Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN MATRIX MATERIALS, DEVICES AND
METHODS OF MAKING AND USING THEREOF
Field of the Invention
The present invention relates to protein matrix materials and devices
and the methods of making and using protein matrix materials and devices. More
specifically the present invention relates to protein matrix materials and
devices that
may be utilized for various medical applications, but not limited to, drug
delivery
devices for the controlled release of pharmacologically active agents,
encapsulated or
coated stent device, vessels, tubular grafts, vascular grafts, wound healing
devices
including protein matrix suture material and meshes, skin/bone/tissue grafts,
clear
protein matrices, protein matrix adhesion prevention barriers, cell
scaffolding and
other biocompatible protein matrix devices. Furthermore, the present invention
relates to protein matrix materials and devices made by forming a film
comprising
one or more biodegradable protein materials, one or more biocompatible
solvents and
optionally one or more
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pharmacologically active agents. The film is then partially dried, rolled or
otherwise shaped, and
then compressed to form the desired protein matrix device.
Background of the Invention
Protein materials are generally present in the tissues of many biological
species.
Therefore, the development of medical devices that utilize protein materials,
which mimic and/or
are biocompatible with the host tissue, have been pursued as desirable devices
due to their
acceptance and incorporation into such tissue. For example the utilization of
protein materials to
prepare drug delivery devices, tissue grafts, wound healing and other types of
medical devices
have been perceived as being valuable products due to their biocompatibility.
The use of dried protein, gelatins and/or hydrogels have previously been used
as
components for the preparation of devices for drug delivery, wound healing,
tissue repair,
medical device coating and the like. However, many of these previously
developed devices do
not offer sufficient strength, stability and support when administered to
tissue environments that
contain high solvent content, such as the tissue environment of the human
body. Furthermore,
the features of such medical devices that additionally incorporated
pharmacologically active
agents often provided an ineffective and uncontrollable release of such
agents, thereby not
providing an optimal device for controlled drug delivery.
A concern and disadvantage of such devices is the rapid dissolving or
degradation of the
device upon entry into an aqueous or high solvent environment. For example,
gelatins and
compressed dry proteins tend to rapidly disintegrate and/or lose their form
when placed in an
aqueous environment. Therefore, many dried or gelatin type devices do not
provide optimal
drug delivery and/or structural and durability characteristics. Also, gelatins
often contain large
amounts of water or other liquid that makes the structure fragile, non-rigid
and unstable.
Alternatively, dried protein devices are often very rigid, tend to be brittle
and are extremely
susceptible to disintegration upon contact with solvents. It is also noted
that the proteins of
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gelatins usually denature during preparation caused by heating, thereby
reducing or eliminating
the beneficial characteristics of the protein. The deficiencies gelatins and
dried matrices have
with regards to rapid degradation and structure make such devices less than
optimal for the
controlled release of pharmacologically active agents, or for operating as the
structural
scaffolding for devices such as vessels, stents or wound healing implants.
Hydrogel-forming polymeric materials, in particular, have been found to be
useful in the
formulation of medical devices, such as drug delivery devices. See, e.g., Lee,
J. Controlled
Release, 2, 277 (1985). Hydrogel-forming polymers are polymers that are
capable of absorbing
a substantial amount of water to form elastic or inelastic gels. Many non-
toxic hydrogel-forming
polymers are known and are easy to formulate. Furthermore, medical devices
incorporating
hydrogel-forming polymers offer the flexibility of being capable of being
implantable in liquid
or gelled form. Once implanted, the hydrogel forming polymer absorbs water and
swells. The
release of a pharmacologically active agent incorporated into the device takes
place through this
gelled matrix via a diffusion mechanism.
However, many hydrogels, although biocompatible, are not biodegradable or are
not
capable of being remodeled and incorporated into the host tissue. Furthermore,
most medical
devices comprising of hydrogels require the use of undesirable organic
solvents for their
manufacture. Residual amounts of such solvents could potentially remain in the
medical device,
where they could cause solvent-induced toxicity in surrounding tissues or
cause structural or
pharmacological degradation to the pharmacologically active agents
incorporated within the
medical device. Finally, implanted medical devices that incorporate
pharmacologically active
agents in general, and such implanted medical devices comprising hydrogel-
forming polymers in
particular, oftentimes provide suboptimal release characteristics of the
drug(s) incorporated
therein. That is, typically, the release of pharmacologically active agents
from an implanted
medical device that includes pharmacologically active agent(s) is irregular,
e.g., there is an initial
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burst period when the drug is released primarily from the surface of the
device, followed by a
second period during which little or no drug is released, and a third period
during which most of
the remainder of the drug is released or alternatively, the drug is released
in one large burst.
It would be desirable to provide a medical device that would biocompatibly
degrade and
resorb into the host tissue for which it is administered. Alternatively, it
would be desirable to
provide a medical device that can be incorporated and remodeled by the host
tissue to remain in
the tissue and provide a prolonged intended function of the device.
Furthermore, it would be
desirable to provide improved medical devices capable of sustained, controlled
local delivery of
pharmacologically active agents when implanted while also being biodegradable
and resorbable
or alternatively capable of being incorporated and remodeled into the host
tissue, such that
removal of the device is not necessary. It would further be desirable to
control the rate of
delivery from such devices to avoid possible side effects associated with
irregular delivery, e.g.,
high drug concentration induced tissue toxicity. Finally, it would be
advantageous if such
devices could be manufactured with biocompatible proteins and solvents so that
the potential for
residual solvent toxicity and immunogenicity is reduced.
Summary of the Invention
The present invention relates to protein matrix materials and devices and the
methods of
making and using protein matrix materials and devices. Embodiments of the
present invention
may include, but are not limited to, drug delivery devices for the controlled
release of
pharmacologically active agents, encapsulated or coated stent devices,
vessels, tubular grafts,
vascular grafts, wound healing devices including protein matrix suture
material and meshes,
skin/bone/tissue grafts, clear protein matrices, protein matrix adhesion
prevention barriers, cell
scaffolding and other biocompatible protein matrix devices.
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Furthermore, the present invention relates to a method of making a protein
matrix
material and devices by forming a coatable composition comprising one or more
biocompatible
protein materials, one or more biocompatible solvents and optionally one or
more
pharmacologically active agents. The coatable composition may also include
additional
5 polymeric materials and/or therapeutic entities that would provide
additional beneficial
characteristics or features to the protein matrix. The coatable composition is
then coated so as to
form a film (preferably a substantially planar body having opposed major
surfaces and preferably
having a thickness between the major surfaces of from about 0.1 millimeters to
about 5
millimeters). Next, the film is at least partially dried until it is cohesive,
and then formed (rolled,
folded, accordion-pleated, crumpled, or otherwise shaped) into a cohesive body
having a surface
area less than that of the film. The cohesive body is then compressed to
provide the desired
protein matrix device in accordance with the present invention.
The protein matrix material is compressed to limit bulk biocompatible solvent,
such as
bulk or trapped water (i.e., iceberg water). The elimination of the bulk
biocompatible solvent by
compressing enhances the strength and durability of the matrix by initiating,
stimulating and
forcing additional intramolecular and intermolecular attraction between the
biocompatible
solvent molecules, such as hydrogen bonding activity, and also initiates,
stimulates and forces
intramolecular and intermolecular activity between the protein molecules, the
biocompatible
solvent molecules and the optional pharmacologically active agents.
The above described process has many advantages if one or more
pharmacologically
active agents are incorporated into the matrix. For example, the controlled
release characteristics
of the protein matrix provides for a higher amount of pharmacologically active
agent(s) that may
be incorporated into the matrix. Additionally, the pharmacologically active
agent(s) is/are
substantially homogeneously distributed throughout the protein matrix material
or device. This
homogenous distribution provides for a more systematic and consistent release
of the
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pharmacologically active agent(s). As a result, the release characteristics of
the
pharmacologically active agent from the protein matrix material and/or device
are enhanced.
As previously suggested, embodiments of the protein matrix devices produced
utilizing
the method of the present invention are capable of the sustainable,
controllable local delivery of
pharmacologically active agent(s), while also providing the advantage of being
capable of being
degraded, and preferably safely resorbed. The resorbable characteristic of
various embodiments
of the present invention eliminates the need for the removal of the drug
delivery device from the
patient once the pharmacologically active agent(s) have been completely
delivered from the
matrix.
Additionally, other embodiments of the present invention may be produced to
remain in
the patient. This may be accomplished by utilizing protein materials that do
not readily degrade
and resorb, but are remodeled by the host tissue, by incorporating an
additional polymeric
material into the protein matrix or by treating the protein matrix material
with a reagent. For
example, the protein matrix material may be partially or totally treated with
a reagent, such as
glutaraldehyde, to create crosslinking of the protein fibers in the matrix.
The crosslinking of the
protein material may be utilized to produce a biocompatible device that has a
desired function,
form or shape, such as a graft, valve or tube, and additionally may retain its
form without
resorbing or degrading into the patient or until the matrix has been
incorporated and/or
remodeled into the host tissue. Examples of protein matrix devices that would
benefit from such
a nonresorbable or nondegradable characteristic include, but are not limited
to, stent covers,
vessels, valves, tissue grafts, electronic implant coverings and other devices
that need a
biocompatible sustaining structure to remain in the patient. Such devices may
further include
one or more pharmacologically agents. The nonresorbable and nondegradable
protein matrix
device would still retain the systematic release of the pharmacological active
agents, thereby
diffusing out of the device rather than releasing upon degradation of the
protein matrix material.
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Whether the device is intended to be entirely resorbable or not, the method of
making the
protein matrix devices is generally the same. In describing the method more
specifically, the
method comprises the steps of preparing a coatable composition comprising one
or more
biodegradable protein materials, one or more biocompatible solvents and
optionally one or more
pharmacologically active agents. Additional biodegradable polymeric materials
may be added in
the preparation of the coatable composition to provide optimum features
desired for the
particular protein matrix device being prepared. For example, polyanhydride
may be added to
the protein matrix to inhibit the absorption of physiological body fluids and
slows the diffusion
and/or degradation of the protein matrix and/or pharmacological active agent.
Preferably, the
biocompatible solvent is water, dimethyl sulfoxide (DMSO), ethanol, an oil,
combinations of
these, or the like. More preferably, the biocompatible solvent comprises
water. The coatable
composition is then coated to form a film and partially dried until the coated
film can be formed
into a cohesive body, e.g., preferably until the film has a solvent content of
from about 50% to
about 70%. The film is then formed into the cohesive body, preferably with a
surface area less
than that of the film. The film is then shaped into a cohesive body, e.g.,
rolled, folded,
accordion-pleated, crumpled, or otherwise shaped into a cylinder or shaped
into a ball, cube and
the like, preferably with a surface area less than that of the film. The
cohesive body is then
compressed to remove as much of the solvent as possible so that the compressed
body remains
cohesive, but without removing so much solvent that the compressed body
becomes brittle or
otherwise lacks cohesiveness. Typically, the resulting protein matrix device
has a solvent
content of from about 10% to about 60%, preferably from about 30% to about
50%. If desired,
the compressed body may next be treated with a crosslinking reagent, such as
glutaraldehyde to
form a compressed body that has additional structural and nonresorbable
features.
As previously suggested, by coating the aforementioned components into a film,
partially
drying the film, forming the film into a cohesive body and subsequently
compressing the
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cohesive body, a protein matrix device, which includes one or more
pharmacologically active
agents, has a substantially homogeneous distribution of the pharmacologically
active agent(s).
Due to this substantially homogeneous distribution, the protein matrix devices
of the present
invention that include one or more pharmacologically active agents provide a
sustainable.and
controllable release of the pharmacologically active agent(s). Furthermore,
the method of the
present invention utilizes biocompatible, and if selected, resorbable and
biodegradable, protein
materials. As a result, protein matrix devices formed in accordance with the
method of the
present invention may include the benefit of remaining in the patient
indefinitely or simply
resorbing and/or degrading into the tissue surrounding it. Finally, since the
protein matrix
material is biocompatible, any solvent remaining in the protein matrix device
after the
manufacture thereof presents a reduced, if not substantially eliminated, risk
of producing
undesirable side effects when implanted into a patient.
The biocompatible protein material incorporated into a device in accordance
with the
present invention generally comprises one or more biocompatible proteins,
which preferably are
a water-absorbing, biocompatible protein. Additionally, the biocompatible
protein may be
synthetic, genetically engineered or natural. In various embodiments of the
present invention, the
genetically engineered protein material comprises silklike blocks and
elastinlike blocks. As
previously indicated, the protein matrix device can incorporate any desired
pharmacologically
active agent or even a second drug delivery device, e.g., corticosteroids,
opioid analgesics,
neurotoxins, local anesthetics, vesicles, lipospheres, microspheres,
nanospheres, enzymes,
combinations of these, and the like.
It has now additionally been discovered that the sustainable release and rate
controllable
characteristics of the present protein matrix device may also been
beneficially utilized to deliver
other drug delivery devices that are either vulnerable to migration from the
delivery site and/or
are potentially undesirably reactive with surrounding bodily fluids or
tissues. That is, not only
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can the protein matrix device of the present invention be beneficially
utilized to deliver a
pharmacologically active agent to a particular site where a therapeutic effect
is desired, but also
the protein matrix device of the present invention may be a "two-stage drug
delivery device"
utilized to deliver a second, migration-vulnerable drug delivery device
comprising a
pharmacologically active agent so that the second, migration-vulnerable and/or
reactive drug
delivery device is held in place, e.g., by the protein matrix provided by the
protein matrix device
of the present invention. In the instance that the two-stage protein matrix
device is used to
deliver a reactive drug delivery device, the protein matrix of the two stage
drug delivery device
reduces, if not substantially prevents the second drug delivery device from
undesirably reacting
with surrounding bodily tissues and/or fluids.
Thus, in another aspect, the present invention provides a protein matrix
device
comprising a compressed matrix comprising at least one biodegradable polymeric
material and at
least one such substance vulnerable to migration and/or reaction with
surrounding tissues or
bodily fluids, wherein said substance is substantially homogeneously
distributed within the
matrix. Examples of such substances include, but are not limited to, vesicles,
such as lipospheres
or liposomes, comprising an encapsulated pharmacologically active agent,
microspheres
comprising an encapsulated pharmacologically active agent, combinations of
these, and the like.
Other examples of such substances include, but are not limited to, stents,
electronic devices and
other non-tissue implant that may illicit an adverse reaction from surrounding
tissues.
Inasmuch as the protein matrix devices of the present invention provide the
sustained
release of one or more pharmacologically active agents in a rate controllable
fashion, they are
also capable of delivering other migration-vulnerable and/or reactive drug
delivery devices and
furthermore are produced in a manner that reduces, if not eliminates, the risk
of residual solvent
toxicity or adverse tissue reaction. Also, the protein matrix devices of the
present invention
provide a method of effecting a local therapeutic response in a patient in
need of such treatment.
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Specifically, the method comprises the step of administering a protein matrix
device in
accordance with the present invention to the site at which a local therapeutic
response is
desired. Additionally, the protein matrix devices may be administered for
systemic
delivery of pharmacologically active agents, including oral, as well as nasal,
5 pulmonary, subcutaneous, or any other parenteral mode of delivery.
Prefereably, the
therapeutic response effected is an analgesic response, an anti-inflammatory
response,
an anesthetic response, a response preventative of an immunogenic response, an
anti-
coagulatory response, a genetic response, a protein assembly response, an
antibacterial
response, a vaccination response, combinations of these, and the like. As used
herein,
10 unless stated otherwise, all percentages are percentages based on upon the
total mass of
the composition being described, e.g., 100% is total.
In accordance with another aspect of the present invention, there is
provided a solvated compressed protein matrix material, comprising at least
one
biocompatible protein material combined with at least one biocompatible
solvent and
formed into a cohesive body having a solvent content of about 20% to 80% prior
to
compression, wherein the cohesive body is compressed at a pressure of about
100 psi to
100,000 psi to reduce bulk solvent and generate additional intermolecular and
intramolecular forces between the protein material(s) and the solvent(s) to
form the
solvated compressed protein matrix material having a solvent content of about
10% to
60%.
In accordance with another aspect of the present invention, there is
provided a solvated cohesive protein body, comprising at least one
biocompatible
protein material combined with at least one biocompatible solvent to form a
coatable
film, wherein the coatable film is partially dried and formed into the
solvated cohesive
body having a solvent content of about 20% to 80% prior to compression.
In accordance with a further aspect of the present invention, there is
provided a method of making a solvated compressed protein matrix material,
comprising the steps of:
(a) preparing a coatable composition comprising at least one biocompatible
protein material and at least one biocompatible solvent;
(b) coating the composition to form a film;
(c) partially drying the coated film until the coated film can be formed into
a cohesive body;
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(d) forming said cohesive body having a solvent content of about 20% to
80% prior to compression; and
(e) compressing the cohesive body at a pressure of about 100 psi to
100,000 psi to form a protein matrix material having a solvent content of
about 10%
to 60%.
In accordance with another aspect of the present invention, there is
provided a solvated compressed drug delivery device comprising at least one
biocompatible protein material combined with at least one biocompatible
solvent and
formed into a cohesive body having a solvent content of about 20% to 80% prior
to
compression, wherein the cohesive body is compressed at a pressure of about
100 psi
to 100,000 psi to reduce bulk solvent and generate additional intermolecular
and
intramolecular forces between the protein material(s), and solvent(s) to form
the
solvated compressed drug delivery device having a solvent content of about 10%
to
60%.
In accordance with a further aspect of the present invention, there is
provided a solvated compressed wound healing device comprising at least one
biocompatible protein material combined with at least one biocompatible
solvent and
formed into a cohesive body having a solvent content of about 20% to 80% prior
to
compression, wherein the cohesive body is compressed at a pressure of about
100 psi
to 100,000 psi to reduce bulk solvent and generate additional intermolecular
and
intramolecular forces between the protein material(s), and solvent(s) to form
the
solvated compressed wound healing device having a solvent content of about 10%
to
60%.
In accordance with another aspect of the present invention, there is
provided a solvated compressed tissue graft comprising at least one
biocompatible
protein material combined with at least one biocompatible solvent and formed
into a
cohesive body having a solvent content of about 20% to 80% prior to
compression,
wherein the cohesive body is compressed at a pressure of about 100 psi to
100,000 psi
to reduce bulk solvent and generate additional intermolecular and
intramolecular
forces between the protein material(s), and solvent(s) to form the solvated
compressed
tissue graft having a solvent content of about 10% to 60%.
In accordance with another aspect of the present invention, there is
provided a solvated compressed stent coating comprising at least one
biocompatible
protein material combined with at least one biocompatible solvent and formed
into a
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cohesive body having a solvent content of about 20% to 80% prior to
compression,
wherein the cohesive body is compressed at a pressure of about 100 psi to
100,000 psi
to reduce bulk solvent and generate additional intermolecular and
intramolecular
forces between the protein material(s), and solvent(s) to form the solvated
compressed
stent coating having a solvent content of about 10% to 60%.
In accordance with a further aspect of the present invention, there is
provided a solvated compressed protein matrix IUD comprising at least one
biocompatible protein material combined with at least one biocompatible
solvent and
formed into a cohesive body having a solvent content of about 20% to 80% prior
to
compression, wherein the cohesive body is compressed at a pressure of about
100 psi
to 100,000 psi to reduce bulk solvent and generate additional intermolecular
and
intramolecular forces between the protein material(s), and solvent(s) to form
the
solvated compressed protein matrix IUD having a solvent content of about 10%
to
60%.
In accordance with another aspect of the present invention, there is
provided an image marker comprising at least one biocompatible protein
material
combined with at least one biocompatible solvent and formed into a cohesive
body
having a solvent content of about 20% to 80% prior to compression, wherein the
cohesive body is compressed at a pressure of about 100 psi to 100,000 psi to
reduce
bulk solvent and generate additional intermolecular and intramolecular forces
between the protein material(s), and solvent(s) to form the solvated
compressed image
marker having a solvent content of about 10% to 60%.
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1Oc
Brief Description of the Figures
The above mentioned and other advantages of the present invention,
and the manner of attaining them, will become more apparent and the invention
itself
will be better understood by reference to the following description of the
embodiments of the invention taken in conjunction with the accompanying
drawing,
wherein:
Figure 1 is a schematic illustration, in partial cross-sectional view, of a
compression molding device that may be used in the method of the present
invention
in a configuration prior to compression;
Figure 2 is a schematic illustration, in partial cross-sectional view, of a
compression molding device that may be used in the method of the present
invention
in a configuration during compression;
Figure 3 is a schematic illustration, in partial cross-sectional view, of a
compression molding device that may be used in the method of the present
invention
in a configuration during ejection; and
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Figure 4 depicts an embodiment of a drug delivery device of the present
invention in
particulate form;
Figure 5 depicts an embodiment of a drug delivery device of the present
invention in
particulate form;
Figure 6 is a schematic illustration, in partial cross-sectional view, of a
compression
molding device that may be used in the method of the present invention in
wherein the inner
insert includes a mandrel that that is engaged with a stent.
Figure 7 depicts various views of an embodiment of the present invention
formulated as a
tubular graft;
Figure 8 depicts various embodiments of an encapsulated stent device with a
silastic tube
and/or angioplasty balloon inserted therein;
Figure 9 depicts various embodiments of an encapsulated stent device ;
Figure 10 depicts various views of a multi-layer vessel;
Figure 11 depicts an embodiment of a tubular graft that illustrates the
capability,
compliancy and capacity of the protein matrix material to accept sutures and
reform to its
original shape;
Figures 12 depicts an embodiment of a compression molding device wherein the
inner
insert includes a mandrel;
Figure 13 depicts the top view of an embodiment of the compression molding
device
without the upper insert or plunger;
Figure 14 depicts an embodiment of a wound healing device shaped in the
configuration of
an ultra-thin skin graft matrix;
Figure 15 depicts an embodiment of a wound healing device comprising a protein
matrix
that is positioned in the center of a non-adhesive strip of material attached
to two adhesive ends;
Figure 16 depicts an embodiment of a protrusion device 34 that includes a port
seal;
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Figure 17 is a graphical illustration of the in vitro release characteristics
of the
pharmacologically active agent, sufentanil, from a drug delivery device in
accordance with the
present invention.
