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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
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BIOACTIVE STENTS FOR TYPE II DIABETICS
AND METHODS FOR USE THEREOF
RELATED APPLICATION
[0001] This application relies for priority under 35 U.S.C. ~ 119(e) upon U.S.
Provisional Application Serial No. 60/559,937 filed April 5, 2004.
FIELD OF THE INVENTION
(0002] The invention relates generally to implantable medical devices, and in
particular to biodegradable polymer coated implantable stems that promote
vascular
healing in diabetics.
BACKGROUND INFORMATION
[0003] The normal endothelium, which lines blood vessels, is uniquely and
completely
compatible with blood. Endothelial cells initiate metabolic processes, like
the secretion
of prostacyclin and endothelium-derived relaxing factor (EDRF), which actively
discourage platelet deposition and thrombus formation in vessel walls.
However,
damaged arterial surfaces within the vascular system are highly susceptible to
thrombus
formation. Abnormal platelet deposition, resulting in thrombosis, is more
likely to occur
in vessels in which endothelial, medial and adventitial damage has occurred.
While
systemic drugs have been used to prevent coagulation and to inhibit platelet
aggregation,
a need exists for a means by which a damaged vessel can be treated directly to
prevent
thrombus formation and subsequent intimal smooth muscle cell proliferation.
[0004] Current treatment regimes for stenosis or occluded vessels include
mechanical
interventions. However, these techniques exacerbate the injuzy, precipitating
new smooth
muscle cell proliferation and neointimal growth. For example, stenotic
arteries are often
treated with balloon angioplasty, which involves the mechanical dilation of a
vessel with
an inflatable catheter. The effectiveness of this procedure is limited in some
patients
because the treatment itself damages the vessel, thereby inducing
proliferation of smooth
muscle cells and reocclusion or restenosis of the vessel. It has been
estimated that
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2
approximately 30 to 40 percent of patients treated by balloon angioplasty
and/or stems
may experience restenosis within one year of the procedure. Damage to the
endothelial
and medial layers of a blood vessel, such as often occurs in the course of
balloon
angioplasty and stmt procedures, has been found to stimulate neointimal
proliferation,
leading to restenosis of atherosclerotic vessels.
[0005] To overcome these problems, numerous approaches have been taken to
providing stems useful in the repair of damaged vasculature. In one aspect,
the stmt itself
reduces restenosis in a mechanical way by providing a larger lumen. For
example, some
stems gradually enlarge over time. To prevent damage to the lumen wall during
implantation of the stmt, many stems are implanted in a contracted form
mounted on a
partially expanded balloon of a balloon catheter and then expanded in situ to
contact the
lumen wall. U. S. Patent No. 5,059,211 discloses an expandable stmt for
supporting the
interior wall of a coronary artery wherein the stmt body is made of a porous
bioabsorbable material. To aid in avoiding damage to vasculature during
implant of such
stems, U. S. Patent No. 5,662,960 discloses a friction-reducing coating of
commingled
hydrogel suitable for application to polymeric plastic, rubber or metallic
substrates that
can be applied to the surface of a'stent.
[0006] A number of agents that affect cell proliferation have been tested as
pharmacological treatments for sfenosis and restenosis in an attempt to slow
or inhibit
proliferation of smooth muscle cells. These compositions have included
heparin,
coumarin, aspirin, fish oils, calcium antagonists, steroids, prostacyclin,
ultraviolet
irradiation, and others. Such agents may be systemically applied or may be
delivered on
a more local basis using a drug delivery catheter or a drug eluting stmt. In
particular,
biodegradable polymer matrices loaded with a pharmaceutical may be implanted
at a
treatment site. As the polymer degrades, a medicament is released directly at
the
treatment site. The rate at which the drug is delivered is to a significant
extent dependent
upon the rate at which the polymer matrix is resorbed by the body. U.S. Patent
No.
5,342,348 to I~aplan and U.S. Patent No. 5,419,760 to Norciso are exemplary of
this
technology. U.S. Patent 5,766,710 discloses a stmt formed of composite
biodegradable
polymers of different melting temperatures.
[0007] Porous stems formed from porous polymers or sintered metal particles or
fibers
have also been used for release of therapeutic drugs within a damaged vessel,
as disclosed
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in U. S. Patent No. 5,843,172. However, tissue surrounding a porous stmt tends
to
infiltrate the pores. In certain applications, pores that promote tissue
ingrowth are
considered to be counterproductive because the growth of neointima can occlude
the
artery, or other body lumen, into which the stmt is being placed.
[0008] Delivery of drugs to the damaged arterial wall components has also been
explored by using latticed intravascular stems that have been seeded with
sheep
endothelial cells engineered to secrete a therapeutic protein, such as t-PA
(D. A. Dichek
et al., Circulation, 80:1347-1353, 1989). However, endothelium is known to be
capable
of promoting both coagulation and thrombolysis.
[0009] To prevent neointimal proliferation that leads to stenosis or
restenosis, U.S.
Patent 5,766,584 to Edelman et al. describes a method for inhibiting vascular
smooth
muscle cell proliferation following injury to the endothelial cell lining by
creating a
matrix containing endothelial cells and surgically wrapping the matrix about
the tuuica
adventitia. The matrix, and especially the endothelial cells attached to the
matrix, secrete
products that diffuse into surrounding tissue, but do not migrate to the
endothelial cell
lining of the injured blood vessel.
[0010] In a healthy individual in response to endothelial damage, the vascular
endothelium participates in many homeostatic mechanisms important for normal
wound
healing, the regulation of vascular tone and the prevention of thrombosis. A
primary
mediator of these functions is endothelium-derived relaxing factor (EDRF).
First
described in 1980 by Furchgott and Zawadzki (Furchgott and Zawadzki, Nature
(Lond.)
288:373-376, 1980) EDRF is either nitric oxide (Moncada et al., Pharnaacol
Rev. 43:109-
142, 1991.) (NO) or a closely related N~-containing molecule (Myers et al.,
Nature
(Lond.), 345:161-163, 1990).
[0011] Removal or damage to the endothelium is a potent stimulus for
neointimal
proliferation, a common mechanism underlying the restenosis of atherosclerotic
vessels
after balloon angioplasty. (Liu et'al., Circulation, 79:1374-1387, 1989);
(Ferns et al.,
Science, 253:1129-1132, 1991). Stent-induced restenosis is caused by local
wounding of
the luminal wall of the artery. Further, restenosis is the result of a
chronically-stimulated
wound-healing cycle.
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[0012] The natural process of wound healing involves a two-phase cycle: blood
coagulation and inflammation at the site of the wound. In healthy individuals,
these two
cycles are counterbalanced, each including a natural negative feedback
mechanism that
prevents over-stimulation. For example, in the coagulation enzyme pathway
thrombin
factor Xa operates upon factor VII to control thrombus formation and, at the
same time
stimulates production of PARs (Protease Activated Receptors) by pro-
inflammatory
monocytes and macrophages. Nitric oxide produced endogenously by endothelial
cells
regulates invasion of the proinflammatory monocytes and macrophages. In the
lumen of
an artery, this two-phase cycle results in influx and proliferation of healing
cells through a
break in the endothelium. Stabilization of the vascular smooth muscle cell
population by
this naturally counterbalanced process is required to prevent neointimal
proliferation
leading to restenosis. The absence or scarcity of endogenously produced nitric
oxide
caused by damage to the endothelial layer in the vasculature is thought to be
responsible
for the proliferation of vascular smooth muscle cells that results in
restenosis following
vessel injury, for example following angioplasty.
[0013] Nitric oxide dilates blood vessels (Vallance et al., Lancet, 2:997-
1000, 1989),
inhibits platelet activation and adhesion (Radomski et al., Br.
JPha~°ryaacol, 92:181-187,
1987) and, in vitro, nitric oxide limits the proliferation of vascular smooth
muscle cells
(Garg et al., J. Clin. Invest. 83:1774-1777, 1986). Similarly, in animal
models,
suppression of platelet-derived mitogens by nitric oxide decreases intimal
proliferation
(Ferns et al., Sciefice, 253:1129-1132, 1991). The potential importance of
endothelium-
derived nitric oxide in the control of arterial remodeling after injury is
further supported
by recent preliminary reports in humans suggesting that systemic NO donors
reduce
angiographic-restenosis six months after balloon angioplasty (The ACCORD Study
Investigators, J. Am. Coll. Caf-diol. 23:59A. (Abstr.), 1994).
[0014] The earliest understanding of the function of the endothelium within an
artery
was its action as a barrier between highly reactive, blood borne materials and
the intima
of the artery. A wide variety of biological activity within the artery wall is
generated
when platelets, monocytes and neutrophils infiltrate intima. These reactions
result from
release of activating factors such as ATP and PDGF from platelets and IL-1, IL-
6, TNFa
and bFGF from monocytes and neutrophils. An important consequence of release
of
these activating factors is a change in the cellular structure of smooth
muscle cells,
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causing the cells to shift from quiescent to migratory. This cellular change
is of particular
importance in vascular medicine, since activation of quiescent smooth muscle
cells in
arteries can lead to uncontrolled proliferation, leading to the blockage or
narrowing of
arteries known as stenosis or restenosis.
[0015) The standard of care for the non-surgical treatment of blocked arteries
is to re-
open the blockage with an angioplasty balloon, often followed by the placement
of a wire
metal structure called a stmt to retain the opening in the artery. An
unfortunate
consequence of this procedure is the nearly total destruction of the
endothelial layer by
expansion of the angioplasty balloon and precipitation of a foreign body
inflammatory
response to the stmt. Therefore, after removal of the balloon catheter used in
the
angioplasty, the artery is rapidly exposed to an influx of activating factors.
Since
mechanical intervention has destroyed the natural blood/artery barrier, in a
significant
number of patients the result is a local uncontrolled proliferative response
by smooth
muscle cells leading to restenosis.
[0016] A disproportionate number of diabetic patients, especially those with
Type II
diabetes, do not benefit from stenting of atherosclerotic arteries to the same
extent as in
equivalent non-diabetic patients. Clinical research has strongly implicated
the generally
impaired healing of the endothelium in patients who suffer from
diabetes'mellitus as a
major contributor to the diminished therapeutic outcome in these patients when
an arterial
stmt has been implanted. Impaired glucose tolerance (IGT) is considered a
transitional
phase to the development of Type II diabetes and many of the changes in health
of
endothelium found in Type II diabetics are prefigured in IGT. IGT and diabetes
are also
independently associated with the occurrence of cardiovascular disease. While
Type II
diabetic patients make up a significant proportion of those patients who
experience such
treatment failure, all Type II diabetics do not experience stmt failure and
the reason why
some do and some do not has not hitherto been studied.
