U.S. patent application number 14/150652 was filed with the patent office on 2014-07-10 for silk-elastin like protein polymers for embolization and chemoembolization to treat cancer.
The applicant listed for this patent is University of Utah Research Foundation. Invention is credited to Joseph Cappello, Hamidreza Ghandehari, Azadeh E. Poursaid.
Application Number | 20140194370 14/150652 |
Document ID | / |
Family ID | 51061416 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140194370 |
Kind Code |
A1 |
Cappello; Joseph ; et
al. |
July 10, 2014 |
SILK-ELASTIN LIKE PROTEIN POLYMERS FOR EMBOLIZATION AND
CHEMOEMBOLIZATION TO TREAT CANCER
Abstract
A chemoembolic agent is disclosed that includes an injectable,
recombinantly synthesized silk-elastin like protein copolymer and
one or more chemotherapeutic agents. Upon injection, the
chemoembolic agent blocks the tumor vasculature, including the
capillary bed, and may optionally release chemotherapeutic agents.
The chemoembolic agent may be used to treat cancer, including
hepatocellular carcinoma.
Inventors: |
Cappello; Joseph; (San
Diego, CA) ; Ghandehari; Hamidreza; (Salt Lake City,
UT) ; Poursaid; Azadeh E.; (Salt Lake City,
UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Utah Research Foundation |
Salt Lake City |
UT |
US |
|
|
Family ID: |
51061416 |
Appl. No.: |
14/150652 |
Filed: |
January 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61848673 |
Jan 8, 2013 |
|
|
|
Current U.S.
Class: |
514/21.2 ;
530/353 |
Current CPC
Class: |
A61K 38/39 20130101;
C07K 14/78 20130101; A61K 9/0019 20130101; A61K 9/0024 20130101;
A61K 45/06 20130101; C07K 14/43586 20130101; A61K 47/42 20130101;
A61K 38/1767 20130101 |
Class at
Publication: |
514/21.2 ;
530/353 |
International
Class: |
A61K 38/39 20060101
A61K038/39; A61K 45/06 20060101 A61K045/06; C07K 14/78 20060101
C07K014/78 |
Claims
1. An injectable embolic agent comprising at least one silk-elastin
like protein copolymer.
2. An embolic agent according to claim 1, wherein the agent is a
liquid at 18-23.degree. C. and wherein the agent transforms to a
cross-linked hydrogel at 37.degree. C.
3. An embolic agent according to claim 1, wherein the at least one
silk-elastin like protein copolymer is produced using recombinant
methods.
4. An embolic agent according to claim 1, wherein the at least one
silk-elastin like protein copolymer comprises one or more of
TABLE-US-00002 (SEQ ID NO: 3)
MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGSGAG
AGS[(GVGVP).sub.4GKGVP(GVGVP).sub.3(GAGAGS).sub.4].sub.12(GVGVP).sub.4GKG
VP(GVGVP).sub.2(GAGAGS).sub.2GAMDPGRYQDLRSHHHHHH and (SEQ ID NO: 4)
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM[GAGS(GA
GAGS).sub.2(GVGVP).sub.4GKGVP(GVGVP).sub.11(GAGAGS).sub.5GA].sub.6GAMDP
GRYQDLRSHHHHHH.
5. An embolic agent according to claim 1, wherein the at least one
silk-elastin like protein copolymer comprises one or more matrix
metalloprotease cleavage sites.
6. An embolic agent according to claim 2, wherein the agent has a
viscosity of equal to or less than 1000 cP at 18-23.degree. C.
7. An embolic agent according to claim 2, wherein the agent has a
viscosity of equal to or less than 150 cP at 18-23.degree. C.
8. An embolic agent according to claim 2, wherein the stiffness of
the cross-linked hydrogel is approximately at least approximately
1.times.10.sup.5 Pa.
9. An embolic agent according to claim 1, wherein the agent
contains a contrast-agent.
10. An injectable chemoembolic agent comprising at least one
silk-elastin like protein copolymer and at least one
chemotherapeutic agent.
11. An injectable chemoembolic agent according to claim 10, wherein
at least one chemotherapeutic agent comprises a biologic
compound.
12. An injectable chemoembolic agent according to claim 10, wherein
the agent is a liquid at 18-23.degree. C. and wherein the agent
transforms to a cross-linked hydrogel at 37.degree. C.
13. An injectable chemoembolic agent according to claim 10, wherein
the at least one silk-elastin-like protein copolymer is produced
using recombinant methods.
14. An injectable chemoembolic agent according to claim 10, wherein
the at least one silk-elastin-like protein copolymer comprises one
or more of TABLE-US-00003 (SEQ ID NO: 3)
MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGSGAG
AGS[(GVGVP).sub.4GKGVP(GVGVP).sub.3(GAGAGS).sub.4].sub.12(GVGVP).sub.4GKG
VP(GVGVP).sub.2(GAGAGS).sub.2GAMDPGRYQDLRSHHHHHH and (SEQ ID NO: 4)
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM[GAGS(GA
GAGS).sub.2(GVGVP).sub.4GKGVP(GVGVP).sub.11(GAGAGS).sub.5GA].sub.6GAMDP
GRYQDLRSHHHHHH.