Figure 18 is a before and after depiction of an embodiment of a protein matrix
device that
includes a release mechanism;
Figure 19 depicts two protein matrix devices that include release mechanisms
contained
in an agar gel;
Figure 20 depicts a time progression illustration of a protein matrix device
that includes a
protein matrix device following release of the mechanism;
Figure 21 is a magnified view of an embodiment of a noncrosslinked wafer;
Figure 22 is a magnified view of an embodiment of a crosslinked wafer;
Figure 23 is a chart of the effect of GA crosslinking and molding pressure on
the
Young's modulus of collagen wafers;
Figure 24 is a chart of the effect of GA crosslinking and molding pressure on
the UTS of
collagen wafers;
Figure 25 is a chart regarding the amount of collagen released into PBS
involving
noncrosslinked embodiments of the present invention;
Figure 26 is a chart regarding the amount of collagen released into PBS
involving various
embodiments of the present invention crosslinked by contacting with 1 %
glutaraldehyde for 1
minute;
Figure 27 is a chart regarding the amount of collagen released into PBS
involving various
embodiments of the present invention crosslinked by contacting with 1%
glutaraldehyde for 10
minutes;
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Figure 28 is a chart regarding the amount of collagen released into PBS
involving various
embodiments of the present invention crosslinked by contacting with 1 %
glutaraldehyde for 30
minutes;
Figure 29 depicts an embodiment of a vascular tube;
Figure 30 depicts an embodiment of a vascular tube tested for durability and
compliance;
Figure 31 depicts views of both sides of an embodiment of a vascular tube
tested for
hydraulic pressure;
Figure 32 depicts, at the arrows, an embodiment of a bulging vascular tube
tested for
pressure strength and durability; and
Figure 33 depicts the results of the hEGF release study from embodiments of
PVA
particles used in the protein matrix wafers of the present invention.
Detailed Description of the Invention
The embodiments of the invention described below are not intended to be
exhaustive or
to limit the invention to the precise forms disclosed in the following
detailed description. Rather,
the embodiments are chosen and described so that others skilled in the art may
appreciate and
understand the principles and practices of the present invention. The present
invention relates to
protein matrix materials and devices and a method of making such protein
matrix materials and
devices. More specifically, the method of the present invention involves
preparing a coatable
composition comprising one or more biocompatible protein materials, one or
more
biocompatible solvents and optionally one or more pharmacologically active
agents. It is noted
that additional polymeric materials and/or therapeutic entities may be
included in the coatable
composition to provide various beneficial features such as strength,
elasticity, structure and/or
any other desirable characteristics. The coatable composition is then coated
to form a film that is
subsequently partially dried, formed into a cohesive body, and then compressed
to provide a
protein matrix device in accordance with the present invention.
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While not wishing to be bound by any theory, it is believed that by preparing
a coatable
composition from the aforementioned components, coating this composition to
form a film that
is subsequently partially dried, and then forming the film into a cohesive
body, a relatively
homogeneous distribution of the components is obtained in the cohesive body.
Furthermore,
when the film has dried enough so as to be cohesive unto itself, e.g., to a
solvent content from
about 50% to about 70%, subsequently formed into a cohesive body and then
compressed many,
if not all, of any distribution anomalies are removed or resolved. Therefore,
when the protein
matrix device includes a pharmacologically active agent, the distribution of
the
pharmacologically active agent is rendered substantially homogenous throughout
the resulting
drug delivery device.
In addition, the removal of such distribution anomalies also includes the
removal of bulk
or trapped biocompatible solvent, such as aqueous solutions, i.e. bulk water
(i.e., iceberg water)
from the matrix. For example, in aqueous solutions, proteins bind some of the
water molecules
very firmly and others are either very loosely bound or form islands of water
molecules between
loops of folded peptide chains. Because the water molecules in such an island
are thought to be
oriented as in ice, which is crystalline water, the islands of water in
proteins are called icebergs.
Furthermore, water molecules may also form bridges between the carbonyl (C=O)
and imino
(NH) groups of adjacent peptide chains, resulting in structures similar to
those of a pleated sheet
((3-sheets) but with a water molecule in the position of the hydrogen bonds of
that configuration.
Generally, the amount of water bound to one gram of a globular protein in
solution varies from
0.2 to 0.5 grams. Much larger amounts of water are mechanically immobilized
between the
elongated peptide chains of fibrous proteins, such as gelatin. For example,
one gram of gelatin
can immobilize at room temperature 25 to 30 grams of water. It is noted that
other
biocompatible solvents may also interact with protein molecules to effect
intra- and inter-
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molecular forces upon compression. The compression of the cohesive body
removes the bulk
solvent from the resulting protein matrix.
The protein matrix of the present invention traps biocompatible solvent
molecules, such
as water molecules, and forces them to interact with the protein to produce a
protein-water
5 matrix with natural physical, biological and chemical characteristics. The
compression of the
cohesive body eliminates the islands of water or bulk water resulting in a
strengthened protein
matrix structure. Furthermore, the elimination of bulk water enhances the
homogenous
characteristics of the protein matrix by reducing the pooling of water and
spacing of the protein
molecules and pharmacologically active agent molecules. Upon compression of
the cohesive
10 body, the remaining water molecules are forced to interact with most to all
protein molecules and
thereby add strength, structure and stability to the protein matrix. The
compression forces out
most of the non-structural bulk water (immobilized water) from the matrix. As
previously
suggested, the bulk water is extra water that is only loosely bound to the
matrix. The water that
interacts with the protein molecules of the protein matrix reduces and/or
prevents the protein
15 from denaturing during compression and facilitates the protein binding with
the water through
intra- and inter- molecular forces (i.e., ionic, dipole-dipole such as
hydrogen bonding, London
dispersion, hydrophobic, etc.). The enhanced binding characteristics of the
protein matrix further
inhibits the loss of non-bulk solvent molecules that interact with protein
molecules. Experiments
have indicated that a protein matrix dries to 25-45% water during overnight
drying processes that
would normally dry over 100 times that same amount of water if it were not in
the matrix.
Furthermore, the resulting protein matrix device preferably has as little
solvent as possible
while still being cohesive and possessing the desired features relevant to the
device's function,
e.g., preferably a solvent content of from about 10% to about 60%, more
preferably a solvent
content of from about 30% to about 50%. It is found that when a protein matrix
device of the
present invention includes a pharmacologically active agent, the partial
drying of the film to
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16
form a cohesive body and subsequent compressing of the cohesive body, forces
more solvent out
of the body, thereby producing a resulting protein matrix device that has a
significantly higher
concentration of pharmacologically active agent relative to other components
of the device than
is obtainable in protein matrix devices produced by other methods. As a result
of the
substantially uniform dispersion of a greater concentration of
pharmacologically active agent, a
sustained, controlled release of the pharmacologically active agent is
achieved, while reducing
the initial high concentration effects that can be associated with other
devices that include
pharmacologically active agents or bolus injections of pharmacologically
active agents.
Reducing the solvent content has the additional effect that the resulting drug
delivery
device is more structurally sound, easy to handle, and thus, easy to insert or
implant. Upon
insertion, the cells of the tissue contacting the implanted protein matrix
holds the protein matrix
device substantially in the desired location. Alternatively, embodiments of
the protein matrix
may be held in the desired location by tissue contact, pressure, sutures,
adhesives and/or tissue
folds or creases. Embodiments of the protein matrix device may biodegrade and
resorbs over
time or retain their structural integrity.
To form the coatable composition, the biocompatible protein material(s), the
biocompatible solvent(s), and optionally the pharmacologically active agent(s)
may be combined
in any manner. It is noted that one or more additional polymeric materials
and/or therapeutic
entities may be added to the coatable composition during the combination step
to provide
additional desirable characteristics to the coatable composition. For example,
the components
may simply be combined in one step, or alternatively, the biocompatible
protein materials may
be dissolved and/or suspended in a biocompatible solvent and an additional
protein material
and/or the pharmacologically active agent may be dissolved and/or suspended in
the same or
another biocompatible solvent and then the resulting two solutions mixed.
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Once prepared, the coatable composition may be coated onto any suitable
surface from
which it maybe released after drying by any suitable method. Examples of
suitable coating
techniques include spin coating, gravure coating, flow coating, spray coating,
coating with a
brush or roller, screen printing, knife coating, curtain coating, slide
curtain coating, extrusion,
squeegee coating, and the like. The coated film (preferably having a
substantially planar body
having opposed major surfaces) is desirably thin enough so as to be capable of
drying within a
reasonable amount of time and also thin enough so that the film can be formed
into a cohesive
body comprising a substantially homogeneous dispersion of the components of
the coatable
composition. For example, a thinner film will tend to form a more homogeneous
cohesive body
when the film is formed into the shape of a cylinder. A typical coated film of
the coatable
composition have a thickness in the range of from about 0.01 millimeters to
about 5 millimeters,
more preferably from about 0.05 millimeters to about 2 millimeters.
Initially, when the film is first coated, it is likely to be non-cohesive,
fluidly-flowable,
and/or non self-supporting. Thus, the coated film is preferably dried
sufficiently so that it
becomes cohesive, i.e., the film preferably sticks to itself rather than other
materials. The film
may simply be allowed to dry at room temperature, or alternatively, may be
dried under vacuum,
conditions of mild heating, i.e., heating to a temperature of from about 25 C
to about 50 C, or
conditions of mild cooling, i.e. cooling to a temperature of from about 0 C to
about 10 C. When
utilizing heat to dry the film, care should be taken to avoid denaturation or
structural degradation
of the pharmacologically active agent incorporated therein.
The specific solvent content at which the film becomes cohesive unto itself
will depend
on the individual components incorporated into the coatable composition.
Generally, films that
have too high of a solvent content will not be cohesive. Films that have too
low of a solvent
content will tend to crack, shatter, or otherwise break apart upon efforts to
form them into a
cohesive body. With these considerations in mind, the solvent content of a
partially dried film
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18
will preferably be from about 20% to about 80%, more preferably from about 30%
to about 65%
and most preferably from about 35% to about 50%.
Once the film is capable of forming a cohesive body, such a cohesive body may
be
formed by any of a number of methods. For example, the film maybe rolled,
folded,
accordion-pleated, crumpled, or otherwise shaped such that the resulting
cohesive body has a
surface area that is less than that of the coated film. For example the film
can be shaped into a
cylinder, a cube, a sphere or the like. Preferably, the cohesive body is
formed by rolling the
coated film to form a cylinder.
Once so formed, the cohesive body is compressed to form a protein matrix
device in
accordance with the present invention. Any manually or automatically operable
mechanical,
pneumatic, hydraulic, or electrical molding device capable of subjecting the
cohesive body to
pressure is suitable for use in the method of the present invention. In the
production of various
embodiments of the present invention, a molding device may be utilized that is
capable of
applying a pressure of from about 100 pounds per square inch (psi) to about
100,000 psi for a
time period of from about 2 seconds to about 48 hours. Preferably, the molding
device used in
the method of the present invention will be capable of applying a pressure of
from about 1000
psi to about 30,000 psi for a time period of from about 10 seconds to about 60
minutes. More
preferably, the molding device used in the method of the present invention
will be capable of
applying a pressure of from about 3,000 psi to about 25,000 psi for a time
period of from about
one minute to about ten minutes.
Compression molding devices suitable for use in the practice of the method of
the present
invention are generally known. Suitable devices may be manufactured by a
number of vendors
according to provided specifications, such as desirable pressure, desired
materials for
formulation, desired pressure source, desired size of the moldable and
resulting molded device,
, and the like. For example, Gami Engineering, located in Mississauga, Ontario
manufactures
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compression molding devices to specifications provided by the customer.
Additionally, many
compression molding devices are commercially available.
An embodiment of a compression molding device 10 suitable for use in the
method of the
present invention is schematically shown in Figure 1. Compression molding
device 10 is
equipped with a mold body 12 in which cohesive body 22 can be subjected to
pressure in order
to compress and mold the cohesive body 22 into a protein matrix device in
accordance with the
present invention. Mold body 12 is shown supported in position on a base plate
20. More
specifically, mold body 12 has provided therein a cavity 16 that preferably
extends all the way
through mold body 12. Within the cavity 16 a molding chamber 17 can be defined
into which a
cohesive body in accordance with the present invention may be inserted. The
molding chamber
17 may be configured in any shape and size depending upon the shape and size
of the protein
matrix device. For example, the chamber may take the shape or form of a tube,
heart valve,
cylinder or any other desired shape. The cavity 16 may comprise a bore of any
shape that may
be machined, formed, cast or otherwise provided into the mold body 12. The
compression
molding device may optionally include one or more apertures of approximately
.004 to .0001
inches for biocompatible solvent to escape the chamber 17 during compression
of the cohesive
body. An inner insert 18 is preferably slidably fit within cavity 16 to be
positioned against one
surface 13 of the base plate 20 to define the molding chamber 17 and support
to cohesive body
22 when positioned within the molding chamber 17. The insert 18 may be any
shape that is
desired for molding the protein matrix device. For example the insert 18 maybe
a solid
cylindrical mandrel that can form the lumen of a tube or vessel. The insert 18
is thus fixed with
respect to the mold body 12 to define the inner extent of the molding chamber
17. An outer
insert 19 is also preferably provided to be slidable within the cavity 16.
Outer insert 19 is used to close the molding chamber 17 of cavity 16 after the
inner insert
18 and the cohesive body 22 are provided in that order within the cavity 16.
The inner and outer
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inserts 18 and 19, respectively, can be the same or different from one
another, but both are
preferably slidably movable within the cavity 16. The inner and outer inserts
18 and 19,
respectively, are configured to create the desired form or shape of the
protein matrix device.
Additionally, the inserts 18 and 19 may be shaped similarly to the shape of
the cavity 16 to slide
5 therein and are sized to effectively prevent the material of the cohesive
body 22 to pass between
the inserts 18 and 19 and the walls of cavity 16 when the cohesive body 22 is
compressed as
described below. However, the sizing may be such that moisture can escape
between the outer
edges of one or both inserts 18 and 19 and the surface walls of the cavity 16
from the cohesive
body 22 during compression. Otherwise, other conventional or developed means
can be
10 provided to permit moisture to escape from the mold cavity during
compression. For example,
small openings could pass through one or both of the inserts 18 and 19 or mold
body 12 which
may also include one-way valve devices. Insert 18 may be eliminated so that
surface 13 of base
plate 20 defines the lower constraint to molding chamber 17. However, the use
of insert 18 is
beneficial, in that its presence facilitates easy removal of the cohesive body
22 after compression
15 (described below) and provides a sufficiently hard surface against which
the cohesive body 22
can be compressed. Moreover, by utilizing a series of differently sized and/or
shaped inner
inserts 18, the volume of the molding chamber can be varied, or different end
features may be
provided to the cohesive body 22. Outer inserts 19 can likewise be varied.
Outer insert 19 is also positioned to be advanced within cavity 16 or
retracted from cavity
20 16 by a plunger 14. Preferably, the contacting surfaces of outer insert 19
and plunger 14 provide
a cooperating alignment structure so that pressure can be evenly applied to
the cohesive body 22.
The plunger 14 may comprise a part of, or may be operatively connected with a
pressure
generation mechanism 24 that has the ability to apply pressure of the type and
force necessary to
achieve the results of the present invention. Conventional or developed
technologies are
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21
contemplated, such as using mechanical, hydraulic, pneumatic, electrical, or
other systems. Such
systems can be manually or automatically operable.
Plunger 14 operates independently of mold body 12 and is operationally coupled
to the
pressure generation mechanism 24. Pressure generation mechanism 24 maybe any
pressure
source capable of applying from about 100 psi to about 100,000 psi for a time
period of from
about 2 seconds to about 48 hours, preferably capable of applying from about
1000 psi to about
30,000 psi for a time period of from about 10 seconds to about 60 minutes, and
more preferably,
capable of applying a pressure of from about 3000 psi to about 25,000 psi for
a time period of
from about 1 minute to about 10 minutes. Preferably, plunger 14 is formulated
of a material
capable of translating substantially all of the pressure applied by pressure
generation mechanism
24 to cohesive body 22.
Mold body 12 may be fabricated from any material capable of withstanding the
pressure
to be applied from pressure generation mechanism 24, e.g., high density
polyethylene, Teflon ,
steel, stainless steel, titanium, brass, copper, combinations of these and the
like. Desirably, mold
body 12 is fabricated from a material that provides low surface friction to
inserts 18 and 19 and
cohesive body 22. Alternatively, surfaces defining the cavity 16 may be coated
with a low
friction material, e.g., Teflon , to provide such low surface friction. Due to
its relatively low
cost, sufficient strength and surface friction characteristics, mold body 12
is desirably fabricated
from brass. Cavity 16, extending substantially through mold body 12, may be of
any shape and
configuration, as determined by the desired configuration of the resulting,
compressed protein
matrix devices. In one embodiment, cavity 16 is cylindrical. However, the
shape of the cavity
16 can be configured to accommodate the shape and size of the resulting,
compressed protein
matrix device. As above, inserts 18 and 19 preferably fit within cavity 16 in
a manner that
allows moisture to escape from mold body 12, and so that inserts 18 and 19 may
be easily
inserted into and removed from cavity 16. Furthermore, it is preferred that
inserts 18 and 19 fit
CA 02401385 2002-08-27
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22
within cavity 16 in a manner that provides adequate support and containment
for cohesive body
22, so that, upon compression, the material of cohesive body 22 does not
escape cavity 16 in a
manner that would produce irregularly shaped edges on the resulting protein
matrix device.
According to one procedure for using compression molding device 10 to carry
out the metho(
the present invention, the mold body 12 is positioned as shown in Figure 1 on
the base plate 20,
which itself may be supported in any manner. Then, an inner insert 18 is
placed into cavity 16
followed by a cohesive body 22 to be compressed and an outer insert 19 as
shown. Plunger 14 is
then positioned so as to be in driving engagement with outer insert 19. Then,
as schematically
illustrated in Figure 2, the pressure generation mechanism 24 is activated to
move plunger 14 in
the direction of arrow A to reduce the volume of the molding cavity 17 to make
a compressed
cohesive body 23. Pressure generation mechanism 24 applies sufficient
pressure, i.e., from
about
100 psi to about 100,000 psi for a time period of from about 2 seconds to
about 48 hours, to
plunger 14, insert 19 and cohesive body 22 against the inner insert 18,
thereby driving moisture
from and compressing cohesive body 22 into a protein matrix device in
accordance with the
present invention.
As shown in Figure 3, the compressed cohesive body 23 can then be ejected from
the mold
body 12 along with inserts 18 and 19 by positioning the mold body 12 on a
support spacer 30
and further advancing the plunger 14 in the direction of arrow A by the
pressure generation
mechanism 24. Generally, base plate 20 is separated from the mold body 12 when
ejecting the
protein matrix device and inserts 18 and 19. The support spacer 30 is
preferably shaped and
dimensioned to provide an open volume 31 for the compressed cohesive body 23
to be easily
removed. That is, when the plunger 14 is sufficiently advanced, the insert 18
and compressed
cohesive body 23 can fall into the open volume 31 within the support spacer
30. After
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23
completion, the plunger 14 can be fully retracted so that the compression
molding device 10 can
be reconfigured for a next operation.
Any biocompatible protein material maybe utilized in the protein matrix
devices and
corresponding methods of the present invention. Preferably, any such material
will at least be
water-compatible, and more preferably will be water-absorbing or hydrogel
forming.
Furthermore, one or more biocompatible protein materials may be incorporated
into the protein
matrix device of the present invention and may desirably be selected based
upon their
biocompatible and/or degradation properties. The combination of more than one
biocompatible
protein can be utilized to mimic the environment in which the device is to be
administered,
optimize the biofunctional characteristics, such as cell attachment and
growth, nonimmuno-
response reaction and/or alter the release characteristics, or duration of an
included
pharmacologically active agent, if a pharmacologically active agent is to be
included in the
device.
The biocompatible protein material comprises one or more biocompatible
synthetic
protein, genetically-engineered protein, natural protein or any combination
thereof. In many
embodiments of the present invention, the biocompatible protein material
comprises a
water-absorbing, biocompatible protein. In various embodiments of the present
invention, the
utilization of a water-absorbing biocompatible protein provides the advantage
that, not only will
the protein matrix device be biodegradable, but also resorbable. That is, that
the metabolites of
the degradation of the water-absorbing biodegradable protein may be reused by
the patient's
body rather than excreted. In other embodiments that do not degrade or resorb
the water
absorbing material provides enhanced biocompatible characteristics since the
device is generally
administered to environments that contain water.
The biocompatible protein utilized may either be naturally occurring,
synthetic or
genetically engineered. Naturally occurring protein that may be utilized in
the protein matrix
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device of the present invention include, but are not limited to elastin,
collagen, albumin, keratin,
fibronectin, silk, silk fibroin, actin, myosin, fibrinogen, thrombin,
aprotinin, antithrombin III and
any other biocompatible natural protein. It is noted that combinations of
natural proteins may be
utilized to optimize desirable characteristics of the resulting protein
matrix, such as strength,
degradability, resorption, etc. Inasmuch as heterogeneity in molecular weight,
sequence and
stereochemistry can influence the function of a protein in a protein matrix
device, in some
embodiments of the present invention synthetic or genetically engineered
proteins are preferred
in that a higher degree of control can be exercised over these parameters.
Synthetic proteins are generally prepared by chemical synthesis utilizing
techniques known
in the art. Examples of such synthetic proteins include but are not limited to
natural protein
made synthetically and collagen linked GAGS like collagen-heparin, collagen-
chondroitin and
the like. Also, individual proteins may be chemically combined with one or
more other proteins
of the same or different type to produce a dimer, trimer or other multimer. A
simple advantage
of having a larger protein molecule is that it will make interconnections with
other protein
molecules to create a stronger matrix that is less susceptible to dissolving
in aqueous solutions.
Additional, protein molecules can also be chemically combined to any other
chemical so
that the chemical does not release from the matrix. In this way, the chemical
entity can provide
surface modifications to the matrix or structural contributions to the matrix
to produce specific
characteristics. The surface modifications can enhance and/or facilitate cell
attachment
depending on the chemical substance or the cell type. The structural
modifications can be used
to facilitate or impede dissolution, enzymatic degradation or dissolution of
the matrix.
Synthetic biocompatible materials maybe cross-linked, linked, bonded or
chemically
and/or physically linked to pharmacological active agents and utilized alone
or in combination
with other biocompatible proteins to form the cohesive body. Examples of such
cohesive body
materials include, but are not limited to heparin-protein, heparin-polymer,
chondroitin-protein,
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chondroitin-polymer, heparin-cellulose, heparin-alginate, heparin-polylactide,
GAGs-collagen,
heparin-collagen.
Specific examples of a -particularly preferred genetically engineered proteins
for use in the
protein matrix devices of the present invention is that commercially available
under the
5 nomenclature "ELP", "SLP "CLP", "SLPL", "SLPF" and "SELP" from Protein
Polymer
Technologies, Inc. San Diego, CA. ELP's, SLP's, CLP's, SLPL's, SLPF's and
SELP's are
families of genetically engineered protein polymers consisting of sillclike
blocks, elastinlike
blocks, collagenlike blocks, lamininlike blocks, fibronectinlike blocks and
the combination of
sillclike and elastinlike blocks, respectively. The ELP's, SLP's, CLP's,
SLPL's, SLPF's and
10 SELP's are produced in various block lengths and compositional ratios.
Generally, blocks
include groups of repeating amino acids making up a peptide sequence that
occurs in a protein.