[0017] Thus, a need exists in the art for new and better methods and devices
for
stimulating and supplementing endothelial healing in patients who suffer from
diabetes
mellitus and who have suffered damage to arterial endothelial lining.
Particularly, the
need exists for better methods and devices for restoring in diabetics the
natural process of
wound healing in damaged arteries and other blood vessels.
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SUMMARY OF THE INVENTION
[0018] The present invention is based on the discovery that endogenous
endothelial
healing processes at a site of vascular damage in patients suffering from Type
II diabetes
can be promoted by coating stems and other implantable devices with
biodegradable,
bioactive polymers bearing covalently attached bioligands that specifically
capture and
activate therapeutic progenitors of endothelial cells from the circulating
blood of such
patients. The polymers, which biodegrade over time, may also release bioactive
agents
that re-establish in patients suffering from Type II diabetes the natural
endothelial healing
process in an artery. The bioactive agents) attached to the polymers (e.g.,
the polymer
backbone) promote endogenous endothelial processes in arteries of diabetics by
specifically recruiting to the stmt surface progenitors of endothelial cells
from circulating
blood at the site of stmt or device implantation in the vasculature. Thus, a
significant
proportion of the healing properties of the stmt in type II diabetics takes
place before
biodegradation of the stmt.
[0019] In one embodiment, the invention provides bioactive implantable stems
including a stmt structure with a surface coating of a biodegradable,
bioactive polymer,
and at least one bioligand that specifically binds to an integrin receptor on
progenitors of
endothelial cells (PECs) in circulating blood. The bioligand is covalently
bonded to the
polymer. This bioligand may itself be bioactive in also activating the PECs,
or it may act
in conjunction with another bioactive PEC-activating agent.
[0020] In still another embodiment, the invention provides a kit that includes
a
biocompatible implantable stmt. .The invention stmt has a stmt structure with
a surface
coating of a biodegradable, biocompatible polymer with at least one bioligand
or first
member of a specific binding pair that binds specifically to an integrin
receptor on PECs.
The bioligand or first member is covalently bound to the biodegradable,
biocompatible
polymer.
(0021] In yet another embodiment, the invention provides a tubular sheath
comprising
a biodegradable, bioactive polymer, wherein the polymer comprises at least one
bioligand
covalently bound to the polymer, wherein the bioligand specifically binds to
an integrin
receptor on PECs in peripheral blood.
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7
[0022] In another embodiment, the invention provides implantable medical
devices
having a biodegradable, bioactive polymer coated upon at least a portion of a
surface. At
least one bioligand that specifically binds an integrin receptor on PECs found
in
peripheral blood is covalently bound to the polymer.
[0023) In still another embodiment, the invention provides methods for
treating
damaged arterial endothelium ina heart or limb in a patient having Type II
diabetes
comprising implanting an invention stmt to promote natural healing of damaged
endothelium in the artery wall of the patient.
[0024] In yet another embodiment, the invention provides methods for using a
polymer as a medical device, a pharmaceutical, or as a carrier for covalent
immobilization
of a bioligand or first member of a specific binding pair that specifically
attaches to an
integrin receptor in PECs in the circulating blood of a patient with Type II
diabetes into
which the polymer is implanted. In this embodiment, a) the bioligand is a
polypeptide
that binds specifically to an integrin receptor on PECs in circulating blood;
b) the
bioligand forms a specific binding pair with an antibody that binds
specifically to the
integrin receptor; or c) the antibody is tagged with a first member of a
specific binding
pair and the bioligand comprises a second member of the specific binding pair.
[0025] In still another embodiment, the invention provides methods for
promoting
natural healing of endothelium damaged by mechanical intervention in an artery
of a
subject having Type II diabetes by implanting into the artery following the
mechanical
intervention an invention stmt to promote natural healing of the artery.
BRIEF DESCRIPTION OF THE FIGURES
[0026] Fig. 1 is a schematic cross-section of an invention multilayered
polymer-coated
stmt.
[0027] Fig. 2 is a flow chart describing the PEC isolation protocol.
[0028] Fig. 3 is a flow chart of the protocol for adhesion assays conducted
with ECs
and SMCs.
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[0029] Fig. 4 is a graph summarizing the results of a representative adhesion
assay
quantitation based on ATP standard curve. At each time point of the adhesion
assay, an
ATP assay was done to determine the number of adherent cells.
[0030] Fig. 5 shows the chemical structure of dansyl, an acronym for 5
dimethylamino-1 naphthalenesulfonyl, a reactive fluorescent dye, linked to
PEA.
[0031] Figs. 6A-B are flowcharts smmnarizing surface chemistry optimization
protocols. Fig. 6A shows a flowchart of the surface chemistry for conjugation
of peptides
to the acid version of the polymers (PEA-H). Fig. 6B shows a flowchart of the
protocol
for surface conjugation of peptides to mixtures of PEA polymers.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In one embodiment, this invention provides stems and methods using such
devices to re-establish an endothelial blood/artery barrier in patients
suffering from
diabetes mellitus, particularly Type II diabetes. The invention is also
designed to
promote endothelial healing at a site of damaged vascular endothelium in
patients having
impaired glucose tolerance, which is considered a transitional phase to the
development
of Type II diabetes. The invention stems comprise a biocompatible, resorbable
polymeric
sheath that encapsulates the stmt structure. In a preferred embodiment of the
invention
methods, the stmt is placed at the conclusion of an angioplasty procedure, or
other
medical procedure that damages arterial endothelium, without allowing a lapse
of time
sufficient for infiltration of inflammatory factors from the blood stream into
the artery
wall. In this method, the stmt is placed at the location of the damage and
preferably
immediately covers and protects the area of damaged endothelium so as to
prevent
infiltration of inflammatory factors from the blood stream into the artery
wall, while
performing its primary function of gathering therapeutic progenitors of
endothelial cells
from the patient's circulating blood so that the natural processes of
endothelial healing
can go forward in the patient suffering from Type II diabetes.
[0033] In other words, the invention stems perform as an artificial
endothelial layer
while promoting the natural cycle of endothelial healing in diabetics as
described herein.
The polymeric sheath may have additional features that contribute to the
healing of the
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artery. In one embodiment, the invention sheath or covering comprises multiple
layers,
each of which can perform a distinct function in re-establishing a stable
lesion and
contributing to healing endothelium of the injured artery wall.
[0034] The terms "diabetes" and "diabetes mellitus" as used herein mean Type
II
diabetes as well as impaired glucose tolerance (IGT), which is widely
considered a
transitional phase to the development of Type II diabetes. Many of the changes
in health
of endothelium found in Type II diabetics are prefigured in IGT.
[0035] The term "progenitors of endothelial cells (PECs)", as used herein with
reference to the blood of subjects with Type II diabetes, encompasses, but is
not limited
to, endothelial progenitor cells (EPCs). There is significant evidence in the
literature that
endothelial progenitor cells (EPCs) can derive from the bone marrow and that
CD133+/VEGFR2+ cells represent a population with endothelial progenitor
capacity
(BZ~od (2000) 95:952-958 and 3106-3112; Circ. Res. (2001) 88:167-174;
Artef°ioscler.
Tl2romb. Yasc. Biol. (2003) 23:1185-89 and Circ. Res. (2004) 95:343-353).
There are,
however, also reports of additional bone-marrow-derived cell populations (i.e.
myeloid
cells and mesenchymal cells) and even non-bone marrow-derived cells that can
also give
rise to endothelial cells (Circulation (2003).107:1164-1169;
Cif°culation (2003)
108:2511-2516; Anat. Res. (2004) Part A 276A:13-21; and Circ. Res. (2004)
95:343-353).
The more differentiated source of endothelial cells in the circulating blood
may be
monocytes or monocytic-like cells, and this is the source of PECs used in the
Examples
herein. The term "precursor endothelial cells" (PECs) is used herein to
encompass and
describe all of these non-"classical" precursors of ECs.
[0036] In another aspect, examples of bioligands suitable for use in capture
of PECs
from circulating blood are monoclonal antibodies directed against a known or
identified
surface marker of therapeutic PECs. Complementary determinants (CDs) that have
been
reported to decorate the surface of endothelial cells include CD31, CD34,
CD102,
CD 1 O5, CD 106, CD 109, CDw 130, CD 141, CD 142, CD 143, CD 144, CDw 145, CD
146,
CD 147, and CD 166. These cell surface markers can be of varying specificity
for a
particular cell/developmental type/stage in EC development. CDs 106, 142 and
144 have
been reported to mark mature endothelial cells with some specificity. CD34 is
presently
known to be specific for progenitor endothelial cells in non-diabetics and
therefore is one
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of the cell surface markers that is believed to be useful for capturing PECs
out of blood
circulating in the vessels in a diabetic patient into which the stmt is
implanted.
[0037] Additional examples of bioligands for the capture of PECs from
circulating
blood are extracellular matrix (ECM) proteins. Within the bone marrow stroma
and in
most areas of the body, interactions between progenitor cells and the ECM
occur. ECM
ligands are important, not only for differentiation and proliferation but also
for
maintenance of the hematopoietic stem cell. Fibronectin is one of the more
ubiquitous
members of the ECM. It is a potential ligand for most cell types and is
recognized by at
least 10 adhesion receptors of the integrin family (Leu7zefnia 1997; 11:822-
829 and Blood
1998; 91 (9):3230-3238). In particular, CSS and REDVDY are both found in the
Type III
connecting segment of fibronectin. The sequence for the CSS peptide is: Gly-
Glu-Glu-
Ile-Gln-Ile-Gly-His-Ile-Pro-~-Glu-Asp-T~al-Asp-Ty~'-His-Leu-Tyr-Pro (SEQ ID
NO:l),
which contains REDVDY (underlined) (SEQ ID N0:2). It has been discovered that
CSS
and REDVDY peptides bind specifically to integrin receptors on PECs.
[0038] The minimal active cell binding amino acid sequence, REDV , is somewhat
related to the RGDs, a major active site in the central cell binding domain of
fibronectin.
However, REDV is novel in its cell type selectivity. The integrin x,4(31 is
known to bind
to the REDV sequence and is found on ECs but not on SMCs (JBC (1991)
266(6):3579-
3585; Am. JofPathology (1994) 145:1070-1081; and Blood (1998) 91(9):3230-
32384).
