
for example, a reference to "a method" includes one or more methods, and/or steps of the
type described herein and/or which will become apparent to those persons skilled in the art
upon reading this disclosure and so forth.
[0085] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which a composition is administered. Carriers can include sterile liquids, such as, for
example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin,
such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like.
[0086] The term "complementary" is used herein to refer to peptides that selfassemble
into a scaffold in which ionic or hydrogen bonding interactions occur between
hydrophilic residues from adjacent peptides in the scaffold. In many embodiments, as
illustrated in Figure 1, each hydrophilic residue in a peptide either interacts (e.g., hydrogen
bonds or ionically pairs) with a hydrophilic residue on an adjacent peptide, or is exposed to
solvent.
[0087] The term "excipient" refers to a non-therapeutic agent added to a
pharmaceutical composition to provide a desired consistency or stabilizing effect. Suitable
pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
[0088] The phrase "physiological conditions" refers to conditions of the external or
internal mileu that may occur in nature for an organism or cell system. As used herein,
physiological conditions are those conditions present within the body of a human or nonhuman
animal, especially those conditions present at and/or within a surgical site. Exemplary
physiological conditions are in contrast to conditions in a laboratory setting, which are
interpreted to be artificial in comparison. Physiological conditions typically include, e.g., a
temperature range of 20 - 40°C, atmospheric pressure of 1, pH of 6 - 8, glucose
concentration of 1- 20 mM, oxygen concentration at atmospheric levels, and gravity as it is
encountered on earth.
[0089] The term "pure" is used to indicate t e extent to which peptide compositions
described herein are free of other chemical species, including deletion adducts of the peptide
in question and peptides of differing lengths. For example, in some embodiments, a peptide
composition is considered to be a "pure" composition of a particular peptide (i.e., of a peptide
having a particular amino acid sequence) if substantially all peptides in the composition have
amino acid sequences that are identical to the particular sequence, or to a truncation thereof
(e.g., a terminal truncation thereof, for example a carboxy-terminal truncation thereof). In
some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
of the peptides in a pure composition of a particular peptide (i.e., of a peptide having a
particular amino acid sequence) have amino acid sequences that are identical to the particular
sequence, or to a truncation thereof (e.g., a terminal truncation thereof, for example an
amino-terminal truncation thereof). In some embodiments, at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more of the peptides in a pure composition of a
particular peptide (i.e., of a peptide having a particular amino acid sequence) are full length.
[0090] By the phrase "therapeutically effective amount" is meant an amount that
produces the desired effect for which it is administered. In some embodiments, the term
refers to an amount that is sufficient, when administered to a population suffering from or
susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing
regimen, to treat the disease, disorder, and/or condition. In some embodiments, a
therapeutically effective amount is one that reduces the incidence and/or severity of, and/or
delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of
ordinary skill in the art will appreciate that the term "therapeutically effective amount" does
not in fact require successful treatment be achieved in a particular individual. Rather, a
therapeutically effective amount may be that amount that provides a particular desired
pharmacological response in a significant number of subjects when administered to patients
in need of such treatment. In some embodiments, reference to a therapeutically effective
amount may be a reference to an amount as measured in one or more specific tissues (e.g., a
tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum,
sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some
embodiments, a therapeutically effective amount of a particular agent or therapy may be
formulated and/or administered in a single dose. In some embodiments, a therapeutically
effective agent may be formulated and/or administered in a plurality of doses, for example, as
part of a dosing regimen.
[0091] As used herein, the term "topical" when used to describe application of a
composition is intended to describe a situation when the composition is applied to body
surfaces such as t e skin or mucous membranes as is typically t e case in the context of
known compositions used in a similar manner, such as, e.g., creams, foams, gels, lotions and
ointments. Topical administration is understood to be epicutaneous, meaning that they are
applied directly to the skin. Topical administration is also intended to include other
formulations that may be applied to the surface of tissues other than the skin, such as eye
drops applied to the conjunctiva, or ear drops placed in the ear, or treatment applied to the
surface of a tooth. As a route of administration, topical administration are contrasted with
enteral (in the digestive tract) and parenteral administration (injected into the circulatory
system).
[0092] As used herein, the phrase "storage and/or drug delivery system" refers to a
system for storing and/or delivering peptide compositions described herein. Exemplary
storage and/or delivery systems suitable for peptide compositions described herein are vials,
bottles, beakers, bags, syringes, ampules, cartridges, reservoirs or LYO-JECTS®. Storage
and/or delivery systems need not be one in the same and can be separate.
[0093] As used herein, the term "nozzle" refers to a generally thin, cylindrical object,
often with a narrow end and a wide end, which is adapted for fixing onto a delivery device
described herein. In some embodiments, the terms "nozzle" and "cannula" are used
interchangeably. Nozzles are composed of two connection points or ends, a first connection
point or end to connect to a delivery system (e.g. a syringe) and a second connection point
which may serve as the point where delivery of pharmaceutical composition is administered
or as a point to connect to a secondary device (e.g., a catheter).
[0094] The term "bore" is used herein to refer to an opening of a nozzle, cannula
and/or catheter that are used in connection with delivery and/or storage systems (e.g., a
syringe) containing peptide compositions of the present invention as described herein.
Typically, a bore is characterized by various measurements or gauge, e.g., an inner wall
diameter thickness, an outer wall diameter and a wall thickness. Exemplary measurements or
the gauge of (e.g., diameter, thickness, etc.) a bore of a nozzle, cannula and/or catheter for
use in connection with delivery and/or storage systems (e.g., a syringe) containing peptide
compositions of t e present invention can be found in any needle gauge system (e.g., a
French scale or French gauge system, Stubs Iron Wire Gauge system also known as t e
Birmingham Wire Gauge).
[0095] The term "structurally compatible" is used herein to refer to peptides that are
capable of maintaining a sufficiently constant intrapeptide distance to allow scaffold
formation. In certain embodiments of the invention the variation in intrapeptide distance is
less than 4, 3, 2, or 1 angstroms. It is also contemplated that larger variations in intrapeptide
distance may not prevent scaffold formation if sufficient stabilizing forces are present.
Intrapeptide distance may be calculated based on molecular modeling or based on simplified
procedures known in the art (see, for example, U.S. Patent Number 5,670,483). In one
exemplary method, intrapeptide distance is calculated by taking the sum of the number of
unbranched atoms on the side-chains of each amino acid in a pair. For example, the
intrapeptide distance for a lysine-glutamic acid ionic pair is 5+4=9 atoms, and the distance
for a glutamine-glutamine hydrogen bonding pair is 4+4=8 atoms. Using a conversion factor
of 3 angstroms per atom, the variation in the intrapeptide distance of peptides having lysineglutamic
acid pairs and glutamine-glutamine pairs (e.g., 9 versus 8 atoms) is 3 angstroms.
[0096] As used herein, the term "subject" means any mammal, including humans. In
certain embodiments of the present invention the subject is an adult, an adolescent or an
infant. In some embodiments, terms "individual" or "patient" are used and are intended to be
interchangeable with "subject". Also contemplated by the present invention are the
administration of the pharmaceutical compositions and/or performance of the methods of
treatment in-utero.
Self-Gelling Peptide Compositions
[0097] The present disclosure provides surgical methodologies that are improved
through the use of certain sterile self-gelling peptide compositions, as described herein. The
present disclosure further provides such compositions specifically prepared for administration
during particular surgical procedures. For example, the present disclosure provides specially
designed delivery systems (e.g., pre-loaded syringes and/or cannulas) containing such selfgelling
peptide compositions.
[0098] Compositions, surgical methods and devices disclosed herein provide various
improvements to existing methodologies.
[0099] Particular exemplary peptides appropriate for use in peptide compositions as
described herein include those with sequences reported in U.S. Patents Nos. 5,670,483,
and/or 5,955,343, and/or in U.S. Patent Application No. 09/778,200, each of which is
incorporated herein by reference. These peptides have amino acid sequences that consist of
alternating hydrophilic and hydrophobic amino acids, and are characterized by an ability to
self-assemble in the present of electrolytes (e.g., monovalent cations) into a stable beta-sheet
macroscopic structure. Exemplary electrolytes are Na+ and K+. These peptide chains are
self-complementary and structurally compatible. When assembled into the beta-sheet
structure, the amino acid side-chains of residues within the peptide partition into one of two
faces, a polar face with charged ionic side chains and a nonpolar face with alanines or other
hydrophobic groups.
[0100] In many embodiments, utilized peptides have amino acid sequences that
consist of alternating positively and negatively charged amino acids. Such peptides are
considered to be self-complementary when the positively charged and negatively charged
amino acid residues can form complementary ionic pairs. Such peptide chains are referred to
as ionic, self-complementary peptides, or Type I self-assembling peptides. If the ionic
residues alternate with one positively and one negatively charged residue (-+-+-+-+), the
peptide chains are described as "modulus I;" if the ionic residues alternate with two positively
and two negatively charged residues (--++--++), the peptide chains are described as "modulus
II." Exemplary peptides for use with the present invention include those whose sequences are
presented in Table 1 (N/A: not applicable; Asterisk: These peptides form a beta-sheet when
incubated in a solution containing NaCl, however they have not been observed to selfassemble
to form macroscopic scaffolds).
TABLE 1
Representative Self-Assembling Peptides
Name Sequence (n~>c) Modulus SEQ ID NO:
RAD16-1 n-RADARADARADARADA-c I 4
RGDA16-I n-RADARGDARADARGDA-c I 5
RADA8-I n-RADARADA-c I 2
RAD 16-11 n-RARADADARARADADA-c II 6
RAD8-II n-RARADADA-c II 7
EAKA16-I n-AEAKAEAKAEAKAEAK-c I 8
EAKA8-I n-AEAKAEAK-c I 9
RAEA16-I n-RAEARAEARAEARAEA-c I 10
RAEA8-I n-RAEARAEA-c I 11
KADA16-I n-KADAKADAKADAKADA-c I 12
KADA8-I n-KADAKADA-c I 13
KLD12 n-KLDLKLDLKLDL-c 14
EAH16-II n-AEAEAHAHAEAEAHAH-c II 15
EAH8-II n-AEAEAHAH-c II 16
EFK16-II n-FEFEFKFKFEFEFKFK-c II 17
EFK8-II n-FEFKFEFK-c I 18
KFE12 n-FKFEFKFEFKFE-c 19
KFE8 n-FKFEFKFE-c 20
KFE16 n-FKFEFKFEFKFEFKFE-c 2 1
KFQ12 n-FKFQFKFQFKFQ-c 22
KIE12 n-IKIEIKIEIKIE-c 23
KVE12 n-VKVEVKVEVKVE-c 24
IEIK9 n- IEIKIEIKI-c 25
IEIK13 n- IEIKIEIKIEIKI-c 26
IEIK17 n- IEIKIEIKIEIKIEIKI-c 27
ELK16-II n-LELELKLKLELELKLK-c II 28
ELK8-II n-LELELKLK-c II 29
EAK16-II n-AEAEAKAKAEAEAKAK-c II 30
EAK12 n-AEAEAEAEAKAK-c IV/II 3 1
EAK8-II n-AEAEAKAK-c II 32
KAE16-IV n-KAKAKAKAEAEAEAEA-c IV 33
EAK16-IV n-AEAEAEAEAKAKAKAK-c IV 34
RAD16-IV n-RARARARADADADADA-c IV 35
DAR16-IV n-ADADADADARARARAR-c IV 36
DAR16-IV* n-DADADADARARARARA-c IV 37
DAR32-IV n-(ADADADADARARARAR) -c IV 38
EHK16 n-HEHEHKHKHEHEHKHK-c N/A 39
EHK8-I n-HEHEHKHK-c N/A 40
VE20* n-VEVEVEVEVEVEVEVEVEVE-c N/A 4 1
RF20* n-RFRFRFRFRFRFRFRFRFRF-c N/A 42
[0101] Previous studies have demonstrated that charged residues in peptides within
Table 1 may be substituted with other residues of the same charge (e.g., substitution of
positively charged lysines with positively charged arginines and/or substitution of negatively
charged glutamates with negatively charged aspartates) without negatively impacting selfassembly.
However, substitution with residues of opposite charge (e.g., substitution of
positively charged lysines and/or arginines with negatively charged residues such as aspartate
and glutamate) disrupts self-assembly.
[0102] Alternatively or additionally, other hydrophilic residues, such as asparagine
and glutamine, that form hydrogen-bonds may be incorporated into the peptide chains instead
of, or in addition to, charged residues. If the alanines in the peptide chains are changed to
more hydrophobic residues, such as leucine, isoleucine, phenylalanine or tyrosine, these
peptide chains have a greater tendency to self-assemble and form peptide matrices with
enhanced strength. Some peptides that have similar compositions and lengths as the
aforementioned peptide chains form alpha-helices and random-coils rather than beta-sheets
and do not form macroscopic structures. Thus, in addition to self-complementarity, other
factors are likely to be important for the formation of macroscopic scaffolds, such as the
chain length, t e degree of intermolecular interaction, and the ability to form staggered
arrays.