Genetically engineered proteins are qualitatively distinguished from
sequential polypeptides
found in nature in that the length of their block repeats can be greater (up
to several hundred
amino acids versus less than ten for sequential polypeptides) and the sequence
of their block
15 repeats can be almost infinitely complex. Table A depicts examples of
genetically engineered
blocks. Table A and a further description of genetically engineered blocks may
be found in
Franco A. Ferrari and Joseph Cappello, Biosynthesis of Protein Polymers, in:
Protein-Based
Materials, (eds., Kevin McGrath and David Kaplan), Chapter 2, pp. 37-60,
Birkhauser, Boston
(1997).
CA 02401385 2010-07-26
26
Table A. Protein polymer sequences
Polymer Name Monomer Amino Acid Sequence
SLP 3 [(GAGAGS)9 GAAGY)]
SLP 4 (GAGAGS)n
SLP F [(GAGAGS)9 GAA VTGRGDSPAS AAGY]õ
SLP L3.0 [(GAGAGS)9 GAA PGASIKVAVSAGPS AGY]õ
SLP L3.1 [(GAGAGS)9 GAA PGASIKVAVSGPS AGY]õ
SLP F9 [(GAGAGS)9 RYVVLPRPVCFEK AAGY]õ
ELP I [(VPGVG)4]n
SELF 0 [(GVGVP)s (GAGAGS)2]õ
SELP 1 [GAA (VPGVG)4 VAAGY (GAGAGS)9]õ
SELF 2 [(GAGAGS)6 GAAGY (GAGAGS)5 ((3VGVP)s]õ
SELF 3 [(GVGVP)s (GAGAGS)s]õ
SELF 4 [(GVGVP)12 (GAGAGS)s]n
SELF 5 [(GVGVP)16(GAGAGS)3]n
SELP 6 [(GVGVP)32 (GAGAGS)s]õ
SELP 7 [(GVGVP)s (GAGAGS)6]n
SELP 8 [(GVGVP)8 (GAGAGS)4]n
KLP 1.2 [(AKI.KI.,AEAKLELAE)4]n
CLP 1 [GAP(GPP)4]n
CLP 2 {[GAP(GPP)4]2 GPAGPVGSP]õ
CLP-CB {[GAP(GPP)4]2 (GLPGPKGDRGDAGPKGADGSPGPA) GPAGPVGSP)õ
CLP 3 (GAPGAPGSQGAPGLQ)n
Repetitive amino acid sequences of selected protein polymers. SLP = silk like
protein, SLPF =
SLP containing the RGD sequence from fibroneetin; SLPL 3/0 and SLPL 3/1 = SLP
containin
two difference sequences from laminin protein; ELP = elastin like protein;
SELP = silk elastin
like protein; CLP = collagen like protein; CLP-CB = CLP containing a cell
binding domain how
human collagen; KLP = keratin like protein
The nature of the elastinlike blocks, and their length and position within the
monomers
influences the water solubility of the SELP polymers. For example, decreasing
the iengili acrd/or
content of the silklike block domains, while maintaining the length of the
elastinlike block
domains, increases the water solubility of the polymers. For a more detailed
discussion of the
production of SLP's, ELP's, CLP's, SLPF's and SLPF's as well as their
properties and
characteristics see, for example, in J. Cappello et al., Biotechnol. Pr og.,
6, 198 (I 990).
One preferred SELP, SELP7, has an elastin:silk ratio of 133, and has 45%
silklike
protein material and is believed to have weight average molecular weight of
80,338.
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The amount of the biocompatible protein component utilized in the coatable
composition
will be dependent upon the amount of coatable composition desired in relation
to the other
components of the device and the particular biocompatible protein component
chosen for use in
the coatable composition. Furthermore, the amount of coatable composition
utilized in the
coating of the film will be determinative of the size of the film, and thus,
the size of the cohesive
body and the resulting protein matrix device. That is, inasmuch as the amounts
of the remaining
components are dependent upon the amount of biocompatible protein component
utilized, the
amount of biocompatible protein component may be chosen based upon the
aforementioned
parameters.
to Any biocompatible solvent may be utilized in the method and corresponding
protein
matrix device of the present invention. By using a biocompatible solvent, the
risk of adverse
tissue reactions to residual solvent remaining in the device after manufacture
is minimized.
Additionally, the use of a biocompatible solvent reduces the potential
structural and/or
pharmacological degradation of the pharmacologically active agent that some
such
pharmacologically active agents undergo when exposed to organic solvents.
Suitable
biocompatible solvents for use in the method of the present invention include,
but are not limited
to, water; dimethyl sulfoxide (DMSO); biocompatible alcohols, such as methanol
and ethanol;
various acids, such as formic acid; oils, such as olive oil, peanut oil and
the like; ethylene glycol,
glycols; and combinations of these and the like. Preferably, the biocompatible
solvent comprises
water. The amount of biocompatible solvent utilized in the coatable
composition will preferably
be that amount sufficient to result in the composition being fluid and
flowable enough to be
coatable. Generally, the amount of biocompatible solvent suitable for use in
the method of the
present invention will range from about 50% to about 500%, preferably from
about 100% to
about 300% by weight, based upon the weight of the biodegradable polymeric
material.
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In addition to the biocompatible protein material(s) and the biocompatible
solvent(s), the
protein matrix devices of the present invention may optionally comprise one.
or more
pharmacologically active agents. As used herein, "pharmacologically active
agent" generally
refers to a pharmacologically active agent having a direct or indirect
beneficial therapeutic effect
upon introduction into a host. Pharmacologically active agents further
includes neutraceuticals.
The phrase "pharmacologically active agent" is also meant to indicate prodrug
forms thereof. A
"prodrug form" of a pharmacologically active agent means a structurally
related compound or
derivative of the pharmacologically active agent which, when administered to a
host is converted
into the desired pharmacologically active agent. A prodrug form may have
little or none of the
desired pharmacological activity exhibited by the pharmacologically active
agent to which it is
converted. Representative examples of pharmacologically active agents that may
be suitable for
use in the protein matrix device of the present invention include, but are not
limited to, (grouped
by therapeutic class):
Antidiarrhoeals such as diphenoxylate, loperamide and hyoscyamine;
Antihypertensives such as hydralazine, minoxidil, captopril, enalapril,
clonidine, prazosin,
debrisoquine, diazoxide, guanethidine, methyldopa, reserpine, trimethaphan;
Calcium channel blockers such as diltiazem, felodipine, amodipine,
nitrendipine,
nifedipine and verapamil;
Antiarrhyrthmics such as amiodarone, flecainide, disopyramide, procainamide,
mexiletene
and quinidine,
Antiangina agents such as glyceryl trinitrate, erythrityl tetranitrate,
pentaerythritol
tetranitrate, mannitol hexanitrate, perhexilene, isosorbide dinitrate and
nicorandil;
Beta-adrenergic blocking agents such as alprenolol, atenolol, bupranolol,
carteolol,
labetalol, metoprolol, nadolol, nadoxolol, oxprenolol, pindolol, propranolol,
sotalol, timolol and
timolol maleate;
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Cardiotonic glycosides such as digoxin and other cardiac glycosides and
theophylline
derivatives;
Adrenergic stimulants such as adrenaline, ephedrine, fenoterol, isoprenaline,
orciprenaline,
rimeterol, salbutamol, salmeterol, terbutaline, dobutamine, phenylephrine,
phenylpropanolamine,
05 pseudoephedrine and dopamine;
Vasodilators such as cyclandelate, isoxsuprine, papaverine, dipyrimadole,
isosorbide
dinitrate, phentolamine, nicotinyl alcohol, co-dergocrine, nicotinic acid,
glycerl trinitrate,
pentaerythritol tetranitrate and xanthinol;
Antimigraine preparations such as ergotanmine, dihydroergotamine,
methysergide,
pizotifen and sumatriptan;
Anticoagulants and thrombolytic agents such as warfarin, dicoumarol, low
molecular
weight hepafins such as enoxaparin, streptokinase and its active derivatives;
Hemostatic agents such as aprotinin, tranexamic acid and protarnine;
Analgesics and antipyretics including the opioid analgesics such as
buprenorphine,
dextromoramide, dextropropoxyphene, fentanyl, alfentanil, sufentanil,
hydromorphone,
methadone, morphine, oxycodone, papaveretum, pentazocine, pethidine,
phenopefidine, codeine
dihydrocodeine; acetylsalicylic acid (aspirin), paracetamol, and phenazone;
Neurotoxins such as capsaicin;
Hypnotics and sedatives such as the barbiturates amylobarbitone, butobarbitone
and
pentobarbitone and other hypnotics and sedatives such as chloral hydrate,
chlormethiazole,
hydroxyzine and meprobamate;
Antianxiety agents such as the benzodiazepines alprazolam, bromazepam,
chlordiazepoxide, clobazam, chlorazepate, diazepam, flunitrazepam, flurazepam,
lorazepam,
nitrazepam, oxazepam, temazepam and triazolam;
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Neuroleptic and antipsychotic drugs such as the phenothiazines,
chlorpromazine,
flupbenazine, pericyazine, perphenazine, promazine, thiopropazate,
thioridazine, trifluoperazine;
and butyrophenone, droperidol and haloperidol; and other antipsychotic drugs
such as pimozide,
thiothixene and lithium;
5 Antidepressants such as the tricyclic antidepressants amitryptyline,
clomipramine,
desipramine, dothiepin, doxepin, imipramine, nortriptyline, opipramol,
protriptyline and
trimipramine and the tetracyclic antidepressants such as mianserin and the
monoamine oxidase
inhibitors such as isocarboxazid, phenelizine, tranylcypromine and moclobemide
and selective
serotonin re-uptake inhibitors such as fluoxetine, paroxetine, citalopram,
fluvoxamine and
10 sertraline;
CNS stimulants such as caffeine and 3-(2-aminobutyl) indole;
Anti-alzheimer's agents such as tacrine;
Anti-Parkinson's agents such as amantadine, benserazide, carbidopa, levodopa,
benztropine, bipefiden, benzhexol, procyclidine and dopamine-2 agonists such
as S (-)-2
15 -(N-propyl -N-2 -thi enyl ethyl amino)-5 -hydroxytetralin (N-0923)-,
Anticonvulsants such as phenytoin, valproic acid, primidone, phenobarbitone,
methylphenobarbitone and carbamazepine, ethosuximide, methsuximide,
phensuximide,
sulthiame and clonazepam,
Antiemetics and antinauseants such as the phenothiazines prochloperazine,
20 thiethylperazine and 5HT-3 receptor antagonists such as ondansetron and
granisetron, as
well as dimenhydrinate, diphenhydramine, metoclopramide, domperidone,
hyoscine,
hyoscine hydrobromide, hyoscine hydrochloride, clebopride and brompride;
Non-steroidal anti-inflammatory agents including their racemic mixtures or
individual
enantiomers where applicable, preferably which can be formulated in
combination with dermal
25 penetration enhancers, such as ibuprofen, flurbiprofen, ketoprofen,
aclofenac, diclofenac,
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aloxiprin, aproxen, aspirin, diflunisal, fenoprofen, indomethacin, mefenamic
acid, naproxen,
phenylbutazone, piroxicam, salicylamide, salicylic acid, sulindac,
desoxysulindac, tenoxicam,
tramadol, ketoralac, flufenisal, salsalate, triethanolamine salicylate,
atninopyrine, antipyrine,
oxyphenbutazone, apazone, cintazone, flufenamic acid, clonixerl, clonixin,
meclofenamic acid,
flunixin, colchicine, demecolcine, allopurinol, oxypurinol, benzydamine
hydrochloride,
dimefadane, indoxole, intrazole, mimbane hydrochloride, paranylene
hydrochloride,
tetrydamine, benzindopyrine hydrochloride, fluprofen, ibufenac, naproxol,
fenbufen, cinchophen,
diflumidone sodium, fenamole, flutiazin, metazamide, letimide hydrochloride,
nexeridine
hydrochloride, octazamide, molinazole, neocinchophen, nimazole, proxazole
citrate, tesicam,
tesimide, tolmetin, and triflumidate;
Antirheumatoid agents such as penicillamine, aurothioglucose, sodium
aurothiomalate,
methotrexate and auranofin;
Muscle relaxants such as baclofen, diazepam, cyclobenzaprine hydrochloride,
dantrolene,
methocarbamol, orphenadrine and quinine;
Agents used in gout and hyperuricaemia such as allopurinol, colchicine,
probenecid and sulphinpyrazone;
Oestrogens such as oestradiol, oestriol, oestrone, ethinyloestradiol,
mestranol,
stilboestrol, dienoestrol, epioestriol, estropipate and zeranol;
Progesterone and other progestagens such as allyloestrenol, dydrgesterone,
lynoestrenol,
norgestrel, norethyndrel, norethisterone, norethisterone acetate, gestodene,
levonorgestrel,
medroxyprogesterone and megestrol;
Antiandrogens such as cyproterone acetate and danazol;
Antioestrogens such as tamoxifen and epitiostanol and the aromatase
inhibitors,
exemestane and 4-hydroxy-androstenedione and its derivatives;
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Androgens and anabolic agents such as testosterone, methyltestosterone,
clostebol
acetate, drostanolone, furazabol, nandrolone oxandrolone, stanozolol,
trenbolone acetate, dihydro-t
17-(a-methyl-19-noriestosterone and fluoxymesterone;
5-alpha reductase inhibitors such as finastride, turosteride, LY- 191704 and
MK-306-1;
Corticosteroids such as betamethasone, betamethasone valerate, cortisone,
dexamethasone, dexamethasone 21-phosphate, fludrocortisone, flumethasone,
fluocinonide,
fluocinonide desonide, fluocinolone, fluocinolone acetonide, fluocortolone,
halcinonide,
halopredone, hydrocortisone, hydrocortisone 17-valerate, hydrocortisone 17-
butyrate,
hydrocortisone 21-acetate, methylprednisolone, prednisolone, prednisolone 21 -
phosphate,
prednisone, triamcinolone, triamcinolone acetonide;
Glycosylated proteins, proteoglycans, glycosaminoglycans such as chondroitin
sulfate;
chitin, acetyl-glucosamine, hyaluronic acid;
Complex carbohydrates such as glucans;
Further examples of steroidal anti-inflammatory agents such as cortodoxone,
fludroracetonide, fludrocortisone, difluorsone diacetate, flurandrenolone
acetonide, medrysone,
amcinafel, amcinafide, betamethasone and its other esters, chloroprednisone,
clorcortelone,
descinolone, desonide, dichlofsone, difluprednate, flucloronide, flumethasone,
flunisolide,
flucortolone, fluoromethalone, fluperolone, fluprednisolone, meprednisone,
methylmeprednisolone, paramethasone, cortisone acetate, hydrocortisone
cyclopentylpropionate,
cortodoxone, flucetonide, fludrocortisone acetate, amcinafal, amcinafide,
betamethasone,
betamethasone benzoate, chloroprednisone acetate, clocortolone acetate,
descinolone acetonide,
desoximetasone, dichlorisone acetate, difluprednate, flucloronide,
flumethasone pivalate,
flunisolide acetate, fluperolone acetate, fluprednisolone valerate,
paramethasone acetate,
prednisolamate, prednival, triamcinolone hexacetonide, cortivazol, formocortal
and nivazoll;
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Pituitary hormones and their active derivatives or analogs such as
corticotrophin,
thyrotropin, follicle stimulating hormone (FSH), luteinising hormone (LH) and
gonadotrophin
releasing hormone (GnRH);
Hypoglycemic agents such as insulin, chlorpropamide, glibenclamide,
gliclazide,
glipizide, tolazamide, tolbutamide and metformin;
Thyroid hormones such as calcitonin, thyroxine and liothyronine and
antithyroid agents
such as carbimazole and propylthiouracil;
Other miscellaneous hormone agents such as octreotide;
Pituitary inhibitors such as bromocriptine;
Ovulation inducers such as clomiphene;
Diuretics such as the thiazides, related diuretics and loop diuretics,
bendrofluazide,
chlorothiazide, chlorthalidone, dopamine, cyclopenthiazide,
hydrochlorothiazide, indapamide,
mefruside, methycholthiazide, metolazone, quinethazone, bumetanide, ethacrynic
acid and
frusemide and potasium sparing diuretics, spironolactone, amiloride and
triamterene;
Antidiuretics such as desmopressin, lypressin and vasopressin including their
active
derivatives or analogs;
Obstetric drugs including agents acting on the uterus such as ergometfine,
oxytocin and gemeprost;
Prostaglandins such as alprostadil (PGEI), prostacyclin (PG12), dinoprost
(prostaglandin F2-alpha) and misoprostol;
Antimicrobials including the cephalospofins such as cephalexin, cefoxytin
and cephalothin;
Penicillins such as amoxycillin, amoxycillin with clavulanic acid, ampicillin,
bacampicillin, benzathine penicillin, benzylpenicillin, carbenicillin,
cloxacillin, methicillin,
phenethicillin, phenoxymethylpenicillin, flucloxacillin, meziocillin,
piperacillin, ticarcillin
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and azlocillin;
Tetracyclines such as minocycline, chlortetracycline, tetracycline,
demeclocycline,
doxycycline, methacycline and oxytetracycline and other tetracycline-type
antibiotics;
Amnioglycoides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin
and
tobramycin;
Antifungals such as amorolfine, isoconazole, clotrimazole, econazole,
miconazole, nystatin, terbinafine, bifonazole, amphotericin, griseofulvin,
ketoconazole,
fluconazole and flucytosine, salicylic acid, fezatione, ticlatone, tolnaftate,
triacetin, zinc,
pyrithione and sodium pyfithione;
Quinolones such as nalidixic acid, cinoxacin, ciprofloxacin, enoxacin and
norfloxacin;
Sulphonamides such as phthalysulphthiazole, sulfadoxine, sulphadiazine,
sulphamethizole and sulphamethoxazole;
Sulphones such as dapsone;
Other miscellaneous antibiotics such as chloramphenicol, clindamycin,
erythromycin, erythromycin ethyl carbonate, erythromycin estolate,
erythromycin
glucepate, erythromycin ethylsuccinate, erythromycin lactobionate,
roxithromycin,
lincomycin, natamycin, nitrofurantoin, spectinomycin, vancomycin, aztreonarn,
colistin IV,
metronidazole, tinidazole, fusidic acid, trimethoprim, and 2-thiopyridine N-
oxide; halogen
compounds, particularly iodine and iodine compounds such as iodine-PVP complex
and
diiodohydroxyquin, hexachlorophene; chlorhexidine; chloroan-tine compounds;
and
benzoylperoxide;
Antituberculosis drugs such as ethambutol, isoniazid, pyrazinamide, rifampicin
and
clofazimine;
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Antimalarials such as primaquine, pyrimethamine, chloroquine,
hydroxychloroquine,
quinine, mefloquine and halofantrine;
Antiviral agents such as acyclovir and acyclovir prodrugs, famcyclovir,
zidovudine, didanosine, stavudine, lamivudine, zalcitabine, saquinavir,
indinavir, ritonavir,
5 n-docosanol, tromantadine and idoxuridine;
Anthelmintics such as mebendazole, thiabendazole, niclosamide, praziquantel,
pyrantel
embonate and diethylcarbamazine;
Cytotoxic agents such as plicamycin, cyclophosphamide, dacarbazine,
fluorouracil and
its prodrugs (described, for example, in International Journal of
Pharmaceutics, 111, 223-233
10 (1994)), methotrexate, procarbazine, 6-mercaptopurine and mucophenolic
acid;
Anorectic and weight reducing agents including dexfenfluramine, fenfluramine,
diethylpropion, mazindol and phentermine;
Agents used in hypercalcaemia such as calcitriol, dihydrotachysterol and their
active
derivatives or analogs;
15 Antitussives such as ethylmorphine, dextromethorphan and pholcodine;
Expectorants such as carbolcysteine, bromhexine, emetine, quanifesin,
ipecacuanha and
saponins;
Decongestants such as phenylephrine, phenylpropanolamine and pseudoephedrine;
Bronchospasm relaxants such as ephedrine, fenoterol, orciprenaline, rimiterol,
?0 salbutamol, sodium cromoglycate, cromoglycic acid and its prodrugs
(described, for example, in
International Journal of Pharmaceutics 7, 63-75 (1980)), terbutaline,
ipratropium bromide,
salmeterol and theophylline and theophylline derivatives;
Antihistamines such as meclozine, cyclizine, chlorcyclizine, hydroxyzine,
brompheniramine, chlorpheniramiine, clemastine, cyproheptadine,
dexchlorpheniramine,
?5 diphenhydramine, diphenylamine, doxylatnine, mebhydrolin, pheniramine,
tripolidine,
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azatadine, diphenylpyraline, methdilazine, terfenadine, astemizole, loratidine
and cetirizine;
Local anaesthetics such as bupivacaine, amethocaine, lignocaine, lidocaine,
cinchocaine, dibucaine, mepivacaine, prilocaine, etidocaine, veratridine
(specific c-fiber
blocker) and procaine;
Stratum corneum lipids, such as ceramides, cholesterol and free fatty acids,
for improved
skin barrier repair [Man, et al. J. Invest. Dermatol., 106(5), 1096, (1996)];
Neuromuscular blocking agents such as suxamethonium, alcuronium, pancuronium,
atracurium, gallamine, tubocurarine and vecuronium;
Smoking cessation agents such as nicotine, bupropion and ibogaine;
Insecticides and other pesticides which are suitable for local application;
Dermatological agents, such as vitamins A, C, B 1, B2, B6, B 12., and E,
vitamin E acetate
and vitamin E sorbate;
Allergens for desensitisation such as house, dust or mite allergens;
Nutritional agents and neutraceuticals, such as vitamins, essential amino
acids and fats;
acromolecular pharmacologically active agents such as proteins, enzymes,
peptides,
polysaccharides (such as cellulose, amylose, dextran, chitin), nucleic acids,
cells, tissues, and the
like; and
Keratolytics such as the alpha-hydroxy acids, glycolic acid and salicylic
acid.
The protein matrix devices as disclosed herein may also be utilized for DNA
delivery,
either naked DNA, plasma DNA or any size DNA delivery. Also, the protein
matrix may be
utilized for delivery of RNA types of senses, or oligonucleotides that may be
man-made portions
of DNA or RNA. The protein matrix could also be utilized for delivery of
compounds, as
explained anywhere herein, in ovum or in embryos, as the site for implantation
of the protein
matrix.
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The DNA, RNA or oligonucleotide may be incorporated into the protein matrix
utilizing
the same process of making the protein matrix device as described above. The
only difference
would be that the pharmacological active agents utilized would be the DNA,
RNA,
oligonucleotides and other such materials. In one example, a cohesive body may
be produced by
making a composition containing one or more biocompatible proteins, one or
more
biocompatible solvents and an antisense type material. In general the
complementary strand of a
coding sequence of DNA is the cDNA and the complementary strand of mRNA is the
antisense
RNA. In various embodiments of the present invention, antisense material
delivered by a protein
matrix device of the present invention binds with mRNA, thereby preventing it
from making the
protein.