15. An injectable chemoembolic agent according to claim 10, wherein
the at least one silk-elastin-like protein copolymer comprises one
or more matrix metalloprotease cleavage sites.
16. An injectable chemoembolic agent according to claim 12, wherein
the agent has a viscosity of equal to or less than 1000 cP at
18-23.degree. C.
17. An injectable chemoembolic agent according to claim 12, wherein
the agent has a viscosity of equal to or less than 150 cP at
18-23.degree. C.
18. An injectable chemoembolic agent according to claim 10, wherein
the stiffness of the cross-linked hydrogel is at least
approximately 1.times.10.sup.5 Pa.
19. An injectable chemoembolic agent according to claim 10, wherein
the at least one chemotherapeutic agent is effective in treating
hepatocellular carcinoma.
20. An injectable chemoembolic agent according to claim 10, wherein
the at least one chemotherapeutic agent comprises one or more of a
drug targeting vascular endothelial growth factor and vascular
endothelial growth factor receptor.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/848,673 filed Jan. 8, 2013, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates generally to the field of
treating cancer by blocking tumor vasculature with a protein
hydrogel embolic agent that may also include a chemotherapeutic
drug. The hydrogel may be configured to release chemotherapeutic
drug into the tumor at a defined rate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1A provides the amino acid sequence of two embodiments
of SELPs as disclosed herein, SELP-47K and SELP-815K.
[0004] FIG. 1B illustrates the assumed network configuration of the
SELPs of FIG. 1A.
[0005] FIGS. 2A, 2B, and 2C, together, present a schematic
depicting TACE treatment in a subject.
[0006] FIG. 2A illustrates the step of gaining vascular access in
the subject.
[0007] FIG. 2B illustrates the step of selecting an artery that
feeds the tumor for injecting the chemoembolic agent.
[0008] FIG. 2C illustrates the step of administering the
chemoembolic to the tumor.
[0009] FIG. 3 depicts a flowchart that defines steps of a proposed
method to identify and test a candidate formulation for a
chemoembolic as disclosed herein.
[0010] FIG. 4A is a graph that illustrates the viscosity profiles
of different concentrations of SELP-815K.
[0011] FIG. 4B illustrates the viscosity of 12% w/w SELP-47K during
a thermal profile that simulated transcatheter injection.
[0012] FIG. 4C is a graph that illustrates the gel stiffness of
varying concentrations of SELP-815K over time as it forms a
hydrogel.
[0013] FIG. 4D is a graph that illustrates the gelation rates of
varying concentrations of SELP-47K over time as they form hydrogels
at 37.degree. C.
[0014] FIG. 5 is a graph that demonstrates the incorporation of
contrast agent into 16% w/w SELP-815K for use in guiding a
transarterial catheter during administration of the embolic
agent.
[0015] FIGS. 6A, 6B, and 6C illustrate the in vitro microfluidic
system used to assess the ability of the embolic agent to occlude
the microvasculature.
[0016] FIG. 6A depicts the in vitro microfluidic device with
simulated blood flowing through the structures that simulate the
vasculature.
[0017] FIG. 6B depicts the in vitro microfluidic device of FIG. 6A
being injected with SELP-815K solution.
[0018] FIG. 6C depicts the in vitro microfluidic device of FIG. 6B
being injected with simulated blood after injected SELP-815K has
formed a hydrogel embolism.
BRIEF DESCRIPTION
[0019] The present disclosure relates generally to the field of
embolics and drug delivery methods for treating cancer. More
specifically, the present disclosure relates to the use of
recombinantly synthesized silk-elastin-like protein (SELP)
copolymers to embolize tumor vasculature, including the small
vessels such as arterioles. The SELP solution is an injectable
liquid at room temperature and forms a hydrogel in the tumor
vasculature at body temperature. The material may be loaded with
one or more chemotherapeutic drugs which may be released into the
tumor from the embolic agent. The copolymers may also have matrix
metalloprotease cleavage sites engineered into the protein
copolymer using recombinant techniques to enable controlled
breakdown of the embolic material. This modification provides
control over the duration of embolization and controlled release of
the chemotherapeutic agent. Therefore, the material may attack the
tumor both by depriving it of blood supply and/or by delivering
chemotherapeutic compounds.
DETAILED DESCRIPTION
[0020] Hepatocellular carcinoma (HCC) is a cancer of the liver
which, due to its relative lack of symptoms, is detected at
advanced stages in 84% of cases. The 1-year survival rate of
symptomatic HCC patients is 22% and at 5 years it is 5%. For these
patients, the only curative option is surgical liver resection and
liver transplantation. The lack of donor livers and the rapid
progression of the disease, however, eliminates this option for
most patients.
[0021] Several palliative treatment options can slow the
progression of HCC and increase the survival time of patients.
Because HCC is generally unresponsive to systemic chemotherapy,
localized treatments such as local chemotherapy, radiotherapy, or
ablative therapy are typically employed. The most widely used is
transcatheter arterial chemoemobolization (TACE). Using
endovascular catheters to selectively access the arteries in the
liver under radiographic imaging, the objectives of TACE are: 1) to
deliver an embolizing agent to the arteries of the tumor(s),
selectively blocking blood flow and causing ischemic necrosis, and
2) to co-deliver a chemotherapeutic agent or cocktail of agents,
which concentrate in the tumor.