[0103] Other self-assembling peptide chains may be generated, for example that have
amino acid sequences that differ from that of any self-assembling peptide chains by a single
amino acid residue or by multiple amino acid residues. Additionally, the incorporation of
specific cell recognition ligands, such as RGD or RAD, into self-assembling peptides may
promote the proliferation of cells in the scaffold, and/or may attract cells into the scaffold.
[0104] In some embodiments, cysteines are included in self-assembling peptides, for
example to permit formation of disulfide bonds. Alternatively or additionally, residues with
aromatic rings may be incorporated into self-assembling peptides, so that cross-links between
peptide chains can be generated by exposure to UV light. Table 2 presents representative
examples of amino acid sequences of peptides that are susceptible to UV crosslinking. The
extent of the cross-linking may be precisely controlled by the predetermined length of
exposure to UV light and the predetermined peptide chain concentration. The extent of crosslinking
may be determined, for example, by light scattering, gel filtration, or scanning
electron microscopy using standard methods. Alternatively or additionally, the extent of
cross-linking may be examined by HPLC and/or mass spectrometry analysis of a selfassembled
peptide structure after digestion with a protease, such as matrix metalloproteases.
The material strength of the scaffold may be determined before and after cross-linking, as
described herein.
TABLE 2
Representative Sequences of Cross-Linkable Peptides
Name Sequence (n->c) SEQ ID NO:
RGDY16 RGDYRYDYRYDYRGDY 43
RGDF16 RGDFRFDFRFDFRGDF 44
RGDW16 RGDWRWDWRWDWRGDW 45
RADY16 RADYRYEYRYEYRADY 46
RADF16 RADFRFDFRFDFRADF 47
RADW16 RADWRWDWRWDWRADW 48
[0105] Combinations of any sequences or alterations described herein may be made
to any particular self-assembling peptide of interest.
[0106] In some embodiments, peptide sequences are selected to achieve a desired
level of stiffness and/or elasticity in the structure formed by self-assembly of the peptides.
While not wishing to be bound by any theory, low elasticity may help allow cells to migrate
into the assembled structure and/or to communicate with one another once resident in the
structure.
[0107] In some embodiments, peptide sequences are selected to assemble into
structures with a low elastic modulus, for example in the range of 1-10 kPa as measured in a
standard cone-plate rheometer. Such low values permit scaffold deformation as a result of
cell contraction, and this deformation may provide t e means for cell-cell communication. In
addition, such moduli allow the scaffold to transmit physiological stresses to cells migrating
therein, stimulating the cells to produce tissue that is closer in microstructure to native tissue
than scar.
[0108] Scaffold stiffness can be controlled by a variety of means including, for
example, changes in peptide sequence, changes in peptide concentration, changes in peptide
length, and combinations thereof. Alternatively or additionally, other methods for increasing
stiffness can be used, such as attaching one or more crosslinkable moieties (e.g., biotin) to the
peptides (e.g., to the amino terminus, to the carboxy terminus, or to an internal site such as to
a side chain) so that they may be cross-linked for example within a self-assembled structure.
[0109] In some embodiments, degradation sites such as one or more aggrecan
processing sites (e.g., as underlined in Table 3), matrix metalloprotease (MMP) cleavage
sites, such as those for collagenase sites, etc. may be introduced into peptides, whether at
their amino termini, their carboxy termini, or elsewhere in their sequence the same manner.
Peptide structures formed from such degradation-site-containing peptides, alone or in
combination with peptides capable of being cross-linked, may be degraded by exposure to
appropriate proteases under appropriate conditions (including time of exposure) as
understood by those skilled in the art. In some embodiments, the in vivo half-life of a
structure formed by assembled peptides may be modulated by incorporation of one or more
degradation sites into utilized peptides, for example allowing the structure to be
enzymatically degraded.
[0110] The rate of degradation of peptide structures may be determined, for example,
by HPLC, mass spectrometry, and/or NMR analysis of released peptide components.
Alternatively or additionally, if radiolabeled peptides are utilized, the amount of released
radioactivity may be measured, for example by scintillation counting. For some
embodiments, t e beta-sheet structure of the assembled peptide chains is degraded
sufficiently rapidly that it is not necessary to incorporate cleavage sites into peptides used for
assembly.
TABLE 3
Representative Peptide Sequences having Aggrecan Processing Sites
Name Sequence (N->C) SEQ ID NO:
REEE RGDYRYDYTFREEE-GLGSRYDYRGDY 49
KEEE RGDYRYDYTFKEEE-GLGSRYDYRGDY 50
SELE RGDYRYDYTASELE-GRGTRYDYRGDY 51
TAQE RGDYRYDYAPTAOE-AGEGPRYDY-RGDY 52
ISQE RGDYRYDYPTISOE-LGORPRYDYRGDY 53
VSQE RGDYRYDYPTVSOE-LGORPRYDYRGDY 54
[0111] In some embodiments, utilized peptides possess an alternating structure of the
hydrophobic amino acid alanine (A) and the hydrophilic amino acids arginine (R) and
aspartate (D), in which the respective positive and negative charges determine the relative
position of the adjoining molecules. Without wishing to be bound by any particular theory, it
is proposed that in such embodiments, self-assembly may be completed by hydrophobic
bonding between neutral amino acid side chains and hydrogen bonding between peptide
backbones. In some such embodiments, utilized peptides have an amino acid sequence that
comprises, or in some embodiments consists of, repeats of arginine-alanine-aspartate-alanine
(RADA). In some embodiments, utilized peptides contain two, three, four or more repeats of
RADA (SEQ ID NO: 1). In some embodiments, utilized peptides contain four RADA repeats
(e.g., have the sequence RADARADARADARADA; SEQ ID NO:4).
[0112] In some embodiments, peptides utilized in peptide compositions as described
herein are at least 12 or 16 amino acids long. In some embodiments, peptides utilized in
peptide compositions as described herein are exactly 12 or 16 amino acids long.
[0113] In some embodiments, peptides utilized in peptide compositions as described
herein are at least 8 or 12 amino acids long. In some embodiments, peptides utilized in
peptide compositions as described herein are exactly 8 or 12 amino acids long.
[0114] In some embodiments, peptides utilized in peptide compositions as described
herein comprise or consist of natural amino acids; in some embodiments they include one or
more non-natural and/or modified amino acids.
[0115] In some embodiments, peptides utilized in peptide compositions as described
herein comprise or consist of D- amino acids. In some embodiments, peptides utilized in
peptide compositions as described herein comprise or consist of L- amino acids. In some
embodiments, peptides utilized in peptide compositions as described include both D- and Lamino
acids.
[0116] In some embodiments, peptides utilized in peptide compositions as described
herein are synthesized, for example using standard f-moc chemistry and purified using high
pressure liquid chromatography.
[0117] In some embodiments, a peptide composition for use in accordance with t e
present invention is or comprises a bioabsorbable aqueous solution having as its main
constituent a peptide. In some embodiments, such a solution is characterized by an ability to
transition between two states: an un-gelled state adopted, for example at a particular pH
and/or when one or more particular ions is substantially absent, and a gelled state adopted at a
particular pH and/or when the one or more ions is present at or above a threshold level.
[0118] In some embodiments, transition from un-gelled to gelled state (e.g., via
peptide self-assembly) occurs when the peptide solution is exposed to pH in the vicinity of
the isoelectric point; in some such embodiments, the isoelectric point is around pH 7 . In
some embodiments, such transition (e.g., via peptide self-assembly) occurs when the peptide
solution is exposed to a pH within a range of about pH 6 to about pH 8, inclusive, for
example about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, or 8.0; in some embodiments, such pH is within the range of about 6.5 to about 7.5,
inclusive; in some embodiments about 6.8 to about 7.2, inclusive; in some embodiments
about 7.0.
[0119] In some embodiments, transition from un-gelled to gelled state (e.g., via
peptide self-assembly) occurs when the peptide solution is exposed to the presence of a lowconcentration
(e.g., about several millimoles, for example within a range of about 1millimole
to about 10 millimoles, inclusive) of univalent alkali metal ions (e.g., Na+, K+) . In some
embodiments, such concentration of univalent alkali metal ions is about 1, 2, 3, 4, 5, 6, 7, 8, 9
or about 10 millimoles. In some embodiments, such concentration is greater than 1
millimole.
[0120] In some embodiments, such transition (and/or peptide self-assembly) occurs
under physiological conditions (i.e., pH around 7 in the presence of salts such as Na+ and K .
In some embodiments, such transition (and/or peptide self-assembly) occurs rapidly (e.g.,
within a time period less than about 5 minutes) upon exposure to appropriate pH and metal
ions; in some embodiments, such transition occurs within a time period of about 1 minute to
about 5 minutes, inclusive, for example, about 1, 2, 3, 4 or 5 minutes; in some embodiments,
such transition occurs within about 5 minutes; in some embodiments such transition occurs
within about 4 minutes; in some embodiments, such transition occurs within about 3 minutes;
in some embodiments, such transition occurs within about 2 minutes; in some embodiments,
such transition occurs within about 1minute.
[0121] In some embodiments, physiological conditions are those present in a body of
a subject, e.g., at a surgical site within or on a subject. For example, in some embodiments,
physiological conditions can be achieved by the presence of bodily fluids, blood, tissues
and/or a combination thereof. In some embodiments, physiological conditions are achieved
in vivo or ex vivo by the addition of a buffer that comprise the ions, e.g., by exogenously
adding one or exemplary ions at a level that induces the formation of the gelled state. For
example, in some embodiments, peptides may be exposed to appropriate physiological
conditions ex vivo, for example prior to or during a surgical procedure. In some
embodiments, such exposure occurs within a subject's body (e.g., during intrabody surgery),
or on a subject's body (e.g., when applied topically, for example, to opening left from a
laparoscope or biopsy.
[0122] In some embodiments, utilized peptides in solution self-assemble into a
structure, for example comprised of a network of fibers, when exposed to an appropriate pH
and ion condition. In some embodiments, utilized peptides self-assemble into a network
structure that includes fibers and pores. In some embodiments, such fibers have a diameter
within the range of about 10 to about 20 nm, inclusive; in some embodiments, such pores
have a diameter within the range of about 50 to about 200 nm. In some particular
embodiments, a utilized peptide self-assembles into a network structured that resembles the
structure of natural collagen (Figure 1).
[0123] In some embodiments, peptide compositions for use in accordance with the
present invention contain peptides in solution in a concentration within the range of about
0 .1% to about 10%, inclusive. In various embodiments, the concentration is within the range
of 0.1 - 10%; 0.2 - 9.9%, 0.3 - 9.8%, 0.4 - 9.7%, 0.5 - 9.6%, 0.6 - 9.5%, 0.7 - 9.4%, 0.8 -
9.3%, 0.9 - 9.2%, 1.0 - 9.1%, 1.1 - 9.0%, 1.2 - 8.9%, 1.3 - 8.8%, 1.4 - 8.7%, 1.5 - 8.6%,
1.6 - 8.5%, 1.7 - 8.4%, 1.8 - 8.3%, 1.9 - 8.2%, 2.0 - 8.1%, 2.1 - 8.0%, 2.2 - 7.9%, 2.3 -
7.8%, 2.4 - 7.7%, 2.5 - 7.6%, 2.6 - 7.5%, 2.7 - 7.4%, 2.8 - 7.3%, 2.9 - 7.2%, 3.0 - 7.1%,
3.1 - 7.0%, 3.2 - 6.9%, 3.3 - 6.8%, 3.4 - 6.7%, 3.5 - 6.6%, 3.6 - 6.5%, 3.7 - 6.4%, 3.8 -
6.3%, 3.9 - 6.2%, 4.0 - 6.1%, 4.1 - 6.0%, 4.2 - 5.9%, 4.3 - 5.8%, 4.4 - 5.4%, 4.6 - 5.3%,
4.7 - 5.2%, 4.8 - 5.1% or 4.9 - 5.0%, inclusive.
[0124] In various embodiments, t e concentration is within t e range of 0.1 - 5%,
0.25 - 4.75%, 0.5 - 4.5%, 0.75 - 4.25%, 1.0 - 4.0%, 1.25 - 3.75%, 1.5 - 3.5%, 1.75 - 3.25%,
2.0 - 3.0%, 2.25 - 2.75%; in a specific embodiment, within a range of 1.0 - 3.0%; in a
specific embodiment, the concentration is about 1%; in a specific embodiment, the
concentration is about 1.5%; in a specific embodiment, the concentration is about 2%; in a
specific embodiment, the concentration is about 2.5%; in a specific embodiment, the
concentration is about 3%.