Two of the advantages of including DNA, RNA or oligonucleotides in a protein
matrix
device is that such a device includes the benefits of local drug delivery to
target cells and to have
a controlled time release component so that there is an extended delivery
period. An additional
advantage to delivery of DNA, RNA or oligonucleotides components is that the
DNA, RNA or
oligonucleotides components can be released in a systematic and controlled
manner over a long
period of time. For example, when the antisense components bind with RNA, the
body tends to
cleave the RNA thereby inhibiting protein production. The biological system
responds by
making more RNA to make proteins. The protein matrix device provides delivery
of additional
antisense components in a location for an extended period of time, thereby
blocking the
production of the undesired protein. Also the biocompatibility of the protein
matrix material
enhances the binding characteristics of the anitsense components to their
proper binding sites.
Since the protein matrix material can be fabricated or produced to resemble
the host tissue, the
host cells are able to better interact with the administered protein matrix
device, thereby
facilitating the binding of the complimentary antisense components delivered
by the protein
matrix with the DNA and RNA in the host cells.
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Additionally, the use of a protein matrix device in an egg or womb could be
very useful
for a number of applications. For example, a vaccine may be delivered in ova
and then released
into the animal, such as mammals, birds or reptiles, even after it's born..
Also, the introduction
of pharmacologically active agents that could be put in the egg or womb, could
be beneficial in
that it could inhibit things like bacteria or viral infection of the egg or
womb during incubation
and promote the healthy development of a mature animal. For example, it would
be possible for
the protein matrix to provide a drug delivery device for growth factors,
neutraceuticals like
vitamins or other agents that would help in the growth of the animal after
it's hatched, or even
during the stage when it is unhatched to facilitate the development of that
animal. Another
example would be the production of livestock, such as domestic animals like
horses, cattle, pigs,
sheep, dogs, cats, chickens or turkeys. If domestic animals would get a head
start on growth, it
may enhance their body weight, which would have a tremendous impact on the
overall
development of the specimen.
Finally, protein matrices may be produced in particulate forms. hese forms
comprise
vaccine particles of all types, including protein particles containing antigen
components that may
be made small enough (2-10 m) to be absorbed by immunogenic cells for enhanced
immune
response via subcutaneous, intraparetaneal, intravenous, intramuscular,
intrathecal, epidural,
intraarticular or any other administration delivery means.
The protein matrix device in accordance with the present invention, as
mentioned
hereinabove, may comprise an amount of a neurotoxin as the pharmacologically
active agent.
Specifically, inasmuch as some cases of chronic pain are the result of
permanent nerve damage,
in some instances it may be desirable to locally deliver an amount of a
neurotoxin to the injured
nerve to destroy that portion of the nerve that is the cause of the
persistent, chronic pain. One
example of a neurotoxin suitable for use in the present invention is
capsaicin, as shown in
Examples 4 and 12, hereinbelow. If a neurotoxin is to be incorporated into the
protein matrix
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39
device of the present invention, it is preferred that it be incorporated in an
amount ranging from
about 0.001 % to about 5%, more preferably, from about 0.05% to about 1 % by
weight, based
upon the weight of the biocompatible protein component.
The protein matrix device of the present invention is particularly
advantageous for the
encapsulation/incorporation of macromolecular pharmacologically active agents
such as
proteins, enzymes, peptides, polysaccharides, nucleic acids, cells, tissues,
and the like.
Immobilization of macromolecular pharmacologically active agents into or onto
a protein matrix
device can be difficult due to the ease with which some of these
macromolecular agents denature
when exposed to organic solvents, some constituents present in bodily fluids
or to temperatures
appreciably higher than room temperature. However, since the method of the
present invention,
as well as the protein matrix device formed by the method utilizes
biocompatible solvents such
as water, DMSO or ethanol, and furthermore does not require heating, the risk
of the
denaturation of these types of materials is reduced. Furthermore, due to the
size of these
macromolecular pharmacologically active agents, these agents are encapsulated
within the
protein matrix upon implantation of protein matrix devices in accordance with
the present
invention, and thereby are protected from constituents of bodily fluids that
would otherwise
denature them. Thus, the protein matrix devices of the present invention allow
these
macromolecular agents may exert their therapeutic effects, while yet
protecting them from
denaturation or other structural degradation.
Examples of cells which can be utilized as the pharmacologically active agent
in the
protein matrix device of the present invention include primary cultures as
well as established cell
lines, including transformed cells. Examples of these include, but are not
limited to pancreatic
islet cells, human foreskin fibroblasts, Chinese hamster ovary cells, beta
cell insulomas,
lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopamine secreting
ventral mesencephalon
cells, neuroblastold cells, adrenal medulla cells, T-cells combinations of
these, and the like. As
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can be seen from this partial list, cells of all types, including dermal,
neural, blood, organ, stem,
muscle, glandular, reproductive and immune system cells, as well as cells of
all species of origin,
can be encapsulated successfully by this method. Examples of proteins which
can be
incorporated into. the protein matrix device of the present invention include,
but are not limited
5 to, hemoglobin, vasporessin, oxytocin, adrenocorticocotrophic hormone,
epidermal growth
factor, prolactin, luliberin or luteinising hormone releasing factor, human
growth factor, and the
like; enzymes such as adenosine deaminase, superoxide dismutase, xanthine
oxidase, and the
like; enzyme systems; blood clotting factors; clot inhibitors or clot
dissolving agents such as
streptokinase and tissue plasminogen activator; antigens for immunization;
hormones;
10 polysaccharides such as heparin; oligonucleotides; bacteria and other
microbial microorganisms
including viruses; monoclonal antibodies; vitamins; cofactors; retroviruses
for gene therapy,
combinations of these and the like.
An efficacious amount of the aforementioned pharmacologically active agent(s)
can
easily be determined by those of ordinary skill in the art taking into
consideration such
15 parameters as the particular pharmacologically active agent chosen, the
size and weight of the
patient, the desired therapeutic effect, the pharmacokinetics of the chosen
pharmacologically
active agent, and the like, as well as by reference to well known resources
such as Physicians'
Desk Reference : PDR--52 ed (1998)--Medical Economics 1974. In consideration
of these
parameters, it has been found that a wide range exists in the amount of the
pharmacologically
20 active agent(s) capable of being incorporated, into, and subsequently
released from or
alternatively allowed to exert the agent's therapeutic effects from within,
the protein matrix
device. More specifically, the amount of pharmacologically active agent that
may be
incorporated into and then either released from or active from within the
protein matrix device
may range from about 0.001 % to about 200%, more preferably, from about 0.05%
to about
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41
100%, most preferably from about 0. 1% to 70%, based on the weight of the
biocompatible
protein material.
In addition to the biocompatible protein material(s), the biocompatible
solvent(s) and
pharmacologically active agent(s), the protein matrix devices of the present
invention
advantageously may themselves incorporate other drug delivery devices that
would otherwise
typically migrate away from the desired delivery site and/or are potentially
undesirably reactive
with surrounding bodily fluids or tissues. Such migration is undesirable in
that the therapeutic
effect of the pharmacological agents encapsulated therein may occur away from
the desired site,
thus eliminating the advantage of localized delivery. When a protein matrix
device incorporating
a migration-vulnerable and/or reactive drug delivery device (hereinafter
referred to as a
"two-stage protein matrix device") is subsequently implanted, the migration-
vulnerable and/or
reactive drug delivery device(s) is/are held in place and protected by the two-
stage protein matrix
device. More particularly, once implanted and/or administered, the
pharmacologically active
agent is released by the biodegradable material of the migration-vulnerable
drug delivery devices
as it degrades. Then the pharmacologically active agents diffuse through the
protein matrix of
the two-stage protein matrix device or is released with the degradation of the
protein matrix
device of the present invention.
Furthermore, the compressed cohesive body of the protein matrix device
reduces, if not
prevents, the potential for undesirable reaction with bodily fluids or tissues
that may otherwise
occur upon implantation of a reactive drug delivery device without the
protective protein matrix
encapsulation. Examples of such drug delivery devices subject to migration for
the delivery site
include, but are not limited to, vesicles, e.g., liposomes, lipospheres and
microspheres. Vesicles
are made up of microparticles or colloidal carriers composed of lipids,
carbohydrates or synthetic
polymer matrices and are commonly used in liquid drug delivery devices.
Vesicles, for example,
have been used to deliver anesthetics using formulations with polylactic acid,
lecithin,
CA 02401385 2010-07-26
42
iophendylate and phosphotidyl choline and cholesterol. For a discussion of the
characteristics and efficiency of drug delivery from vesicles, see, e. g.,
Wakiyama et
al., Chem., Pharm. Bull., 30, 3719 (1982) and Haynes et al., Anesthiol, 74,
105
(1991).
Liposomes, the most widely studied type of vesicle, can be formulated to
include a wide variety of compositions and structures that are potentially non-
toxic,
biodegradable and non-immunogenic. Furthermore, studies are in progress to
create
liposomes that release more drug in response to changes in their environment,
including the presence of enzymes or polycations or changes in pH. For a
review of
the properties and characteristics of liposomes see, e.g., Langer, Science,
249, 1527
(1990); and Langer, Ann. Biomed. Eng, 23,101 (1995).
Lipospheres are an aqueous microdispersion of water insoluble, spherical
microparticles (from about 0.2 to about 100 um in diameter), each consisting
of a
solid core of hydrophobic triglycerides and drug particles that are embedded
with
phospholipids on the surface. Lipospheres are disclosed in U. S. Patent No.
5,188,837, issued to Domb.
Microspheres typically comprise a biodegradable polymer matrix
incorporating a drug. Microspheres can be formed by a wide variety of
techniques
known to those of skill in the art. Examples of microsphere forming techniques
include, but are not limited to, (a) phase separation by emulsification and
subsequent
organic solvent evaporation (including complex emulsion methods such as oil in
water emulsions, water in oil emulsions and water-oil-water emulsions); (b)
coacervation-phase separation; (c) melt dispersion; (d) interfacial
deposition; (e) in
situ polymerization; (f) spray drying and spray congealing; (g) air suspension
coating;
and (h) pan and spray coating. These methods, as well as properties and
characteristics of microspheres are disclosed in, e.g., U.S. Pat. No.
4,652,441; U.S.
Pat. No. 5,100,669; U.S. Pat. No. 4,526,938;
CA 02401385 2010-07-26
43
WO 93/24150; EPA 0258780 A2-U.S. Pat. No. 4,438,253; and U.S. Patent
5,330,768.
Inasmuch as the migration-vulnerable and/or reactive drug delivery devices
will desirably further encapsulate a pharmacologically active agent, the
amount of
these devices to be utilized in the two-stage protein matrix device may be
determined
by the dosage of the pharmacologically active agent, as determined and
described
hereinabove. Inasmuch as such migration-vulnerable and/or reactive drug
delivery
devices represent solid matter that may change the ability of the coatable
composition
to be coated, the amount of such devices to be included in a two-stage drug
delivery
device desirably ranges about 10,000 to about 1 billion, more preferably
ranges from
about 1 million to about 500 million, and most preferably ranges from about
200
million to about 400 million.
Additionally, the protein matrix devices formed according to the method of the
present invention may optionally comprise one or more additives. Such
additives
may be utilized, for example, to facilitate the processing of the protein
matrix devices,
to stabilize the pharmacologically active agents, to facilitate the activity
of the
pharmacologically active agents, or to alter the release characteristics of
the protein
matrix device. For example, when the pharmacologically active agent is to be
an
enzyme, such as xanthine oxidase or superoxide dismutase, the protein matrix
device
may further comprise an amount of an enzyme substrate, such as xanthine, to
facilitate the action of the enzyme.
Additionally, hydrophobic substances such as lipids can be incorporated into
the protein matrix device to extend the duration of drug release, while
hydrophilic,
polar additives, such as salts and amino acids, can be added to facilitate,
i.e., shorten
the duration of, drug release. Exemplary hydrophobic substances include
lipids, e.g.,
tristeafin, ethyl stearate, phosphotidycholine, polyethylene glycol (PEG);
fatty acids,
e. g., sebacic acid erucic acid; combinations of these and the like. A
particularly
preferred hydrophobic additive useful to
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extend the release of the pharmacologically active agents comprises a
combination of a dimer of
erucic acid and sebacic acid, wherein the ratio of the dimer of erucic acid to
sebacic acid is 1:4.
Exemplary hydrophilic additives useful to shorten the release duration of the
pharmacologically
active agent include but are not limited to, salts, such as sodium chloride;
and amino acids, such
as glutamine and glycine. If additives are to be incorporated into the
coatable composition, they
will preferably be included in an amount so that the desired result of the
additive is exhibited.
Generally, the amount of additives may vary between from about 0% to about
300%, preferably
from about 100% to 200% by weight, based upon the weight of the biocompatible
protein
material.
Manufacturing protein matrix devices with the method of the present invention
imparts
many advantageous qualities to the resulting protein matrix devices. First of
all, by compressing
the cohesive body in such a manner, the resulting protein matrix device is
substantially cohesive
and durable, i.e., with a solvent content of from about 10% to about 60%,
preferably of from
about 30% to about 50%. Thus, administration of the protein matrix device is
made easy,
inasmuch as it may be easily handled to be injected or implanted. Furthermore,
once implanted,
the biocompatible protein material may absorb water and swell, thereby
assisting the protein
matrix device to stay substantially in the location where it was implanted or
injected.
Additionally, since the protein material maybe biodegradable and the
pharmacologically active
agent is distributed substantially homogeneously therein, the release kinetics
of the
pharmacologically active agent are optimized. Indeed, the components and the
amounts thereof
to be utilized in the protein matrix device may be selected so as to optimize
the rate of delivery
of the pharmacologically active agent depending upon the desired therapeutic
effect and
pharmacokinetics of the chosen pharmacologically active agent.
Finally, since biocompatible solvents are used in the manufacture of the
protein matrix
devices, the potential for adverse tissue reactions to chemical solvents are
reduced, if not
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substantially precluded. For all of these reasons, protein matrix devices in
accordance with the
present invention may advantageously be used to effect a local therapeutic
result in a patient in
need of such treatment. More specifically, the protein matrix devices of the
present invention
may be injected, implanted, or administered via oral, as well as nasal,
pulmonary, subcutaneous,
5 or any other parenteral mode of delivery. The protein matrix device may be
delivered to a site
within a patient to illicit a therapeutic effect either locally or
systemically. Depending on the
desired therapeutic effect, the protein matrix devices may be used to
regenerate tissue, repair
tissue, replace tissue, and deliver local and systemic therapeutic effects
such as analgesia or
anesthesia, or alternatively, may be used to treat specific conditions, such
as coronary artery
10 disease, heart valve failure, cornea trauma, skin wounds and other tissue
specific conditions.
Protein matrix devices that include pharmacologically active agents may be
utilized in instances
where long term, sustained, controlled release of pharmacologically active
agents is desirable,
such as in the treatment of surgical and post-operative pain, cancer pain, or
other conditions
requiring chronic pain management.
15 Furthermore, the protein matrix devices of the present invention may
incorporate
multiple pharmacologically active agents, one or more of which may be agents
that are effective
to suppress an immune and/or inflammatory response. In this regard, the
protein matrix devices
will deter, or substantially prevent the encapsulation that typically occurs
when a foreign body is
introduced into a host. Such encapsulation could potentially have the
undesirable effect of
20 limiting the efficacy of the protein matrix device.
Additionally, one or more polymeric materials may be included in the coatable
composition to add or enhance the features of the protein matrix device. For
example, one or
more polymeric materials that degrades slowly may be incorporated into an
embodiment of the
protein matrix device that degrades in order to provide controllable release
of a
25 pharmacologically active agent that is also incorporated into the protein
matrix device. That is,
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while a protein matrix device that includes a relatively fast-degrading
protein material without a
particular polymeric material will readily degrade thereby releasing drug
relatively quickly upon
insertion or implantation, a protein matrix device that includes a particular
polymeric material,
such as polyanhydride, will degrade slowly, as well as release the
pharmacologically active
agent(s) over a longer period of time. Examples of biodegradable and/or
biocompatible
polymeric materials suitable for use in the drug delivery device of the
present invention include,
but are not limited to epoxies, polyesters, acrylics, nylons, silicones,
polyanhydride,
polyurethane, polycarbonate, poly(tetrafluoroethylene) (PTFE),
polycaprolactone, polyethylene
oxide, polyethylene glycol, poly(vinyl chloride), polylactic acid,
polyglycolic acid,
polypropylene oxide, poly(akylene)glycol, polyoxyethylene, sebacic acid,
polyvinyl alcohol
(PVA), 2-hydroxyethyl methacrylate (HEMA), polymethyl methacrylate,
1,3-bis(carboxyphenoxy)propane, lipids, phosphatidylcholine, triglycerides,
polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyethylene oxide)
(PEO), poly
ortho esters, poly (amino acids), polycynoacrylates, polyphophazenes,
polysulfone, polyamine,
poly (amido amines), fibrin, graphite, flexible fluoropolymer, isobutyl-based,
isopropyl styrene,
vinyl pyrrolidone, cellulose acetate dibutyrate, silicone rubber, copolymers
of these, and the like.
Other materials that may be incorporated into the matrix that are not
considered polymers, but
provide enhanced features include, but are not limited to, ceramics,
bioceramics, glasses
bioglasses, glass-ceramics, resin cement, resin fill; more specifically, glass
ionomer,
hydroxyapatite, calcium sulfate, A1203, tricalcium phosphate, calcium
phosphate salts, alginate
and carbon. Additional other materials that may be incorporated into the
matrix included alloys
such as, cobalt-based, galvanic- based, stainless steel- based, titanium-
based, zirconium oxide,
zirconia, aluminum- based, vanadium- based, molybdenum- based, nickel- based,
iron- based, or
zinc- based (zinc phosphate, zinc polycarboxylate).
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Embodiments of the protein matrix device may also be crosslinked by reacting
the
components of the protein matrix with a suitable and biocompatible
crosslinking agent.
Crosslinking agents include, but are not limited to glutaraldehyde, p-
Azidobenzolyl Hydazide,
N-5-Azido-2-nitrobenzoyloxysuccinimide, 4-[p-Azidosalicylamido]butylamine, any
other
suitable crosslinking agent and any combination thereof. A description and
list of various
crosslinking agents and a disclosure of methods of performing crosslinking
steps with such
agents may be found in the Pierce Endogen 2001-2002 Catalog which is hereby
incorporated by
reference.
Furthermore, it is noted that embodiments of the protein matrix device of the
present
invention may include crosslinking reagents that may initiated and thereby
perform the
crosslinking process by LTV light activation or other radiation source, such
as ultrasound or
gamma ray or any other activation means.
The protein matrix may be crosslinked by utilizing methods generally known in
the art.
For example, a protein matrix may be partially or entirely crosslinked by
exposing, contacting
and/or incubating the protein matrix device with a gaseous crosslinking
reagent, liquid
crosslinking reagent, light or combination thereof. In one embodiment of the
present invention a
tube be crosslinked on the outside surface by exposing the only the outside
surface to a
crosslinking reagent, such as glutaraldehyde. Such a matrix has the advantages
of including an
outer exterior that is very pliable and possesses greater mechanical
characteristics, but includes
an interior surface that retains higher biofunctional features. For example,
cell growth may be
controlled on portions of the protein matrix by exposing such areas to
crosslinking reagents
while still having portions of the same protein matrix that are not
crosslinked, and thereby
producing biofunctional selective features for the entire protein matrix
device. For example
crosslinking portions of the protein matrix may be used to change, modify
and/or inhibit cell
attachment. It is also noted that the pharmacologically active agent may also
be crosslinked,
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bonded and/or chemically and/or physically linked to protein matrix either
partially or in totality
such that the surface of the protein matrix and/or the interior of the protein
matrix is linked to the
protein matrix material. For example, glutaraldehyde may cross-link heparin to
a single surface
of a protein matrix device.
Embodiments of the present invention may include the addition of reagents to
properly
pH the resulting protein matrix device and thereby enhance the biocompatible
characteristics of
the device with the host tissue of which it is to be administered. When
preparing the protein
matrix device, the pH steps of the biocompatable material and biocompatable
solvent occur prior
to the partial drying preparation of the cohesive body. The pH steps can be
started with the
addition of biocompatable solvent to the protein material or to the mixture of
protein material
and optional biocompatible materials, or the pH steps can be started after
mixing the material(s)
and solvent(s) together before the cohesive body is formed. The pH steps can
include the
addition of drops of 0.05N to 4.ON acid or base to the solvent wetted material
until the desired
pH is reached as indicated by a pH meter, pH paper or any pH indicator. More
preferably, the
addition of drops of 0.1N-0.5 N acid or base are used. Although any acid or
base maybe used,
the preferable acids and bases are HCl and NaOH, respectively. If known
amounts of
biocompatable material are used it may be possible to add acid or base to
adjust the pH when the
biocompatable material is first wetted, thereby allowing wetting and pH
adjustments to occur in
one step.
The patient to which the protein matrix device is administered may be any
patient in need
of a therapeutic treatment. Preferably, the patient is a mammal, reptiles and
birds. More
preferably, the patient is a human. Furthermore, the protein matrix device can
be implanted in
any location to which it is desired to effect a local therapeutic response.
For example, the protein
matrix device may be administered, applied, sutured, clipped, stapled, gas
delivered, injected
and/or implanted vaginally, in ova, in utero, in uteral, subcutaneously, near
heart valves, in
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periodontal pockets, in the eye, in the intracranial space, next to an injured
nerve, next to the
spinal cord, etc. The present invention will now be further described with
reference to the
following non-limiting examples and the following materials and methods were
employed. It is
noted that any additional features presented in other embodiments described
herein may be
incorporated into the various embodiments being described.
DRUG DELIVERY DEVICES:
As previously suggested, various embodiments of the protein matrix device of
the present
invention may be utilized as drug delivery devices. A drug delivery device
produced and
administered as previously disclosed or suggested includes the biocompatible
features of the
components of the protein matrix and thereby reduces or prevents the
undesirable effects of
toxicity and adverse tissue reactions that may be found in many other types of
drug delivery
devices. Furthermore, the controlled release characteristics of this type of
drug delivery device
provides for a higher amount of pharmacologically active agent(s) that may be
incorporated into
the matrix. The controlled release of such a drug delivery device is partially
attributed to the
homogenous distribution of the pharmacologically active agent(s) throughout
the drug delivery
device. This homogenous distribution provides for a more systematic,
sustainable and consistent
release of the pharmacologically active agent(s) by gradual degradation of the
matrix or diffusion
of the pharmacologically active agent(s) out of the matrix. As a result, the
release characteristics
of the pharmacologically active agent from the protein matrix material and/or
device are
enhanced.