[0022] While TACE is the recommended first-line treatment option to
increase survival times of patients with unresectable HCC, its
effectiveness is dependent on a number of factors. Foremost among
these factors is the physical and chemical nature of the embolizing
agent. Liquid embolizing agents are the most easily injected
through the smallest diameter catheters, consequently accessing the
smaller, more tumor-selective arteries. Their drawback, at times,
is that they may not be stably maintained in the arteries as
liquids after injection as in the case of Lipiodol.RTM., an iodized
esterified oil, or as insoluble masses, as in the case of
Onyx.RTM., a liquid suspension of polyethylenevinylalcohol
dissolved in DMSO. Embolizing agents consisting of particulate
solids form more stable emboli, but are often more difficult to
inject. They may require larger diameter catheters due to their
large size (typically 200-1000 .mu.m diameter particles), which
limits the selectivity of the embolization.
[0023] TACE has been used to treat HCC with some success. However,
there are several limitations to the current state of this
technique. Collateral damage to healthy liver can arise from
excessive non-tumor selective embolization or chemotherapeutic
toxicity. For this reason, TACE is contraindicated for treatment of
patients with multiple tumors (>2 tumors) or large tumors (>3
cm diameter). Such damage could be avoided and TACE treatment
offered to more patients if embolization could be more selectively
performed and chemotherapeutic delivery better controlled.
Moreover, advances in the understanding of the physiology and
pharmacology of hepatocellular carcinoma have led to the
development of new potential drug therapies targeting the
vascularization of HCC tumors. Attractive among these are the
anti-angiogenic drugs targeting vascular endothelial growth factor,
VEGF, and its receptor. However, these include high molecular
weight therapeutics which cannot be effectively delivered using
existing embolizing agents. An example is the biologic,
bevacizumab, an anti-VEGF monoclonal antibody, which has a
molecular weight of approximately 149 kD. An embolizing agent that
is compatible with these drugs and capable of providing sustained
delivery of high-molecular weight agents is needed.
[0024] Another factor affecting the efficacy of TACE is the
duration of embolization. Ischemic necrosis as a result of
embolization is important in controlling tumor growth. Using
embolic agents composed of the synthetic polymers polyvinyl alcohol
(PVA) or ethylene vinyl acetate (EVA) provides a permanent
embolization. These polymers are non-degradable and can remain in
tissues indefinitely. If an effective occlusion occurs immediately
upon embolization and the occlusion is physically maintained (lack
of recanalization), then blood flow to the target tissue will be
permanently blocked. However, clinical outcomes are seldom
clear-cut. After TACE, tumors have been found to respond to
treatment for periods of up to several weeks to months, and then
resume growth. Regardless of the reason, the opportunity to retreat
a patient that experiences tumor rebound is the hallmark of
sustained cancer treatment. Especially for unresectable HCC, which
inherently responds poorly to systemic chemotherapy and for which
retreatment options are limited, the blockage of blood flow from a
previous TACE procedure further restricts these options in that
intravascular access to the rebounding tumor is blocked. Restored
blood flow to a previously embolized tumor in a treatment-relevant
fashion would be clinically beneficial in treating unresectable
HCC.
[0025] To be effective, an embolizing agent must be able to be
selectively delivered to tumor arteries where it forms stable
arterial occlusions. Ideal embolizing agents would likely take the
form of a liquid with a viscosity low enough for injection through
the smallest endovascular catheters (in some instances .ltoreq.500
.mu.m inner diameter), enabling its flow into the smallest
arteries, but high enough to restrict its flow through the
capillaries and into systemic circulation. After injection, such
liquid embolizing agents would transition to a solid hydrogel with
enough physical strength to prevent its wash-out into the venous
blood flow.
[0026] Such liquid embolizing agents and their hydrogels would
also, ideally, be completely aqueous and compatible with the
delivery of anti-cancer drugs, including high-molecular-weight
biotherapeutics, which are unable to be effectively delivered with
current drug-eluting embolizing agents. Localized delivery to the
tumor is important because therapeutics that effectively treat HCC
and other tumors also often have undesired effects on other
tissues. For example, new anti-angiogenic agents suppress tumor
revascularization and regrowth, but may also suppress wound healing
responses in patients with underlying wound pathologies such as
extremity wounds in diabetic patients. Potent anti-proliferative
drugs that target cells experiencing hypoxia could have significant
effects on embolized liver tumors, however, they may also
exacerbate the deterioration of heart and vascular tissues in
patients with cardiovascular disease. Avoiding the off-target
effects of these and other drugs by concentrating and localizing
their release by delivering TACE according to the present
disclosure could significantly advance new therapeutic options for
HCC.
[0027] Novel arterial embolizing agents are disclosed herein which
may elute drugs such as chemotherapeutic agents and which may
possess one, two, or more of the properties of an ideal embolizing
agent described above. The embolizing agents are injectable as a
liquid, able to penetrate into the smallest arteries, and transform
to an insoluble gel in-situ forming a substantially durable
occlusion. The embolizing liquids are completely aqueous and
compatible with drugs and biotherapeutics thus enabling their
localized controlled release. The compositions are even compatible
with live cells. The embolic agents are composed of the protein
polymer, silk-elastin-like protein (SELP), a class of genetically
engineered protein polymers which have been investigated for use in
several different applications. SELPs are made up of repeating
"blocks" of amino acids, referred to as "silk blocks"
(Gly-Ala-Gly-Ala-Gly-Ser) (SEQ ID NO: 1) and "elastin
blocks"(Gly-Val-Gly-Val-Pro) (SEQ ID NO: 2). The silk blocks
consist of the sequence Gly-Ala-Gly-Ala-Gly-Ser (SEQ ID NO: 1), and
are based on the naturally occurring fibrillar silk of b. mori, the
common silkworm.