[0125] In some embodiments, peptide compositions contain peptides in solution at a
concentration within the range of about 0.5% to about 5%. In some embodiments, peptide
compositions for use in accordance with the present invention contains peptides in solution at
a concentration of about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%,3.5%, 4%, 4.5%, 5%, or more.
[0126] The present invention provides methods of using compositions comprising the
peptide solutions described herein, in particular, in methods of performing surgical
procedures. In some embodiments, the surgical procedures may be intrabody surgical
procedures. In some embodiments, the surgical procedures may be may be superficial or
topical.
Surgical Methods
[0127] Peptide compositions described herein may be used in various surgical
procedures to control and arrest bleeding encountered by surgeons while performing surgical
tasks in a more effective and efficient manner. Exemplary surgical procedures are provided
that are performed in shorter time and/or involve less bleeding than typically occurs in
standard procedures by use of the peptide compositions described herein.
[0128] The present invention provides the insight that peptide compositions as
described herein are particularly useful in, and/or provide particular advantages when utilized
in certain surgical procedures. For example, among other things, the present invention
encompasses the recognition that the peptide compositions described herein provide an
effectiveness advantage in arresting bleeding during various surgical procedures. Exemplary
advantages are faster completion of one or more surgical tasks during a surgical procedure
and, as a result, a decrease in the overall duration of a surgical procedure. In particular, the
various examples describe the efficacy and safety of a composition comprising a peptide
solution, wherein the peptide comprises an amino acid sequence of RADA repeats; and
wherein the solution is characterized by an ability to transition between two states: an ungelled
(or aqueous) state adopted when one or more particular ions is substantially absent,
and a gelled state adopted when t e one or more ions is present at or above a threshold level,
wherein the one or more ions is or becomes present in the site (or location) of administration.
[0129] In some embodiments, the present invention provides the recognition, among
other things, that peptide compositions described herein provide clinical advantages
compared to existing materials used in a similar manner for arresting bleeding during surgical
procedures.
[0130] In some embodiments, the present invention provides the recognition, among
other things, that peptide compositions may be manufactured from artificial synthesis without
the use of any animal-derived products, negating any risk of infection.
[0131] In some embodiments, the present invention provides the recognition that,
compared with existing materials, methods of performing a surgical procedure on a subject
comprising applying peptide compositions described herein require minimal, or substantially
no, preparation and operation, thereby providing an advantage in application.
[0132] In some embodiments, the present invention provides the recognition that
existing materials (e.g., fibrin glue), in contrast to peptide compositions described herein, are
difficult to remove from application sites after hardening. For example, peptide compositions
may be washed with saline, allowing for repeated use during surgery.
[0133] In some embodiments, the present invention provides the recognition that
peptide compositions described herein are colorless and remain transparent once in the gelled
state has been adopted during application, thereby maintaining a clear surgical field of view.
Such is essential for ascertaining effective control and/or arrest of bleeding from a surgical
site.
[0134] In some embodiments, the present invention provides the recognition that
upon stoppage of bleeding during a surgical procedure or once all or substantially all tasks
associated with the a surgical procedure have been completed, excess peptide composition
described herein can simply be removed by washing with water. In specific embodiments,
after removal of peptide compositions that have been applied to one or more sites on or
within a surgical site, secondary bleeding is impeded, inhibited and/or ameliorated by the
coagulation system of the subject.
[0135] In some embodiments, the present invention provides the recognition that
gelation of peptide compositions described herein after contact with blood at or on an
application site, rather than solidification within a delivery device, e.g. a nozzle attached to a
pre-filled syringe, allows use in specific surgical procedures, e.g., endoscopy and
laparoscopy, and thereby eliminates difficulties by using existing materials, which can
solidify leading to complications.
[0136] In some embodiments, t e present invention provides the recognition that
peptide compositions described herein provide a contrasting mechanism of action. In certain
embodiments, application of peptide compositions described herein to one or more bleeding
sites provides a surface pressure on the one or more bleeding sites. Such surface pressure
provides normal coagulation to occur beneath the layer of the applied peptide composition
once a gelled state is adopted, thereby closing the bleeding site and stopping bleeding.
Existing materials require additional manual pressure for compression.
[0137] In some embodiments, the present invention provides the recognition that
peptide compositions described herein provide a decrease in the time to perform one or more
tasks associated with a surgical procedure.
[0138] Thus, among other things, the present invention provides improved surgical
methods that utilize peptide compositions as described herein. In some embodiments, a
provided surgical method is improved relative to a reference or standard of care method in
that it is performed in shorter period of time. In some embodiments, a provided surgical
method is improved relative to a reference or standard of care method in that recovery of a
patient is improved relative to a patient on whom the same surgical method was performed
without utilizing peptide compositions described herein.
[0139] In some embodiments, peptide compositions described herein are utilized in
surgical methods that are performed on the exterior or interior of the body of a subject. In
certain embodiments, peptide compositions described herein are utilized in surgical methods
that are performed on the vasculature, internal organs and/or bone(s) of a subject.
[0140] In some embodiments, peptide compositions described herein are utilized in
surgical methods to graft vessels within a surgical site. In certain embodiments, vascular
surgical methods comprise bypass surgery (e.g., coronary artery bypass).
[0141] In some embodiments, peptide compositions described herein are utilized in
surgical methods that are performed to resect or dissect an organ in whole or in part.
Virtually an organ may be a candidate in a given surgical procedure, however, without
wishing to be bound by theory, exemplary organs may include, e.g., liver, spleen, gall
bladder, pancreas, stomach or lung. In certain embodiments, peptide compositions described
herein are utilized in surgical methods that are performed to remove cancerous or otherwise
malignant tissue from an organ in whole or in part. In certain embodiments, peptide
compositions described herein are utilized in surgical methods that are performed to resect
benign tissue of an organ in whole or in part.
[0142] In some embodiments, peptide compositions described herein may be utilized
in surgical methods performed to repair a fracture of one or more bone(s) of a subject. In
certain embodiments, peptide compositions are utilized by injecting into a fracture site of one
or more bones in a subject. In certain embodiments, peptide compositions are utilized by
applying onto a fracture site of one or more bones in a subject.
[0143] In some embodiments, application of peptide composition described herein to
a surgical site may vary, e.g., depending upon t e application site, patient-specific factors,
surgical procedure, application site conditions, route of administration, and the like. When
peptide compositions described herein are used for treating various bleeding sites associated
with a given surgical procedure, including intrabody surgery in a subject, it is advantageous
to administer directly, normally in an amount necessary to arrest bleeding (e.g., a
therapeutically effective amount). In some embodiments, the frequency and duration of
administering peptide compositions as described herein can be adjusted depending on the
severity of the condition(s) or application site.
[0144] In some embodiments, peptide compositions described herein utilized in a
surgical method are provided in an injectable preparation. Such means for providing peptide
compositions for use in arresting bleeding during a surgical procedure is advantageous over
existing materials, which may require mixing or otherwise mechanical manipulation on the
part of the administrator or surgeon. The injectable preparations may be used for any type of
application to a bleeding site of a subject (human or non-human) in need of treatment. A
pharmaceutical composition comprising the peptide compositions described herein may be
delivered to a bleeding site or surgical site with a syringe and nozzle.
[0145] In some embodiments, a subject undergoing a surgical procedure, intrabody or
otherwise, may be administered a therapeutically effective amount of peptide compositions as
described herein to a bleeding site using a pre-filled syringe. Exemplary techniques include
placing a nozzle fixed to the pre-filled syringe in close proximity to one or more bleeding
sites as desired.
Pharmaceutical Compositions
[0146] Peptide compositions for use in accordance with the present invention
comprise peptides as described herein, optionally together with one or more with suitable
carriers, excipients, and/or other agents that are incorporated into formulations; in some
embodiments, components of utilized compositions are selected to provide improved transfer,
delivery, tolerance, performance, and the like.
[0147] In many embodiments, peptide compositions for use in the present invention
comprise peptides in aqueous solution (i.e., in a water-based and/or water-miscible carrier).
Exemplary aqueous carriers for such compositions include, for example, pharmaceutical
grade water, sucrose (e.g., sucrose water), and combinations thereof. In some certain
embodiments, peptide compositions for use in t e present invention comprise peptides in
aqueous solution, wherein the aqueous solution comprises a carrier that is an organic
compound that is characterized by an ability to confer solubility and/or bodying effects to the
peptides in aqueous solution.
[0148] In many embodiments, peptide compositions for use in accordance with the
present invention are sterile and/or are prepared aseptically.
[0149] In some embodiments, peptide compositions for use in accordance with the
present invention, including aqueous formulations, can be stored in an oxygen-deprived
environment. Oxygen-deprived environments can be generated, for example, by storing the
aqueous solution under an inert gas (e.g., nitrogen or argon).
[0150] In some embodiments, peptide compositions for use in accordance with the
present invention may be stored in dry form, for example in dry powder form, for example as
is achieved by lyophilization.
[0151] In some embodiments, peptide compositions for use in accordance with the
present invention, specifically including aqueous formulations, are suitably stored at a
temperature within the range of about 0°C to about 10°C, inclusive, for example about 0.5°C,
1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C,
7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, or 10.0°C; in some embodiments, such temperature is
within the range of about 2.0°C to about 8.0°C, inclusive. In some embodiments, such
temperature is above 0°C and lower than 10°C.
[0152] In some embodiments, peptide compositions for use in accordance with the
present invention are provided in unit dose forms, for example together with a delivery
system.
[0153] In some embodiments, an appropriate unit dose of a peptide composition in
accordance with the present invention, delivers an amount of peptide within the range of
about 0.1% to about 10% (w/v) of peptide, inclusive; for example about 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,
1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%,
3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%,
4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%,
6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%,
7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%,
8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, or 10.0%. In
some embodiments, delivers such an amount of peptide within the range of about 1.0% to
about 5.0%. In some embodiments, within the range of about 1.0% to about 3%. In some
embodiments, about 1.0%; in some embodiments, about 1.5%; in some embodiments, about
2.0%; in some embodiments, about 2.5%; in some embodiments, about 3.0%. In some
embodiments, an appropriate unit dose of a peptide composition that is a solution is within
the range of about 1.0 mL to about 50.0 mL, inclusive, for example about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mL. In some embodiments,
an appropriate unit dose of a peptide composition that is a solution is within the range of
about 1.0 mL to about 10 mL. In some embodiments, within a range of about 20 mL to about
30 mL. In some embodiments, within a range of about 30 mL to about 40 mL. In some
embodiments, within a range of about 40 mL to about 50 mL. In some embodiments, about 5
mL; in some embodiments, about 10 mL; in some embodiments about 30 mL.
[0154] In some embodiments, an appropriate unit dose of a peptide solution having a
concentration within the range of about 0 .1% to about 10% (w/v), as described herein. In
some embodiments, an appropriate unit dose is about 1mL to about 5 mL of a 1.0% to 3.0%
(w/v) aqueous solution, or about 30 mL of a 1.0% to 3.0% (w/v) aqueous solution.
[0155] In some embodiments, a peptide composition as described herein is provided
together with (e.g., within) an appropriate storage or delivery container such as for example, a
vial, bottle, beaker, bag, syringe, ampule, cartridge, reservoir or LYO-JECT®. In some
embodiments, the amount of peptide composition included in such an appropriate storage or
delivery container is at least a unit dose of the peptide composition. In some embodiments,
the amount is a unit dose, or a multiple thereof. The storage or delivery container may be
formed from a variety of materials such as glass or plastic. In some embodiments, peptide
compositions for use in accordance with t e present invention are provided in a pre-filled
syringe, and optionally together with one or more nozzles as described herein for delivery of
a peptide solution from such a pre-loaded syringe or other storage container.
[0156] Suitable pre-filled syringes include, but are not limited, to, borosilicate glass
syringes with baked silicone coating, borosilicate glass syringes with prayed silicone, plastic
resin syringes without silicone, or cyclo-olefin-polymer syringes, polypropylene syringes and
polyethylene syringes.
[0157] In some embodiments, the form of peptide composition that is provided
together with (e.g., within) an appropriate storage or delivery container is a solution as
described herein; in some embodiments, the form is a dry form as described herein (e.g., a
dry powder form).
[0158] In various embodiments, peptide compositions for use in accordance with the
present invention are suitable for administration to a subject during a surgical procedure. In
various embodiments, surgical procedures are performed within the body of a subject, e.g.,
intrabody. Exemplary intrabody surgical procedures are procedures to correct vascular
abnormalities (e.g., a bypass), resection or dissection (e.g., to remove damaged or diseased
tissue from an organ in whole or in part), or to repair a damaged organ, tissue or bone (e.g.,
repair a lacerated spleen, repair a bone fracture, repair torn muscle or ligaments, etc.). In
various embodiments, surgical procedures are performed on the exterior of a body of a
subject, e.g., topical. Exemplary topical surgical procedures are procedures to repair an
opening in the skin of a subject (e.g., sutures to close an opening in the skin made from a
puncture or other protrusion). In some embodiments, the subject is a human. In some
embodiments, the subject is an non-human animal (e.g., a horse, dog, cat, etc.).