Additionally, the systematic, sustainable and consistent release of the drug
delivery
device may be attributed to the cohesive and interaction features present in
the drug delivery
device. As previously described, the protein matrix is compressed to eliminate
part or all of the
bulls water present in the cohesive body. This compression also compels and
influences
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additional attracting forces amongst the protein molecules, solvent molecules
and
pharmacologically active agent molecules included in the matrix that would not
be found if
compression was not undertaken. Also other optional biocompatible materials,
if included in the
matrix, will be compelled and influenced to interact with the
pharmacologically active agents to
5 augment their release characteristics. This additional binding
characteristic provides for a more
systematic and controllable release of the pharmacologically active agents
that are either trapped
by interacting protein, optional biocompatible material and solvent molecules
or that are also
interacting with the protein, optional biocompatible material and solvent
molecules themselves.
Augmentation may include inhibiting or enhancing the release characteristics
of the
10 pharmacologically active agent(s). For example, a multi-layered drug
delivery device may
comprise alternating layers of protein matrix material that have sequential
inhibiting and
enhancing biocompatible materials included, thereby providing a pulsing
release of
pharmacologically active agents. A specific example may be utilizing glutamine
in a layer as an
enhancer and polyanhydride as an inhibitor. The inhibiting layer may include
drugs or no drugs.
15 As previously suggested, embodiments of the drug delivery devices, produced
and
administered utilizing the methods of the present invention, are capable of
the sustainable,
controllable local delivery of pharmacologically active agent(s), while also
providing the
advantage of being capable of being degraded, and preferably safely resorbed
and/or remodeled
into the surrounding host tissue. The resorbable characteristic of various
embodiments of the
20 present invention eliminates the need for the removal of the drug delivery
device from the patient
once the pharmacologically active agent(s) have been completely delivered from
the matrix.
Alternatively, the drug delivery device may be produced to remain in the
patient and provide a
systematic and controllable diffusion of the pharmacololgically active
agent(s) as described and
suggested previously.
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The drug delivery device of present invention may be formed into any shape and
size,
such as a cylinder, a tube, a wafer, particles or any other shape that may
optimize the delivery of
the incorporated pharmacologically active agent. For example, the drug
delivery device may be
administered to a patient in the form of particles. Figures 4 and 5 depict
embodiments of the'
drug delivery device in particulate form. Particles may be produced by
pulverizing the protein
matrix following the freezing of the matrix in liquid nitrogen or by utilizing
other freeze fracture
or particle forming techniques. A characteristic of the protein particles is
that they no longer
aggregate when in the particulate state. The protein matrix in particulate
form may be
administered to a patient in many ways, but have the proper characteristics
which allow it to be a
very good injectible. Furthermore, cells, can be attached to particles and/or
may be incorporated
into the larger matrix. Any types cells such as eukaryotic cells, organ cells,
such as live islets of
the pancreas (for production of insulin) may be included in a particulate drug
delivery device.
Furthermore, the particles may include a mixture of drugs incorporated within
the protein matrix
and may be taken orally or through nasal mucosa, wherein the particles may
interact with cellular
membranes and/or body fluids.
Also, a release mechanism may be included in the protein matrix drug delivery
device for
the release of the one or more pharmacologically active agents. The release
mechanism may be
a material that encapsulates a larger drug delivery device, such as a cylinder
or the release
mechanism may be within a protein matrix material that includes encapsulated
particles of either
the drug delivery device or particles of one or more pharmacologically active
agents.
Additionally, the protein matrix may also encapsulate an drug delivery device
larger and/or
different than a particle that is covered by the release mechanism material.
Figure 5A depicts and embodiment of a protein matrix device that includes a
release
mechanism. The release mechanism 40 is positioned within a protein matrix
material 42.
Generally, the mechanism 40 is a material that creates a shell around the
pharmacologically
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52
active agents 44 and inhibits their release until opened by some outside
stimuli 46. Normally,
the pharmacologically active agent can be released by a pulse of energy,
radiation or a chemical
reagent acting upon the encapsulating substance. For example, a drug delivery
device
comprising a pharmacologically active agent encapsulated in a polyanhydride
coating inhibits
release of the pharmacologically active agent and/or its interaction with the
host tissue. In this
example, the pharmacologically active agents can be released when the
polyanhydride surface is
contacted with an ultrasound pulse. Such an embodiment has many advantages in
treating
afflictions that may require an extended time period before release of the
pharmacologically
active agent is necessary.
Treatment of cancer or chronic pain may be examples of afflictions that may
benefit from
such an embodiment. The retention of chemotherapy drugs localized in an area
of the patient
that includes cancerous tissue may be beneficial to the long term treatment of
the patient. The
treatment may include implantation of a drug delivery device that includes a
release mechanism
in a position of the body wherein cancerous tissues has been previously
resected. Upon
determination that cancerous cell growth may be ongoing or occurring again,
the drug deliver
device can be released by some stimuli, such as a ultrasound pulse or chemical
reagent. The
stimuli opens the release mechanism material and allows the host tissue to
interact with the
pharmacologically active agents.
ENCAPSULATED OR COATED STENT DEVICES:
Other embodiments of the present invention include the utilization of the
protein matrix
material in encapsulated or coated stent devices. A stent is a tube made of
metal or plastic that is
inserted into a vessel or passage to keep the lumen open and prevent closure
due to a stricture or
external compression. Stents are commonly used to keep blood vessels open in
the coronary
arteries, into the oesophagus for strictures or cancer, the ureter to maintain
drainage from the
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kidneys, or the bile duct for pancreatic cancer or cholangiocarcinoma. Stents
are also commonly
utilized in other vascular and neural applications to keep blood vessels open
and provide
structural stability to the vessel. Stents are usually inserted under
radiological guidance and can
be inserted percutaneously. Stents are commonly made of gold or stainless
steel. Gold is
considered more biocompatible. However, stents constructed of any suitable
material may be
utilized with the protein matrix of the present invention.
Encapsulation or coating of a stent with the protein matrix material of the
present
invention produces a device that is more biocompatible with the host tissue
than the stent device
alone. Such encapsulation or coating of the stent reduces or prevents adverse
immuno-response
reactions to the stent device being administered and further enhances
acceptance and remodeling
of the device by the host tissue. Furthermore, encapsulated or coated stent
devices may also
include one or more pharmacologically active agents, such as heparin, within
or attached to the
protein matrix material that may assist in the facilitation of tissue
acceptance and remodeling as
well as inhibit additional adverse conditions sometimes related to
implantation of stents, such as
blockage of the vessel from platelet aggregation. In addition to anti-platelet
aggregation drugs,
anti-inflammatory agents, gene altering agents such as antisense, and other
pharmacologically
active agents can be administered locally to the host tissue.
The protein matrix material may completely encapsulate or otherwise coat the
exterior of
the stent. Generally, the encapsulated or coated stent device is made in a
similar process as
described above. Figure 6 depicts a compression molding device wherein the
inner insert 18
includes a mandrel 29 that extends upward from the insert 18 into the chamber
17. Following
preparation of the cohesive body 23, inner insert 18 is inserted into the
cavity 16. A stent 32 is
positioned over the mandrel 29 and the cohesive body 22 is placed in the
cavity and compressed.
Encapsulation or coating of the stent 32 is determined by the size of the
mandrel 29 utilized in
the compression molding device. A stent 32 that fits snuggly over the mandrel
29 will allow for
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only a coating upon the exterior of the stent 32. A smaller mandrel 29 that
does provide a snug
fit for the stent 32 will allow protein matrix material to move between the
mandrel 29 and the
stent 32 thereby creating an encapsulation of the stent 32. The encapsulated
or coated stent
device is then removed from the compression molding device in a similar way as
described
above and shown in Figure 3. The stent device, either encapsulated or coated
generally has a
wall thickness of approximately 0.05mm to 2 mm and preferably has a wall
thickness of .15 to
0.50 mm.
As previously described additional polymeric and other biocompatible materials
may be
included in the protein matrix material to provide additional structural
stability and durability to
the encapsulated or coated stent device. Also, other structural materials,
such as proteoglycans,
can be used in this process to add greater tissue imitation and
biocompatibility. The
proteoglycans can replace or be mixed with the protein material in the
production of the protein
matrix material.
Additionally, the protein matrix material included in the encapsulated or
coated stent
cover may be cross-linked to provide additional desirable features such as the
inhibition of cell
growth or to provide additional structural durability and stability. For
example the protein
matrix material of the encapsulated or coated stent device may be crosslinked
by contacting the
material with a chemical reagent, such as glutaraldehyde, or other type of
crosslinking reagent.
Figure 7 depicts various views of a tube made of elastin which has been
crosslinked by being
20. exposed to a 1% solution of glutaraldehyde for 5 minutes.
Figures 8 and 9 depict additional embodiments of encapsulated and coated
stents. Figure
8 depicts an encapsulated stent device including a protein matrix material
comprising a 1:1 ratio
of elastin to albumen (bovine serum albumin). Figure 8 further depicts the
encapsulated stent
device inserted within a silastic tube. The encapsulated stent device in
Figure 8 is further shown
being expanded by insertion and expansion of an angioplasty balloon within the
interior of the
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device. Furthermore, the stent device of Figure 8 illustrates that the protein
matrix material
remains engaged to the stent struts and does not separate from the stent after
the stent device is
opened by the angioplasty balloon.
Other embodiments of the stent device of the present invention maybe produced
by
5 preparing a stent device that includes a ratio of 2:1:2 collagen to elastin
to albumen, 4:1 collagen
to elastin, 1:4:15 heparin to elastin to collagen, 1:4:15 condroitin to
elastin to collagen. Each
embodiment depicted in the Figures illustrates the uniform distribution of the
protein matrix
material around the stent and also depicts the strength and durability of the
stent after expansion
by a balloon.
10 Furthermore, the stent devices can also be used to incorporate peptides and
other
materials that have the ability to inhibit cell migration. A disadvantage of
utilizing stents in a
vessel is that the expansion of the vessel upon insertion of stent weakens the
vessel and may
allow smooth muscle cells to enter into the vessels thereby occluding or
restinosing the vessel.
Occlusion of the vessel and restinosis can be treated by utilizing the stent
device and vessels or
15 tube grafts of the present invention. Vessels and tubular grafts will be
explained later in the text
of this disclosure. It is important to note that inserting a stent with or
without drugs can prevent
such breakdown and growth of cells into the diseased or damaged vessel.
TISSUE GRAFTS:
20 Additional embodiments of the present invention include the utilization of
the protein
matrix material in producing tissue grafts such as vessels; tubular grafts
like tracheal tubes,
bronchial tubes, catheter functioning tubes, lung, gastrointestinal segments;
clear matrix grafts;
valves; cartilage; tendons; ligaments skin; pancreatic implant devices; and
other types of tissue
that relate to the heart, brain, nerve, spinal cord, nasal, liver, muscle,
thyroid, adrenal, pancreas,
25 and surrounding tissue such as connective tissue, pericardium and
peritoneum. It is noted that a
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tube does not necessarily have to be cylindrical in shape, but is generally
found in that
configuration.
Vessels and tubular grafts may be synthesized utilizing the protein matrix
material.
Generally, a vessel is a tubular graft made of the protein matrix material
that includes the growth
of cells on and/or within the matrix. For example, vessels may be produced
utilizing the protein
matrix material by growing endothelial cells on the inside of the protein
matrix tube and smooth
muscle cells on the outside of the tube. Alternatively, a multi-layered vessel
may be created with
two or more separate tubes, wherein a smaller tube with endothelial cells
grown on the inside of
the tube is inserted into a larger tube with smooth muscle cells grown on the
outside of the tube.
Both tubes may then be crosslinked on the surface that does not include cell
growth to add
further durability and stability to the vessel. Additional tubular layers may
be included in the
vessel that may or may not include the growth of cells on the surfaces or
within the protein
matrix. Figure 10 depicts various views of a multi-layer vessel by
illustrating the multi-layer
vessel the various tubes inserted within each other and also side by side.
These layers may also
contain pharmacologically active agents and/or more structural components,
such as polymeric
materials or stents. The layers will generally stay in position through
adhesives, fasteners like
sutures, cell interaction, pressure fitting, crosslinking, protein matrix
intermolecular forces and
other layer alignment means and may adhere or may not adhere to each other. It
is also noted that
layers that include cell growth may also include pharmacologically active
agents.
Once prepared the tubular graft or vessel maybe administered to the patient as
a
replacement to a damaged vessel or as a scaffolding device that can be
inserted into or mounted
around the damaged vessel. Vascular tubes, known as STUNTS (Support Tube Using
New
Technology Stent) can be used for placement within a blood vessel. (A support
tube without the
wire stent that can "stunt" the growth of smooth muscle cells into the lumen
of the vessel to
prevent restenosis.) Embodiments of the tubular grafts have form memory and
will reform if cut
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or severed back to its original form and shape. Figure 11 depicts an
embodiment of the present
invention that illustrates the capability, compliancy and capacity of the
protein matrix material to
accept sutures and reform to its original shape.
A vessel structure of the present invention will meet the mechanical and
histological
requirements of a blood vessel, while providing the biological and biochemical
functions that are
necessary for its success. One embodiment that ensures mechanical integrity
and biological
compatibility is a scaffold comprising collagen and elastin. These proteins
are the primary
components of a typical arterial wall. This will create the natural
environment for the endothelial
cells, while providing the structural characteristics of these proteins.
Endothelialization of the
cylindrical matrices will provide the critical hemocompatibility, while also
providing the
thrombolytic characteristics. This feature will allow for the creation of
small-diameter vascular
grafts with a reduction in thrombosis. Embodiments of the tubular structure
will have a diameter
of approximately 2-4 mm due to the small-diameters of native coronary
arteries. Due to the
prevalence of coronary disease and the need for effective treatments, the
proposed tubular
structure would be embraced as a compatible vascular graft.
Additionally, the tubular grafts prepared by using the methods of the present
invention
can provide the similar function as the previously described encapsulated or
coated stent devices.
The difference between the tubes and the stent device would be the elimination
of the stent. The
tubes of the present invention have been shown to provide sufficient strength
and durability and
may be utilized as a scaffolding in diseased vessels thereby inhibiting the
narrowing of vessels in
all regions of the patient, such as the cardiovascular and neural regions. The
vessels or tubular
grafts may also be inserted under radiological guidance and can be inserted
percutaneously.
Similar to the encapsulated or coated stent devices, the vessels or tubular
grafts that include the
protein matrix material of the present invention are biocompatible and reduce
or prevent
immunogenicity with the host tissue. Additionally, since the vessels or
tubular grafts of the
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present invention are produced with a biocompatible protein matrix material
and may include the
growth of cells from the patient or compatible cells, the vessel or tubular
graft administered to
the host tissue further enhances acceptance and remodeling of the vessel or
tubular graft by the
host tissue. It is again noted that remodeling of the protein matrix device of
the present
invention is the modifying, adapting and/or transforming the device into an
interwoven and/or
functioning part of the host tissue.
Furthermore, the vessels and/or tubular grafts may also include one or more
pharmacologically active agents within or attached to the protein matrix
material that may assist
in the facilitation of tissue acceptance and remodeling, as well as inhibit
additional adverse
conditions sometimes related to implantation of vessels, such as platelet
aggregation causing
blockage of the vessel. In addition to antiplatelet aggregation drugs, anti-
inflammatory agent,
gene altering agents, enzymes, growth factors and other additional
pharmacologically active
agents can be included in the vessel and/or tubular graft for localized
administration to or near
the host tissue.
Embodiments of the protein matrix vessels and/or tubular grafts maybe prepared
by
methods similar to those described and suggested above. Figures 12 and 13
depict a compression
molding device wherein the inner insert 18 includes a mandrel 29 that extends
upward from the
insert 18 into the chamber 17. Figure 13 depicts a top view of the compression
molding device
without the upper insert 19 or plunger 14. Following the insertion of a
sufficient amount of
cohesive body 22 the upper insert 19 and plunger 14 are applied to the
cohesive body 22. As
with the previous compression molding device embodiments the pressure applied
by the plunger
14 and surfaces of the chamber 17 and mandrel 26 to the cohesive body 23
removes the bulk
water within the cohesive body 23 thereby resulting in the protein matrix
device. The vessel
and/or tubular graft is then removed from the compression molding device in a
similar way as
described above and shown in Figure 3. The vessel and/or tubular graft
generally has a wall
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thickness of approximately 0.05 mm to 1 cm and preferably has a wall thickness
of 0.15 to 0.50
mm.
Furthermore, other tissue grafts may be made by including in the compression
molding
device a cavity 16 and inserts 18 and 19 that are configured to produce the
size and shape of the
tissue graft desired. For example valves such as heart valves; bone;
cartilage; tendons; ligaments
skin; pancreatic implant devices; and other types repairs for tissue that
relate to the heart, brain,
abdomen, breast, palate, nerve, spinal cord, nasal, liver, muscle, thyroid,
adrenal, pancreas, and
surrounding tissue such as connective tissue, pericardium and peritoneum may
be produced by
forming the cavity 16 and inserts 18 and 19 of the molding compression chamber
into the
[0 corresponding size and shape of the particular tissue part. It is noted,
that the above mentioned
tissue parts may optionally include one or more pharmacologically active
agents or other
structural materials, such as metal, polymeric and/or biocompatible materials
including wire,
ceramic, nylon.or polymeric meshes.
As previously described additional polymeric and other biocompatible materials
may be
included in the protein matrix material of the tissue grafts to provide
additional structural
stability and durability. Also, other structural materials, such as
proteoglycans, can be used in
this process. The proteoglycans can be mixed with one or more protein
materials in the
production of tissue grafts.
Additionally, the protein matrix material included in the tissue grafts may be
cross-linked
>0 to provide additional desirable features such as to inhibit cell growth,
reduce immunogenicity or
provide additional structural durability and stability. For example the
protein matrix material of
the vessels or tubular grafts may be crosslinked by contacting the material
with a chemical
reagent, such as glutaraldehyde, or other type of crosslinking reagent similar
to the procedure
performed on the stent device of Figure 7.
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In another embodiment of the present invention, vessels can be used to bring
blood to
cell-support constructs made of the protein matrix material and bring the
blood acted on by these
cells back into the body's circulation. The cell support constructs provides
the biological
environment for the growth and maintenance of various cell types e.g. a
protein matrix cell
5 scaffold for hepatocytes or islet cells can be placed in a direct blood
link. Such a device will
provide the hepatocytes or islet cells with adequate access to the blood
supply. For example, the
cell support construct can act similar to a functioning pancreas, liver or
other viable organ in a
biological system. In other words a cell support construct can be produced and
incorporated
within a biological system as an organ or partial organ replacement.
10 Another embodiment of the present invention is a protein matrix device that
is clear. The
procedure for making a clear protein matrix comprises making a mold of
collagen and/or elastin
as described herein and putting it through a spinning process that aligns the
fibers. The clear
protein matrix may be utilized in cornea transplants. More, specifically, the
procedure includes
putting a protein matrix material inside a device that spins upon its axis,
similar to a nuclear
15 magnetic resonance or NMR type machine. The spinning device will spin this
material at a very
high rate around its own axis so that the center of the protein matrix is
thrown outward so that
the fibers and/or molecules of the protein matrix are aligned.
Since the protein matrix contains water, the protein matrix, at this high rate
of spin, starts
to act like a fluid and slowly moves the protein matrix molecules into
alignment. The greater
lO amounts of water incorporated into the matrix, the easier to align the
protein and the other
molecules. The process may be enhanced if other molecules, such as
proteoglycans like heparin,
are incorporated in the matrix to make the protein fibers more slippery. As
previously
mentioned a clear material, such as this, could be used as a cornea transplant
upon growing the
requisite cells on the clear matrix.
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In preparation of a clear protein matrix material, a sample of protein matrix
material, as
prepared by the methods described or suggested above, was placed in a probe
and inserted into
an NMR device. Once inside the NMR machine the protein matrix is spun for 48-
72 hours,
thereby aligning the fibers and/or molecules and producing the clear matrix.
In another embodiment of making the clear protein matrix material it may be
possible to
create a device that spins on its axis for this process. The NMR is just
spinning the protein
matrix around its own axis, so it's possible to create such a device wherein
the protein matrix
may be placed in the center of the spinning device so that it also would spin
on its own axis and
create the alignment of the fibers and/or molecules of the protein matrix
material.
The protein matrix utilized for making a clear protein matrix could be any
shape or size.
However, if you're spinning the protein matrix around its own axis, more
homogenous force
may be applied to all parts of the matrix if it were circular or cylindrical.
Furthermore if the
circle was made big enough, it could then be cut out into any shape and size,
with the idea that
all parts of that shape received the same kind of force when produced.
Also, the protein matrix material contains water, typically somewhere between
10-60%
water depending upon how it's made. At this high rate of spin, it is possible
to get some flow of
material and provide forces between the protein molecules that make them
correspond to each
other in a certain way. Moreover, this water environment gives them a lot of
motion and the
spinning gets that motion to align so that when you're done, the fibers align.
This alignment
0 produces a clear protein material much like the cornea.
WOUND HEALING DEVICES:
Other embodiments of the present invention include wound healing devices that
utilize
the protein matrix material. The wound healing devices may be configured in
any shape and size
to accommodate the wound being treated. Moreover, the wound healing devices of
the present
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invention may be produced in whatever shape and size is necessary to provide
optimum
treatment to the wound. These devices can be produced in the forms that
include, but are not
limited to, plugs, meshes, strips, sutures, or any other form able-to
accommodate and assist in the
repair of a wound. The damaged portions of the patient that may be treated
with a device made
of the protein matrix material include skin, tissue (nerve, brain, spinal
cord, heart, lung, etc.) and
bone. Moreover, the wound healing device of the present invention maybe
configured and
formed into devices that include, but are not limited to, dental plugs and
inserts, skin dressings
and bandages, bone inserts, tissue plugs and inserts, vertebrae, vertebral
discs, joints (e.g., finger,
toe, knee, hip, elbow, wrist,), tissue plugs to close off airway, (e.g.,
bronchial airway from
resected tissue site), other similar devices administered to assist in the
treatment repair and
remodeling of the damaged tissue and/or bone.
In one embodiment of the wound healing device of the present invention, a
protein matrix
material may be formed into a dressing or bandage, to be applied to a wound
that has penetrated
the skin, that utilizes a very thin amount of protein matrix material. Figure
14 depicts an ultra-
thin collagen/elastin matrix that is approximately 0.1 mm in thickness. Thin
matrices may be
made of one or more suitable biocompatible protein materials, one or more
biocompatible
solvents and optionally one or more pharmacologically active agents.
Furthermore, the protein
matrix materials formed into a thin dressing or bandage may be approximately
0.05-5 mm in
thickness.