[0028] The design of the elastin blocks is based on mammalian
elastin, a very common connective tissue in the body which gives
skin its elasticity. With appropriate sequence and composition,
SELPs transform from a liquid at room temperature (approximately
18-23.degree. C.) to a physically cross-linked hydrogel network at
body temperature (approximately 37.degree. C.). SELPs have been
described previously, including in PCT publication no. WO
2013/181471, which is incorporated herein by reference in its
entirety. The viscosity and gelation rate of the SELP fluids are
adjusted by specifying the composition and the concentration of the
SELPs. The physical properties of the hydrogels, their polymer
network densities and its stiffness, can be controlled by the SELP
compositions (the silk to elastin ratio and the length of the silk
and elastin block domains) and their solution concentrations.
[0029] FIG. 1A illustrates the amino acid sequences of SELP-47K and
SELP-815K, two embodiments of SELPs that may comprise the
chemoembolics as disclosed herein. The silk units forming the rigid
backbone are in grey font and the flexible elastin units are shown
underlined and in grey font. The elastin units allow pore formation
which is required for drug release. FIG. 1B illustrates the assumed
network configurations of SELP-47K and SELP-815K. As shown in FIG.
1B, the pore size of SELP-815K is larger than that of SELP-47K.
Because pores size impacts the rate of drug release from the
polymer, the size of the one or more chemotherapeutic molecule
intended to be included in the chemoembolic agents may impact the
optimal pore size for the hydrogels and, consequently, the optimal
SELP compositions for drug delivery.
[0030] In addition to SELP structure, pore size of the network is
affected by the concentration of the polymer. Therefore, both SELP
structure and concentration can be optimized for drug release by
adjusting either the choice of SELP polymer, its concentration, or
both. In addition, the pore size, and thus the drug release rate,
may be adjusted by blending different SELPs in combination. For
example, a mixture of SELP-47K and SELP-815K may release a
combination of low and high molecular weight drugs more effectively
than either polymer alone.
[0031] In addition, the present disclosure describes novel
therapeutic methods comprising the steps of using endovascular
catheters to selectively access the arteries in the tumor tissue
under radiographic imaging, delivering the embolizing agents and/or
chemoembolizing agents to the arteries of the tumor(s) by injecting
the agent into the tumor vasculature, thus, selectively blocking
blood flow causing ischemic necrosis, and, optionally,
co-delivering a chemotherapeutic agent or cocktail of agents, which
concentrate in the tumor(s). FIGS. 2A, 2B, and 2C illustrate a
schematic of an embodiment of the methods of the present
disclosure. FIG. 2A illustrates the step of gaining vascular access
using an endovascular catheter 102. FIG. 2B illustrates the step of
identifying and selecting the artery 104 in the liver 106 that
feeds the tumor 108. The chemoembolic agent 110 will be injected
into this artery 104. FIG. 2C illustrates the step of administering
the chemoembolic agent 110 that comprises a drug or cocktail of
drugs by injecting the chemoembolic agent 110 into the tumor
vasculature through the selected artery 104. The tip 112 of the
endovascular catheter 102 is shown within the selected artery 104
that feeds the tumor 108.
[0032] The present disclosure provides methods of treating cancer
comprising the step of injecting a chemoembolic agent into the
vasculature of a subject in need thereof using techniques
including, but not limited to, that illustrated in FIGS. 2A, 2B,
and 2C. The methods include a method of treating a cancer, wherein
the cancer is hepatocellular carcinoma. Furthermore, the method may
include the step of injecting a chemoembolic agent, wherein the at
least one chemotherapeutic agent is effective against
hepatocellular carcinoma.
[0033] In an alternative embodiment of the method illustrated in
FIGS. 2A, 2B, and 2C, the SELP embolic agent is administered
without chemotherapeutic agents or cocktails thereof. Because the
SELPs in the disclosed agents are biodegradable, the agents may be
administered repeatedly. Each time the embolic agent is
administered, it may either include one or more chemotherapeutic
agents or exclude such agents. In some embodiments, the steps of
the disclosed method may alternate between administration of an
embolic agent with one or more chemotherapeutic agents and
administration of an embolic agent without a chemotherapeutic
agent. Additionally, the one or more chemotherapeutic agents that
comprise the chemoembolic agent may vary with each
administration.