This becomes even more important in recruiting PECs versus smooth muscle
progenitor
cells (SPCs) in peripheral blood. Recent studies have shown that PECs express
the x,4[31
integrin while the SPCs do not (Circ. (2002) 106:1199-1204; and Circ. (2004)
110(17):2673-26775). This preference of REDV for ECs provides a significant
advantage to a stmt with a polymer coating containing REDV as a bioligand
acting as a
PEC cell recruitment factor. Even if an integrin receptor bioligand is not
considered to
significantly increase cell adhesion to the stmt, it has been discovered that
such
bioligands still confer an advantage to the recruitment of ECs by stimulating
more rapid
adhesion with better cell spreading of ECs on stmt surfaces.
[0039] The investigations into cell binding regions described in the Examples
herein
identified the importance of integrin receptors found on the surface of
numerous cell
types. Bioligands (e.g., peptides and polypeptides) that bind specifically to
integrin
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receptors in PECs are incorporated into (e.g, covalently bonded to) a
biodegradable
polymer as described herein for coating at least a portion of the surface of
an
interventional implantable device, such as a vascular stmt, to endow the
coating with the
property of preferential and specific recruitment of a subpopulation of PECs
from the
circulating bloodstream of a diabetic patient into which the device is
implanted. The
resulting localized concentration of PECs throughout the stmt will enhance
endothelial
wound healing of the arterial wall of the diabetic patient.
[0040] In one embodiment, the bioligand is an antibody, such as a monoclonal
antibody, and is specific for an integrin receptor identified on PECs as
described above.
A stmt having a polymer coating to which the capture antibody is bound will,
when
implanted in a Type II diabetic, in turn bind to and hold captured PECs near
the polymer
surface for activation and subsequent migration.
[0041] As used herein, the term "antibody" is used in its broadest sense to
include
polyclonal and monoclonal antibodies, as well as antigen binding fragments of
such
antibodies. An antibody useful in a method of the invention, or an antigen-
binding
fragment thereof, is characterized, for example, by having specific binding
activity for an
epitope of a target molecule.
[0042] The antibody, for example, includes naturally occurring antibodies as
well as
non-naturally occurring antibodies, including, for example, single chain
antibodies,
chimeric, bifunctional and humanized antibodies, as well as antigen-binding
fragments
thereof. Such non-naturally occurring antibodies can be constructed using
solid phase
peptide synthesis, can be produced recombinantly or can be obtained, for
example, by
screening combinatorial libraries consisting of variable heavy chains and
variable light
chains (see Huse et al., Science 246:1275-1281 (1989)). These and other
methods of
making, for example, chimeric, humanized, CDR-grafted, single chain, and
bifunctional
antibodies are well known to those skilled in the art (Winter and Harris,
Immunol. Today
14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane,
Antibodies:
A laboratofy mafazcal (Cold Spring Harbor Laboratory Press, 1988); Hilyard et
al., Protein
Etagineering: A pf°actical approach (IRL Press 1992); Borrabeck,
Antibody Efagineering,
2d ed. (Oxford University Press 1995)). Examples of antibodies that can be
used in the
invention devices and methods include single-chain antibodies, chimeric
antibodies,
monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab
fragments, IgA,
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12
IgG, IgM, IgD, IgE and humanized antibodies. Monoclonal antibodies suitable
for use as
bioligands may also be obtained from a number of commercial sources. Such
commercial
antibodies are available against a wide variety of targets. Antibody probes
can be
conjugated to molecular backbones using standard chemistries, as discussed
below.
[0043] The term "binds specifically" or "specific binding activity," when used
in
reference to an antibody means that an interaction of the antibody and a
particular epitope
has a dissociation constant of at least about 1 x 10-6, generally at least
about 1 x 10-x,
usually at least about 1 x 10-8, and particularly at least about 1 x 1 O-9 Or
1 x 10-1° or less.
As such, Fab, F(ab')2, Fd and Fv fragments of an antibody that retain specific
binding
activity for an epitope of an antigen, are included within the definition of
an antibody.
[0044] In an alternative embodiment, a pair of biocompatible specific binding
partners, A and B, can be used to specifically capture PECs from the
circulating blood'of
Type II diabetics. In this embodiment, one of the specific binding pair acts
as the
bioligand covalently attached to the polymer coating of the stmt or other
implantable
device. The other member of the pair of specific binding partners is attached
or allowed
to attach to an integrin receptor on the PECs of the diabetic patient to be
treated (either ex
vivo or in vivo by administration to the blood of the patient). For example,
if the pair of
biocompatible specific binding partners is biotin (molecule A) and
streptavidin (molecule
B), a Mab that binds specifically to a PEC cell surface marker, such as CD
144, can be
conjugated with molecule A at a site on the Mab that does not interfere with
the Mab
binding to its cognate PEC cell surface marker. Alternatively, the roles of
the specific
binding partners, A and B, can be reversed, with biotin, for example, being
attached to the
polymer of the stmt and streptavidin being attached to a monoclonal antibody
administered to the patient for specific attachment to the integrin receptor
on the patient's
PECs.
(0045] In one embodiment of the invention, Mab-A conjugates are added to the
patient's blood either in vivo (e.g., parenterally) or ex vivo (e.g., by
extracorporeal
circulation of the patient's blood) either prior to, contemporaneously with,
or immediately
following installation of the stmt or other therapeutic device. As a result,
circulating
therapeutic EPC-Mab-A complexes are preferentially recruited to binding
partner B,
streptavidin, which is covalently attached to the device coating, enhancing
the local
concentration of therapeutic PECs at the site of intervention and injury. A
monoclonal
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antibody administered to the blood of a human is preferably a "humanized
monoclonal
antibody" and suitable antigen-binding fragments can be commissioned
commercially or
can readily be produced recombinantly using well known techniques. Although
this
aspect of the invention is illustrated by reference to specific binding
partners biotin and
streptavidin, any biocompatible pair of specific binding partners can be used
in an
analogous way.
[0046] Alternatively, the biocompatible bioligand can further comprise one
member of
a specific binding pair, such as a biotin-streptavidin, and the other member
of the specific
binding pair can be pre-attached to the polymer. In use, in this alternative
case, the
bioligand is administered to the patient's blood stream, either in vivo or ex
vivo, and
allowed to bind to its specific target on therapeutic PECs therein, via a
specific binding
pair bridge. If the bioligand is administered to the patient's blood stream
in~vivo (e.g.,
parenterally), the PECs in the blood stream become bound to the polymer in
vivo via the
bioligand-specific binding pair-polymer complex.
(0047] In addition, small proteinaceous motifs, such as the B domain of
bacterial
Protein A and the fixnctionally equivalent region of Protein G, are known to
form a
specific binding pair with, and thereby capture Fc-containing antibodies.
Accordingly, in
further embodiments, the antibody administered to the diabetic patient's blood
is an Fc-
containing antibody that is specific for an integrin receptor on PECs in blood
and the
bioligand attached to the polymer of the stmt is a "sticky" peptide or
polypeptide, such as
Protein A and Protein G, which will capture the antibody and hold it near to
the polymer
surface of the stmt to aid in recruiting PECs to the area of endothelium
damage.
However, these "sticky" peptides or polypeptides may also capture other
circulating, Fc-
containing, native antibodies, thereby reducing specificity of the reaction
for the
therapeutic purposes.
[0048] Protein A is a constituent of staphylococcus A bacteria that binds the
Fc region
of particular antibodies or immunoglobulin molecules. For example, the Protein
A
bioligand can be or contain the amino acid sequence:
MTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVD
GVWTYDDATKTFTVTE (SEQ ID N0:3)
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or a functionally equivalent peptidic derivative thereof, such as, by way of
an example,
the functionally equivalent peptide or polypeptide having the amino acid
sequence:
TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
ATKTFTVTE (SEQ ID N0:4)
[0049] Protein G is a constituent of group G streptococci bacteria, and
displays similar
activity to Protein A, namely binding the Fc region of particular antibody or
immunoglobulin molecules. For example, the Protein G bioligand can be, or
contain
Protein G having an amino acid sequence:
MTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDG
VWTYDDATKTFTVTE (SEQ ID NO:S)
or a functionally equivalent peptide derivative thereof, such as, by way of an
example, the
functionally equivalent polypeptide having the amino acid sequence:
TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
ATKTFTVTE (SEQ ID N0:6)
[0050] Such Protein A and Protein G molecules can be covalently attached as
bioligands to the bioactive polymer coatings on the stmt structure (e.g., the
inner layer of
a multilayered stmt as described herein) and will act as bioligands to capture
out of the
patient's circulating blood stream Fc-containing antibodies that have been
complexed
with the patients' therapeutic PECs. Bioligands are selected and conjugated to
the
polymer backbone while avoiding steric hindrance to binding of the ligand to
its
biological target.
[0051] Other bioactive agents that activate the progenitor endothelial cells
and are
contemplated for attachment to the polymer backbone in the polymer coatings
covering
the invention medical devices (e.g., surface coatings of stems and sheaths for
covering the
stmt structure) include the bradykinins. Bradykinins are vasoactive
nonapeptides formed
by the action of proteases on kininogens, to produce the decapeptide kallidin
(KRPPGFSPFR) (SEQ ID N0:7), which can undergo further C-terminal proteolytic
cleavage to yield the bradykinin 1 nonapeptide: (KRPPGFSPF) (SEQ ID NO: S), or
N-
terminal proteolytic cleavage to yield the bradykinin 2 nonapeptide:
(RPPGFSPFR) (SEQ
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ID NO: 9). Bradykinins 1 and 2 are functionally distinct as agonists of
specific
bradykinin cell surface receptors B 1 and B2 respectively: both kallidin and
bradykinin 2
are natural bioligands for the B2 receptor; whereas their C-terminal
metabolites
(bradykinin 1 and the octapeptide RPPGFSPF (SEQ ID NO:10) respectively) axe
bioligands for the B 1 receptor. A portion of circulating bradykinin peptides
can be
subject to a further post-translational modification: hydroxylation of the
second proline
residue in the sequence (Pro3 to Hyp3 in the bradykinin 2 amino acid
numbering).
Bradykinins are very potent vasodilators, increasing permeability of post-
capillary
venules, and acting on endothelial cells to activate calmodulin and thereby
nitric oxide
synthase.
[0052] Bradykinin peptides are incorporated into the bioactive polymers used
in the
invention stems by attachment at one end of the peptide. The unattached end of
the
bradykinin extends freely from the polymer as a bioligand to contact
endothelial cells in
the vessel wall as well as progenitor endothelial cells captured from the
blood in the
vessel into which the stmt is implanted. Thereby the endothelial cells with
which contact
is made become activated.