Storage and/or Delivery Systems
[0159] In some embodiments, in addition to providing improved surgical methods as
described herein, the present invention provides storage and/or delivery systems particularly
adapted for delivery of peptide compositions as described herein In some embodiments,
storage systems are separate from delivery systems for peptide compositions described
herein. In some embodiments, storage of peptide compositions described herein is provided
in delivery systems. For example, peptide compositions described herein may be stored in a
delivery system, e.g., a pre-filled syringe, until time for application during a surgical method.
[0160] In some embodiments, storage and/or delivery systems as described herein can
be utilized in one or more surgical methods. In some embodiments, storage and/or delivery
systems as described herein may be utilized in methods for arresting bleeding so as to
decrease the duration of a surgical method performed on a subject.
[0161] In some embodiments, provided storage and/or delivery systems are
particularly adapted for delivery of peptide compositions as described herein to intrabody
sites including for example surgical sites. In some embodiments, the present invention
provides nozzles and/or cannulas for delivery of compositions such as peptide compositions.
[0162] In some embodiments, such nozzles and/or cannulas are adapted for
attachment to a syringe or other storage or delivery vessel, which may, for example, be pre
loaded with a composition for delivery. Examples of such nozzles and/or cannulas are
depicted in Figures 7 and 8.
[0163] In some embodiments, provided nozzles differ from traditional needles in one
or more of a variety of features. For example, in some embodiments, exemplary nozzles are
made from a non-metal material, in contrast to standard metal needles.
[0164] In some embodiments, provided nozzles and/or cannulas are formed from a
plastic material (e.g., polypropylene). In certain embodiments, provided nozzles and/or
cannulas are formed from a flexible material. In some embodiments, provided nozzles and/or
cannulas are formed from a stiff (e.g., non-flexible) material. In some embodiments,
provided nozzles and/or cannulas are formed from a material susceptible to sterilization, e.g.,
by autoclaving.
[0165] In some embodiments, provided nozzles and/or cannulas have a blunt end, in
contrast to many standard needles, which have a pointed end. For example, standard
hypodermic or suture needles, typically have a pointed end, which may be further
characterized by a bevel. Exemplary types of bevels include standard, short or true short
bevels.
[0166] In some embodiments, provided nozzles and/or cannulas have a relatively
wide bore as compared with many standard needles. In some embodiments, such nozzles
and/or cannulas have an inner bore diameter, an outer bore diameter and a bore wall
thickness. For example, in some embodiments, provided nozzles and/or cannulas have an
inner bore diameter within the range of about 4.00 mm to about 0.05 mm, inclusive; for
example about 4.00 mm, 3.90 mm, 3.80 mm, 3.70 mm, 3.60 mm, 3.50 mm, 3.40 mm, 3.30
mm, 3.20 mm, 3.10 mm, 3.00 mm, 2.90 mm, 2.80 mm, 2.70 mm, 2.60 mm, 2.50 mm, 2.40
mm, 2.30 mm, 2.20 mm, 2.10 mm, 2.00 mm, 1.90 mm, 1.80 mm, 1.70 mm, 1.60 mm, 1.50
mm, 1.40 mm, 1.30 mm, 1.20 mm, 1.10 mm, 1.00 mm, 0.90 mm, 0.80 mm, 0.70 mm, 0.60
mm, 0.50 mm, 0.40 mm, 0.30 mm, 0.20 mm, 0.10 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06
mm, or 0.05 mm; in some embodiments, such inner bore diameter is about 3.810 mm, 3.429
mm, 2.997 mm, 2.692 mm, 2.388 mm, 2.159 mm, 1.803 mm, 1.600 mm, 1.372 mm, 1.194
mm, 1.067 mm, 0.838 mm, 0.686 mm, 0.603 mm, 0.514 mm, 0.413 mm, 0.152 mm, 0.337
mm, 0.311 mm, 0.260 mm, 0.127 mm, 0.210 mm, 0.184 mm, 0.159 mm, 0.133 mm, 0.108
mm, or 0.0826 mm; in some embodiments, such an inner bore diameter is within the range of
about 1.200 mm to about 0.400 mm, inclusive; in some embodiments, about 1.194 mm, in
some embodiments, about 1.067; in some embodiments, about 0.838 mm; in some
embodiments, about 0.686 mm; in some embodiments, about 0.603 mm; in some
embodiments, about 0.514 mm.
[0167] In some embodiments, such an outer bore diameter is within the range of
about 5.00 mm to about 0.15 mm, inclusive; for example about 5.00 mm, 4.90 mm, 4.80 mm,
4.70 mm, 4.60 mm, 4.50 mm, 4.40 mm, 4.30 mm, 4.20 mm, 4.10 mm, 4.00 mm, 3.90 mm,
3.80 mm, 3.70 mm, 3.60 mm, 3.50 mm, 3.40 mm, 3.30 mm, 3.20 mm, 3.10 mm, 3.00 mm,
2.90 mm, 2.80 mm, 2.70 mm, 2.60 mm, 2.50 mm, 2.40 mm, 2.30 mm, 2.20 mm, 2.10 mm,
2.00 mm, 1.90 mm, 1.80 mm, 1.70 mm, 1.60 mm, 1.50 mm, 1.40 mm, 1.30 mm, 1.20 mm,
1.10 mm, 1.00 mm, 0.90 mm, 0.80 mm, 0.70 mm, 0.60 mm, 0.50 mm, 0.40 mm, 0.30 mm,
0.20 mm, or 0.10 mm; in some embodiments, such an inner bore diameter is about 4.572 mm,
4.191 mm, 3.759 mm, 3.404 mm, 3.048 mm, 2.769 mm, 2.413 mm, 2.108 mm, 1.829 mm,
1.651 mm, 1.473 mm, 1.270 mm, 1.067 mm, 0.9081 mm, 0.8192 mm, 0.7176 mm, 0.6414
mm, 0.5652 mm, 0.5144 mm, 0.4636 mm, 0.4737 mm, 0.4128 mm, 0.3620 mm, 0.3366 mm,
0.3 112 mm, 0.2604 mm, 0.2350 mm, 0.2096 mm, or 0 .1842 mm; in some embodiments, such
outer bore diameter is within the range of about 1.650 mm to about 0.750 mm, inclusive; in
some embodiments, about 1.651; in some embodiments, about 1.473; in some embodiments,
about 1.270 mm; in some embodiments, about 1.067 mm; in some embodiments, about
0.9081 mm; in some embodiments, about 0.8192.
[0168] In some embodiments, such a bore wall thickness is within the range of about
0.400 mm to about 0.025 mm, inclusive; for example about 0.400 mm, 0.375 mm, 0.350 mm,
0.325 mm, 0.300 mm, 0.0275 mm, 0.250 mm, 0.225 mm, 0.200 mm, 0.175 mm, 0.150 mm,
0.125 mm, 0.100 mm, 0.075, 0.050 mm, or 0.025 mm; in some embodiments, such a bore
wall thickness is about 0.381 mm, 0.356 mm, 0.330 mm, 0.305 mm, 0.254 mm, 0.229 mm,
0.203 mm, 0.216 mm, 0.191 mm, 0.1524 mm, 0.2826 mm, 0.1524 mm, 0.1270 mm, 0.1016
mm, 0.1734 mm, 0.1016 mm, 0.0889 mm, 0.0762 mm, 0.0635 mm, or 0.0508 mm; in some
embodiments, such a bore wall thickness is within the range of about 0.250 mm to about
0.150 mm; in some embodiments, about 0.229 mm; in some embodiments, about 0.216 mm;
in some embodiments, about 0.203 mm; in some embodiments, about 0.191 mm; in some
embodiments, about 0.1524 mm.
[0169] In some embodiments, provided nozzles and/or cannulas may have a tapered
bore. In some embodiments, such provided nozzles and/or cannulas taper substantially
evenly between their large and small bore portions. In some embodiments, provided nozzles
and/or cannulas taper to a small bore portion at their delivery end, which may for example be
a blunt end as described herein.
[0170] In some embodiments, provided nozzles and/or cannulas have a length within
a range of about 6 inches to about 0.25 inches; inclusive, for example, about 6.0 inches, 5.9
inches, 5.8 inches, 5.7 inches, 5.6 inches, 5.5 inches, 5.4 inches, 5.3 inches, 5.2 inches, 5.1
inches, 5.0 inches, 4.9 inches, 4.8 inches, 4.7 inches, 4.6 inches, 4.5 inches, 4.4 inches, 4.3
inches, 4.2 inches, 4.1 inches, 4.0 inches, 3.9 inches, 3.8 inches, 3.7 inches, 3.6 inches, 3.5
inches, 3.4 inches, 3.3 inches, 3.2 inches, 3.1 inches , 3.0 inches, 2.9 inches, 2.8 inches, 2.7
inches, 2.6 inches, 2.5 inches, 2.4 inches, 2.3 inches, 2.2 inches, 2.1 inches, 2.0 inches, 1.9
inches, 1.8 inches, 1.7 inches, 1.6 inches, 1.5 inches, 1.4 inches, 1.3 inches, 1.2 inches, 1.1
inches, 1.0 inch, 0.9 inches, 0.8 inches, 0.7 inches, 0.6 inches, 0.5 inches, 0.4 inches, 0.3
inches, or 0.2 inches; in some embodiments, about 0.50 inches to about 1.5 inches.
[0171] In some embodiments, provided nozzles and/or cannulas are specially adapted
for application in a particular surgical procedure. For example, nozzles may be engineered
based on type of surgery for which they are used (e.g., endoscopy, laparoscopy, etc.); other
factors consideration are size, length and flexibility (e.g., adapted for range of motion, ability
to use such that surrounding tissue is not disrupted or damaged), geometry and other formats.
Further, as may be appropriate, the addition of or coupling with an optical system and/or light
system, thereby allowing for visual confirmation of application to a site otherwise occluded
from a surgeon's view. In some embodiments, nozzles and/or cannulas adapted for coupling
to a catheter are provided. An example of such adaptor is provided in Figure 8.
[0172] In some embodiments, provided nozzles and/or cannulas adapted for coupling
to a catheter have a first connection end for connection to a catheter that has a diameter
within the range of about one millimeter to about four millimeters. In some embodiments, a
diameter within the range of about one millimeter to about 2 millimeters. In certain
embodiments, a diameter of about 1.5 millimeters.
[0173] In some embodiments, provided nozzles and/or cannulas adapted for coupling
to a catheter have a second connection end for connection to a storage and/or delivery device,
e.g., a pre-filled syringe, that has a diameter within the range of about four millimeters to
about 8 millimeters. In some embodiments, a diameter within t e range of about five
millimeters to about 7 millimeters. In certain embodiments, a diameter of about five
millimeters. In a specific embodiment, a diameter of about 5.21 millimeters. In certain
embodiments, a diameter of about seven millimeters. In a specific embodiment, a diameter
of about 6.9 millimeters.
[0174] Alternatively or additionally, provided nozzles and/or cannulas may be
designed in the context of pressure when applying the compositions from a syringe.
[0175] In some embodiments, provided nozzles and/or cannulas are reusable, for
example, being adapted to be removed from a first storage and/or delivery vehicle (e.g., after
delivery of composition from the storage and/or delivery vehicle) and attached to a second
(and/or subsequent) storage and/or delivery vehicle. In some embodiments, provided nozzles
and/or cannulas are single-use.
[0176] In some embodiments, pharmaceutical packages that contain a storage and/or
delivery system described herein are provided. Suitable pharmaceutical packages are sterile
and acceptable for use in a surgical setting. Examples of pharmaceutical packages are blister
packs, bubble packs or clamshell packages. Pharmaceutically acceptable packages, for
example, may be performed packaging and made from a various of materials, such as, e.g.,
cyclic olefin copolymers ( ), polychlorotrifluoroethylene, or polyvinyl chloride. An example
of a pharmaceutical package (e.g., a blister pack) comprising a storage and/or delivery device
comprising peptide compositions (e.g., a pre-filled syringe) described herein and a
nozzle/cannula is provided in Figure 9 .