[0034] Embolization with SELPs may offer important advantages over
the use of existing embolic agents. Unlike products composed of
synthetic polymers, SELPs are proteins composed solely of natural
amino acids and they will ultimately degrade to their constituent
amino acids, which are non-toxic and biocompatible. Unlike the
currently-available liquid embolics, such as Lipiodol.RTM. and
Onyx.RTM., the SELP formulations disclosed herein transition from
liquids upon injection at room temperature (approximately
18-23.degree. C.) to elastic hydrogels at body temperature
(approximately 37.degree. C.), forming stable biomaterials. The
transition is not associated with any thermal release, nor is there
a change of volume. Furthermore, the transition does not involve
any chemical reaction, thus there is no possibility of chemically
altering the chemotherapeutic agent(s). Unlike currently-available
preformed particles, SELPs may be injected through finer catheters,
enabling access to distal tumor-specific arteries. This increased
precision of transcatheter delivery using a SELP liquid embolic may
translate into more selective embolizations, potentially reducing
collateral damage to the healthy tissue. Consequently, the novel
TACE treatment disclosed herein may be applicable to a greater
number of patients, including those with a greater number of tumors
and/or greater tumor size than those currently treated with TACE.
Furthermore, the SELP hydrogels eventually biodegrade, enabling
subsequent TACE treatments, if necessary.
[0035] This disclosure also provides a method to further improve
the drug delivery capability of SELPs by adding one or more matrix
metalloprotease (MMP)-responsive peptide sequences to the monomer
unit. Drug delivery rate is proportional to the rate the SELP
polymer degrades. Adding MMP-responsive sequences may increase the
rate of SELP polymer degradation and, thus, increase the drug
delivery rate when the chemoembolic agent reaches the tumor.
[0036] MMPs are a family of structurally-related endopeptidases,
which exist in a dynamic balance with tissue inhibitors of
metalloproteases (TIMPs) to control myriad biological functions
requiring extracellular matrix degradation. Proper function and
regulation of MMPs is responsible for diverse biological functions
such as angiogenesis, embryonic development, and wound healing.
There are over 20 known specific MMPs, divided into subgroups based
on their additional domains and known biological functions. The
main classes of MMPs are collagenases, gelatinases, stromelysins,
matrilysins, membrane-type MMPs, and other unclassified MMPs.
[0037] MMPs-2 and -9 are known as gelatinase type A and B,
respectively, due to their known ability to degrade gelatin
(denatured collagen). In normal situations, MMPs-2 and -9
contribute to many processes involving cell migration and
signaling, including, for example, angiogenesis and
inflammation/innate immunity. However, these MMPs have also been
shown to be overexpressed in certain disease states relative to
their expression in healthy tissue. The expression and activity of
MMPs are increased in almost every type of human cancer, and this
correlates with advanced tumor stage, increased invasion and
metastasis, and shortened survival. HCC cells have been shown to
produce MMPs including, but not limited to, MMPs-2 and -9.
[0038] The one or more MMP-specific cleavage sites may be chosen to
correspond to the enzyme expressed by the relevant tumor. The
sequence of each MMP-specific cleavage site will depend on the
relevant MMP, regardless of the protein polymer used, and may be
inserted in advantageous locations within the protein polymers.
[0039] In one embodiment of the disclosure, the chemoembolic agent
is a SELP-815K copolymer including MMP cleavage sites. The one or
more MMP cleavage sites in the SELP-815K protein polymer may
comprise a cleavage site of either MMP-2, MMP-9, or of both MMP-2
and MMP-9. In some embodiments of the chemoembolic agent, the SELP
copolymer comprises the following structure with the MMP-responsive
sequence indicated by bold font:
TABLE-US-00001 (SEQ ID NO: 5)
[GAGS(GAGAGS).sub.2(GVGVP).sub.3GVGGPQGIFGQPGKGVP(GVGVP).sub.11
(GAGAGS).sub.5GA].sub.6.
[0040] Various embodiments of the disclosed protein polymer are
within the scope of this disclosure. FIG. 3 presents a flowchart
that shows the steps that may be taken to determine a candidate
formulation of the chemoembolic agent as disclosed herein. The
flowchart also includes steps that may be used to move the
chemoembolic through feasibility testing of occlusive abilities.
FIG. 3 also discloses steps that may be used to determine the drug
delivery capabilities of the chemoembolic agent.
[0041] The method depicted in FIG. 3 comprises two phases. Phase I
is designed to demonstrate the feasibility of a particular SELP
formulation as an effective embolizing agent. Phase I begins with
steps to be taken to identify SELP copolymer candidates that
include (1) testing viscosity of varying concentrations of the
SELPs to determine whether they may be injectable through a
catheter, (2) rheological characterization to assess gelation time
and gel stiffness so as to assess their ability to remain liquid at
room temperature and transform to a transarterial embolism at body
temperature, and (3) directly testing the feasibility in an in
vitro system that mimics the vasculature. SELP solutions that are
identified as candidates after being tested in Phase I proceed to
Phase II, which is designed to test the ability of SELP
formulations to deliver drugs to tumors. During Phase II, the
manufacturing process of the selected SELP will be scaled up, the
formulation optimized, product sterilization and packaging
optimized, and drug release profiles evaluated. Implant safety and
performance studies in suitable animal models may also be
conducted.
[0042] The disclosure also describes a kit that may provide the
components of the embolic and/or chemoembolic disclosed herein. The
kit may comprise a SELP copolymer that is formulated to be used as
an embolic as disclosed herein. One or more chemotherapeutic
compounds may also be included in the kit. The SELP copolymer may
be provided in liquid form or provided as a freeze dried or
lyophilized powder along with a vial or ampoule of sterile water
for reconstitution. The chemotherapeutic agent may also be provided
in liquid form or provided as a freeze dried or lyophilized powder
along with a vial or ampoule of sterile water for reconstitution.