[0053] In a still further aspect, the bioactive agent can be a nucleoside,
such as
adenosine, which is also known to be a potent activator of endothelial cells
to, produce
nitric oxide endogenously. Endothelial cells activated in this way activate
further
progenitor endothelial cells with which they come into contact. Thus, a
cascade of
endothelial cell activation at the site of the injury is caused to result in
endogenous
production of nitric oxide and development of an endothelial lining on the
surface of the
stmt that contacts blood.
[0054] In another embodiment, the invention stmt has a multilayered polymer
covering that encapsulates a stmt structure. Fig. 1 shows a schematic cross-
section of an
example of an invention stmt 11 with stmt struts 10 and a multilayered sheath
or
covering. When the multilayered stmt is implanted, the outer layer 16 of the
stmt
covering or sheath lies directly next to the artery wall. Bioactive agents and
additional
bioactive agents, as described herein, are incorporated into the outer layer
of the stmt
covering or sheath to promote healing of the epithelium. An optional diffusion
barrier
layer 14 can be placed between and in contact with outer layer 16 and inner
layer 12.
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[0055] The inner layer 12 of the multilayered stmt covering is exposed to the
circulating blood with its PECs and has bioligands for recruitment of PECs
covalently
attached thereto. A biocompatible polymer of the type specifically described
herein (e.g.,
having a chemical structure described by structures I and III herein) is used
for inner layer
12. One or more bioligands that bind specifically to PECs, such as those
having an amino
acid sequence as set forth in SEQ ID NOS:l, 2, or 1 l, or a member of a
specific binding
pair for which the other member is contained within or conjugated with a
specifically
binding bioligand, are covalently attached to the polymer in the inner layer
using
techniques of covalent attachment described herein. For example, streptavidin
can be
bound to the polymer of the inner layer of the sheath for use with a biotin-
tagged antibody
that specifically binds the target on PECs in the circulating blood (which
biotin-tagged
antibody will be administered to the patient's blood stream). Optionally, one
or more
"bioactive agent," as described herein, but not "an additional bioactive
agent" can also be
covalently bound to the polymer in the inner layer of the multilayered stmt.
As in other
embodiments of the invention stems, the bioactive agent is selected to
activate PECs
attracted to the inner layer of the sheath from the circulating blood of
diabetic patients by
the bioligands attached to the inner layer of the stmt covering. Thus the stmt
takes an
active role in the process of re-establishing the natural endothelial cell
layer at the site of
one or more damaged areas of arterial endothelium.
[0056] The outer layer 16 comprises a polymer layer loaded with a bioactive
agent
and/or an additional bioactive agent, or combination thereof, specifically
including those
that limit cellular proliferation or reduce inflammation as disclosed herein.
These cellular
proliferation limiting and/or inflammation reducing drugs and bioactive agents
can be
solubilized in the polyner solid phase and, hence, are preferably not bound to
the
polymer of the outer layer. Rather such bioactive agents and additional
bioactive agents
are loaded into the polymer and sequestered there until the stmt is put into
place. Once
implanted, the bioactive agents in the outer layer 16 are eluted and diffuse
into the artery
wall.
[0057] Preferred bioactive agents for incorporation into the outer layer of
invention
multilayered stems include rapamycin and any of its analogs or derivatives,
such
aseverolimus (also known as sirolimus), paclitaxel or any of its analogs or
derivatives,
and statins, such as simvastatin. In the outer layer, non-covalently bound
bioactive agents
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and/or additional bioactive agents can be intermingled with or "loaded into"
any
biocompatible biodegradable polymer as is known in the art since the outer
layer in this
embodiment of the invention does not come into contact with blood, except
during
placement of the stmt.
[0058] Optionally, lying along and covering the interior surface of the outer
layer of
the covering is a diffusion barrier layer 14 of resorbable polymer that acts
as a diffusion
barrier to the bioactive agent or additional bioactive agent contained in the
outer layer.
The purpose of this diffusion barrier is to direct elution of the
drug/biologic into the artery
wall to prevent proliferation of smooth muscle cells, while limiting or
preventing passage
of the drug/biologic into the inner layer. The diffusion barner layer 14 can
accomplish its
purpose of partitioning of the drug through hydrophobic/hydrophilic
interaction related to
the solubility of the bioactive agent in the polymer solid phase. For example,
if the
bioactive agent or additional bioactive agent in the outer layer is
hydrophobic, the
polymer barrier layer is selected to be less hydrophobic than the agent(s),
and if the
bioactive agent or additional bioactive agent in the outer layer is
hydrophilic, the barrier ,
layer is selected to be hydrophobic. For example, the barrier layer can be
selected from
such polymers as polyester, poly(amino acid), polyester amide), polyester
urethane),
polyurethane, polylactone, polyester ether), or copolymers thereof, whose
charge
properties are well known by those of skill in the art. The barrier layer is
considered
optional because the inner layer of the stmt may itself prove an effective
diffusion
barrier, depending upon the properties of the polymers and various active
agents
contained in the inner and outer layers of the stmt.
[0059] In one embodiment, the stmt structure used in manufacture of the
invention
multilayered stmt as well as the stems comprising a single layer of polymer
covering
described herein is made of a biodegradable and absorbable material with
sufficient
strength and stiffness to replace a conventional stmt structure, such as a
stainless steel or
wire mesh stmt structure. A cross-linked polyester amide), polycaprolactone,
or
polyester urethane) as described herein can be used for this purpose so that
the stmt
structure as well as its coverings) is completely bioabsorbable, for example,
over a
period of three months to years. In this case, over time, each of the layers,
and the stmt
structure as well, will be re-absorbed by the body through natural processes,
including
enzymatic action, allowing the re-established endothelial cell layer to resume
its dual
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function of acting as a blood/artery barrier and providing natural control and
stabilization
of the intra-cellular matrix within the artery wall through the production of
nitric oxide.
[0060] As used herein, "biodegradable" means that at least the polymer coating
of the
invention stmt is capable of being broken down into innocuous and
biocompatible
products in the normal functioning of the body. In one embodiment, the entire
stmt,
including the stmt structure is biodegradable. The preferred biodegradable,
biocompatible polymers have hydrolyzable ester and/or amide linkages, which
provide
the biodegradability, and are typically chain terminated with carboxyl or
capping groups.
[0061] Biodegradable, blood compatible polymers suitable for use in the
practice of
the invention of the type specifically described herein (e.g., having a
chemical structure
described by structures I and III herein) bear functionalities that allow for
facile covalent
attachment of bioactive agents to the polymer. For example, a polymer bearing
carboxyl
groups can readily react with a bioactive agent having an amino moiety,
thereby
covalently bonding the bioactive agent to the polymer via the resulting amide
group. As
will be described herein, the biodegradable, biocompatible polymer and the
bioligands
and bioactive agents can contain numerous complementary functional groups that
can be
used to covalently attach the bioactive agent to the biodegradable,
biocompatible
polymer.
[0062] The term "bioactive agent", as used herein, means agents that play an
active
role in the endogenous healing processes at a site of stmt implantation by
holding
bioligands or members of a specific binding pair, and/or releasing a bioactive
or
therapeutic agent during biodegradation of the polymer. Bioactive agents,
include those
specifically described herein as having properties that capture (i.e.,
"bioligands"), attract
and activate captured circulating PECs, and are contemplated for covalent
attachment to
the polymers used in coating the invention stems. Such bioactive agents
include, but are
not limited to, agents that, when freed from the polymer backbone during
polymer
degradation, promote endogenous production of a therapeutic natural wound
healing
agent, such as nitric oxide endogenously produced by endothelial cells.
Alternatively the
"bioactive agents" released from the polymers during degradation may be
directly active
in promoting natural wound healing processes by endothelial cells while
controlling
proliferation of smooth muscle cells in the vessel at the locus of the damage.
These
bioactive agents can include any agent that donates, transfers, or releases
nitric oxide,
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elevates endogenous levels of nitric oxide, stimulates endogenous synthesis of
nitric
oxide, or serves as a substrate for nitric oxide synthase or that inhibits
proliferation of
smooth muscle cells. Such agents include, for example, aminoxyls, furoxans,
nitrosothiols, nitrates and anthocyanins; nucleosides such as adenosine, and
nucleotides
such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP);
neurotransmitter/neuromodulators such as acetylcholine and 5-hydroxytryptamine
(serotonin/5-HT); histamine and catecholamines such as adrenalin and
noradrenalin; lipid
molecules such as sphingosine-1-phosphate and lysophosphatidic acid; amino
acids such
as arginine and lysine; peptides such as the bradykinins, substance P and
calcium gene-
related peptide (CGRP), and proteins such as insulin, vascular endothelial
growth factor
(VEGF), and thrombin.
[0063] A wide variety of other bioactive agents are optionally covalently
attached to
the bioactive polymers used in the coverings of the invention stems and
devices.
Aminoxyls contemplated for use as bioactive agents have the structure:
O'
[0064] Exemplary aminoxyls include the following compounds:
-O. ~ -O.
3,
2,2,6,6-tetramethylpiperidine-1-oxy (1); 2,2,5,5-tetramethylpyrrolidine-1-oxy
(2); and
2,2,5,5-tetramethylpyrroline-1-oxy-3-carbonyl (3). Further aminoxyls
contemplated for
use include 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy (TEMPAMINE); 4-(N,N-
dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxy, iodide
(CAT16);
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4-(N,N-dimethyl-N-(2-hydroxyethyl))ammonium-2,2,6,6-tetramethylpiperidine- 1-
oxy(TEMPO choline); 4-(N,N-dimethyl-N-(3-sulfopropyl)ammonium-2,2,6,6-
tetramethylpiperidine-1- oxy; N-(4-(iodoacetyl)amino-2,2,6,6-
tetramethylpiperidine-1-
oxy(TEMPO 1A); N-(2,2,6,6-tetramethylpiperidine-1-oxy-4-yl)maleimide(TEMPO
maleimide, MAL-6); and 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-
oxy,
iodide (CAT 1); 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxy; and N-(3-
(iodoacetyl)amino)-2,2,5,5-tetramethylpyrrolidine-1-oxy(PROXYL 1A);
succinimidyl
2,2,5,5-tetramethyl-3-pyrroline-1-oxy-3-carboxylate and 2,2,5,5-tetramethyl-3-
pyrroline-
1-oxy-3-carboxylic acid, and the like.