[0177] In some embodiments, a blister pack is provided that contains a cavity or
pocket that provides a custom, formable location to accept a storage and/or delivery device
comprising peptide compositions described herein. Additionally or optionally, in some
embodiments, a blister pack is provided that contains a cavity or pocket that provides a
custom, formable location to accept a nozzle or cannula described herein. Additionally or
optionally, in some embodiments, a blister pack is provided that contains a cavity or pocket
that provides a custom, formable location to accept an adaptor described herein. In various
embodiments, individual blister packs contain a cover (e.g., a tyvek sheet) fixed to the cavity
or pocket to maintain sterile conditions.
[0178] In various embodiments, provided pharmaceutical packages, e.g., blister
packs, are sterile. Sterilization (i.e. aseptic processing of storage and/or delivery devices
described herein) may be accomplished by methods know in t e art and acceptable for
pharmaceutical products and/or packages. Examples of sterilization techniques for
pharmaceutical packages described herein are pressurized steam, hot air, ionizing radiation
(e.g., gama and/or electron beam), and gas (e.g., ethylene oxide or formaldehyde).
[0179] In some embodiments, multiple storage and/or delivery devices may be
provided in a single pharmaceutical package. For example, a unit or multiple doses provided
in pre-filled syringes may be packaged in multiple blister packs, or optionally, in a clamshelltype
container suitable for packaging multiple syringes. In some embodiments, pre-filled
syringes comprising a unit dose, or multiple doses, are provided in blister packs. In some
embodiments, multiple pre-filled syringes are provided in clamshell-type packages and are
acceptable for use in a surgical setting (i.e., sterile). Suitable sterilization techniques are
employed as described above to ensure sterilized pharmaceutical products are provided in
various surgical settings.
EXAMPLES
[0180] The following examples are put forth so as to provide those of ordinary skill in
t e art with examples of how to make and use the methods and compositions of the invention,
and are not intended to limit the scope of the invention. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some
experimental deviations are to be expected as is known to one of skill in the art. Unless
indicated otherwise, parts are parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1. RADA-16 Solution
[0181] The inventors have determined that peptide compositions described herein
arrest bleeding in a surgical procedure by an entirely different mechanism than that of
existing materials used to control and/or stop bleeding during surgery. Typically, coagulation
factors are used. However, through the rather rapid gelation under physiological conditions,
peptide compositions described herein block the bleeding site without the use of a
coagulation factor and can stop bleeding without pharmacological action. The inventors have
employed peptide compositions described herein in various surgical procedures on human
and non-human subjects and discovered that peptide gels intertwine with blood cells at a
given bleeding site at the superficial portion of t e blood vessels thereby allowing blood
coagulation to occur below the top layer of the gel. Other materials, e.g., fibrin glue, act by
activating the blood coagulation system by mobilizing coagulation factors, thereby blocking
bleeding from a given site. Exemplary properties of peptide compositions described herein
include starting materials of an artificial synthetic peptide and water for injection without
including any animal-derived materials. This drastically reduces, if not eliminates altogether,
the risk of infection by using peptide compositions described herein in a surgical procedure.
Further, because peptide compositions described herein are provided in an aqueous solution it
can be supplied in pre-filled syringes and used directly on or within a surgical site. There is
no need for special preparation before application, as is typically encountered with other
materials, e.g., fibrin glue. Also, a large component of the aqueous solution is water, which
allows for repeated use in an almost unlimited manner, unlike other materials, especially
adhesive-based materials.
[0182] The present Example describes a particular peptide composition, referred to
herein as "Composition 1" utilized in various surgical methods as described herein.
Composition 1 is a bioabsorbable aqueous solution containing 2 - 2.5% of RADA-16 in
water.
[0183] Composition 1 is manufactured by preparing peptides consisting of chemically
synthesized amino acids using solid-phase synthesis, dissolving the peptides in water for
injection, filtering the solution with a bacterial filter (0.2 mm), and filling the resulting filtrate
in a sterile manner into a syringe. As such, manufacture is completed without using any
animal-derived materials, eliminating any risk of infection by biological materials.
[0184] Composition 1 is a clear, colorless liquid and retains this transparent quality
upon application to a surgical site upon which the peptide solution adopts a gelled state by the
formation of a hydrogel and has the ability to stop bleeding during the performance of a
surgical procedure. This transparent quality makes Composition 1 uniquely suited for use in
surgical procedures over other materials in terms of its ease of use and ability to maintain a
clear surgical field. Composition 1 can be provided in a pre-filled syringe and thus is unique
compared to other materials, e.g., fibrin glue, which needs to be prepared and mixed from
separate liquids. There is no such requirement with Composition 1 as it is made from
peptides and can be completely broken down by washing. The inventors have realized a
number of advantages in employing Composition 1 in surgical procedures: virtually
unlimited frequency of application, faster and more efficient control and stoppage of
bleeding, maintenance of clear surgical field and bleeding site due to transparent quality,
easily removed by irrigation, shortens duration of bleeding control measures during surgery,
overall shortening of time required to complete surgical procedure, and may improve the rate
of patient recovery by contributing to overall decrease in blood loss during surgery.
[0185] Cell culture experiments have demonstrated that Composition l's main
constituent peptide (CH3CO-(Arg-Ala-Asp-Ala)4-NH2, see below) does not exhibit
bioactivity by acting on the signal transduction system of living organisms (data not shown).
A search of t e European Molecular Biology Laboratory (EMBL) and Kyoto University's
GenomeNet Database Resources for protein sequence motifs for all amino acid sequences in
which the main constituent peptide can be generated by cleavage did not reveal any
sequences indicating a high degree of homology with known motifs. Once Composition 1
forms a gel, the peptides resist degradation even when exposed to digestive enzymes such as
trypsin, a-chymotrypsin, papain, protease K and pronase.
weight 2
[0186] Unlike other measures to control and/or stop bleeding during surgery, e.g. ,
oxidized cellulose or starch-based absorbent topical preparations that stem blood flow by the
formation of clots, the mechanism of action of Composition 1 is realized by modification of
physical properties upon a change in pH to seal off the bleeding point.
Example 2. Surgical procedures in non-human animals employing Composition 1
[0187] This Example describes certain animal studies based on results of an efficacy
validation study of an approved material for controlling bleeding during surgery. The oozing
needle hole hemorrhage model of prosthetic vascular graft implantation in a beagle dog was
designed to mimic the oozing needle hole hemorrhage from anastomotic sites at t e
autologous vascular.
[0188] All animals received humane care in compliance with the Principles of
Laboratory Animal Care formulated by the National Society for Medical Research and the
Guidefor the Care and Use of Laboratory Animals prepared by the Institute of Laboratory
Animal Research (ILAR), published by the National Academies Press (1996).
[0189] Rabbit abdominal aortic puncture. Laparotomy was performed to expose
approximately 10 cm of the abdominal aorta of each rabbit. Heparin sodium (500 IU) was
administered intravenously. The bleeding model was established by puncturing the
abdominal aorta using an injection needle (23-26G). After bleeding was confirmed,
peripheral and central blood flow was stopped with clamps and Composition 1 was
immediately applied to the wound site using a syringe. Blood flow was allowed to resume
after 1-2 min, and the puncture site was visually inspected for bleeding. Rabbit abdominal
aorta used in the present study was fixed in formalin, and vascular cross sections of both
Composition 1-treated and untreated sites (control) were used to make pathology specimens
that were then observed under a microscope.
[0190] The results demonstrated that total cessation of bleeding was observed in all
animals following administration with a > 2% peptide concentration of Composition 1, with
the exception of one animal treated with a 2% peptide concentration of Composition 1. The
structure-less and eosinophilic gelatinized Composition 1 was observed at the vessel puncture
site and surface. Further, gelatinized Composition 1 was observed to have formed a coating
on the tissue surface that physically occluded the puncture.
[0191] Beagle abdominal aortic graft replacement. Male beagles (n=2) weighing
13.1 kg and 11.4 kg were employed for an aortic graft replacement surgical procedure using
Composition 1. The abdominal aorta was exposed via laparotomy under general anesthesia.
Heparin sodium was intravenously infused at 1000 IU. After confirmation that the active
coagulation time (ACT) had exceeded 200 seconds, the abdominal aorta was clamped, and an
end-to-end graft replacement procedure was performed. Exudative bleeding (an oozing-type
bleeding) from the graft anastomosis and needle hole were observed. Composition 1 was
applied to the needle hole to evaluate the efficacy and cessation of bleeding.
[0192] The results demonstrated that oozing-type bleeding from anastomosis site was
stopped, and about 1 min after applying approximately 2 mL of 2.5% Composition 1,
stoppage of the anastomotic oozing was confirmed. Further, the oozing-type bleeding from
t e needle hole was also arrested. The abdominal aorta puncture bleeding model was
prepared by piercing the artificial vascular graft with the same-sized 26-G injection needle
used on the rabbits (as described above), and spurting of blood was consequently observed.
Peripheral and central blood flow was stopped, and approximately 1mL of 2.5%
Composition 1was applied. Blood flow was allowed to resume after about 1 minute.
Complete stoppage of bleeding at the wound site was confirmed. This procedure was
repeated three times at three separate sites on the graft. Postoperative observation was
performed up to three days.
[0193] Mouse intravenous administration. Composition 1 forms a gel as soon as it
comes in contact with blood from a bleeding site. Through application at a bleeding site, it is
possible for an amount of gelatinized Composition 1 to enter the blood stream. [[Note to
client: what can we make of this: "There is also an undeniable risk of gelatinized
Composition 1 entering the blood stream as a result of erroneous intravascular administration.
To evaluate these risks, we carried out tests on mice and rabbits simulating accidental IV
administration of Composition 1."]] In this example, the safety of intravenous administration
of Composition 1was demonstrated using mice.
[0194] Briefly, gelatinized Composition 1 in suspension was dosed at a concentration
that would presumed to have an adverse biological effect. The results demonstrated that
death in mouse subjects was observed up to a 40-fold dilution. While direct causes were not
determined, it was suspected that death was due to pulmonary embolism. No autopsy was
performed on the mice. However, the following abnormal behaviors indicate pulmonary
infarction: reduction of spontaneous behavior, squatting position, and accelerated respiration.
No deaths were observed at an 80-fold dilution, although abnormal findings (inactivity and
tachypnea) were observed. At a 160-fold dilution, no abnormalities were observed.
[0195] In a similar experiment, guinea pigs were administered 0.2 mL of
Composition 1 in a 160-fold diluted suspension. No abnormal behaviors were observed in
any of the animals.
[0196] Without wishing to be bound by theory, these results may be extrapolated to
humans assuming the following conditions: the subject is an adult weighing 60 kg having a
total blood volume of 4.6 L. Assuming this criteria and using the data observed using the
mice (body weight of about 40 g and a total blood volume of about 3 mL), the expected
amount of Composition 1 gel administered via intravenous injection that would likely cause
death in a manner similar to that observed in the mice would be a 4- to 40-fold dilution of
approximately 770 mL, or approximately 19.3-193 mL of Composition 1. Such a volume is
much larger than that employed in a surgical procedure. As shown by this example,
Composition 1 is a safe and effective solution to controlling bleeding in surgical models in
various animals.
[0197] As shown in this example, application of Composition 1 exhibited efficacy at
arresting bleeding in the exemplary animal surgical procedures described above. Further,
from the intravenous administration experiments, a single dose of 5 mL Composition 1 via
syringe is unlikely to cause pulmonary embolism or other adverse events resulting in death,
even in the case of a mistaken administration, directly into a blood vessel.
Example 3. Coronary Artery Bypass Graft (CABG)
[0198] The present Example describes a Coronary Artery Bypass Graft surgery
utilizing peptide compositions described herein and, in particular, steps of the surgical
method at which peptide compositions described herein can be applied. An exemplary
peptide composition provided is Composition 1 (described above).
[0199] Coronary Artery Bypass Graft, or CABG, begins with exfoliating the internal
thoracic artery or collecting the great saphenous vein, which are to be used as grafts. The
internal thoracic artery is anastomosed to the outer area of the heart away from blocked
coronary arteries. The collected great saphenous vein is anastomosed to the base of the
coronary artery and to outer area in the heart away from lesion (blocked) coronary arteries.
[0200] Typically in CABG surgery, the internal thoracic artery as graft would be the
first choice because of the historical success rate. Optionally, the great saphenous vein can
be used in the event more grafts are necessary. Bleeding can occur during multiple stages of
CABG surgery, for example, exfoliating internal thoracic artery or collecting great saphenous
vein, which are used as grafts, rebleeding on above areas due to heparinization, anastomosis
sites of various heart arteries and grafts, and connection sites of the heart and the cannula
(tube) of an oxygenator (Figure 3).