The SELP formulation and the one or more chemotherapeutic agents
may be provided in the same or separate containers. A microcatheter
for use in injection may be provided as may instructions for use of
the kit.
EXAMPLES
[0043] The SELP-47K and SELP-815K copolymers used in the following
examples were synthesized according to methods known in the art.
While the examples disclosed herein characterize the use of
SELP-47K and SELP-815K for use as chemoembolics, one of skill in
the art will understand that these are but two embodiments of the
protein polymers according to the present disclosure that may be
formulated for use as embolics and/or chemoembolics.
Example 1
[0044] Viscosity of SELP-815K Formulations at Increasing
Temperatures
[0045] Viscosity of the SELPs was determined using an AR 550
stress-controlled rheometer (TA Instruments, New Castle, Del.) with
a cone-and-plate configuration using a 20 mm diameter, 4 degree
cone. SELP copolymers were dissolved in phosphate buffered saline
(PBS) at concentrations of 12%, 16%, 18%, or 20% w/w. The polymer
solutions were mixed via vortex and manual inversion incrementally
with cooling in ice every 30 s until dissolved (3-4 min.), followed
by centrifugation at for 3 minutes at high speed in a clinical
centrifuge (International Equipment Co.). Prepared polymer
solutions were kept on ice until transfer to the Peltier plate of
the rheometer. Generally, the elapsed time from which the PBS was
added to the protein to the time in which the rheometer was started
was about 30 to 45 minutes. A temperature ramp method was run
starting at 1.5.degree. C. and ending at 50.degree. C., duration of
15 min and controlled angular velocity of 6.283 rad/s.
[0046] FIG. 4A illustrates the effect of temperature and
concentration on viscosity of SELP-815K. Viscosity levels that are
compatible with injection were determined using silicone oil
standards injected manually through 2.8.degree. F. microcatheters
using 1 cc and 3 cc syringes. Viscosity of all formulations
increased as the temperature approached 37.degree. C. The ideal
viscosity is that which is injectable through a catheter of a
desired size. A less viscous formulation may be injectable through
a larger catheter. The viscosity of the formulation is optimally
less than 1000 cP at room temperature (18-23.degree. C.). A
formulation of less than 500 cP may be used in situations where,
for example, a somewhat smaller injection catheter is employed.
However, it is desirable that the formulation maintain an even less
viscous liquid form at room temperature (18-23.degree. C.) in order
to be able to pass through a microinjection catheter. Therefore,
formulations that demonstrated a viscosity of equal to or less than
150 cP (indicated by the dashed and dotted line in FIG. 4A) at
temperatures of 18-23.degree. C. (identified as the box in FIG. 4A)
were deemed most desirable as injectable embolic materials, at
least in procedures employing microinjection catheters. One of
skill in the art will readily optimize the viscosity of the liquid
for the procedure at hand. As shown in FIG. 4A, solutions of 12,
16, and 18% w/w SELP-815K demonstrated a viscosity of equal to or
less than 150 cP at temperatures of 18-23.degree. C. while 20% w/w
SELP-815K did not.
Example 2
[0047] Assessment of Suitability of Viscosity of SELP-47K
Formulation for Injection through Intravascular Catheter
[0048] A rheometer evaluation was conducted to determine if the
viscosity of a SELP-47K solution could be obtained in the range
suitable for catheter injection. The results of this experiment are
shown in FIG. 4B. The viscosity of a 12% w/w SELP-47K solution for
injection through a 1 m length.times.0.5 mm internal diameter
intravascular catheter using a 1 cc syringe with moderate hand
pressure was determined empirically to be 50 cP. Rheometric
analysis determined the viscosity of the SELP-47K fluid remained
.ltoreq.46 cP at room temperature for up to 30 minutes (FIG. 4B, 1
cP=0.001 Pas). After 30 minutes, the temperature was shifted from
room temperature to 37.degree. C. The viscosity increased rapidly
following the temperature shift but remained .ltoreq.50 cP for 4.8
minutes afterwards. The catheter had a hold -up volume of
approximately 200 .mu.l. At a minimum injection rate of 0.1 ml/min,
the fluid residence time in the catheter would typically be 2
minutes. Therefore, the fluid in-transit through the catheter at
37.degree. C. would remain fluid and injectable at a viscosity
.ltoreq.50 cP throughout an anticipated 30-minute injection
process.
Example 3
[0049] Assessment of Stiffness of Gels Formed by Formulations of
SELP-815K
[0050] FIG. 4C illustrates the results of an experiment that was
conducted to assess gel stiffness (G') of varying concentrations of
SELP-815K over time as it formed a hydrogel. A rheometer evaluation
was conducted as in Example 1 to assess the strength of hydrogels
formed from SELP-815K at concentrations of 12, 16, or 20% w/w.