?0 The protein matrix, upon application, adheres to the skin and will remain
for days
depending upon the conditions. If protected, embodiments of the protein matrix
dressing will
remain on the skin for a considerable period of time. Moreover, if the protein
matrix is acting as
a wound dressing and therefore interacting with a wound it will stick very
tightly. The protein
matrix is also acts as an adhesive when wet and as it dries. It is also noted
that the protein matrix
?5 of the present invention incorporated into a wound dressing would help
facilitate or lessen
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scarring by helping to close the wound. Furthermore, protein matrix dressings
or bandages may
be prepared to administer beneficially healing and repairing pharmacologically
active agents, as
well as, act as a device that may be incorporated and remodeled into the
repairing tissue of the
wound.
In another embodiment of the present invention, the protein matrix can also be
protected
with a tape barrier that is put over the matrix and over the wound. A plastic
and/or cellophane-
like section of material may be used as a tape barrier that does not stick to
the protein matrix
material but holds it in place and provides more protection from the
environment. Tape barriers
that are utilized in bandages existing in the art may be used with the
dressing of the present
invention.
Figure 15 depicts a wound dressing comprising a protein matrix that is
positioned in the
center of a non-adhesive strip of material attached to two adhesive ends. The
protein matrix can
be made from a number of different protein materials including, but not
limited to, a
collagen/elastin protein mixture (4:1; 4 parts collagen, 1 part elastin). In
one embodiment the
elastin utilized may be an insoluble elastin made soluble using DMSO. However,
a soluble
elastin could be used as well. Either type of elastin works well, however, the
insoluble is a much
cheaper raw material, and it may have some advantages, such as greater
potential matrix strength
due to it's insoluble characteristics.
Embodiments of the protein matrix wound healing device, also provide a device
wherein
pharmacologically active agents can be impregnated into it. The matrix or
wound dressing may
include, but are not limited to, substances that help clotting, such as
clotting factors, substances
which are helpful for wound healing, such as vitamin E, as well as, anti-
bacterial or anti-fungal
agents to reduce the chance of infection. Other groups of pharmacologically
active agents that
may be delivered by the protein matrix wound dressing are analgesics, local
anesthetics, other
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therapeutics to reduce pain, reduce scarring, reduce edema, and/or other type
of drugs that would
have very specific effects in the periphery and facilitate healing.
The inclusion of such pharmacologically active agents in the protein matrix
dressing also
facilitates the controlled release of substances, which would assist in
healing and/or treat and
prevent infection. Furthermore, the protein matrix interacts with the cells
that migrate to the
wound to facilitate the healing process and that require a matrix and/or blood
clotting before they
can actually start working to close and remodel the wound area.
The collagen/elastin matrix is made very similar to the cylinders of the
protein matrix
drug delivery devices explained in the present application, except that only
enough material is
utilized to produce a thin wafer. Pressure is placed upon this material to
flatten it out. Examples
of the wound dressings have produced wafers of approximately .1 mm in
thickness.
Because insoluble elastin is present in the production of the protein matrix a
solvent is utilized.
Examples of solvents utilized in this process are DMSO and ethanol. The
insoluble elastin is
mixed into the collagen with a judicious amount of solvent to make the protein
matrix.
An embodiment of the present invention utilizes DMSO as the solvent. DMSO has
some
properties, which provide some benefits. However, any solvent, which dissolves
or sufficiently
wets the insoluble elastin may be used in the present invention. The
properties that DMSO has
are that it actually was used for some time by athletes to help relax muscle
tissue. Athletes
utilized DMSO after a long day of working out or playing in competitions;
rubbing it on the skin
over the muscle tissue that was bothering them would relieve the pain from
their muscle tissue.
DMSO is inexpensive to make and purchase. Additional advantages of using DMSO
in the
present invention are that it may assist in the reduction of muscle pain that
might occur,
depending on the location and type of the wound and it also may allow for the
use of proteins
that are very insoluble in a water environment, but assists in the production
of a strong protein
matrix wound dressing.
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Another feature of the wound dressing is that only the part of the protein
matrix dressing
that is needed will integrate with the cells of the wound and be utilized.
Generally, over a period
of time, a wound will remodel and close utilizing only the amount of the
protein matrix material
necessary to assist in the process. Any remaining protein matrix not utilized
in the mending of
5 the wound will flake away in similar fashion as the way dead skin,
surrounding and covering the
healed wound, dries and flakes off.
The protein matrix wound dressing could also help people who require more
assistance
than normal for a wound to actually close. Individuals who have problems with
wound healing
may find that their wound takes longer to close due to their wound not being
able to develop a
10 clot and/or set up a matrix for cells to close the wound. In these
situations, such as a person with
diabetics or ulcers, the protein matrix may be utilized to assist in healing.
The protein matrix
provides a material that assists the wound in closing, especially if clotting
factors and maybe
some other factors that are known in the art and are important to wound care
are incorporated
into the protein matrix.
15 Again, the incorporation into the protein matrix of substances, such as
biochemicals, that
would naturally be incorporated into the wound during healing may be of
benefit in the healing
process. The protein matrix itself comes in contact with the wound and
supplies a scaffold for the
cells to interact with and thereby assists in healing the wound. Therefore,
the incorporation of the
previously mentioned biochemicals, which can be uniformly dispersed and
impregnated into the
20 matrix, can further assist in the healing process and increase the
prevention of infection,
reduction of pain, remodeling of the damaged tissue and all other overall
healing results.
The biochemicals, previously referred to, such as factor 14, factor 8 and
other similar
biochemicals are most crucial to the beginning steps of wound care. The
impregnation of such
biochemicals into a protein matrix will translate to a faster closing process
and hence a faster
25 healing process. These biochemicals are present in our blood at all times
and are immediately
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prepared to function when they come in contact with a wound site. However,
sometimes for
various reasons a patient's blood does not have enough of these biochemicals
or cannot
satisfactorily supply a sufficient amount to effectively repair a wound.
Therefore, the application
of a protein matrix as described herein which is impregnated with such
biochemicals can have a
beneficial role in stimulating and enhancing the healing process.
It is also possible to extend delivery of chemicals or drugs using this
protein matrix as a
wound dressing. In one embodiment this can be accomplished by providing a
protein matrix
wound dressing that includes a patch delivery system adjoined immediately
behind the protein
matrix dressing. In this example a strip, wrap or patch that includes a larger
dosage of the
chemical or pharmaceutical active component may be applied behind the protein
matrix not in
immediate contact with the wound. By administering such a wound healing
device, the delivery
of chemicals and/or pharmaceuticals could be extended until the wound was
healed or the
desired amount of chemicals and/or pharmaceuticals were applied. In
application, the protein
matrix would continue to absorb more chemicals and/or pharmaceuticals from the
patch as the
initial material impregnated in the matrix was being utilized in the wound.
Therefore, the protein
matrix would provide a controlled release of the chemical and/or
pharmaceutical component and
would prevent the administration of too much chemical and/or pharmaceutical
component from
entering a patient's wound prematurely. Additionally, the protein matrix with
adjoining patch
may be very beneficial for patients who are compromised in some way from
internally supplying
the biological substances needed to reduce or prevent them from healing
quickly. Examples of
such situations where such a protein matrix wound healing device would be
beneficial are in
cases of diabetes, hemophilia, other clotting problems or any other type
affliction that inhibits
the adequate healing of a wound. Furthermore, individuals with such conditions
may require a
great deal more than the clotting agents that can be incorporated into a thin
protein matrix.
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Therefore, the patch may contain more than one additional chemical and/or
pharmaceutical
components that may benefit from extended contact with the wound in the
healing process.
Additionally, embodiments of a moistened protein matrix dressing that includes
a patch
may be configured to allow a varying controlled release of pharmaceuticals
through the matrix
by providing a matrix that release molecules at varying rates based on
molecule size. This
provides a tremendous means for controlling administration of more than one
pharmacologically
active agent that vary in size. Such controlled release facilitates the
administration of
pharmaceutical molecules into the wound when they may be needed. For example,
the protein
matrix dressing may be layered with different types of protein material and
biocompatible
polymeric material mixtures that control the release of molecules based on
size. For example, the
protein matrix material may include physical and/or chemical restraints that
slow the migration
of various size molecules from the patch and through the protein matrix
dressing. Furthermore,
the larger molecules that are proteins and other macromolecules that need to
be in contact with
the wound can be impregnated into the protein matrix itself.
Furthermore, the protein matrix dressing may be set up with pores that allow
fluid flow
through that matrix and also enhances movement of the pharmacologically active
agents through
the matrix. Pores may be created in the matrix by incorporating a substance in
the cohesive body
during its preparation that may be removed or dissolved out of the matrix
before administration
of the device or shortly after administration. Porosity may be produced in a
protein matrix device
by the utilization of materials such as, but not limited to, salts such as
NaCl, amino acids such as
glutamine, microorganisms, enzymes, copolymers or other materials, which will
be leeched out
of the protein matrix to create pores. Other functions of porosity are that
the pores create leakage
so that cells on outside can receive fluids that include the contents of the
matrix and also that
cells may enter the matrix to interact and remodel the matrix material to
better incorporate and
function within the host tissue.
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As described herein a protein matrix maybe made porous by the utilization of
salts or
other such materials. However, it is also possible to produce a porous protein
matrix by the
incorporation of a solution saturated or supersaturated with a gaseous
substance, such as carbon
dioxide. In one embodiment, carbonated water may be utilized in a sealed and
pressurized
environment during the production of the protein matrix. The utilization of
carbonated water
creates bubbles within the protein matrix during the production process. Once
the matrix has
been shaped into the desired form and removed from the sealed and pressurized
environment, the
gaseous bubbles escape from the matrix leaving a porous material.
Another embodiment for producing a porous protein matrix makes use of
polyvinyl
[0 alcohol (PVA or other water soluble polymers). Polyvinyl alcohol (PVA) or
other water-soluble
polymers can be made into particles that correspond to a specific size. The
particles are made by
first producing a gel following standard techniques for that polymer. For
example, PVA is made
into a 4% solution in 100ml and placed into a vacuum oven at 40 C for 24
hours. The resulting
dried gel is pulverized after freezing with liquid nitrogen. The particles are
then separated by a
sieve into specific sizes. The water-soluble polymer particles are
incorporated into the protein
matrix so that they can be dissolved by aqueous solutions to provide a protein
matrix that is a
three dimensional scaffold for cells to migrate and grow within. The PVA
particles will dissolve
at rates that are directly proportional to the size and thickness of the
protein matrix. The PVA
particles can be made with cell enhancing agents or chemicals to act as
therapeutics so that
?0 residual particles can facilitate cell migration, growth and/or
proliferation from the pore
structures.
The protein matrix material of the present invention may also be utilized as
port seals for
protrusion devices entering and or exiting the patient. Figure 16 depicts one
embodiment of a
protrusion device 34 that includes a port seal 36 comprising the protein
matrix material of the
?5 present invention. The port seal 26 may be included around the point of
insertion of a protrusion
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device, such as an electrical lead or a catheter. Generally, the port seal 36
surrounds the
protrusion device 34 and insulates it from the host tissue. One or more tabs
38 may optionally be
included on the port seal 36 to assist in the retention of the protrusion
device and further seal the
opening in the patients skin. The tabs 38 maybe inserted under the skin or may
remain on the
outside of the patient's skin. Also, the biocompatible seal comprising the
protein matrix material
of the present invention provides stability, reduces the seeping of bodily
fluid from around the
protrusion and reduces or prevents immunogenicity caused by the protrusion
device.
Furthermore, the port seal may include pharmacologically active agents that
may be produced to
deliver anti-bacterial, analgesic, anti-inflammatory and/or other beneficial
pharmacologically
active agents.
Other embodiments of the present invention include wound-healing devices
configured
and produced as protein matrix biological fasteners, such as threads, sutures
and woven sheets.
Threads and sutures comprising various embodiments of the protein matrix
material provide a
biocompatible fastening and suturing function for temporarily treating and
sealing an open
wound. Additionally, the biological fasteners may include pharmacologically
active agents that
may assist in the healing and remodeling of the tissue within and around the
wound.
One method of preparing the biocompatible biological fasteners is to
manufacture sheets
of protein matrix material. Once the sheets of protein matrix material are
prepared each sheet
may cut into strips, threads or other shapes to form sutures, threads and
other biological fasteners
(e.g., hemostats). The sheets may be cut using cutting techniques Known in the
art. Also, the
protein matrix threads may be woven into sheets and used as a strengthened
protein matrix
material that has desired porosity. For example, this woven protein matrix may
also be used
with cohesive body to form a protein matrix that has a woven protein matrix
encapsulated or
filled by protein matrix.
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Additionally, fibers (large or small, e.g., macro, micro, nano) of a known
suturing
material, such as nylon, may incorporated in the cohesive body and compressed
to make a sheet
of protein matrix material. It is noted that the protein matrix forms a
cohesive body around the
biocompatible thread/fibers during compression to encapsulate the
biocompatible fibers into the
5 protein matrix. Once the sheet is prepared it may be cut by methods common
to the art to
produce a thread/suture that has biocompatible and durable characteristics.
Additional embodiments of wound healing devices that include the protein
matrix
material of the present invention include but are not limited to dental
inserts, dental plugs, dental
implants, dental adhesives, and other devices utilized for dental
applications. Wounds and dental
10 complications, such as dry socket, present within the interior of the mouth
are generally slow to
heal, are painful and/or are susceptible to bacterial and other forms of
infection. The dental
inserts or implants of the present invention may be utilized to remedy such
problems since they
are biocompatible with the surrounding host tissue and may be manufactured to
release
appropriate pharmacologically active agents that may assist in healing,
relieve pain and/or reduce
15 bacterial attack of the damaged region. Furthermore, the dental plugs,
inserts or implants of the
present invention include one or more biocompatible protein material and one
or more
biocompatible solvent that may be incorporated into and remodeled by the
surrounding tissue,
thereby hastening the healing of the damaged region and/or returning the
damaged region to its
original state. For example, dental plugs or implants may be administered to
open wounds
20 within the mouth region of the patient following tooth extraction, oral
surgery or any other type
of injury to the interior of the mouth to assist in the healing and
regeneration of the damaged
region.
In general, the dental plugs, implants or inserts may be administered to the
damaged area
by any method known in the art. For example a dental plug may be administered
to the socket of
25 a tooth after removal by placing a properly sized and shaped dental plug
that includes the protein
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matrix of the present invention into the socket. The dental plug may
optionally be fastened to the
surrounding tissue of the socket by any means known in the art such as
adhesives or sutures.
However, it may not be necessary to use any fastening means since the cells of
the host tissue
may be found to readily interact with the plug and begin to incorporate the
plug into the host
tissue. As previously suggested, such a dental plug may also include analgesic
antibacterial, and
other pharmacologically active agents to reduce or prevent pain and infection
and to promote the
reconstruction of the damaged region.
OTHER PROTEIN MATRIX DEVICES:
The protein matrix material of the present invention may also be utilized in
other medical
devices to enhance their biocompatibility, provide medical functionality
and/or deliver
pharmacologically active agents. One example, of other devices that utilized
the protein matrix
material of the present invention may be as an intrauterine device (IUD). An
IUD is a
contraceptive device that is placed within the uterus for the purpose of
inhibiting conception.
Generally, the protein matrix may be produced into any IUD like configuration
known in the art
and inserted into the uterus. The protein matrix mesh may be prepared by
utilizing methods
previously described or suggested in the application. Upon insertion of
protein matrix mesh
and/or particles of any shape into the uterus, the mesh and/or particles
interact with the uterine
wall cells to create a natural fibrotic meshwork that closes the uterus by
fusing the uterine walls
together to thereby inhibit the endometrial lining from forming inhibiting
menstruation and
conception. The IUD protein matrix device may also include pharmacologically
active agents
that aid in the production of the fibrotic meshwork and/or locally treat the
surrounding tissue.
Additionally other protein matrix device embodiments include a protein matrix
that has
incorporated into it a marker system that allows the matrix to be located and
imaged using
ultrasound, MRI, X-Ray, PET or other imaging techniques. The image marker can
be made with
air bubbles or density materials that allow easy visualization of the protein
matrix by ultrasound.
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The incorporated materials can be metallic, gaseous or liquid in nature.
Specific materials that
may be utilized as image markers incorporated into the protein matrix material
include, but are
not limited to, Gd-DPTA. It may be possible to cause the material to react to
an imaging
technique, i.e., ultrasound to make bubbles or through the addition of another
chemical or
substance to the system (e.g., peroxide addition to a protein matrix that
contains peroxidase as an
intrauterine marker that can be monitored by ultrasound). Also, the addition
of a harmless
unique salt solution, or enzyme, may promote gas production by the protein
matrix as an
ultrasound maker. ,
The protein matrix can contain agents that can be seen by ultrasound, MRI,
PET, x-ray or
any imaging device that is either known, in development or developed in the
future.
Other embodiments of the present invention are protein matrices, which can
include
imprints that provide for specific site location for attachment of substances,
such as chemicals,
cells or enzymes, or for preventing or reducing attachment of such substances.
Examples of
materials that may be targeted for specific attachment sites on the protein
matrix may be cell
adhesion molecules or electro-conductive molecules.
The protein matrix can be of any size, shape or form and can be imprinted with
any
pattern desired depending upon the application. For example, an embodiment of
the imprinted
protein matrix may take the form of a blood vessel. The exterior of the blood
vessel may be
imprinted with a pattern that limits the attachment of cellular material that
facilitates capillary
growth to the exterior. This promotion of angiogenesis provides a number of
benefits including
the reduction of inflammation to the vessel surroundings and the further
promotion of the
surrounding tissue's acceptance and incorporation of the vessel.
Another embodiment includes the protein matrix in the form of a sphere. Such a
matrix
may be imprinted in areas with a substance that inhibits the binding of
biological tissue upon
implantation in only these predetermined areas. More specifically, a protein
matrix may be
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impregnated with an adhesive substance, which would facilitate binding to
tissue. Therefore, the
portions of the protein matrix imprinted with the nonbonding substance are
thereby prevented
from adjoining the surrounding tissue. However, the regions not imprinted
would adhere to the
tissue and perform the intended functions.
Methods of imprinting the protein matrix with a desired pattern can be
performed by any
means known in the art. For example, the utilization of UV light can produce a
crosslinking
pattern upon the protein matrix. Many difference crosslinking agents can be
used but
crosslinking agents that are only active upon W activation can selectively
attach chemical
substances to the protein matrix. This crosslinking can occur either on the
surface or within the
protein matrix. One function of such a crosslinking pattern would be to
inhibit the attachment of
cells. Alternately, it is also possible to attached molecules that will allow
attachment of cells.
Chemicals, enzymes, short peptides or large peptide segments can be
crosslinked to selected
areas of the protein matrix. Such substances can be utilized to attract and
enhance the
attachment and/or growth of various cells.
Another embodiment of the present invention relating to an imprinting method
is the use
of masking systems to create the imprinted pattern. The pattern on a protein
matrix may be
produced by covering the protein matrix with a mask that has the desired
pattern and exposing
the covered matrix to a chemical substance, such as glutaraldehyde or any
crosslinking agent
(e.g., UV-activated chemical). The chemical substance contacts the portions of
the protein
?0 matrix not covered by the mask and crosslinking occurs. Alternatively, when
utilizing IN-
activated chemicals, the mask blocks the light thereby inhibiting crosslinking
so that crosslinking
only occurs at unmasked sites. The mask is then removed thereby providing a
protein matrix
with both crosslinked and non-crosslinked portions. The non-crosslinked areas
can provide
locations for the attachment or access to chemicals, cells, enzymes,
oligonucleotides, other
Z5 proteins, etc. Furthermore, these site-specific attachment areas of the
protein matrix maybe
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utilized for diagnostic reasons, the growth of cells or as access points for
other chemicals or
enzymes.
Finally the imprinted protein matrix has applications in the protein chip
technology
described above. The imprinting of patterns upon the protein matrix chip may
produce chips,
which provide a number of similar characteristics as a silicon chip or silicon
coated substance.
As previously suggested, such an embodiment may be beneficial in various
diagnostic
applications.
EXAMPLES:
The drug delivery devices of the present invention will now be further
described with
reference to the following non-limiting examples and the following materials
and methods that
were employed.
Xanthine oxidase, superoxide dismutase, capsaicin and dexamethasone were
obtained
from (Sigma Chemical Company, St. Louis MO). The silklike, elastinlike polymer
SELP7 was
obtained from Protein Polymer Technologies, San Diego, CA.
Test method 1. Thermal Sensitivity Test.
The thermal sensitivity tests referred to herein below were conducted as
follows.
Thermal sensitivity was measured by the time required for each rat to withdraw
its hind paw from a 56 C hot plate (commercially available under the trade
designation
35-D from IITC Life Science Instruments, Woodland Hills CA). Specifically the
rats were
positioned to stand with one hind paw on a hot plate and the other on a room
temperature
board. Latency to withdraw each hind paw from the hot plate was recorded by
alternating
paws and allowing at least 15 seconds of recovery between each measurement. If
no
withdrawal occurred from the hot plate within 15 seconds, the trial was
terminated to
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prevent injury and the termination time was recorded. Testing ended after
three
measurements per side and the mean was calculated for each side.
Test method 2. Motor Capaci Test.
5 The motor capacity tests referred to herein below were conducted as follows.
The rat is
held in the same manner as during the thermal sensitivity testing so that it
is positioned to stand
on one leg against an electronic balance. The resistance of the rat's leg is
measured as the force
against the balance in grams. Previous results from control experiments show
that a 200-275
gram rat exerts about 150-225 grams of force with a normal leg. However, if
the leg is showing
10 a lack of motor capacity from local anesthetic action, then forces of only
from about 30 to about
70 grams are expected. Thus, a lack of motor capacity resulting in the rat
exerting only from
about 30 to about 70 grams of force against the balance shows that the
administered drug
delivery device has delivered enough of a pharmacologically active agent to
produce local
anesthetic action.
Example 1: Preparation of a Drug Delivery Device Comprising a Biodegradable
Protein and an Enzyme
The enzyme xanthine oxidase was dissolved in deionized water to 0.28
units/100 l. This xanthine oxidase solution was mixed in with 50 mg protein
(SELP 7) to
form a coatable composition. The composition was then coated on a glass
surface to form
a film with a thickness of from about 0.1 to about 0.3 mm. The coated film was
allowed to dry
at room temperature until dry enough so as to be cohesive, i.e., to a solvent
content of from about
50% to about 70%. The resulting film was rolled up, placed in a 3.5 mm
diameter mold and
compressed at 1750 psi for 2 minutes to form a 3.5 mm diameter cylinder,
approximately 5 mm
long, utilizing the compression molding device discussed hereinabove. The
resulting cylinder
had a solvent content of approximately 30% to about 60%. This cylinder was cut
into four equal
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pieces so that each piece contained approximately 0.07 xanthine oxidase
units/piece. These
pieces were frozen at -80 C until used within 4 weeks.