[0201] Bleeding at exfoliation or graft collection sites. Exfoliation or collection of
grafts is performed using standard surgical instruments or an electrosurgical knife. Bleeding
from collection sites are usually the result of using an electrosurgical knife and the patient
undergoing this surgery is heparinized after this procedure to prevent blood from clotting in
preparation for connecting an oxygenator. After heparinization, oozing type of rebleeding
frequently occurs from collection sites, at which time an electrosurgical knife is usually
applied to stop such rebleeding. This can take extra time that prolongs the CABG procedure
and causes damage to tissues. This prolongs the healing process leading to a slower recovery
following the procedure. Surgeons desire to minimize overall surgery time required to
perform the surgery as well as steps to minimize bleeding during the surgery as t e target of
the surgery is bypass, not collecting grafts. Existing surgical methods at controlling bleeding
are inadequate and are not typically and widely used to control the bleeding during this
procedure. Instead, an electrosurgical knife is used and the tissue is burned to stop any
bleeding.
[0202] In a CABG surgery, Composition 1 can be applied at multiple steps during the
procedure to control bleeding and decrease the overall time necessary to complete the
surgery. Composition 1 can be applied to a collection site to effectively prevent bleeding.
Due to its ease of use, Composition 1 can be applied before and during the collection
procedure. Further, it can also be applied before heparinization. By preventing bleeding
during the procedure and of rebleeding after heparinization, a surgeon can reduce the total
amount of bleeding and shorten the overall time needed to complete the surgery. Further, this
time savings is recognized in time required for anesthesia as well.
[0203] In one CABG surgery procedure in a patient in need of such surgery, four
grafts are required to perform a successful bypass. Time per graft is decreased by 5 to 20
minutes and overall time for the surgery to be performed is decreased by 20 to 80 minutes.
This decrease in time is due, in part, to the removal of or reduced need for the electrosurgical
knife in the procedure. Further, a decrease risk of infection is observed. As a result in the
decreased time for performance of the CABG procedure, a reduction of total hospitalization
time is expected, e.g., one 24 hour period. In some cases, where the surgeon encounters
difficult rebleeding during the procedure, additional 2 or 3 days of hospitalization are
required.
[0204] Peptide compositions such as Composition 1 are applied to bleeding sites
during and after exfoliating or graft collection in a wide area around the target bleeding sites,
and permitted to remain in the area untouched. This allows the solution to form into a gelled
state on the target site. Manual manipulation of peptide compositions is not advised, e.g., by
rubbing with one's fingers, as this lead to break down of the gel. Peptide compositions
remain transparent despite t e change in state from a solution to a gel. This unique property
allows for the maintenance of a clear surgical field as well as improved and superior control
of bleeding from multiple sites during the procedure. The use of a pre-filled syringe
containing the solution and a specialized nozzle adapted for use in such a procedure
contributes to the reduced time for performing the various steps of the procedure as well as
the procedure as a whole. Peptide compositions can be washed from the area at the end of
the CABG procedure.
[0205] SURGICEL® is made of an oxidized cellulose polymer with a low pH and is
used to control post-surgical bleeding by inducing clotting of blood. It has been associated
with incidents of neurotoxicity. For example, SURGICEL® is used extensively in oral and
maxillofacial surgery to control intrabody arterial bleeds from the inferior alveolar artery.
When placed in the mandibular canal with the inferior alveolar nerve exposed there have
been reports of neurotoxic effects.
[0206] In CABG surgery, SURGICEL® could be applied to bleeding sites, however,
surgeons typically prefer an electrosurgical knife. Preferred use electrosurgical is due, in
part, to the time needed to use SURGICEL® as it has a cotton-wool or sheet-type property
and surgeons have to cut it using tweezers and subsequently apply it to bleeding sites after
removing blood. This is a difficult task since it is easy SURGICEL® to stick to tweezers. It
is necessary for SURGICEL® to absorb blood to become sticky and thereby control bleeding.
Further, applied pressure may be needed or time for it to remain in place to allow for
absorption of blood. In this case, surgeons typically leave it as applied until the conclusion of
the surgery since a graft area is not the main target of this surgery. SURGICEL® turns a
black color and, as a consequence, makes arteries within the target sites black. This
decreases the visibility of the surgical site. Since it has some adhesive properties,
SURGICEL® must be removed by tweezers after application. This step increases time for
completion of the procedure and, on occasion, when it is removed, damage to the surrounding
tissue may lead to rebleeding. Further, a surgeon is unable to confirm that bleeding is
controlled and has stopped from all areas until the SURGICEL® is removed.
[0207] Fibrin glue may be applied in the same manner as SURGICEL®, however, as
stated above, surgeons prefer using an electrosurgical knife. When fibrin glue is applied in a
CABG surgery, it is typically sprayed by attaching a spray nozzle. After removing blood on
the target area, it is sprayed by a larger applied pressure or by using compressed air. Fibrin
glue requires five to ten minutes to become sticky enough to remain in the location and stop
any bleeding. Occasionally, fibrin glue requires pressure using gauze, etc. Fibrin glue does
not possess the efficacy to heparinized blood and cannot be applied in advance against
bleeding. If it is applied and does not control bleeding sufficiently, it must be removed in
order to reapply. This incurs more time on the procedure and the step at which the fibrin glue
is being applied. This can also lead to rebleeding. As with t e use of SURGICEL®,
surgeons cannot confirm that bleeding is controlled and has stopped from all areas until t e
fibrin glue is removed.
[0208] Bleeding from coronary arteries and after systemic circulation. After
exfoliating the internal thoracic artery, one end is anastomosed to the periphery end of a
blocked coronary artery. To accomplish this, the targeted coronary artery needs to be
identified from the surface of the heart. However, the heart is covered by adipose tissue and
surgeons have to dig into the adipose tissue to find the coronary artery. This can lead to
bleeding. If the point of bleeding can be identified, a hemo-clip is applied, and if not,
bleeding is widely astricted by gauze, which must be pressed for around a minute and
bleeding is controlled. The procedure has to be stopped during this time. Alternatively,
peptide compositions described herein can be applied to the area of the coronary artery in
advance to prevent bleeding, or when any bleeding is found on the area, since it is transparent
and the applied area can be operated by surgical instruments. SURGICEL® and Fibrin glue
cannot be applied in advance since they do not accept additional surgical procedures to the
area once applied. When SURGICEL® and fibrin glue are applied after bleeding is found,
additional time is required due to the need to stop the procedure for application.
[0209] Bleeding from anastomosed arteries. When the great saphenous vein is
anastomosed to a coronary artery, it is performed by thread and needle or by an autoanastomosing
device and bleeding is typically encountered. An electrosurgical knife cannot
be used to control this type of bleeding since the burn causes damage to the anastomosed
artery. If the bleeding is projectile in nature, additional sutures are made to the appropriate
areas. If necessary, fibrin glue, SURGICEL®, or astriction with gauze are applied.
SURGICEL® is typically applied to smaller bleeding sites than fibrin glue. In these cases,
since the artery has a round shape, application is first conducted to one side of the artery and
then, the artery is turned around and application is conducted to the other side. By putting
pressure on fibrin glue, the efficacy can be enhanced. However, the mechanism of fibrin glue
depends on coagulation of blood itself, which may require more than ten minutes even under
pressure.
[0210] Peptide compositions described herein are applied easily to the anastomosed
arteries. It can be once applied to a finger or gauze then be pasted on the unseen area of
anastomosed artery. In this case, instant astriction is possible, unlike fibrin glue, and
rebleeding by blood pressure after t e application is prevented. This minimizes time under
astriction (to 2-5 minutes) and enhances the process of controlling bleeding.
[0211] Bleeding at connecting sites of heart and cannula/tube of an oxygenator.
In CABG surgery when it is necessary to prevent the heart from beating, an oxygenator is
connected through a cannula/tube to an artery and heart in order to circulate blood to the rest
of the patient's body. The cannula is directly inserted and fixed by sutures. Bleeding is
sometimes identified on the suture site during the circulation or on t e removal site of
cannula after it has been removed. Fibrin glue is typically not applied because it fixes the
area and makes removal of cannula difficult. Further, SURGICEL® is difficult to apply due
to its sheet-like characteristics. Typically, gauze is pressed and placed on the bleeding point.
If the bleeding remains or gest stronger, additional anastomosis is performed. The unique
non-glue/non-sheet properties of peptide compositions described herein make it especially
applicable to this situation and these types of bleeding encountered during surgery.
[0212] Clinical Study of Composition 1 in human cardiovascular surgery. Study
protocols were approved by the Institutional Review Board of Toho University Medical
Center Sakura Hospital and Omori Hospital. Informed consent was obtained from all
patients. In this clinical study, 33 application sites in 25 patients (22 men, 3 women) were
targeted for application of Composition 1. Patients that satisfied specific criteria and
underwent CABG, vascular surgery for abdominal aortic aneurysm (AAA), or arteriosclerosis
obliterans (ASO) between January 2010 and April 201 1.
[0213] The following exclusion criteria was used: (1) individuals with past medical
history of hypersensitivity to peptide drugs or protein preparations, (2) individuals with
serious complications other than diseases indicated for surgery that may hinder the study, (3)
individuals who were unable to discontinue drugs that may affect the use of Composition 1 in
the surgical procedure, e.g., blood-clotting drugs (blood coagulation accelerators; i.e.,
hemocoagulase) and antifibrinolytic agents (e.g., drugs with antifibrinolytic action; epsilon
aminocaproic acid, tranexamic acid, aprotinin preparations, etc.), (4) individuals with child's
classification of B or C, and (5) individuals otherwise deemed unsuitable for the study by the
investigator.
[0214] All procedures were performed while the patient was under general anesthesia.
CABG was performed without cardiopulmonary bypass. Heparin sodium was administered
during the procedure at 200 IU/kg, and protamine sulfate after t e procedure for achieving a
target ACT of 200 seconds. Prosthetic vascular graft replacement surgery to treat AAA was
performed with a woven Dacron graft (J-graft; Japan Lifeline, Tokyo, Japan). Graft bypass
surgery or autologous vein patch plasty to treat ASO was performed with an ePTFE ringed
Gore-Tex vascular graft (WL Gore & Associates; Flagstaff, AZ, USA) and saphenous vein,
respectively. Heparin sodium was administered during the surgical procedures at 5000 IU,
but protamine sulfate was not typically used after the surgical procedure.
[0215] In the CABG procedure, target sites designated for application of Composition
1 were vessel-to-vessel anastomotic sites. For surgical procedures to treat AAA and ASO,
target sites for application of Composition 1 included the graft anastomotic site and
autologous vein patch plasty site. Types of bleeding targeted for application were (1) blood
oozing that typically would be arrested with fibrin glue and collagen materials, and (2) blood
oozing during typical treatment using other methodology such as ligation, clips, and
coagulation that were ineffective or could not be performed. If copious blood spurting or
gushing bleeding was encountered, other treatment methodology were typically performed
including ligation, clips, or coagulation. Composition 1was not applied in these situations.
[0216] After anastomotic blood was removed with gauze, Composition 1was evenly
applied gently without break down the gelated Composition 1 and smeared into each of the
target sites before the administration of protamine sulfate. Specifically, approximately 1mL
of 2.5% Composition 1was applied to coronary anastomotic sites, approximately 2 mL was
applied to aortic anastomotic sites, and approximately 1mL was applied to other peripheral
vascular anastomotic sites.
[0217] The primary endpoint of Composition 1that was evaluated was intraoperative
bleeding. It was determined as follows: complete response (CR), total arresting of bleeding
at the target site; partial response (PR), temporary total arresting of bleeding confirmed, but
permanent stoppage of bleeding only observed after reapplying Composition 1to application
sites due to intraoperative secondary bleeding requiring treatment; minor response (MR),
temporary stoppage of bleeding confirmed, but permanent stoppage of bleeding only
observed after using a procedure other than Composition 1 due to intraoperative secondary
bleeding from application sites requiring treatment; no response (NR), bleeding from target
sites not reduced and stoppage of bleeding not achieved.
[0218] A secondary endpoint of post-operative bleeding was recorded and determined
as follows: CR, no post-operative bleeding observed during post-operative examination; PR,
post-operative bleeding from Composition 1 application sites inferred from the post-operative
examination, without requiring reoperation; and NR, post-operative bleeding originating from
Composition 1 application sites observed during t e post-operative examination requiring
reoperation.
[0219] Adverse events including any abnormal findings or adverse reactions were
recorded concerning symptoms, severity, duration, treatment, course and outcome, and
association with the study drug (as well as the rationale for determining any association).