[0065] Furoxans contemplated for use as bioactive agents have the structure:
f' ~, /
r' 'n,
n a/N,b
[0066] An exemplary furoxan is 4-phenyl-3-furoxancarbonitrile, as set forth
below:
CN
N~ ~N~O
O
[0067] Nitrosothiols include compounds bearing the -S-N=O moiety, such as the
exemplary nitrosothiol set forth below:
COOH
O=N-S
NHCOCH3,
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[0068] Anthocyanins are also contemplated for use as bioactive agents.
Anthocyanins
are glycosylated anthocyanidins and have the structure:
3'
5'
OH
wherein the sugars are attached to the 3-hydroxy position. Anthocyanins are
known to
stimulate NO production in vivo and therefore are suitable for use as
bioactive agents in
the practice of the invention.
[0069] Bioactive agents for dispersion into and release from the surface
coverings of
the invention stems and medical devices also include anti-proliferants,
rapamycin and any
of its analogs or derivatives, paclitaxel or any of its taxene analogs or
derivatives,
everolimus, Sirolimus, tacrolimus, or any of its -limns named family of drugs,
and statins
such as simvastatin, atorvastatin, fluvastatin, pravastatin, lovastatin,
rosuvastatin,
geldanamycins, such as 17AAG (17-allylamino-17-demethoxygeldanamycin);
Epothilone
D and other epothilones, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin
and
other polyketide inhibitors of heat shock protein 90 (Hsp90)> Cilostazol, and
the like.
[0070] Polymers contemplated for use in forming the blood-compatible,
hydrophilic
coating or inner layer in the invention multilayered stems include polyesters,
poly(amino
acids), polyester amides, polyurethanes, or copolymers thereof. In particular,
examples of
biodegradable polyesters include poly(oc-hydroxy C1 -CS alkyl carboxylic
acids), e.g.,
polyglycolic acids, poly-L-lactides, and poly-D,L-lactides; poly-3-hydroxy
butyrate;
polyhydroxyvalerate; polycaprolactones, e.g., poly(s-caprolactone); and
modified poly(oc-
hydroxyacid)homopolyrners, e.g., homopolymers of the cyclic diester monomer, 3-
(S)[alkyloxycarbonyl)methyl]-1,4-dioxane-2,5-dione which has the formula 4
where R is
lower alkyl, depicted in I~imura, Y., "Biocompatible Polymers" in Bior~aedical
Applications of Polyf3aeric Mates°ials, Tsuruta, T., et al, eds., CRC
Press, 1993 at page
179.
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(0071] Examples of biodegradable copolymer polyesters useful in forming he
blood-
compatible, hydrophilic coating or inner layer in the invention stems include
copolyester
amides, copolyester urethanes, glycolide-lactide copolymers, glycolide-
caprolactone
copolymers, poly-3-hydroxy butyrate-valerate copolymers, and copolymers of the
cyclic
diester monomer, 3-(S)[(alkyloxycarbonyl)methyl]-1,4-dioxane-2,5-dione, with L-
lactide.
The glycolide-lactide copolymers include poly(glycolide-L-lactide) copolymers
formed
utilizing a monomer mole ratio of glycolic acid to L-lactic acid ranging from
5:95 to 95:5
and preferably a monomer mole ratio of glycolic acid to L-lactic acid ranging
from 45:65
to 95:5. The glycolide-caprolactone copolymers include glycolide and E-
caprolactone
block copolymer, e.g., Monocryl or Poliglecaprone.
[0072] The biodegradable polymers useful in forming the coatings for the
invention
biocompatible polymer coated stems and medical devices also include those
comprising
at least one amino acid conjugated to at least one non-amino acid moiety per
repeat unit.
The term "non-amino acid moiety" as used herein includes various chemical
moieties, but
specifically excludes amino acid derivatives and peptidomimetics as described
herein. In
addition, the polymers containing at least one amino acid are not contemplated
to include
poly(amino acid) segments, including naturally occurring polypeptides, unless
specifically described as such. In one embodiment, the non-amino acid is
placed between
two adjacent amino acids in the repeat unit. The polymers may comprise at
least two
different amino acids per repeat unit.
[0073] Preferred for use in forming the biocompatible polymer surface
coverings of the
invention stems and medical devices (and the inner layers of invention
multilayered stems)
are polyester amides (PEAs) and polyester urethanes (PEURs) that have built-in
functional
groups on PEA or PEUR side chains, and these built-in functional groups can
react with other
chemicals and lead to the incorporation of additional functional groups to
expand the
functionality of PEA or PEUR further. Therefore, such polymers used in the
invention
compositions and methods are ready for reaction with other chemicals having a
hydrophilic
structure to increase water solubility and with bioactive agents and
additional bioactive
agents, without the necessity of prior modification.
[0074] In addition, the polymers used in the invention polymer coated stems
and medical
devices display minimal hydrolytic degradation when tested in a saline (PBS)
medium, but in
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an enzymatic solution, such as chymotrypsin or CT, a uniform erosive behavior
has been
observed.
[0075] In one embodiment the PEAS and PEURs have a chemical formula described
by the general structural formula (I):
0 0
PEA or -PEUR G-R~-C-N-CH-(CH2)4 NH
m ~ -o pJ n
ORS
(I)
where
O O H H ~ ~ H H
-PEA 1S -C-R~-C-N-C-C-O-R4 O-C- i -N-
I
R3 R3
and
0II 0 0 0
-PEUR- 1S ~O-Rq-O-C-C-N-C-O-R4 O-C-CH-N
I
R3 R3
and wherein n ranges from about 50 to about 150, m ranges about 0.1 to about
0.9: p
ranges from about 0.9 to about 0.1; wherein Rl is selected from the group
consisting of
(C2 - CZO) alkylene or (C2-C2o) alkenylene; RZ is hydrogen or (C6-CIO)aryl (CI-
C6) alkyl
or t-butyl or other protecting group; R3 is selected from the group consisting
of hydrogen,
(C~-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl and (C6-Coo) aryl (CI-C6)
alkyl; and R4 is
selected from the group consisting of (C2-CZO) alkylene, (C2-C2o) alkenylene
or alkyloxy,
and bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II):
Formula I~
CH O
H2C / H2
\O CH
(II),
except that for unsaturated polymers having the structural formula (I), R~ and
R4 are
selected from (C2-C2o) alkylene and (CZ-CZO) alkenylene; wherein at least one
of RI and
R4 is (C2-C2o) alkenylene; n is about 5 to about 150; each RZ is independently
hydrogen,
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or (C6-CIO)aryl(Cl-C6)alkyl; and each R3 is independently hydrogen, (C1-
C6)alkyl, (C2-
C6)alkenyl, (C2-C6)alkynyl, or (C~-G~o)aryl(C1-C6)alkyl.
[0076] In one alternative, R3,is CHZPh and the alpha amino acid used in
synthesis is L-
phenylalanine.
[0077] In alternatives wherein R3 is CH2-CH(CH3)2, the polymer contains the
alpha-
amino acid, leucine. By varying R3, other alpha amino acids can also be used,
e.g.,
glycine (when R3 is H), alanine (when R3 is CH3), valine (when R3 is
CH(CH3)2),
isoleucine (when R3 is CH(CH3)-CH2-CH3), phenylalanine (when R3 is CHZ-C6H5) ,
or
lysine (when R3 = (CHZ)4 NH2).
[0078) The polymer molecules may also have the active agent attached thereto,
optionally via a linker or incorporated into a crosslinker between molecules.
For
example, in one embodiment, the polymer is contained in a polymer-bioactive
agent
conjugate having the structural formula (III):
o-R~-C-N-C-C-O-R4 O-OC-C-N OC-R~-OC-N-CH-(CH2)4 NH
Rs R3 m ~ i =O p n
R5
..
R6
Formula (III)
wherein n, m, p, R~, R3, and Rø are as above, RS is selected from the group
consisting of
-O-, -S-, and -NR8-, wherein R8 is H or (C1-C~) alkyl; and R6 is a bioactive
agent.
[0079] In yet another embodiment, two molecules of the polymer can be
crosslinked
to provide an -RS-R6-RS- conjugate. In another embodiment, as shown in
structural
formula IV below, the bioactive agent is covalently linked to one molecule of
the polymer
through the -RS-R6- RS- conjugate and RS is independently selected from the
group
consisting of-O-, -S-, and -NR$-, wherein R8 is H or (C~-C$) alkyl.
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R3 R3
C-R~-C-N-H-C-O-R4-O-C-CIH-N C-R~-C-N-C-(CH2)4-NH
0 0 0 ° m o o~ Jn
0
R5 R6 R5
R3 R3 H
-(CH2)4-C-NH-C-R~-C NH-C-C-O-R4-O-C-CH-N-C-R~-C
.... H ii ii ~ H ~~ n n ii
0 o p o 0 0 o~n
Formula (IV)
[0080] Alternatively still, as shown in structural formula (V) below, a
linker, X-Y-,
can be inserted between RS and bioactive ,agent R6 in the molecule of
structural formula
III, wherein X is selected from the group consisting of (CI-CI8) alkylene,
substituted
alkylene, (C3-Cg) cycloalkylene, substituted cycloalkylene, 5-6 membered
heterocyclic
system containing 1-3 heteroatoms selected from the group O, N, and S,
substituted
heterocyclic, (Cz-C~8) alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, C6 and
CIO aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl,
substituted
alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted
arylalkenyl,
arylalkynyl, substituted arylkynyl and wherein the substituents are selected
from the
group H, F, Cl, Br, I, (C~-C6) alkyl, -CN, -NOz. -OH, -O(CI-C4) alkyl), -S(CI-
C6) alkyl),
-S[(=O)(CI-C6) alkyl)], -S[(Oz)(CI-C6) alkyl], -C[(=O)(CI-C6) alkyl], CF3, -
O[(CO)-( CI_
C6) alkyl)], -S(Oz)[N(R9RIO), -NH[(C=O)(CI-C6) alkyl],
-NH(C=O)N(R9RIO), and -N(RgRIO); where R9 and RIO are independently H or (CI-
C6)alkyl; and
Y is selected from the group consisting of-O-, -S-, -S-S-, -S(O)-,-S(Oz)-,
-NR8-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)NH-, -NRBC(=O)-, -C(=O)NR8-,
-NRBC(=O)NRg-, -NRgC(=O)NR8-, and -NRBC(=S)NR8-.
(0081] Alternatively, one molecule of the polymer is covalently linked to a
bioactive
agent through an RS-R6-Y-X- RS- bridge (Formula VI).