Example 2: Preparation of a Drug Delivery Device Comprising a Biodegradable
Protein and an Enzyme
The enzyme superoxide dismutase (SOD) was dissolved in deionized water to 30.0
units/100 l. This SOD solution was mixed with 50 mg (SELP7) to form a
coatable
composition. The composition was then coated on a glass surface to form a film
with a thickness
of from about 0.1 mm to about 0.3 mm. The coated film was allowed to dry at
room temperature
until dry enough so as to be cohesive, i.e., to a solvent content of from
about 50% to about 70%.
The resulting film was rolled up, placed in a 3.5 mm diameter mold and
compressed at 1750 psi
for 2 minutes to form a 3.5 mm diameter cylinder, approximately 5 mm long,
utilizing the
compression molding device discussed hereinabove. The resulting cylinder had a
solvent
content of from about 30% to about 60%. This cylinder was cut into four equal
pieces so that
each piece contained approximately 7.5 units of SOD per /piece. These pieces
were frozen at
-80 C until used within 4 weeks.
Example 3: Preparation of a Drug Delivery Device Comprising a Biodegradable
Protein and Lipospheres
Lipospheres with 3.6% of the local anesthetic bupivacaine were made as
described in
U.S. Patent No. 5,188,837. From about 200 million to about 400 million of
these lipospheres
were then suspended in 150 gl deionized water. This suspension was then mixed
with 30 mg
SELP7 to form a coatable composition. The composition was then coated onto a
glass surface to
form a film with a thickness of from about 0.1 to about 0.3 mm. The coated
film was allowed to
dry at room temperature until the film was dry enough so as to be cohesive,
i.e., to a solvent
content of from about 50% to about 70%. The resulting film was rolled up,
placed in a 3.5 mm
diameter mold and compressed at 1750 psi for 2 minutes to form a 3.5 mm
diameter cylinder,
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approximately 4 mm long, utilizing the compression molding device discussed
hereinabove. The
resulting cylinder had a solvent content of from about 30% to about 50%. Four
cylinders were
made according to this procedure. These cylinders were refrigerated at 4 C
until used within 4
weeks.
Example 4: Preparation of a Drug Delivery Device Comprising a Biodegradable
Protein and two Pharmacologically Active Agents
Drug delivery devices were prepared with differing concentrations of the two
pharmacologically active agents capsaicin and dexamethasone as follows.
Specifically, first
drug delivery devices were prepared comprising 6 mg of capsaicin and 6 mg
dexamethasone by
dissolving these amounts in 100 gl ethanol. This solution was then added to a
solution of 128
mg SELP7 dissolved in 150 gl water to form a coatable composition. This
composition was then
coated onto a glass surface to form a film with a thickness of from about 0.1
mm to about 0.3
mm film. The coated film was allowed to dry at room temperature until dry
enough so as to be
cohesive, i.e., to a solvent content of from about 50% to about 70%. The
resulting film was
rolled up, placed in a 3.5 mm diameter mold and compressed at 7600 psi
overnight to form a 3.5
mm diameter cylinder, approximately 5 mm long, utilizing the compression
molding device
discussed. The cylinder was dried to a solvent content of from about 30% to
about 50% in a
vacuum and then cut into three equal pieces. From initially added quantities,
each pellet was
calculated to contain approximately 2 mg capsaicin and 2 mg dexamethasone,
weighing
approximately 35 mg each.
Second drug delivery devices were prepared comprising 6 mg of capsaicin and
1.2 mg
dexamethasone by dissolving these amounts of these agents in 25 l ethanol.
This solution was
then added to a solution of 120 mg SELP7 dissolved in 200 gl deionized water
to form a
coatable composition. This composition was then coated onto two glass surfaces
to form two
films with thicknesses of from about 0.1 mm to about 0.3 mm. The films were
allowed to dry at
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room temperature until dry enough sous to be cohesive, i.e., to a solvent
content of from about
50% to about 70%. The resulting films were rolled up, each placed in a 3.5 mm
diameter mold
and compressed at 7600 psi overnight to form two 3.5 mm diameter cylinders,
approximately 5
mm long, utilizing the compression molding device discussed. The resulting
cylinders had a
solvent content of from about 30% to about 60%. These cylinders were cut into
5 equal pellets.
From initially added quantities, each pellet was calculated to contain
approximately 2.4 mg
capsaicin and 0.24 mg dexamethasone, weighing approximately 30 mg each.
Example 5: Preparation of an Injectable Drug Delivery Device Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
Injectable drug delivery devices comprising a biodegradable protein, an
additive and an
analgesic were made as follows. The opioid analgesic, sufentanil citrate
(obtained from National
Institute on Drug Abuse) was desalted by adding ammonium hydroxide and
extracted with
n-hexane, collection of solvent and evaporation. The desalted sufentanil was
reconstituted in 20
l of 90% ethanol containing approximately 4,500,000 cpm of tritiated
sufentanil (obtained from
Jannsen Pharmaceutica, Belgium) to 2.0 mg/20 l. The biodegradable protein
SELP7 was
dissolved in deionized water to 20 mg SELP7/30 gl and spread into a thin layer
approximately 5
cm by 5 cm in area. Immediately thereafter, 10 mg of finely pulverized powder
of an additive,
fatty acid dimer:sabacic acid (FAD:SA in 1:4 ratio), was added to the center
of the protein
solution area. Immediately thereafter, the sufentanil dissolved in the ethanol
was added very
slowly to the mound of FAD:SA over a time period of a few minutes, i.e., from
about 1 to about
5 minutes. After the sufentanil solution had soaked into the FAD:SA powder,
the components
were thoroughly mixed to form a coatable composition. The composition was then
coated onto a
glass surface to form a film with a thickness of approximately 0. 1 - 0.2 mm.
The film was
allowed to dry at room temperature until capable of forming a cohesive body,
i.e., to a solvent
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content of from about 50% to about 70%. The resultant was rolled up and cut
into many small
pieces. Each piece was placed in a 0.63 mm diameter mold and compressed at
3,000 psi for 2
minutes to form 0.63 mm diameter cylinders, approximately 1.5 mm long and
weighing about
0.85 mg to 1.05 mg, utilizing the compression molding device discussed
hereinabove. The drug
delivery devices were then exposed to gamma irradiation (60-90 KRads) for
sterilization and
stored in a refrigerator (4 C) until used within 8 weeks.
Example 6: Preparation of an Implantable Drug Delivery Device Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
Implantable drug delivery devices comprising a biodegradable protein, an
additive and an
analgesic were made as follows. The opioid analgesic sufentanil citrate
(obtained from National
Institute on Drug Abuse), was desalted by adding ammonium hydroxide, extracted
with
n-hexane, collection of solvent and evaporation. The desalted sufentanil was
reconstituted in 20
gl of 90% ethanol containing approximately 4,500,000 cpm of tritiated
sufentanil (obtained from
Jannsen Pharmaceutica, Belgium) to 2.0 mg/20 l. The biodegradable protein
SELP7 was
dissolved in deionized water to 42.3 mg (SELP7)/200 l and spread into a thin
layer
approximately 6 cm by 6 cm in area. Immediately thereafter, 22.5 mg of finely
pulverized
powder of an additive, the fatty acid dimer:sabacic acid (FAD:SA in 1:4 ratio)
was added to the
center of the protein solution area. Immediately thereafter, the sufentanil
dissolved in the ethanol
was added very slowly to the mound of FAD:SA over a period of a few minutes,
i.e., from about
1 minute to about 5 minutes. After the sufentanil solution had soaked into the
FAD:SA powder,
the components were thoroughly mixed to form a coatable composition. The
composition was
then coated onto a glass surface to form a film with a thickness of
approximately 0. 1 - 0.2 mm.
The film was allowed to dry at room temperature until capable of forming a
cohesive body, i.e.,
to a solvent content of from about 50% to 70%. The resultant cohesive body was
rolled up and
placed in a 3.5 mm diameter mold and compressed at 8500 psi for 2 minutes to
form a 3.5 mm
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diameter cylinder, approximately 4 mm long and weighing 54.1 mg, utilizing the
compression
molding device discussed hereinabove. This device was then exposed to gamma
irradiation
(60-90 KRads) for sterilization and stored in a refrigerator (4 C) until used
within 8 weeks.
5 Example 7: Preparation of an Implantable Drug Delivery Device Comprising,
Biodegradable Protein, an Additive and an Opioid Analgesic
Implantable drug delivery devices comprising a biodegradable protein, an
additive and an opioid analgesic were made as follows. The opioid analgesic
sufentanil citrate
10 (obtained from National Institute on Drug Abuse) was desalted by adding
ammonium hydroxide,
extracted with n-hexane, collection of solvent and evaporation. The desalted
sufentanil was
reconstituted in 20 gl of 90% ethanol containing approximately 3,500,000 cpm
of tritiated
sufentanil (obtained from Jannsen Pharmaceutica, Belgium) to 2.0 mg/20 l. The
biodegradable
protein SELP7 was dissolved in deionized water to 15 mg (SELP7)/200 gl and
spread into a thin
15 layer approximately 6 cm by 6 cm in area. Immediately thereafter, 35.0 mg
of finely pulverized
powder of the additive glutamine, was added to the center of the protein
solution area.
Immediately thereafter, the sufentanil dissolved in the ethanol was added very
slowly to the
mound of glutamine over a time period of a few minutes. After the sufentanil
solution had
soaked into the glutamine powder, the components were thoroughly mixed to form
a coatable
20 composition. The composition was then coated onto a glass surface to form a
film with a
thickness of approximately 0.1-0.2 mm. The cast film was allowed to dry at
room temperature
until capable of forming a cohesive body, i.e., to a solvent content of from
about 50% to 70%.
The resultant cohesive body was rolled up and placed in a 3.5 mm diameter mold
and
compressed at 8500 psi for 2 minutes to form 3.5 mm diameter cylinders,
approximately 2 mm
25 long and weighing 39.1 mg, utilizing the compression molding device
discussed hereinabove.
This device was then exposed to gamma irradiation (60-90 KRads) for
sterilization and stored in
a refrigerator (4 C) until used within 8 weeks.
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Example 8: Preparation of an Implantable Drug Delivery Device Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
Implantable drug delivery devices comprising a biodegradable protein, an
additive and an
analgesic were made as follows. The opioid analgesic sufentanil citrate
(obtained from National
Institute on Drug Abuse), was desalted by adding ammonium hydroxide, extracted
with n-hexane,
collection of solvent and evaporation. The desalted sufentanil was
reconstituted in 20 d of 90%
ethanol containing approximately 4,500,000 cpm of tritiated sufentanil
(obtained from Jannsen
Pharmaceutica, Belgium) to 2.0 mg/20 l. The biodegradable protein SELP7 was
dissolved in
deionized water to 42.3 mg (SELP7)/200 gl and spread into a thin layer
approximately 6 cm by 6
cm in area. Immediately thereafter, 22.5 mg of finely pulverized powder of an
additive, the fatty
acid dimmer:sabacic acid (FAD:SA in 1:4 ratio) was added to the center of the
protein solution
area. Immediately thereafter, the sufentanil dissolved in the ethanol was
added very slowly to
the mound of FAD:SA over a period of a few minutes, i.e., from about 1 minute
to about 5
minutes. After the sufentanil solution had soaked into the FAD:SA powder, the
components
were thoroughly mixed to form a coatable composition. The composition was then
coated onto a
glass surface to form a film with a thickness of approximately 0.1-0.2 mm. The
film was
allowed to dry at room temperature until capable of forming a cohesive body,
i.e., to a solvent
content of from about 50% to 70%. The resultant cohesive body was rolled up
and placed in a
3.5 mm diameter mold and compressed at 8500 psi for 2 minutes to form 3.5 mm
diameter
cylinders, approximately 4 mm long and weighing 54.1 mg, utilizing the
compression molding
device discussed hereinabove. This device was then exposed to gamma
irradiation (60-90
KRads) for sterilization and stored in a refrigerator (4 C) until used within
8 weeks.
Example 9: In vitro Experiment with a Drug Delivery Device Comprising a
Biodegradable Protein and an Enzyme
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A single cylinder piece, prepared as described above in Example 1, was added
to a reaction
chamber in a spectrophotometer containing xanthine, cytochrome C and other
reactants
according to previously described superoxide dismutase protocol (Sigma Quality
Control Test
Procedure EC 1.15.1.1 "Enzymatic Assay of Superoxide Dismutase") enzyme
activity of the
enzyme xanthine oxidase in the piece was calculated at 0.0005 delta absorbance
min (absorbance
measured at 550 mm where no enzyme activity produces 0.00000 change in
absorbance). In
comparison to a 0.01 unit solution of xanthine oxidase, which produced 0.0250
delta
absorbance/min, the activity of the xanthine oxidase in the piece equaled 1%
of the control
solution in a time period of only 3 minutes. Thus, this result indicates that
the diffusional barrier
provided by the biodegradable polymeric matrix of the drug delivery device
allows the enzyme
to remain active from within the drug delivery device.
Example 10: In Vitro Experiment with a Drug Delivery Device Comprising a
Biodegradable Protein and an Enzyme
In this assay system, xanthine oxidase, xanthine, cytochrome C and other
reactants were
added together to produce a delta absorbance of 0.0250/min. (Sigma Quality
Control Test
Procedure EC 1.15.1.1 "Enzymatic Assay of Superoxide Dismutase"). SOD activity
is measured
as the inhibition of the rate of reduction of ferricytochrome C by superoxide,
observed at 550
nm, as described by J. McCord, I. J. Biol Chem., 244, 6049 (1969). The
addition of a SOD
containing piece, produced as described in Example 2 hereinabove, reduced the
reaction to
0.0233 delta absorbance/min. Since 1 unit SOD will inhibit the reaction of
cytochrome C by
50% in a coupled system using xanthine oxidase, it can be determined that the
activity of the
SOD pellet equaled 0.14 units of SOD. This activity represents about 2% of the
SOD loaded
into the biodegradable protein matrix of the drug delivery device. Thus, this
result indicates that
the diffusional barrier provided by the biodegradable polymeric matrix of the
drug delivery
device allows the enzyme to remain active from within the drug delivery
device.
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Example 11: In Vivo Experiment with a Drug Delivery Device Comprising a
Biodegradable Protein and Lipospheres
The drug delivery devices comprising a biodegradable protein and lipospheres
produced
according to Example 3 hereinabove were surgically implanted next to the
sciatic nerve of one
young adult male Sprague Dawley rat (200-250 g) as described previously by
Masters in D.B.
Masters et al., Anesthesiol., 79, 340 (1993). Briefly, the rat was
anesthetized with 50-75 mg/kg
pentobarbital to allow faster recovery for behavioral measurements. Bilateral
posterolateral
incisions were made in the upper thighs and the sciatic nerves were visualized
with care to avoid
direct trauma. Drug delivery devices prepared as described in Example 3 were
injected around
the nerve on one leg, while no drug delivery device was inserted in the
contralateral leg to serve
as a control. The fascia and muscle surrounding the administration site was
closed over to
partially restrict egress of the drug delivery device and the entire wound
area was lavaged with
0.5 cc of an antibiotic solution (5000 units/ml penicillin G sodium and 5000
gl/ml streptomycin
sulfate). The experimenter performing subsequent thermal sensitivity testing
and motor capacity
tests was unaware of which side received the drug delivery device and which
side received
nothing.
After having the drug delivery device implanted, the rat was subjected to
periodic thermal
sensitivity and motor capacity testing according to the protocol described
above. As shown in
Table 1, the drug delivery devices so implanted produced at least 4 days of
local anesthetic
block, i.e., a reduction in thermal sensitivity with a concurrent reduction in
motor capacity tests
compared to the control leg.
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Table 1
In vivo local anesthetic block produced by a drug delivery device comprising
lipospheres (they themselves break down within the matrix)
Time (hr) Thermal Sensitivity Tests Motor capacity (weight bearing)
0 100% 5% 100% 2%
2 427% 41%
4 560% 44%
20 196% 56%
26 216% 62%
42 195% 79%
48 180% 77%
96 126% 75%
120 105% 76%
Example 12: In Vivo Experiment with a Drug Delivery Device Comprising a
Biodegradable Protein and two Pharmacologically Active Agents
Three "first drug delivery devices" prepared according to Example 4, i.e.
comprising 6
mg of capsaicin and 6 mg dexamethasone were implanted next to the sciatic
nerve of one young
adult male Sprague Dawley rat using the procedures described above in Example
7. The rat was
monitored for a period of 624 hours. The results of this experiment are shown
in Table 2, below.
The first drug delivery devices produced strong thermal sensitivity, but no
reduced motor
capacity, for 6 days. Because the rat showed some weight loss, the devices
were removed on day
6. The rat continued to show a strong reduction in thermal sensitivity for the
next 14 days before
returning to baseline response levels. In comparison to the contralateral
control leg, no reduced
motor capacity was detected. Therefore, a very strong sensory neural blockade
(analgesia) was
obtained by placement of these matrices without associated motor deficits.
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Table 2
In vivo local anesthetic block produced by a drug delivery device
incorporating 6 mg
Capsaicin and 6 mg Dexamethasone
5 Time (hr) Thermal Sensitivity Test Motor Capacity (weight bearing)
(experimental/control)
-48 0.98 nd
-24 0.98 0.99
-1 1.02 1.01
10 2 2.47 nd
4 2.04 0.97
24 1.80 0.95
48 2.72 1.01
96 1.94 0.86
15 144 2.86 0.91
168 2.34 0.97
192 2.19 0.99
216 3.04 1.00
264 2.59 1.00
20 288 1.76 1.05
312 1.58 0.99
318 2.55 0.99
336 2.06 1.01
360 1.65 0.98
25 384 1.65 0.99
432 2.16 0.99
456 1.35 1.01
480 0.92 0.99
504 1.10 1.01
30 528 0.98 1.02
552 1.38 1.00
624 1.07 1.01
Five "second drug delivery devices," i.e., comprising 6 mg of capsaicin and
1.2 mg
35 dexamethasone, prepared as described above in Example 4 were implanted next
to the sciatic
nerve of individual rats, where they produced a strong reduction in thermal
sensitivity with no
concurrent reduction in motor capacity for several days to weeks. All 5 rats
showed some
weight loss, but far less than that observed with implantation of the first
devices.
The results of this experiment are shown in Table 3, below. As shown, a very
strong
40 reduction in thermal sensitivity was obtained by implantation of these
devices without a
concurrent reduction in motor capacity. As is shown, all rats showed similar
effects with various
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durations, i.e., no rats showed motor deficits. Lower doses of capsaicin and
dexamethasonc
showed similar results.
Table 3
In vivo local anesthetic block produced by a drug delivery device
incorporating 6 mg
Capsaicin and 1.2 mg Dexamethasone
Time (hr) Thermal Sensitivity Tests Motorcapacity- veightbearin
-48 1.13 1.00
-24 0.96 0.99
-1 1.02 1.02
2 2.72 1.02
4 3.77 1.00
24 2.50 1.17
48 2.86 1.00
96 2,72 0.96
120 1.78 1.01
144 3.05 1.01
168 2.06 0.98
192 1.82 1.00
216 1.74 1.03
288 3.14 1.00
312 2.88 1.00
336 2.17 1.01
360 1.83 0.99
456 1.33
480 1.22 0.99
504 1.85 1.01
528 1.72 0.99
552 1.92 1.01
624 2.42 0.99
672 2.13 0.97
792 1.50 1.01
840 1.24 0.99
888 1.49 1.01
984 1.36
Example 13: In Vitro Experiment with au Injectable Drub; Delivery Device
Comprising a Biodegradable Protein, an Additive and an Opioid Analgesic
Four pellets, prepared as described in Example 5, were each added to separate
glass vials treated with a silicone coating (commercially available under the
trade
designation "Sigmacote1M" from Sigma Chemical Company, St. Louis, Missouri) to
prevent loss of tritiated sufentanil. The pellets were added to the glass
vials filled with 15)
mI of 0.1 M phosphate buffered saline (pH
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7.4), and then were incubated at 37 C with agitation. At specific time
intervals, 20 l samples
were taken in triplicate from each glass vial and measured for radioactive
sufentanil using a
scintillation counter. As shown in Figure 8, each of the four matrices
produced at least 9 days of
sufentanil release following a first order release rate.
Example 14: In Vitro Experiment with an Implantable Drug Delivery Device
Comprising a Biodegradable Protein, an Additive and an Opioid Analgesic
A single pellet, prepared as described in Example 6 was added to a glass
vial treated with a silicone coating (commercially available under the trade
designation
"Sigmacote" from Sigma Chemical Company, St. Louis, Missouri) to prevent loss
of
tritiated sufentanil. The glass vial was filled with 15 ml of 0.1 M phosphate
buffered saline
(pH 7.4), and incubated at 37 C with agitation. At specific time intervals, 20
gl samples
were taken in triplicate and measured for radioactive sufentanil using a
scintillation counter. As
shown in Table 4, this 3.5 mm diameter cylinder matrix produced at least 75
days of sufentanil
release following near zero-order release rate kinetics.
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Table 4
In Vitro Release Study of Implantable Drug Delivery Device Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
Time (hr) Scintillation Counts (cpm) Cumulative Release (%)*
1 59050 1.48
4 26883 3.17
228667 5.72
10 28 263650 6.59
49 415150 10.38
73 455000 11.38
120 561517 14.04
200 583333 14.58
251 619283 15.48
299 653517 16.34
428 751517 18.79
603 901483 22.54
793 1281183 32.03
1030 1645650 41.14
1199 1810450 45.26
1368 2093083 52.33
1536 2532467 63.31
1704 3205867 80.15
1899 3446133 86.15
2003 3528650 88.22
2239 3689717 92.24
*Based on total expected counts = 4,500,000
Example 15: In Vitro Experiment With An Implantable Drug Delivery Device
Comprising a Biodegradable Protein, an Additive and an Opioid Analgesic
A single pellet, prepared as described in Example 7 was added to a glass vial
treated with
a silicone coating (to prevent loss of tritiated sufentanil commercially
available under the trade
designation "Sigmacote," from Sigma Chemical Company, St. Louis, Missouri).
The glass vial
was filled with 15 ml of 0.1 M phosphate buffered saline (pH 7.4), and
incubated at 37 C with
agitation. At specific time intervals, 20 gl samples were taken in triplicate
and measured for
radioactive sufentanil using a scintillation counter. As shown in Table 5,
this 3.5 mm diameter
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cylinder matrix produced approximately 2 days of sufentanil release. The
addition of glutamine
facilitated the release of sufentanil out of the matrix.