Oscillatory time sweeps were performed on each sample consisting of
an equilibration time sweep at 23.degree. C. and angular frequency
of 6.283 rad/s and 1.0% strain for 1 minute followed by a 16 hour
sweep at 37.degree. C. and angular frequency of 6.283 rad/s and
0.1% strain. Briefly, individual polymer samples previously
prepared to the correct concentration and kept on ice were
immediately transferred to the Peltier plate pre-heated to
23.degree. C. at a volume of 150 .mu.l. The equilibration step ends
with a temperature ramp up to 37.degree. C. ranging 30-60 seconds
before start of the 16 hour run. The time sweep result in traces
for G' and G'', the storage and loss moduli respectively. The G'
plateau represents dynamic gel strength, formulations of 12 and 16%
w/w SELP-815K showed similar stiffness within the time assessed in
the experiment. In contrast, 20% w/w SELP-815K formed a more stiff
gel. This experiment demonstrates that the stiffness of the
hydrogels formed by the SELP solutions may be modified and
optimized by varying the concentration of the solutions.
Example 4
[0051] Assessment of Relationship between SELP Concentration and
Solution Viscosity
[0052] The relationship between SELP concentration and solution
viscosity as it relates to catheter injectability was determined by
measuring the solution viscosity of SELP-47K at various
concentrations ranging from 7.5 to 20% w/w. Viscosity was measured
as described in Example 1. The storage modulus (G') for each sample
was measured as a function of time at 37.degree. C. This
concentration range yielded SELP-47K solutions that are injectable
through hypodermic needles and that undergo hydrogel formation
(FIG. 4D).
Example 5
[0053] Assessment of Contrast Agent Incorporation into SELP-815K
Solution
[0054] During administration, the embolic and/or chemoembolic
solutions will be injected into a tumor vasculature using a
transarterial microcatheter. Contrast agent may be added to the
solutions to guide the transarterial catheter during this process.
Consequently, in some methods, SELP formulations that retain their
gel strength when mixed with contrast agent are desirable. To
assess the effect of contrast agent on gel strength, a solution of
SELP-815K at a concentration of 16% w/w was prepared. Contrast
agent was added to one sample of the solution at a concentration of
20% w/w contrast agent. The stiffness of the SELP-815K solution
with and without contrast agent was assessed as in Example 3. FIG.
5 depicts a graph that demonstrates the incorporation of contrast
agent into 16% w/w SELP 815K and its impact on gel stiffness. The
gel formed from the SELP-815K solution that included contrast agent
was similar in stiffness to that formed from SELP-815K solution
without contrast agent. Consequently, the SELP-815K formulation
tested is compatible with the addition of contrast agent for use in
guiding the transarterial catheter during embolic and/or
chemoembolic agent administration.
Example 6
[0055] In Vitro Evaluation of Embolic Capabilities of SELP-815K
[0056] An in vitro test system was developed to evaluate the
performance of a 16% w/w SELP-815K solution in embolization. This
system comprised a custom microfluidic device to simulate
arterio-capillary geometry and flow. It consists of a tapered
occlusion channel with a proximal internal diameter of 1 mm at the
entry and a distal internal diameter of 0.05 mm at the center. FIG.
6A illustrates the geometry of the microfluidic device. The device
has two entry ports 610, a Luer Lok port for injection of the SELP
test solution using a syringe 620 and microcatheter 640 and a
second entry port for delivery of saline via a syringe pump 630
(see FIG. 6A). The delivery channels merge and enter the occlusion
channel at the proximal end. A pressure gauge monitored the
internal hydrostatic pressure.
[0057] The experiment was conducted to verify that the SELP
solutions have sufficient viscosity to prevent their flow through
the occlusion channel. Three devices were connected in parallel as
described above were set up to create a low pressure system
mimicking hepatic vasculature. Colored saline designed to simulate
blood was injected into one system at a rate of 3.4 ml/min.
Internal hydrostatic pressure was maintained below 20 mm Hg. The
fluid was able to permeate through the devices without blockage
(see FIG. 6B, note that no SELP solution has entered the system as
indicated by the detached microcatheter 640). The second device was
injected with SELP-815K using syringe 620 and microcatheter 640
(see FIG. 6C) under flow conditions. The SELP-815K solution gelled,
and blocked flow of the colored saline. The SELP hydrogel
effectively blocked the solution from proceeding through the system
(FIG. 6C). This result suggests that the SELP-815K solution (16%
w/w) is sufficient to embolize small arteries such as those within
tumor vasculature.
[0058] All publications cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein and as though fully set forth.
[0059] Modifications and improvements of the embodiments
specifically disclosed herein are within the scope of the following
claims. Without further elaboration, it is believed that one
skilled in the area can, using the preceding description, utilize
the present disclosure to its fullest extent. Therefore the
Examples herein are to be construed as merely illustrative and not
a limitation of the scope of the present invention in any way. The
embodiments disclosed in which an exclusive property or privilege
is claimed are defined as follows.