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R3 R3
C-R~-C-N-C-C-O-R4-O-C-CIH-N C-R~-C-N-C-(CH2)4-NH
.. .. H .. ,. ~~ .. ~ J
~0 0 0 0 , m o o n
0
~ R5 Rs Y X R5
Rs R3 H
-(CH2)4-C-NH-C-R~-C NH-C-C-O-Rq-O-C-CH-N-C-R~-C
H ii ii ~ H ~ O 0
0 o p n
Formula (VI)
wherein, X is selected from the group consisting of (CI-C18) alkylene,
substituted
alkylene, (C3-C$) cycloalkylene, substituted cycloalkylene, 5-6 membered
heterocyclic
system containing 1-3 heteroatoms selected from the group O, N, and S,
substituted
heterocyclic, (C2-C18) alkenyl 1, substituted alkenyl, alkynyl, substituted
alkynyl, C6 and
Clo) aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl,
substituted
alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted
arylalkenyl,
arylalkynyl, substituted arylkynyl, wherein the substituents are selected from
the group
consisting of H, F, Cl, Br, I, (C1-C6) alkyl, -CN, -NOZ, -OH, -O(C1-Cøalkyl), -
S(C1-
C6)alkyl), -S[(=O)(C~-C6 alkyl)], -S[(02)(Cl-C6) alkyl], -C[(=O)(C1-C6)
alkyl], CF3, -
O[(CO)-(C1-C6)alkyl)],
-S(Oa)[N(R9R~o), -NH[(C=O)(C1-C6)alkyl], -NH(C=O)N(R9Rlo), and -N(RnR~z),
wherein Rl is independently (C2-C2o) alkylene and (C2-Cao) alkenylene, and
Rl1, R12 are
independently H or (C~-C6)alkyl.
[0082] In another embodiment, the polymer coating on the invention stems and
medical devices comprises four partially crosslinked molecules of the polymer
of
structural fonnula (III), except that only two of the four molecules omit R6
and are
crosslinked to provide a single -RS-X-RS- conjugate, wherein X is selected
from the
group consisting of (C1-C18) alkylene, substituted alkylene, (C3-C8)
cycloalkylene,
substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3
heteroatoms
selected from the group consisting of O, N, and S, substituted heterocyclic,
(C2-Cls)
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C6 and Coo aryl,
substituted aryl,
heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl,
arylalkynyl, substituted
arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, and
substituted arylkynyl
and wherein the substituents are selected from the group consisting of H, F,
Cl, Br, I, (C1-
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C6) alkyl, -CN, -N02,-OH, -O(C1-Cø) alkyl), -S(CI-Cg) alkyl), -S[(=O)(C1-C6)
alkyl)], -
S[(OZ)(Cl-C6) alkyl], -C[(=O)(C1-C6) alkyl], CF3, -O[(CO)-( C1-C6) alkyl)], -
S(02)[N(R9R~o), NH[(C=O)(Cl-C6) alkyl],
-NH(C=O)N(R9Rlo), and -N(R9Rlo); wherein R9 and Rlo are independently H or (C1-
C6)
alkyl).
[0083] In yet another embodiment, four molecules of the polymer of structural
formula III can be partially crosslinked by omitting R6 on two of the
molecules and
forming instead a single -RS-X-R~- conjugate, wherein X, R5, R6 and R~ are as
described
above and as shown in structural formula (VII), wherein q + s = n:
I
O O H H O O-H3 H~0 O H H O O H H O O-H3 H~0 O H
C-Ri~C-N-C-C-O-RQ O-C C-N C-Ri C-N-CH-(CHZ)y-N C-R~~C-N-C-C-O-Rq O-C C-N C-R~
C-N-CH-(CHZ)4-NH
f23 R m C:O P 9 R3 R m C:O p s
i i
Rs~RS Rs
X
i
R~ Rs R~
i
H R R3 H~ ~ H C;O H H R3 R3 H~ ~ H ~'O H
C-R~ C-N-C-C-O-R4 O-C-C-N~C-R~ C-N-CH-(CHZ)4 N C-RyC-N-C-C-O-R4 O-C-C-N~C-R~ C-
N-CH-(CH2)q-N
~O O HO OH O O pJq[~O O HO OH m 0 O p s
Formula (VII) (~ + S = Il
[0084] The term "aryl" is used with reference to structural formulae herein to
denote
a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about
nine to ten
ring atoms in which at least one ring is aromatic. In certain embodiments, one
or more of
the ring atoms can be substituted with one or more of nitro, cyano, halo,
trifluoromethyl,
or trifluoromethoxy. Examples of aryl include, but are not limited to, phenyl,
naphthyl,
and nitrophenyl.
[0085] The term "alkenylene" is used with reference to structural formulae
herein to
mean a divalent branched or unbranched hydrocarbon chain containing at least
one
unsaturated bond in the main chain or in a side chain.
[0086] The molecular weights and polydisperities herein are determined by gel
permeation chromatograph using polystyrene standards. More particularly,
number and
weight average molecular weights (Mn and MW) are determined, for example,
using a
Model 510 gel permeation chromatograph (Water Associates, Inc., Milford, MA)
equipped with a high-pressure liquid chrorriatographic pump, a Waters 4~6 UV
detector
and a Waters 2410 differential refractive index detector. Tetrahydrofuran
(THF) is used
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2~
as the eluent (1.0 mL/min). The polystyrene standards have a narrow molecular
weight
distribution.
[0087] Methods for making the polymers of structural formula (I-VII),
containing a a-
amino acid in the general formula are well known in the art. For example, for
the
embodiment of the polymer of structural formula (I) wherein R is incorporated
into an a-
amino acid, for polymer synthesis the a-amino acid can be converted into a bis-
a-amino
acid, for example, by condensing the a-amino acid with a diol HO-RZ-OH. As a
result,
ester fragments are formed. Then, the bis-a-amino acid is entered into a
polycondensation reaction with a di-acid such as sebacic acid, to obtain the
final polymer
having both ester and amide bonds. Alternatively, instead of the di-acid, a di-
acid
derivative such as activated di-ester, e.g., di pare-nitrophenoxy. di-acid
chloride can also
be used.
[0088] More particularly, synthesis of the unsaturated polyester-amides
(UPEAs)
useful as biodegradable polymers of the structural formula (I) as described
above will be
described wherein
O
O O
II , II ~~\ ~C\ /
(a) C R C is C C
H
O
and/or (b) R4 is -CHZ-CH=CH-CHI,-. In cases where (a) is present and (b) is
not present,
R4 in (I) is -C4H8- or -C6Hla-. In cases where (a) is not present and (b) is
present, Rl in
(I) is -C~HB- or -C8H16-.
[0089] The unsaturated PEAS can be prepared by solution polycondensation of
either
(1) di-p-toluene sulfonic acid salt of diester of alpha-a.~nino acid and
unsaturated diol and
di-p-nitrophenyl ester of saturated dicarboxylic acid or (2) di-p-toluene
sulfonic acid salt
of alpha-amino acid and saturated diol and di-nitrophenyl ester of unsaturated
dicarboxylic acid or (3) di-p-toluene sulfonic acid salt of diester of alpha-
amino acid and
unsaturated diol and di-nitrophenyl ester of unsaturated dicarboxylic acid.
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[0090] The aryl sulfonic acid salts are used instead of the free amine base
because the
aryl sulfonic acid group is a very good leaving group which can promote the
condensation
reaction to move to the right of the reaction equation so product is obtained
in high yield
and because the p-toluene sulfonic acid salts are known for use in
synthesizing polymers
containing amino acid residues.
[0091] The di-p-nitrophenyl esters of unsaturated dicarboxylic acid can be
synthesized
from p-nitrophenyl and unsaturated dicarboxylic acid chloride, e.g., by
dissolving
triethylamine and p-nitrophenyl in acetone and adding unsaturated dicarboxylic
acid
chloride drop wise with stirring at -78°C and pouring into water to
precipitate product.
Suitable acid chlorides included acrylic methacrylic, crotonic, isocrotonic,
angelic, tiglic,
sorbic, cinnamic, allocinnamic, phenylpropiolic, fumaric, malefic, mesaconic,
citraconic,
glutaconic, itaconic, ethenyl-butane dioic and 2-propenyl-butanedioic acid
chlorides.
Additional compounds that can be used in the place of di-p-nitrophenyl esters
of
unsaturated dicarboxylic acid include those having structural formula (VIII):
0 0
Rs_O_C_O_Ri_O_C_O_Rs
(VIII)
wherein each RS is independently (C~ -C~o)aryl optionally substituted with one
or more
nitro, cyano, halo, trifluoromethyl, or trifluoromethoxy; and RI is
independently (CZ -
C2o)alkylene or (C2 -C8)alkyloxy(Ca -C~o)alkylene.
[0092] The di-aryl sulfonic acid salts of diesters of alpha-amino acid and
unsaturated
diol can be prepared by admixing alpha-amino acid, e.g., p-aryl sulfonic acid
monohydrate and saturated or unsaturated diol in toluene, heating to reflux
temperature,
until water evolution is minimal, then cooling. The unsaturated diols include,
for
example, 2-butene-1,3-diol and 1,18-octadec-9-en-diol.
[0093] Saturated di-p-nitrophenyl esters of dicarboxylic acid and saturated di-
p-
toluene sulfonic acid salts of bis-alpha-amino acid esters can be prepared as
described in
U. S. Patent No. 6,503,538 Bl.
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[0094] Synthesis the unsaturated polyester-amides (UPEAs) useful as
biodegradable
polymers of the structural formula (II) as described above will now be
described.
Compounds having the structural formula (II) can be made in similar fashion to
the
compound (VII) of U. S. Patent No. 6,503,538 B1, except that R4 of (III) of
6,503,535
and/or RI of (V) of 6,503,538 is C2-C2o alkenylene as described above. The
reaction is
carried out, for example, by adding dry triethylamine to a mixture of said
(III) and (IV) of
6,503,538 and said (V) in dry N,N-dimethylacetamide, at room temperature, then
increasing the temperature to 80°C and stirring for 16 hours, then
cooling the reaction
solution to room temperature, diluting with ethanol, pouring into water,
separating
polymer, washing separated polymer with water, drying to about 30°C
under reduced
pressure and then purifying up to negative test on p-nitrophenyl and p-toluene
sulfonic
acid. A preferred reactant (IV) is p-toluene sulfonic acid salt of benzyl
ester, the benzyl
ester protecting group is preferably removed from (II) to confer
biodegradability, but it
should not be removed by hydrogenolysis as in Example 22 of U.S. Patent No.
6,503,538
because hydrogenolysis would saturate the desired double bonds; rather the
benzyl ester
group should be converted to an acid group by a method that would preserve
unsaturation, e.g., by treatment with fluoroacetic acid or gaseous HF.