[0220] Results. Subjects comprised 25 patients (23 men, 2 women) with an age
range of 54-80 years. Of these patients, 9 underwent CABG surgery, 4 underwent AAA
surgery, and 12 underwent surgery for ASO. Composition 1 was used on 33 sites,
specifically at areas of the internal thoracic artery-coronary artery anastomosis (n = 1),
saphenous vein-coronary anastomosis (n = 4), ascending aorta-saphenous vein anastomosis (n
= 4), graft anastomosis (n = 15), and autologous vein patch plasty (n = 9). Mean area of the
application was 3.03 cm2 (ranging from 0.25-10 cm2) . Mean amount of Composition 1
applied was 1.5 mL (ranging from 0.5-3 mL). The efficacy rate observed was 87.9% for the
primary end-point (intraoperative bleeding) and 100% for the secondary endpoint (occurrence
of post-operative after bleeding; Table 4). For heparin treatment, the efficacy rate observed
was 85.2% (23/27), and time for stoppage of bleeding was 153.6 ± 38.7 seconds (mean ±
S.E.). For the protamine treatment, the efficacy rate was 100% (6/6), and the time for
stoppage of bleeding was 195.0 ± 130.1 seconds (mean ± SE). No adverse events (including
serious adverse events) having a causal relationship to the application of Composition 1 were
observed.
TABLE 4
Application site No. 1° Endpoint 2° Endpoint
Internal thoracic arter -coronary artery anastomosis 1 1 1
Saphenous vein-coronary anastomosis 4 3 4
Ascending aorta-saphenous vein anastomosis 4 4 4
Graft anastomosis 15 14 15
Patch suture 9 7 9
Total 33 29 (87.9%) 33 (100%)
[0221] Evaluation of the efficacy of Composition 1 in the clinical study described
above was implemented as suggested by Stark et al. (Stark J et al. 1984, Ann Thorac Surg
38:411-413). Previous have reported total stoppage of bleeding on oozing bleeding at rates
of 23.1%-100%. As shown in this example, Composition 1 performs at the top end of this
range. Further, what is not assessed in this number is the added benefit of the use of an
infection-free material that does not include animal-derived products or human blood
components. Because Composition 1 is entirely synthetic, it provides an alternative material
that poses no risk of infection.
[0222] As shown in this example, Composition 1 was applied to 33 sites in 25
patients and exhibited an efficacy and safety rate of 87.9% (29/33; Table 1). No differences
in the efficacy of Composition 1 in heparin- and protamine- treated individuals was observed
(data not shown). No post-operative bleeding or other adverse events of any kind were
observed. Based on these findings, Composition 1 provides a safe and useful alternative
material that demonstrates excellent local stoppage of bleeding on oozing bleeding during
cardiovascular surgery.
Example 4. Thoracic Aorta Replacement
[0223] This example illustrates the surgical procedure to replace the region of an
aortic aneurysm from the arch to distal region of the aortic aneurysm by total replacement
surgery utilizing peptide compositions described herein and, in particular, steps of the
surgical method at which peptide compositions described herein can be applied. An
exemplary peptide composition provided is Composition 1 (described above).
[0224] Briefly, the surgical procedure comprises five steps, (1) aortic cross-clamping
and establishment of cardiopulmonary bypass (including cerebral protective reflux), (2)
anastomosis of the descending aorta (peripheral side), (3) anastomosis of the ascending aorta
(central side), (4) anastomosis of the left subclavian artery, left common carotid artery, and
innominate artery, and (5) withdrawal of cardiopulmonary bypass.
[0225] Aortic blockage and establishment of a heart-lung machine (including
cerebral protective reflux). Median sternotomy and pericardiotomy are performed followed
by the extirpation of the ascending aorta and heart. Heparin is then administrated. A tube is
inserted into the right axillary artery, innominate artery, left common artery, left subclavian
artery, and descending aorta in order to reflux blood between a heart-lung machine and the
patient. Afterwards, reflux of blood by a heart-lung machine is started during the blockage of
each vessel. The heart is stopped by injection of a myocardial protection liquid.
[0226] Descending aortic anastomosis (peripheral side). The descending aorta is
cut by electric scalpel and is anastomosed with a vessel graft with 3-0 or 4-0 Proline thread.
Certainty of anastomosis is required as the field view is limited in deep area and it is difficult
to stop bleeding after starting reflux.
[0227] Ascending aortic anastomosis (central side). The ascending aorta is cut by
electric scalpel and is anastomosed with a vessel graft with 3-0 or 4-0 Proline thread. The
vent tube is inserted into the vessel graft in order to remove air bubbles prior to the restart of
blood reflux. The application of fibrin glue on the entire region of anastomosis prior to
declamping would decrease the risks of exudative and gushing hemorrhage, however, this
procedure may cause bleeding from between the fibrin glue and anastomosed region. In this
case, the reapplication of fibrin glue will be required instead of stripping of fibrin glue, since
there is a risk of increased bleeding. It is difficult to apply fibrin glue between the
anastomosed region and the region previously applied with the glue. Repeated application of
SURGICEL® in this instance may not be sufficient to stop bleeding and the final step would
be astriction for an extended period of time long time. In the instance that liquid fibrin glue
cannot stop bleeding, sheet type of fibrin glue is employed. If there is further bleeding, fibrin
glue is stripped off and then second anastomosis is performed with needle and thread. These
steps in the surgical procedure not only increase the volume of bleeding due to the
disturbance of anastomosed region where fibrin glue is removed, but also further increases
the time of surgery since anastomosis is complicated by the remaining fibrin glue (about 20-
90 minutes).
[0228] Alternatively, peptide compositions described herein (e.g., Composition l)can
be applied by swab and/or injection on the region where bleeding has not stopped with fibrin
glue and the particular site of anastomosis. Application of, e.g., Composition 1prior to
clamping allows for Composition 1to blend into a shallow layer of blood vessels on the site
of anastomosis. A suitable volume of Composition 1 is applied and kept at the site so as not
to fall off due to gravity or pushed away from the site due to blood pressure after declamping.
During declamping, forceps are slowly removed and about 30 to 60 seconds is elapsed to
allow for Composition 1to gelatinize with blood. The flow of blood vessels from the inferior
side is visible, since Composition 1 is a transparent material both in solution and once
gelatinized. If Composition 1 is washed or pushed away due to blood pressure, it can be
reapplied in repeated fashion until bleeding is stopped while the clamp is retained. Once
bleeding is stopped completely, declamping is performed.
[0229] The anastomosis of innominate artery, left common artery, and left
subclavian artery. The innominate artery, left common artery, and left subclavian artery are
cut by electrical scalpel and are anastomosed to a vessel graft by 5-0 Proline thread followed
by protamine administration. Composition 1may be used during this procedure as described
above.
[0230] The withdrawal from a heart-lung machine (reopened circulation by
cardiac beat).The insertion site of the tube connecting the heat-lung machine and a patient is
closed by 6-0 Proline thread. The blood circulation of innominate artery, left common artery,
and left subclavian artery is reopened and systemic circulation is resumed.
Example 5. Lymph Node Dissection
[0231] The present Example describes a lymph node dissection utilizing peptide
compositions described herein and, in particular, steps of the surgical method at which
peptide compositions described herein can be applied. An exemplary peptide composition
provided is Composition 1 (described above).
[0232] Lymph node dissection of pulmonary hilum and mediastinum is known to be a
standard treatment of lung cancer and requires the dissection of the lymph node and
surrounding tissue within the anatomical site.
[0233] Left periaortic lymph node dissection. The mediastinal pleura is incised on
the site of left main pulmonary artery into the top of the aortic arch by an electric scalpel.
The mediastinal pleura with surrounding tissue is stripped off by using an electric scalpel,
scissors and forceps with gauze ball. Lymph node is dissected from the top site of aortic
hiatus. In inferior side, while pulmonary artery is exteriorized, the lymph node around the
tissue is dissected. The entire lymph node is wrapped with Alice forceps and pulled out.
Next, the surrounding vascular and connective tissues are stripped off by electric scalpel,
scissors and forceps with a gauze ball. The lymph node is then incised and extirpated by
electric scalpel. In cases when the lymph node is adhering to vascular wall or invasively
integrating into vascular wall, it remains difficult to follow standard dissection procedure.
Thus, the lymph node should be detached under the circumstance that has oozing and gushing
hemorrhage, after the astriction for 5-15 minutes and the ligature suture. Otherwise, the
lymph node is extracted by clamping blood vessels that would require the reconstruction of
blood vessels. In a worst case, the surgery procedures should be converted for the cases such
as total extirpation of lung. The conversion of these surgical procedures would f t e time of
surgery but also increase the risk of postoperative bleeding. Thus, it is ideal to follow
standard procedure of dissection.
[0234] It is possible to apply peptide compositions described herein (e.g.,
Composition 1) for the prevention of hemorrhage on the stripped surface. Composition 1has
physical specificity that the gravity slowly pulls it downward, so it is possible to apply
Composition 1 not only on the stripped surface but also specifically on the entire lymph node.
If desired, a large amount may be used. It is not suitable to use fibrin glue which is solidified.
Likewise, SURGICEL®, which covers entire stripped surface in advance of the dissection, is
not optimal.
[0235] In the case of the dissection without treatment for bleeding beforehand, the
dissection can commence immediately after application of Composition 1. If fibrin glue is
applied to the region, it is hard to detach and might promote bleeding when stripped off.
SURGICEL® cannot be used in this instance since it hides the application site and thus
detachment cannot be performed. Further SURGICEL® requires an extend period of time
completely stop bleeding.
[0236] There is possibility that bleeding is not stopped even after a sufficient period
of time and SURGICEL® is removed. This is due to the fact that the stoppage of bleeding
cannot be confirmed when SURGICEL® is applied. In this case, it is necessary for
reapplication. The bleeding may be mild, however, extensive bleeding can begin again and
therefore extend the time of surgery.
Example 6. Application of peptide compositions described herein in Orthopedic Surgery
[0237] The following example illustrates the application of peptide compositions
described herein, e.g., Composition 1, in a surgery to repair an intertrochanteric hip fracture
(Figure 4). The site of fracture is fixed by metal nail plate or gamma nail that can bear 3 to 5
times of weight. In recent years, with modernization of surgical techniques and metal
fixation devices, the compression forces on the fracture site are made by screws and plates (or
triangular nail) that are introduced into fractured bone to prevent the fracture site on the plane
from sliding.
[0238] Gamma Nail for intertrochanteric fracture with application of
Composition 1. First, a skin incision is made at the fracture site. A small incision is made
deep through the fascia lata, splitting the abductor muscle to reach the targeted femur. When
encountering bleeding during incision, normally, astriction by gauze is performed. Five to
ten minutes is necessary for to stop the bleeding and the procedure should be stagnant. Also,
control of bleeding via additional coagulation by high frequency wave electro device needs
substantial procedure time to apply frequent times. Alternatively, Composition 1 presents an
advantage for faster control of bleeding by application over a wide area of bleeding, which
may include multiple bleeding points. Further, due to the transparent nature of Composition
1, there is no obstacle or impairment in the surgical field and thus the procedure can proceed
as normal without any delay.
[0239] After exposing the femur by incision of muscle, a guide wire is introduced into
the canal of femur from the top of the great trochanter before the introduction of a gamma
nail into the appropriate position. Then, a hole is made by a reamer along with the guide wire
with a suitable diameter for introduction of the nail. At this point in the procedure, bone wax
is typically used for controlling any bleeding from the femoral canal. Bone wax is a clay-like
material that requires kneading and/or warming prior to use. Typically this can be
accomplished manually by the surgeon's fingers. Alternatively, Composition 1 can be used
instead of bone wax. Preparation before application such as for bone wax is not necessary for
Composition 1 and faster control of bleeding is achieved. Specifically, Composition 1 can be
applied by a pre-filled syringe with a nozzle adapted for the hole in the bone or fracture
depending upon the type of fracture or repair made to the bone. Further, Composition 1
provides the added benefit of not stopping the procedure for its transparent quality in
maintaining a clear surgical field and easy removal by irrigation.
[0240] Although the applications of both bone wax and Composition 1 are similar
regarding their application on a given bone or bone fracture site, bone wax tends to delay
bone synostosis, whereas Composition 1 promotes bone synostosis and is expected to have
higher efficacy of healing than bone wax. Further, bone wax can cause inflammation
whereas Composition 1 does not, due, in part, to its high biocompatibility.
[0241] A gamma nail is introduced into the femoral canal opened by the reamer. Bone
wax is used when bleeding from the canal during this procedure. Alternatively, Composition
1 is applied at this point and no stoppage in the surgical procedure is incurred. Further,
control of bleeding is achieved application of Composition 1 onto the surface of the gamma
nail before introduction.

We claim:
1. In a method of performing an intrabody surgical procedure on a patient in which an
incision is made in a body so that a) access to a site including a damaged portion of an
internal organ or tissue is provided for a first period of time, b) removal, repair, or
replacement of some or all of t e damaged portion is performed during the first period of
time, and c) the incision is closed at the first period of time's end, the improvement
comprising:
within the first period of time, performing at least one application within the site of a
composition comprising a of 0.1 - 10% peptide solution, wherein the peptide comprises an
amino acid sequence of RADA repeats; and wherein the solution is characterized by an
ability to transition between two states: an un-gelled state adopted when one or more
particular ions is substantially absent, and a gelled state adopted when the one or more ions is
present at or above a threshold level, wherein the one or more ions is or becomes present in
the location; and
permitting the composition to remain in the site for a second period of time, sufficient
for the peptides in the composition to transition to their gelled state.