Sequence CWU 1
1
516PRTBombyx mori 1Gly Ala Gly Ala Gly Ser 1 5 25PRTHomo sapiens
2Gly Val Gly Val Pro 1 5 3877PRTArtificial SequenceCombined
copolymers of b. mori silk blocks and h. sapien elastin blocks 3Met
Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10
15 Thr Gln Leu Val Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30 Met Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro 35 40 45 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 50 55 60 Lys Gly Val Pro Gly Lys Gly Val Pro Gly
Lys Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly 85 90 95 Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro 100 105 110 Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 Lys Gly
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val 130 135 140
Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 145
150 155 160 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro 165 170 175 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 180 185 190 Lys Gly Val Pro Gly Lys Gly Val Pro Gly
Lys Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly 210 215 220 Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro 225 230 235 240 Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 245 250 255 Lys
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val 260 265
270 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
275 280 285 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro 290 295 300 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 305 310 315 320 Lys Gly Val Pro Gly Lys Gly Val Pro
Gly Lys Gly Val Pro Gly Val 325 330 335 Gly Val Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly 340 345 350 Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 355 360 365 Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 Lys
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val 385 390
395 400 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly 405 410 415 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val
Gly Val Pro 420 425 430 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly 435 440 445 Lys Gly Val Pro Gly Lys Gly Val Pro
Gly Lys Gly Val Pro Gly Val 450 455 460 Gly Val Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly 465 470 475 480 Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 485 490 495 Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 500 505 510
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val 515
520 525 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly 530 535 540 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val
Gly Val Pro 545 550 555 560 Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 565 570 575 Lys Gly Val Pro Gly Lys Gly Val
Pro Gly Lys Gly Val Pro Gly Val 580 585 590 Gly Val Pro Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 595 600 605 Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 610 615 620 Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 625 630 635
640 Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val
645 650 655 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly 660 665 670 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
Val Gly Val Pro 675 680 685 Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 690 695 700 Lys Gly Val Pro Gly Lys Gly Val
Pro Gly Lys Gly Val Pro Gly Val 705 710 715 720 Gly Val Pro Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 725 730 735 Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 740 745 750 Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 755 760
765 Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val
770 775 780 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly 785 790 795 800 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly Val Gly Val Pro 805 810 815 Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly 820 825 830 Lys Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Ala 835 840 845 Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Met Asp Pro Gly 850 855 860 Arg Tyr Gln
Asp Leu Arg Ser His His His His His His 865 870 875
4820PRTArtificial SequenceCombined copolymers of b. mori silk
blocks and h. sapien elastin blocks 4Met Asp Pro Val Val Leu Gln
Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln Leu Asn Arg
Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 35 40 45 Ser
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 50 55
60 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
65 70 75 80 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val 85 90 95 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly 100 105 110 Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val 115 120 125 Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly 130 135 140 Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 145 150 155 160 Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 165 170 175 Ser
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 180 185
190 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
195 200 205 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val 210 215 220 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly 225 230 235 240 Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val 245 250 255 Pro Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly 260 265 270 Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 275 280 285 Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 290 295 300 Ser
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 305 310
315 320 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro
Gly 325 330 335 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val 340 345 350 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly 355 360 365 Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val 370 375 380 Pro Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly 385 390 395 400 Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 405 410 415 Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 420 425 430
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 435
440 445 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro
Gly 450 455 460 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val 465 470 475 480 Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly 485 490 495 Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val 500 505 510 Pro Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly 515 520 525 Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 530 535 540 Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 545 550 555
560 Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
565 570 575 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
Pro Gly 580 585 590 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val 595 600 605 Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly 610 615 620 Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val 625 630 635 640 Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly 645 650 655 Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 660 665 670 Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 675 680
685 Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
690 695 700 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
Pro Gly 705 710 715 720 Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val 725 730 735 Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly 740 745 750 Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 755 760 765 Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly 770 775 780 Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 785 790 795 800
Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser His His 805
810 815 His His His His 820 5810PRTArtificial SequenceCombined
copolymers of b. mori silk blocks and h. sapien elastin blocks with
matrix metalloprotease (MMP)- responsive cleavage site 5Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 1 5 10 15 Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 20 25
30 Val Gly Gly Pro Gln Gly Ile Phe Gly Gln Pro Gly Lys Gly Val Pro
35 40 45 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly 50 55 60 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly 85 90 95 Val Pro Gly Val Gly Val Pro Gly
Ala Gly Ala Gly Ser Gly Ala Gly 100 105 110 Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 115 120 125 Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 130 135 140 Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val 145 150 155
160 Pro Gly Val Gly Val Pro Gly Val Gly Gly Pro Gln Gly Ile Phe Gly
165 170 175 Gln Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val 180 185 190 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro 195 200 205 Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 210 215 220 Val Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Ala 225 230 235 240 Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 245 250 255 Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 260 265 270 Gly
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val 275 280
285 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
290 295 300 Gly Pro Gln Gly Ile Phe Gly Gln Pro Gly Lys Gly Val Pro
Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val 340 345 350 Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 355 360 365 Gly Val Gly Val Pro
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 370 375 380 Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly 385 390 395 400
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 405
410 415 Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly 420 425 430 Val Gly Val Pro Gly Val Gly Gly Pro Gln Gly Ile Phe
Gly Gln Pro 435 440 445 Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly 450 455 460 Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val 465 470 475 480 Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly 485 490 495 Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala 500 505 510 Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 515 520 525
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 530
535
540 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val
545 550 555 560 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Gly Pro 565 570 575 Gln Gly Ile Phe Gly Gln Pro Gly Lys Gly Val
Pro Gly Val Gly Val 580 585 590 Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro 595 600 605 Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly 610 615 620 Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val 625 630 635 640 Gly Val
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 645 650 655
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 660
665 670 Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
Gly 675 680 685 Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly 690 695 700 Val Pro Gly Val Gly Gly Pro Gln Gly Ile Phe
Gly Gln Pro Gly Lys 705 710 715 720 Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly 725 730 735 Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val 740 745 750 Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 755 760 765 Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser 770 775 780
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 785
790 795 800 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 805 810
* * * * *