Alternatively, the
lysine reactant (IV) can be protected by a protecting group different from
benzyl which
can be readily removed in the finished product while preserving unsaturation,
e.g., the
lysine reactant can be protected with t-butyl (i.e., the reactant can be t-
butyl ester of
lysine) and the t-butyl can be converted to H while preserving unsaturation by
treatment
of the product (II) with dilute acid.
[0095] A working example of the compound having structural formula (II) is
provided
by substituting p-toluene sulfonic acid salt of L-phenylalanine 2-butene-1,4-
diester for
(III) in Example 1 of 6,503,538 or by substituting di-p-nitrophenyl fumarate
for (V) in
Example 1 of 6,503,538 or by substituting p-toluene sulfonic acid salt of L-
phenylalanine
2-butene-1,3-diester for III in Example 1 of 6,503,538 and also substituting
de-p-
nitrophenyl fumarate for (V) in Example 1 of 6,503,538.
[0096] In unsaturated compounds having either structural formula (I-VII), the
following hold: Aminoxyl radical (e.g., 4-amino TEMPO) can be attached using
carbonyldiimidazol as a condensing agent. Bioactive agents, as described
herein, can be
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attached via the double bond functionality. Hydrophilicity can be imparted by
bonding to
polyethylene glycol) diacrylate.
[0097] In yet another aspect, polymers contemplated for use in forming the
invention
polymer coated stems and medical devices include those set forth in U.S.
Patent Nos.
5,516, 881; 6,338,047; 6,476,204; 6,503,538; and in U.S. Application Nos.
10/096,435;
10/101,408; 10/143,572; and 10/194,965; the entire contents of each of which
is
incorporated herein by reference.
[0098] The PEA/PEUR polymers described herein may contain up to two amino
acids
per monomer and preferably have weight average molecular weights ranging from
10,000
to 125,000; these polymers and copolymers typically have inherent viscosities
at 25 °C,
determined by standard viscosimetric methods, ranging from 0.3 to 4.0,
preferably
ranging from 0.5 to 3.5.
[0099] Polymers contemplated for use in the practice of the invention can be
synthesized by a variety of methods well known in the art. For example,
tributyltin (IV)
catalysts are commonly used to form polyesters such as poly(caprolactone),
poly(glycolide), poly(lactide), and the like. However, it is understood that a
wide variety
of catalysts can be used to form polymers suitable for use in the practice of
the invention.
[0100] Such poly(caprolactones) contemplated for use have an exemplary
structural
formula (IX) as follows:
0
(IX).
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[0101] Poly(glycolides) contemplated for use have an exemplary structural
formula
(X) as follows:
0
)~
[0102] Poly(lactides) contemplated for use have an exemplary structural
formula (XI)
as follows:
O
Me
(XI).
[0103] An exemplary synthesis of a suitable poly(lactide-co-s-caprolactone)
including
an aminoxyl moiety is set forth as follows. The first step involves the
copolymerization
of lactide and s-caprolactone in the presence of benzyl alcohol using stannous
octoate as
the catalyst to form a polymer of structural formula (XII). .
O
O
0
o _
CH20H + +
O
O
O
O O
i n y m
(XII)
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[0104] The hydroxy terminated polymer chains can then be capped with malefic
anhydride to form polymer chains having structural formula (XIII):
O O O
O O O OH
I n ,O m
(XIII)
[0105] At this point, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy can be
reacted with
the carboxylic end group to covalently attach the aminoxyl moiety to the
copolymer via
the amide bond that results from the reaction between the 4-amino group and
the
carboxylic acid end group. Alternatively, the malefic acid capped copolymer
can be
grafted with polyacrylic acid to provide additional carboxylic acid moieties
for
subsequent attachment of further aminoxyl groups.
[0106] The PEAIPEUR polymers described herein can be fabricated in a variety
of
molecular weights, and the appropriate molecular weight for use with a given
bioactive
agent is readily determined by one of skill in the art. Thus, e.g., a suitable
molecular
weight will be on the order of about 5,000 to about 300,000, for example about
5,000 to
about 250,000, or about 75,000 to about 200,000, or about 100,000 to about
150,000.
[0107] Polymers useful in the making the invention polymer coated stems and
medical
devices, such as PEA/PEUR polymers, biodegrade by enzymatic action at the
surface.
Therefore, the polymers administer the bioactive agent to the subject at a
controlled
release rate, which is specific and constant over a prolonged period.
Additionally, since
PEA/PEUR polymers break down in vivo via hydrolytic enzymes without production
of
adverse side products, the polymer coatings on the invention stems and medical
devices
are substantially non-inflammatory.
[0108] As used herein "dispersed" means at least one bioactive agent as
disclosed
herein is dispersed, mixed, dissolved, homogenized, and/or covalently bound
("dispersed") in a polymer, for example attached to the surface of the polymer
or polymer
coating.
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[0109] While the bioactive agents can be dispersed within the polymer matrix
without
chemical linkage to the polymer carrier, it is also contemplated that the
bioactive agent or
additional bioactive agent can be covalently bound to the biodegradable
polymers via a
wide variety of suitable functional groups. For example, when the
biodegradable polymer
is a polyester, the carboxyl group chain end can be used to react with a
complimentary
moiety on the bioactive agent or additional bioactive agent, such as hydroxy,
amino, thio,
and the like. A wide variety of suitable reagents and reaction conditions are
disclosed,
e.g., in Advanced Ofganic Chemistry, Reactions, Mechanisms, and Structure,
Fifth
Edition, (2001); and Compf°ehensive Organic Ti°ansformations,
Second Edition, Larock
( 1999).
[0110] In other embodiments, a bioactive agent can be linked to any of the
polymers
of structures (I)-(VII) through an amide, ester, ether, amino, ketone,
thioether, sulfinyl,
sulfonyl, or disulfide linkage. Such a linkage can be formed from suitably
functionalized
starting materials using synthetic procedures that are known in the art.
[0111] For example, in one embodiment a polymer can be linked to the bioactive
agent or additional bioactive agent via a carboxyl group (e.g., COOH) of the
polymer.
Specifically, a compound of structures (I) and (III) can react with an amino
functional
group or a hydroxyl functional group of a bioactive agent to provide a
biodegradable
polymer having the bioactive agent attached via an amide linkage or carboxylic
ester
linkage, respectively. In another embodiment, the carboxyl group of the
polymer can be
benzylated or transformed into an acyl halide, aryl anhydride/"mixed"
anhydride, or
active ester. In other embodiments, the free NH2 ends of the polymer molecule
can be
acylated to assure that the bioactive agent will attach only via a carboxyl
group of the
polymer and not to the free ends of the polymer.
[0112] Alternatively, the bioactive agent or additional bioactive agent can be
attached
to the polymer via a linker molecule, for example, as described in structural
formulae (V
- VII). Indeed, to improve surface hydrophobicity of the biodegradable
polymer, to
improve accessibility of the biodegradable polymer towards enzyme activation,
and to
improve the release profile of the biodegradable polymer, a linker may be
utilized to
indirectly attach the bioactive agent and/or adjuvant to the biodegradable
polymer. In
certain embodiments, the linker compounds include polyethylene glycol) having
a
molecular weight (MW) of about 44 to about 10,000, preferably 44 to 2000;
amino acids,
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such as serine; polypeptides with repeat number from 1 to 100; and any other
suitable low
molecular weight polymers. The linker typically separates the bioactive agent
from the
polymer by about 5 angstroms up to about 200 angstroms.
[0113] In still further embodiments, the linker is a divalent radical of
formula W-A-Q,
wherein A is (C1-C24)alkyl, (C2-C24)alkenyl, (C2-C24)alkynyl, (C3-
C8)cycloalkyl, or (C6-
CIO) aryl, and W and Q are each independently N(R)C(=O)-, -C(=O)N(R)-, -OC(=O)-
, -
C(=O)O, -O-, -S-, -S(O), -S(O)Z-, -S-S-, -N(R)-, -C(=O)-, wherein each R is
independently H or (C1-C6)alkyl.
[0114] As used to describe the above linkers, the term "alkyl" refers to a
straight or
branched chain hydrocarbon group including methyl, ethyl, n-propyl, isopropyl,
n-butyl,
isobutyl, tert-butyl, n-hexyl, and the like.
[0115] As used herein, "alkenyl" refers to straight or branched chain
hydrocarbyl
groups having one or more carbon-carbon double bonds.
[0116] As used herein, "alkynyl" refers to straight or branched chain
hydrocarbyl
groups having at least one carbon-carbon triple bond.
[0117] As used herein, "aryl" refers to aromatic groups having in the range of
6 up to
14 carbon atoms.
[0118] In certain embodiments, the linker may be a polypeptide having from
about 2
up to about 25 amino acids. Suitable peptides contemplated for use include
poly-L-
glycine, poly-L-lysine, poly-L-glutamic acid, poly-L-aspartic acid, poly-L-
histidine, poly-
L-ornithine, poly-L-serine, poly-L-threonine, poly-L-tyrosine, poly-L-leucine,
poly-L-
lysine-L-phenylalanine, poly-L-arginine, poly-L-lysine-L-tyrosine, and the
like.
[0119] In one embodiment, the bioactive agent can covalently crosslink the
polymer,
i.e. the bioactive agent is bound to more than one polymer molecule. This
covalent
crosslinking can be done with or without additional polymer-bioactive agent
linker.
(0120] The bioactive agent molecule can also be incorporated into an
intramolecular
bridge by covalent attachment between two polymer molecules.
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[0121] A linear polymer polypeptide conjugate is made by protecting the
potential
nucleophiles on the polypeptide backbone and leaving only one reactive group
to be
bound to the polymer or polymer linker construct. Deprotection is performed
according
to methods well known in the art for deprotection of peptides (Boc and Fmoc
chemistry
for example).
[0122] In one embodiment of the present invention, a polypeptide bioactive
agent is
presented as retro-inver~so or partial f°etro-inverso peptide.
Accordingly, the terms
"peptide" and "polypeptide," as used herein, include peptides, wholly peptide
derivatives
(such as branched peptides) and covalent hetero- (such as glyco- and lipo- and
glycolipo-)
derivatives of peptides.
[0123] The peptides described herein can be synthesized using any technique as
is
known in the art. The peptides and polypeptides can also include "peptide
mimetics."
Peptide analogs are commonly used in the pharmaceutical industry as non-
peptide
bioactive agents with properties analogous to those of the template peptide.