2 . The method of claim 1, wherein the improvement further comprises performing,
within the second period of time, at least one other medical procedure in the site.
3. The method of claim 1 or 2, wherein the second period of time is less than five
minutes.
4 . The method of any one of the preceding claims, wherein the second period of time is
greater than five minutes.
5. The method of any one of the preceding claims, wherein the one or more ions are
selected from potassium (K+) and sodium (Na+) .
6. The method of any one of the preceding claims, wherein the one or more ions are
potassium (K+) and sodium (Na+) .
7. The method of any one of the preceding claims, wherein the intrabody surgical
procedure is a resection of or at least a portion of the liver.
8. The method of any one of the preceding claims, wherein t e improvement further
comprises completing t e liver resection within a first period of time that is less than four
hours and therefore reduced as compared with the standard first period of time absent the
improvement, which standard first period of time is within the range of five to six hours.
9. The method of any one of the preceding claims, wherein the improvement further
comprises not applying fibrin glue or SURGICEL® or a combination thereof within the site
during the first period of time.
10. The method of any one of the preceding claims, wherein the improvement comprises
applying the composition comprising a solution of peptides in addition to fibrin glue or
SURGICEL® or a combination thereof within the site during the first period of time.
11. The method of any one of the preceding claims, wherein at least one first application
is completed prior to any other surgical activity within the site.
12. The method of clam 11, wherein the intrabody surgical procedure is a coronary artery
bypass.
13. The method of any one of the preceding claims, wherein the patient is dosed with an
anti-coagulant prior to surgery.
14. The method of any one of the preceding claims, wherein the intrabody surgical
procedure is a coronary artery bypass in which the improvement further comprises
completing the surgical procedure within a first period of time that is at least 20 minutes per
graft shorter as compared with the standard first period of time absent the improvement.
15. The method of any one of the preceding claims, wherein the peptide solution has a
concentration of 1- 3%.
16. The method of any one of the preceding claims, wherein the peptide comprises an
amino acid sequence that comprises two, three or four repeats of RADA.
17. The method of claim 16, wherein the peptide comprises an amino acid sequence
comprising four repeats of RADA.
18. A method of performing an intrabody surgical procedure on a patient comprising
exposing a location within the patient's body to access a damaged portion of an
internal organ or tissue for a first period of time in order to remove, repair, or replace at least
some portion of the organ or tissue during the first period of time,
applying, to a site within t e location, a composition comprising a 0.1 - 10% peptide
solution, wherein the peptide comprises an amino acid sequence of RADA repeats; and
wherein the solution is characterized by an ability to transition between two states: an ungelled
state adopted when one or more particular ions is substantially absent, and a gelled
state adopted when the one or more ions is present at or above a threshold level, wherein the
one or more ions is or becomes present in the location,
retaining the composition in the location for a second period of time, wherein the
peptides in the composition transitions to a gelled state.
19. The method of claim 18, further comprising, performing, during the second period of
time, at least one other medical procedure in the location.
20. The method of claim 18 or 19, wherein the second period of time is less than five
minutes.
21. The method of claim 18 or 19, wherein the second period of time is greater than five
minutes.
22. The method of any one of claims 18 - 21, wherein the one or more ions are selected
from potassium (K+) and sodium (Na+) .
23. The method of claim 22, wherein the one or more ions are potassium (K+) and sodium
(Na+) .
24. The method of any one of claims 18 - 23, wherein the surgical procedure is a liver
resection and the method is completed within a first period of time that is less than four hours
and therefore reduced as compared with the standard first period of time absent the
application, which standard first period of time is within the range of five to six hours.
25. The method of any one of claims 18 - 24, wherein the method excludes application of
fibrin glue or SURGICEL® or a combination thereof within the site during the first period of
time.
26. The method of any one of claims 18 - 24, wherein the method includes applying the
composition comprising a solution of peptides in addition to fibrin glue or SURGICEL® or a
combination thereof within the site during the first period of time.
27. The method of any one of claims 18 - 26, wherein at least one first application is
completed prior to any other surgical activity within the site.
28. The method of any one of claims 18 - 27, wherein the patient is dosed with an anti
coagulant prior to surgery.
29. The method of any one of claims 18 - 28, wherein t e peptide solution has a
concentration of 1 - 3%.
30. The method of any one of claims 18 - 29, wherein t e peptide comprises an amino
acid sequence that comprises two, three or four repeats of RADA.
31. The method of claim 30, wherein the peptide comprises an amino acid sequence that
comprises four repeats of RADA.
32. A method of treating exudative bleeding during liver surgery in a patient, the method
comprising the steps of
(a) applying to a location of exudative bleeding in a subject a composition comprising
a 0 .1 - 10% peptide solution, wherein the peptide comprises an amino acid sequence of
RADA repeats; and wherein the solution is characterized by an ability to transition between
two states: an un-gelled state adopted when one or more particular ions is substantially
absent, and a gelled state adopted when the one or more ions is present at or above a
threshold level, wherein the one or more ions is or becomes present in the location;
(b) retaining the applied composition in the location, with the one or more ions, for a
period of time sufficient for the composition to gel; and
(c) performing one or more liver surgery tasks in the location without first removing
the composition.
33. The method of claim 32, wherein the exudative bleeding is caused by
electrocauterization.
34. The method of claim 32, wherein the subject is dosed with an anticoagulant prior to
the start of the liver surgery.
35. The method of claim 32, wherein the composition comprising the solution of peptides
is applied endoscopically.
36. The method of claim 32 or 35, wherein the one or more liver surgery tasks is
performed endoscopically.
37. The method of claim 32, wherein the one or more liver surgery tasks is performed
laproscopically.
38. The method of claim 32, wherein the one or more liver surgery tasks include liver
separation.
39. The method of claim 32, wherein the one or more liver surgery tasks include vascular
exfoliation.
40. The method of any one of claims 32 - 39, wherein t e peptide solution has a
concentration of 1- 3%.
4 1. The method of any one of claims 32 - 40, wherein t e peptide comprises an amino
acid sequence that comprises two, three or four repeats of RADA.
42. The method of claim 41, wherein the peptide comprises an amino acid sequence that
comprises four repeats of RADA.
43. The method of any one of claims 32 - 42, wherein the one or more ions are selected
from potassium (K+) and sodium (Na+) .
44. The method of claim 43, wherein the one or more ions are potassium (K+) and sodium
(Na+) .
45. A method of treating bleeding during graft collection during coronary artery bypass
surgery in a patient comprising:
(a) applying to a graft collection site a composition comprising a 0 .1 - 10% peptide
solution, wherein the peptide comprises an amino acid sequence of RADA repeats; and
wherein the solution is characterized by an ability to transition between two states: an ungelled
state adopted when one or more particular ions is substantially absent, and a gelled
state adopted when the one or more ions is present at or above a threshold level, wherein the
one or more ions is or becomes present in the location; and
(b) retaining the composition in the location, with the one or more ions, for a period of
time sufficient for the composition to gel.
46. The method of claim 45, wherein the bleeding is caused by electrocauterization.
47. The method of claim 45, wherein applying the composition is performed prior to
initiation of graft collection.
48. The method of claim 45, wherein applying the composition is performed after
initiation but prior to completion of graft collection.
49. The method of claim 48, wherein retaining the composition comprises retaining
through performance of at least one step graft collection step.
50. The method of claim 49, wherein retaining the composition comprises retaining
through completion of graft collection steps.
51. The method of any one of claims 45 - 50, wherein the peptide solution has a
concentration of 1- 3%.
52. The method of any one of claims 44 - 51, wherein the peptide comprises an amino
acid sequence that comprises two, three or four repeats of RADA.
53. The method of claim 52, wherein the peptide comprises an amino acid sequence that
comprises four repeats of RADA.
54. The method of any one of claims 45 - 53, wherein the one or more ions are selected
from potassium (K+) and sodium (Na+) .
55. The method of claim 54, wherein the one or more ions are potassium (K+) and sodium
(Na+) .
56. A method of performing a coronary artery bypass graft procedure in a patient
comprising:
(a) applying to a cardiac location in the patient a composition comprising a 0.1 - 10%
peptide solution, wherein the peptide an amino acid sequence of RADA repeats; and wherein
the solution is characterized by an ability to transition between two states: an un-gelled state
adopted when one or more particular ions is substantially absent, and a gelled state adopted
when the one or more ions is present at or above a threshold level, wherein the one or more
ions is or becomes present in the location.
57. The method of claim 56, wherein the location is an anastomy site on a coronary
artery.
58. The method of claim 56, wherein the location is an anastomy site on a graft vessel.
59. The method of claim 56, wherein the location is an annula connection site for an
oxygenator.
60. The method of any one of claims 56 - 59, wherein the composition is applied without
additional pressure.
6 1. The method of claim 56, wherein applying t e composition is performed prior to
initiation of graft collection.
62. The method of claim 56, wherein the applying the composition is performed after
initiation but prior to completion of graft collection.
63 . The method of claim 62, wherein retaining the composition comprises retaining
through performance of at least one step graft collection step.
64. The method of claim 63, wherein retaining the composition comprises retaining
through completion of graft collection steps.
65. The method of claim 56, wherein the method excludes application of fibrin glue or
SURGICEL® within the site.
66. The method of claim 56, wherein the method includes application the peptide solution
in addition to application of fibrin glue or SURGICEL® within the site.
67. The method of any one of claims 56 - 66, wherein the peptide solution has a
concentration of 1 - 3%.
68. The method of any one of claims 56 - 67, wherein the peptide comprises an amino
acid sequence that comprises two, three or four repeats of RADA.
69. The method of claim 68, wherein the peptide comprises an amino acids sequence that
comprises four repeats of RADA.
70. The method of any one of claims 56 - 69, wherein the ions are selected from
potassium (K+) and sodium (Na+) .
71. The method of claim 70, wherein the one or more ions are potassium (K+) and sodium
(Na+) .
72. A pre-filled syringe for use in a surgical procedure comprising:
a barrel comprising a 0 .1 - 10% peptide solution, wherein the peptide comprises an
amino acid sequence of RADA repeats; and wherein the peptide solution is characterized by
an ability to transition between two states: an ungelled state adopted when one or more
particular ions is substantially absent, and a gelled state adopted when the one or more ions is
present at or above a threshold level, wherein the one or more ions is or becomes present in
the location; and, a non-metal nozzle; wherein said barrel and non-metal nozzle are capable
of forming a secure connection in a liquid-tight manner.
73 . The pre-filled syringe of claim 72, wherein the surgical procedure is selected from the
group consisting of coronary artery bypass graft (CABG), hepatectomy, pure laparoscopic
hepatectomy (PLH), endoscopic mucosal resection (EMR), endoscopic sub mucosal
dissection (ESD), thoracoscopic partial lung resection, lymph node dissection, open partial
nephrectomy, laparoscopic partial nephrectomy, aorta replacement and orthopedic bone
surgery.
74. The pre-filled syringe of claim 72, wherein t e non-metal nozzle is rigid.
75. The pre-filled syringe of claim 72, wherein the non-metal nozzle is flexible.
76. The pre-filled syringe of claim 75, wherein the non-metal nozzle is flexible such that
it is capable for use in an endoscopic surgical procedure.
77. The pre-filled syringe of claim 75, wherein the non-metal nozzle is flexible such that
it is capable for use in a laparoscopic surgical procedure.
78. The pre-filled syringe of any one of claims 72 - 77, wherein the solution has a volume
within the range of about 1 - 50 mL.
79. The pre-filled syringe of claim 78, wherein the solution has a volume of about 1 mL.
80. The pre-filled syringe of claim 78, wherein the solution has a volume of about 3 mL.
81. The pre-filled syringe of claim 78, wherein the solution has a volume of about 5 mL.
82. The pre-filled syringe of claim 78, wherein the solution has a volume of about 30 mL.
83. The pre-filled syringe of any one of claims 72 - 82, wherein the solution has a peptide
concentration within the range of about 0.1% to about 10.0%.
84. The pre-filled syringe of claim 83, wherein the solution has a peptide concentration of
about 1%.
85. The pre-filled syringe of claim 83, wherein the solution has a peptide concentration of
about 2%
86. The pre-filled syringe of claim 83, wherein the solution has a peptide concentration of
about 3%.
87. A kit comprising one or more pre-filled syringes according to any one of claims 72 -
86.
88. A pharmaceutical package comprising a pre-filled according to any one of claims 72 -
86 and a blister pack specifically formed to accept the pre-filled syringe.