Table 5
In Vitro Release Study of Implantable Drug Delivery Device Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
Time (hr) Scintillation Counts (cpm) Cumulative Release
1 59050 1.48
2 671133 19.29
4 1495667 43.00
10 2230283 64.11
28 .2908267 83.61
49 3346450 96.20
73 3422867 98.40
120 3439183 98.87
200 3430783 98.63
leftover cpm in pellet 47792
*total cpm 3478575
Example 16: In Vivo Experiment with Drug Delivery Devices Comprising a
Biodegradable Protein, an Additive and an Opioid Analgesic
The drug delivery devices comprising a protein (SELP7), an additive (FAD:SA),
and an
opioid analgesic (sufentanil), produced according to Example 5 hereinabove,
were injected into
the left side of the epidural space adjacent to spinal cord at the fifth
lumbar vertebrae in 2 young
adult male Sprague Dawley rats. All rats underwent pre-testing for thermal
sensitivity tests and
motor capacity tests as described hereinabove. The rats were anesthetized with
halothane (4%
induction, 2% maintenance) and prepared for spinal injection by creating a
sterile surgical field
over the dorsal aspect of the lower lumbar vertebral column. The placement of
the drug delivery
devices was in close proximity to the left dorsal root ganglion and nerve root
at lumbar level 5,
which is associated with nerve input from the left hind paw via the sciatic
nerve. After needle
insertion validation, drug delivery devices were loaded into an 18 gauge Tuohy
epidural
needle for injection, most commonly used by anesthesiologists for spinal
administration of
drug solutions. Before injection of the implants into the epidural space,
validation of the
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space was carried out by x-ray techniques to locate the tip of the needle
using an opaque
catheter and small x-ray machine. Aspiration of the space occupied by the
catheter was
also used to validate that it was in the dry epidural space and not the
subdural space which
is filled with cerebrospinal fluid. The dosage delivered from the drug
delivery devices was
5 adjusted by administering more than one implant into the epidural space. To
test for a dose
response effect, rat F043 received two drug delivery devices containing
sufentanil and rat
F045 received 6 drug delivery devices containing sufentanil. In this
experiment a third rat,
F046, was used as a control and received two control devices via the same
epidural
administration technique. The control devices were made by the same coatable
composition
10 technique using the same quantities of biodegradable protein (SELP7),
additive FAD:SA,
deionized water and ethanol without the presence of sufentanil. The results of
this experiment
are shown in Table 6, below, where time is in hours relative to epidural
administration of the
drug delivery devices. Rats F043 and F045 showed prolonged opioid analgesia
for
approximately 9-12 days in thermal sensitivity tests, performed as described
hereinabove, i.e.,
15 increased latency (seconds) to remove their paws from a heated surface.
Epidural injections of
sufentanil citrate at highest possible doses without becoming toxic (5-7
g/kg), only produced 2
hours of measurable effects to thermal sensitivity testing in three control
rats.
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Table 6
In Vivo Thermal Sensitivity Latency Tests for Drug Delivery Devices Comprising
a
Protein, and a Polyanhydride Copolymer With and Without an Opioid
Time hr F043 (2 devices) F045 (6 devices) F046(2 control devices)
Left Paw Right Paw Left Paw Right Paw Left Paw Right Paw
-48 2 1.8 1.9 2 2.5 2.4
-24 2.4 2.1 2 2 2.7 2.3
-1 1.8 1.9 1.9 2.1 2.2 2.2
1 2.8 2.4 12 3.7 3.5 3.2
4 3 2 5.6 2.7 2.9 2.7
22 2.8 2.1 5.7 2.9 2.3 2.2
46 3.4 2.1 8.4 3 2.3 2.4
74 2.9 2.1 7.1 2.5 nd nd
119 2.8 1.8 6.8 2.4 2.5 2.4
144 nd nd 9.7 2 2.4 2.3
166 nd nd 7.5 2.2 2.5 2.4
189 2.7 1.9 10.1 2.1 nd nd
211 3.1 2.1 5.6 2.5 nd nd
289 2.9 2.3 2.6 1.8 nd nd
314 2.8 2 2.3 1.9 nd nd
337 2.5 1.8 1.9 1.9 nd nd
391 3 2.1 1.8 1.9 nd nd
435 2 2.1 1.8 1.7 nd nd
457 2.1 1.8 1.8 1.9 nd nd
482 nd nd 2 2 nd nd
*nd = not determined; Testing was stopped after rat returned to pre-device
response level.
Example 17: Experiment with Drug Delivery Devices Comprising a
Biodegradable Protein Matrix that includes a Controlled Release Mechanism
Two types of drug delivery devices were prepared by compressing crystals of
Blue
dextran or Gadolinium gadopentetate dimeglumine (Gd-DPTA) (Magnevist) in a
polyanhydride
copolymer of a 5:1 fatty acid dimer of erucic acid to sebacic acid and then
coated by the same
copolymer to produce an insert. Following production of the insert, the insert
was encapsulated
by compression molding in a protein matrix of collagen. The blue dextran or Gd-
DPTA & MRI
was utilized to verify that the ultrasound triggered device was releasing its
drug agents. Each
drug delivery device had a diameter of 6 mm and a length of 7 mm. The drug
delivery device
including blue dextran was submerged in water and held in place with
monofilament. Once
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positioned in water, the drug deliver device was triggered by a focused
ultrasound pulse of 50
watts for 5 seconds and was visually observed. Figure 18 is a before and after
depiction of the
drug delivery device that includes a release mechanism. The top panel of
Figure 18 is an end and
side view of the drug delivery device before ultrasound triggering of the blue
dextran polymer
insert. The bottom panel is a view of the drug delivery device after
ultrasound triggering.
Figures 19 and 20 depict the ultrasound triggering of a drug delivery device
including a
Gd-DPTA copolymer insert. Figure 19 is an illustration of two Gd-DPTA drug
delivery devices
contained in an agar gel positioned 5 mm apart. The figure depicts the
triggering of the targeted
drug delivery device with a focused ultrasound pulse of 50 watts for 5
seconds. The Gd-DPTA
was observed by Magnetic Resonance Imaging. The Gadolinium is shown to release
from the
drug delivery device in greater amounts over time.
Figure 20 illustrates a time progression depiction of a drug delivery device
including a
Gd-DPTA copolymer insert that has been triggered by a focused ultrasound pulse
of 50 watts
for 5 seconds. The first frame at 0 min is taken immediately before the
ultrasound pulse. The
following frames sequentially illustrate the release progression of the Gd-
DPTA into the agar
gel.
Example #18: Preparation of Collagen:Elastin (4:1 ratio) tubular grafts:
In the preparation of the vascular tubes, Collagen:Elastin was used in a 4:1
ratio and
mixed with sterilized saline in amount equal to 600% the weight of the
combined collagen and
elastin (e.g., 80mg collagen + 20mg elastin in 600microliters of water). The
material was mixed
together and immediately thereafter, the pH was adjusted with drops of 0.1N
and 0.5N NaOH
until pH indicator strips read 7.4 pH. The material was then partially dried
at room temperature
until it was to a state where it was cohesive unto itself and was- then
subsequently formed into a
cohesive body. The cohesive body was loaded into the mold were a mandrel
insert would
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receive the cohesive body as mechanically applied pressure forced the cohesive
body over the
mandrel with a final pressure equal to 5,000 psi for a period of 10 minutes.
The result was the
formation of a tube around the mandrel where the tube wall thickness was 0.2
mm and the length
of the tube was 1 cm. While the protein matrix tube was still on the mandrel
insert, it was
submersed in 1 % glutaraldyhde solution for 2 minutes, resulting in partial
cross linking of the
outside of the tube. After 2 minutes, the tube-mandrel insert was submersed in
saline for 1
minute then it was subjected to a 15 minute submersion in a 0.1 M phosphate
buffered saline
solution containing 1% glutamine and 1% glycine. The tube was then slipped off
the mandrel,
where the mandrel was made with a slope of .001 inches over the 1 cm length to
ease the
removal of the protein matrix tube. Also, before the mandril was placed in the
mold it was
coated with a slippery substance (e.g., glycol or Triton-X100). Finished tubes
were stored in
saline and sterilized with 10-20 KRADS of gamma irradiation from a cesium
source.
The following table includes vascular tubes with various compositions prepared
by
following the procedure described above.
Composition Ratio Solvent pH Pressure Len./Dia.*/Wall Cross-Linking
A) collagen:elastin (4:1) saline 7.4 15000 psi Icm/2nun/0.2mm outside surface
B) collagen:elastin (4:1) saline 7.4 15000 psi lcm/2mm/0.2mm none
C) collagen:elastin (4:1) saline 7.4 15000 psi lcm/2.4mm/0.2mm inside tube
D) collagen:elastin (4:1) 9%NaCI 7.4 15000 psi 1cn /2mm/0.2mm outside surface
E) collagen:elastin:heparin (4:1:1) saline 7.4 15000 psi lcm/2mm/0.2nun
outside surface
F) collagen:elastin (1:1) saline 7.4 15000 psi lcm/2mm/0.2mm outside surface
G) heparin:elastin:collagen (1:4:15) saline 7.4 15000 psi lcm/2nun/0.2mm
outside surface
H) elastin:albumin:collagen (4:1:1) saline 7.4 15000 psi 1cm/2nun/0.2mm
outside surface
1) collagen (1) saline 5 15000 psi lcm/2mm/0.2mm outside surface
J) chondroitin :elastin:collagen: (1:4:15) saline 7.4 15000 psi 1cm/2nun/0.2mm
outside surface
K) collagen: albuniin:elastin (2:2:1) saline 7.4 15000 psi lcm/2mm/0.2mm
outside surface
L) collagen: albuniin:elastin (2:1:2) saline 7.4 15000 psi 1cm/2.4nun/0.2mm
inside surface
M) collagen: albumin:elastin:glutamine (2:2:1) saline 5 15000 psi 1cn
/2mm/0.2mm outside surface
N) elastin:albumin (1:3) saline 7.4 15000 psi lcm/2mm/0.2mm outside surface
diameter of interior of tube
Example #19: Endothelial cell seeding of tubular vessels:
For this experiment, the protein tubes were produced by the method described
in method
#18. The endothelial used in the culture are human umbilical vein endothelial
cells (HUVEC).
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The tubes were seeded with these cells in order to obtain a confluent
endothelial monolayer
within the lumen of the protein tubes. To obtain a high-density culture, the
tubes were first
cultured with these cells using standard culturing techniques that are known
in the discipline.
The cells were cultured on a plastic dish that is two times lager than the
surface area of the
protein tube's lumen. Next, the cells were detached from the culture dish
using a trypsin/EDTA
solution obtained from ICN Pharmaceuticals, Inc. The cells are then seeded
into the lumen of
the protein tube. Four hours after seeding, the nonattached cells were be
removed. Tubes were
then incubated at 37 C under 5% C02 and 95% air atmosphere in a standard
solution of DMEM.
The medium was replaced at least every other day for 4-7 days. Cells have been
found to adhere
and grow to a confluent monolayer on tubes made of collagen and elastin (4:1
ratio), 100%
collagen, and heparin:elastin:collagen (1:4:15).
Example #20: Preparation of Wound Healing Device(Tissue graft; wafer)
Dried bovine type I collagen (ICN Biomedicals, Aurora, OH) was solubilized
using
vitrogen and distilled water added in a dropwise manner. Vitrogen was
continually added to
ensure that the collagen did not dry out before all of the collagen had
solubilized. Once the
collagen had dissolved, the mixture was allowed to dry until it attained a
cohesive state. The
collagen was then rolled into a cylinder and placed in a brass mold between
two stainless steel
inserts. The collagen cylinder was then compressed at 5700 psi for 10 minutes
using a
pneumatic press. The cylinder was removed and divided into wafers using a
razor blade: Wafers
were approximately 0.5 mm thick and were 6 mm in diameter unless otherwise
stated. Wafers
were then recompressed using the pneumatic press for 10 minutes at either 5740
or 28700 psi
(henceforth referred to as low and high pressure, respectively). Some wafers
were then removed
from the brass mold and stored at 4 C until they were crosslinked. After
crosslinking and prior
to use in cell culture experiments, all wafers were sterilized using a Cesium
irradiator. Figure 21
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is a magnified view of a noncrosslinked wafer after it has been incubating
overnight in phosphate
buffered saline. Figure 22 is a magnified view of a crosslinked wafer after it
has been incubating
overnight in phosphate buffered saline.
5 Example #21: Glutaraldehyde Crosslinking Wound Healing Device(Tissue graft;
wafer):
A 1% glutaraldehyde solution (Sigma, St. Louis, MO) was used for crosslinking
wafers.
A single wafer was incubated for 1, 3, 5, 15, or 30 minutes in 1 ml of 1%
glutaraldehyde solution
in 1X PBS. Samples were then washed in 1 ml of 1 PBS for 10 minutes. This
washing
procedure was repeated two more times. A revised washing protocol was
developed in light of
10 evidence that the cells were dying due to cytotoxic effects of
glutaraldehyde. In this new
process, glutaraldehyde was removed from the samples and then wafers were
transferred to a
clean plastic tube. They were then washed in 5 ml of 1X PBS for 4 hours. The
PBS was
removed and 5 ml of fresh 1X PBS was added for a second washing for 8 hours
(overnight). The
PBS was again removed and the wafers were washed for 2 hours prior to cell
seeding in a
15 modified 1X PBS solution, which consisted of 1 mM glycine, and 1:100
dilution of vitrogen.
This last wash was intended to bind up any residual glutaraldehyde and thereby
eliminate the
cytotoxic effects of free glutaraldehyde. Collagen wafers that did no undergo
crosslinking were
washed in the same buffers and used as controls.
20 Example #22: Mechanical Testing System (MTS) of Protein Matrix Material:
MTS Testing:
Six wafers from each experimental group were tested to determine structural
and
mechanical properties. Sample thickness was measured using a Fowler micrometer
(accurate to
0.1mm). Cross-sectional areas were calculated by assuming a rectangular cross-
section. The
25 UTS and modulus (slope of the stress-strain curve) were determined from the
stress-strain curves
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of the collagen wafers. Stress was calculated by dividing the force by initial
cross-sectional area.
Stress-strain curves for wafers were determined an MTS Microbionix
biomechanical tester
controlled by TestStar/TestWare software. Wafers were tested using a gauge
length of 0.5 mm
and a strain rate of 0.8 mm/s after rehydration for 10 minutes in phosphate
buffered saline. The
instrument was operated in a dynamic mode at room temperature. The wafers were
removed
from the solution immediately before testing and mounted onto the screw
clamps. A wafer was
mounted using two parallel screw clamps such that each clamp secured a segment
of the wafer
with a gauge length of 0.5 mm. The clamps were connected to the actuator and a
5-Newton
force transducer of the MTS Microbionix testing system allowing continuous
measurement of
the stress response to a constant strain rate in the radial direction in
extension by separating the
screw clamps at a constant speed. Stress was calculated by dividing the force
generated during
extension by the initial wafer cross-sectional area (approximated by
multiplying wafer thickness
by the wafer diameter). Strain was calculated as the natural log of the ratio
of the extended
distance over the gauge length. The Young's modulus was determined by
measuring the slope
of the stress/strain curve between strains of 0.2 and 0.8. Ultimate Tensile
Strength (UTS)
represents that largest stress value sustained by the wafer during testing.
Crosslinking and Pressure Effects on the Mechanical Properties of Collagen
Devices:
Young's modulus and UTS were assessed used to characterize the mechanical
properties
of the collagen DDS. An increase in Young's modulus was seen as the duration
of
glutaraldehyde crosslinking increased for both low (5700 psi) and high (28,700
psi) psi
compressive loads (Figure 23). For the low psi wafers the increase was
significant between 0
and 3, 0 and 15, and 0 and 30, 1 and 15, 1 and 30, 3 and 30, 5 and 30 minutes,
and 15 and 30
minutes based on ANOVA analysis. For the high psi wafers, the increase was
significant
between all paired time points except 1 and 3, 1 and 5, and 3 and 5 minutes.
In addition, there
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was no significant difference between the high and low psi systems at any of
the crosslinking
times based on ANOVA analysis.
The UTS of the collagen systems also increased as the length of crosslinking
time
increased for both the low psi and high psi load levels (figure 24). For the
low psi wafers this
difference was significant between all pairs of time points except 1 and 3, 1
and 5, 1 and 15, 3
and 5, and 3 and 15 minutes. For the high psi wafers, the increase was
significant between all
pairs of time points except 1 and 3 minutes based on ANOVA analysis. There was
no significant
difference between the low psi and high psi system at any of the crosslinking
times.
Example #23: Dissolution of Collagen Protein from Collagen Wafers:
Figures 25-28 depict the results of tests performed regarding dissolution of
collagen from
collagen wafers made with medium (12,000 psi), high (20,000 psi) and high
(28,000 psi)
pressures in a compression chamber and with various amounts of crosslinking.
The wafers were
crosslinked with 1% glutaraldehyde for 0, 1, 10, and 30 minutes corresponding
to Figures 25-28,
respectively. The collagen wafers were analyzed by placing them in phosphate
buffered saline in
a 15 ml conical Falcon tube (pH 7.4, 37 Q. The Falcon tube was then place in a
shaking
incubator at 37 _ C and set to slow agitation. At various time points samples
of the solution were
tested by BCA protein assay (Pierce Company) for protein content and recorded.
Example #24: Mechanical Testing System (MTS) of Protein Matrix Material
(Vascular
Tubes :
MTS Testing:
A vascular tube was tested to determine structural and mechanical properties.
Sample
thickness was measured using a Fowler micrometer (accurate to 0.lmm). Stress-
strain curves for
tubes were determined an MTS Microbionix biomechanical tester controlled by
TestStar/TestWare software. The tube was wet with a phosphate buffered saline.
The
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instrument was operated in a dynamic mode at room temperature. The tube was
mounted onto
prongs made to fit the inside diameter of the tube. The prongs were mounted to
the actuator and
a 5-Newton force transducer of the MTS Microbionix testing system allowing
continuous
measurement of the stress response to a constant strain rate in the radial
direction in extension by
separating the prongs at a constant speed. Stress was calculated by dividing
the force generated
during spreading of the tube walls (approximated by multiplying wall thickness
by the tube wall
diameter). Strain was calculated as the natural log of the ratio of the
extended distance over the
gauge length. Ultimate Tensile Strength (UTS) represents that largest stress
value sustained by
the wafer during testing. The UTS that resulted was equal to 192.6 mmHg.
Example #25: Mechanical and Hydraulic Testing System (MTS) of Protein Matrix
Material (Tubular Grafts):
A vascular tube was prepared as described above using a mixture of
collagen: albumin:elastin (ratio 2:2:1) (pH 7.4; 2mm inner diameter). Figure
29 depicts an
embodiment of the vascular tube. The tube was placed over polyethylene hose,
tied with silk
suture material and cemented with adhesive. The tube was then visually tested
for durability and
compliance by twisting. Figure 30 depicts the vascular tube tested for
durability and
compliance. Figure 31 depicts both sides of a vascular tube tested for
hydraulic pressure. The
polyethylene hose was attached to a Tygon S-50-HL class V1 hose that was
attached to a
peristaltic pump that circulated phosphate buffered saline (PBS) through the
hose and tube at 3.5
ml /min. It was found that back pressure of over 200 mm Hg could be generated
several times
without damaging the vascular tube. Figure 32 depicts, at the arrows, the
vascular tube bulging
in response to over 200 mm Hg back pressure. Figure 32 also illustrates that
the back pressure
could have been greater, but for leakage occurring at the vascular tube and
polyethylene hose
junction. In similar replicate tubes, it was found that no leaking occurred
after 72 hours of
constant circulation of PBS fluid.
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Example #26 Preparation of Poly(vinyl-alcohol)(PVA) Particles in Protein
Matrix Wafers:
In this study PVA super hydrolyzed (99.3 % M.W. 106,000-110,000, viscosity of
4%
aqueous solution 55-65 cps at 20 C) and recombinant human epidermal growth
factor (hEGF)
(R&D System) were used. A 4% solution of PVA (J.T.Baker) in distilled water
was dissolved 1
hour at 85 C and added to a hEGF solution, which was dissolved into the
distilled water (50
gglml) and dried at 40 C at vacuum oven over night. The film was pulverized
and then sieved to
separate EGF- PVA particles into various groups by size. The size of final
particles was 250-500
gm in diameter.
Formulation of protein matrix containing collagen:
Collagen (80 mg) (Type I, calf skin) (ICN Biomedicals Inc.) was dissolved in
700 RI
vitrogen and 200 l distilled water and spread and dried entirely on glass
plate until spread
protein became cohesive. Once cohesive body was formed the EGF-PVA particles
(6mg) were
added to the cohesive body and rolled into a cylinder and made into a protein
matrix wafer form
by compression molding at 2000 psi. The wafers were cross-linked for 0, 3, 15,
30, or 60 minute
in a 1% glutaraldehyde solution and subsequently rinsed 3 times in a 5 ml
buffer solution (PBS)
for three minute each time. Then EGF - PVA particles and cross-linked and non-
cross-linked
matrices were sterilized 30 minute by Cesium-137 irradiation (> 10K RADS).
Release study:
EGF - PVA particles, cross-linked wafers and non- cross-linked wafers were
incubated
on the thermal rocker at 37 C in 1 ml of PBS or William's E medium solution.
One ml samples
were collected and replaced with fresh medium solution from each tube at 1, 4,
8, 24, 48, 72, 96,
120, 144, 192, 240 hour time intervals. The EGF release was monitored in vitro
using a specific
enzyme linked immunosorbent assay (ELISA) for both particle and matrices.
ELISA Assay:
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The release of hEGF was measured using ELISA. The cytokine antibody pairs were
used
for construction of ELISAs. The captured antibody was monoclonal anti-human
EGF antibody
(MAB 636)(500 g) (R&D System) and detection antibody was biotinylated anti-
human EGF
antibody (BAF236) (50 g)) (R&D System). The wells of a 96 -well titertek
plate
(Polysorb,Nunc Plasticware) were coated with monoclonal anti-human EGF
antibody in PBS
solution. Sample or standards were added in an appropriate diluent per well.
The biotinylated
detection antibody was diluted in the appropriate diluent (0.1 % BSA, 0.05%
Tween 20 in Tris-
buffered Saline pH 7.3 (20mM Trizma base, 150 mM NaC1), and added to each
well. The plate
was covered with an adhesive strip and incubated 2 hours at room temperature.
Streptavidin
HRP(Zymed ) 1/2500 of a 1.25 mg/ml solution or equivalent) was then added to
each well
followed by a substrate solution (H202) and developer ABTS (2,2'Azino-di[3-
ethylbenzthiazoline-6-sulfonate]) (Boehringer-Mannheim). The assay was
incubated for 20-30
minutes at 37 C. The optical density was determined for each well in the plate
within 30
minutes, using microplate reader 450 nm. Figure 33 depicts the results of the
hEGF release study
from the PVA particles used in the protein matrix wafers. The results of this
study show that
crosslinking the protein matrix decreases the release of hEGF. It was also
determined from
subsequent studies that the hEGF released has biological activity in in-vitro
cell culture studies
using hepatocytes.
While the invention has been described in conjunction with specific
embodiments
thereof, it is evident that many alternatives, modifications, and variations
will be apparent to
those skilled in the art in light of the foregoing description. Accordingly,
it is intended to
embrace all such alternatives, modifications, and variations, which fall
within the spirit and
broad scope of the invention.