These types
of non-peptide compound are termed "peptide mimetics" or "peptidomimetics."
Fauchere, J. (1986) Adv. Bioactive agent Res., 15:29; Veber and Freidinger
(1985) TINS
p. 392; and Evans et al. (1987) .I. Med. Claena., 30:1229; and are usually
developed with
the aid of computerized molecular modeling. Generally, peptidomimetics are
structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical
property or
pharmacological activity), but have one or more peptide linkages optionally
replaced by a
linkage selected from the group consisting of.' - -CHZNH--, --CH2S--, CH2-CH2--
, --
CH=CH-- (cis and trans), --COCH2--, --CH(OH)CHa--, and --CH2S0--, by methods
known in the art and further described in the following references: Spatola,
A.F. in
"Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B.
Weinstein,
eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Trega Data (March
1983),
Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley,
J.S., Trends.
Plaaf°n~. Sci., (1980) pp. 463-468 (general review); Hudson, D. et al.,
Int. J. Pept. Pf~ot.
Res., (1979) 14:177-185 (--CH2NH--, CH2CH2--); Spatola, A.F. et al., Life
Sci., (1986)
38:1243-1249 (--CHZ-S--); Harm, M. M., J. Claem. Soc. Pef°kin Trans 1
(1982) 307-314
(--CH=CH--, cis and trans); Ahnquist, R.G. et al., J-Med. Claefn., (1980)
23:2533 (--
COCH2--); Jennings-Whie, C. et al., Tetrahedron Lett., (1982) 23:2533 (--COCHa-
-);
Szelke, M. et al., European Appln., EP 45665 (1982) CA: 97:39405 (1982)
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37
(--CH(OH)CH2--); Holladay, M. W. et al., Tetrahedron Lett., (1983) 24:4401-
4404 (--
C(OH)CH2--); and Hruby, V.J., Life Sci., (1982) 31:189-199 (--CH2-S--). Such
peptide
mimetics may have significant advantages over polypeptide embodiments,
including, for
example: more economical production, greater chemical stability, enhanced
pharmacological properties (half life, absorption, potency, efficacy, etc.),
altered
specificity (e.g., a broad-spectrum of biological activities), reduced
antigenicity, and
others
[0124] Additionally, substitution of one or more amino acids within a peptide
or
polypeptide (e.g., with a D-Lysine in place of L-Lysine) may be used to
generate more
stable peptides and peptides resistant to endogenous proteases. Alternatively,
the
synthetic peptide or polypeptide, e.g., covalently bound to the biodegradable
polymer, can
also be prepared from D-amino acids, referred to as inverso peptides. When a
peptide is
assembled in the opposite direction of the native peptide sequence, it is
referred to as a
f°etro peptide. In general, peptides prepared from D-amino acids are
very stable to
enzymatic hydrolysis. Many cases have been reported of preserved biological
activities
for retf°o-inverso or partial f°etro-invef so peptides (US
patent, 6,261,569 B1 and
references therein ; B. Fromme et al, Endocrinology (2003)144:3262-3269).
[0125] The linker can be attached first to the polymer or to the bioactive
agent or
additional bioactive agent. During synthesis, the linker can be either in
unprotected form
or protected form, using a variety of protecting groups well known to. those
skilled in the
art. In the case of a protected linker, the unprotected end of the linker can
first be
attached to the polymer or the bioactive agent or additional bioactive agent.
The
protecting group can then be de-protected using Pd/H2 hydrogenation, mild acid
or base
hydrolysis, or any other common de-protection method that is known in the art.
The de-
protected linker can then be attached to the bioactive agent or additional
bioactive agent,
or to the polymer
[0126] An exemplary synthesis of a biodegradable polymer according to the
invention
(wherein the molecule to be attached is an aminoxyl) is set forth as follows.
A polyester
can be reacted with an aminoxyl, e.g., 4-amino-2,2,6,6-tetramethylpiperidine-1-
oxy, in
the presence of N,N'-carbonyl diimidazole to replace the hydroxyl moiety in
the carboxyl
group at the chain end of the polyester with imino linked to aminoxyl-
containing radical,
so that the imino moiety covalently bonds to the carbon of the carbonyl
residue of the
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carboxyl group. The N,N'-carbonyl diimidazole converts the hydroxyl moiety in
the
carboxyl group at the chain end of the polyester into an intermediate product
moiety
which will react with the aminoxyl, e.g., 4-amino-2,2,6,6-
tetramethylpiperidine-1-oxy.
The aminoxyl reactant is typically used in a mole ratio of reactant to
polyester ranging
from 1:1 to 100:1. The mole ratio of N,N'-carbonyl diimidazole to aminoxyl is
preferably
about 1:1.
[0127] A typical reaction is as follows. A polyester is dissolved in a
reaction solvent
and reaction is readily carried out at the temperature utilized for the
dissolving. The
reaction solvent may be any in which the polyester will dissolve; this
information is
normally available from the manufacturer of the polyester. When the polyester
is a
polyglycolic acid or a poly(glycolide-L-lactide) (having a monomer mole ratio
of glycolic
acid to L-lactic acid greater than 50:50), highly refined (99.9+% pure)
dimethyl sulfoxide
at 115 °C to 130 °C or DMSO at room temperature suitably
dissolves the polyester.
When the polyester is a poly-L-lactic acid, a poly-DL-lactic acid or a
poly(glycolide-L-
lactide) (having a monomer mole ratio of glycolic acid to L-lactic acid 50:50
or less than
50:50), tetrahydrofuran, dichloromethane (DCM) and chloroform at room
temperature to
40 ~50 °C suitably dissolve the polyester.
Polymer - Bioactive went Linkage
[0128] ~ In one embodiment, the polymers used to make the surface covering for
the
invention stems and other medical devices as described herein have one or more
bioactive
agent directly linked to the polymer. The residues of the polymer can be
linked to the
residues of the one or more bioactive agents. For example, one residue of the
polymer
can be directly linked to one residue of the bioactive agent. The polymer and
the
bioactive agent can each have one open valence.
[0129] Alternatively, more than one bioactive agent, multiple bioactive
agents, or a
mixture of bioactive agents and additional bioactive agents having different
therapeutic or
palliative activity can be directly linked to the polymer. However, since the
residue of
each bioactive agent can be linked to a corresponding residue of the polymer,
the number
of residues of the one or more bioactive agents can correspond to the number
of open
valences on the residue of the polymer.
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[0130] As used herein, a "residue of a polymer" refers to a radical of a
polymer having
one or more open valences. Any synthetically feasible atom, atoms, or
functional group
of the polymer (e.g., on the polymer backbone or pendant group) of the present
invention
can be removed to provide the open valence, provided bioactivity is
substantially retained
when the radical is attached to a residue of a bioactive agent. Additionally,
any
synthetically feasible functional group (e.g., carbonyl) can be created on the
polymer
(e.g., on the polymer backbone or pendant group) to provide the open valence,
provided
bioactivity is substantially retained when the radical is attached to a
residue of a bioactive
agent. Based on the linkage that is desired, those skilled in the art can
select suitably
functionalized starting materials that can be derived from the polymer of the
present
invention using procedures that are known in the art.
[0131] As used herein, a "residue of a compound of structural formula (*)"
refers to a
radical of a compound of polymer formulas (I - VII) as described herein having
one or
more open valences. Any synthetically feasible atom, atoms, or functional
group of the
compound (e.g., on the polymer backbone or pendant group) can be removed to
provide
the open valence, provided bioactivity is substantially retained when the
radical is
attached to a residue of a bioactive agent. Additionally, any synthetically
feasible
functional group (e.g., carboxyl) can be created on the compound of formulas
(I - VII)
(e.g., on the polymer backbone or pendant group) to provide the open valance,
provided
bioactivity is substantially retained when the radical is attached to a
residue of a bioactive
agent. Based on the linkage that is desired, those skilled in the art can
select suitably
functionalized starting materials that can be derived from the compound of
formulas (I -
VII) using procedures that are known in the art.
[0132] For example, the residue of a bioactive agent can be linked to the
residue of a
compound of structural formula (I - VII) through an amide (e.g., -N(R)C(=O)-
or
-C(=O)N(R)-), ester (e.g., -OC(=O)- or-C(=O)O-), ether (e.g., -O-), amino
(e.g., -N(R)-
), ketone (e.g., -C(=O)-), thioether (e.g., -S-), sulfinyl (e.g., -S(O)-),
sulfonyl (e.g., -S(O)2-
), disulfide (e.g., -S-S-), or a direct (e.g., C-C bond) linkage, wherein each
R is
independently H or (C1-C6) alkyl . Such a linkage can be formed from suitably
functionalized starting materials using synthetic procedures that are known in
the art.
Based on the linkage that is desired, those skilled in the art can select
suitably functional
starting material that can be derived from a residue of a compound of
structural formula (I
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- VII) and from a given residue of a bioactive agent or adjuvant using
procedures that are
known in the art. The residue of the bioactive agent or adjuvant can be linked
to any
synthetically feasible position on the residue of a compound of structural
formula (I -
VII). Additionally, the invention also provides compounds having more than one
residue
of a bioactive agent or adjuvant bioactive agent directly linked to a compound
of
structural formula (I - VII).
[0133] The number of bioactive agents that can be linked to the polymer
molecule can
typically depend upon the molecular weight of the polymer. For example, for a
compound of structural formula (I), wherein n is about 5 to about 150,
preferably about 5
to about 70, up to about 150 bioactive agent molecules (i.e., residues
thereof) can be
directly linked to the polymer (i.e., residue thereof) by reacting the
bioactive agent with
side groups of the polymer. In unsaturated polymers, the bioactive agents can
also be
reacted with double (or triple) bonds in the polymer.
[0134] Stents according to the invention are typically cylindrical in shape.
The walls
of the stmt structure can be formed of metal or polymer with openings therein,
e.g., a
mesh. The stmt is implanted into a body lumen, such as a blood vessel, where
it stays
permanently or biodegrades, to keep the vessel open and to improve blood flow
to the
heart muscle and promote natural wound healing processes at a location of
damaged
endothelium. Stems can also be positioned in vasculature in other parts of the
body, such
as the kidneys or the brain. The stenting procedure is fairly common, and
various types of
stems have been developed and used as is known in the art.
[0135] The polymers described herein can be coated onto the surface of a
porous stmt
structure or other medical device as described here in many ways, such as dip-
coating,
spray-coating, ionic deposition, and the like, as is well known in the art. In
coating a
porous stmt, care must be taken not to occlude the pores in the stmt
structure, which are
needed to allow access and migration from the interior of the vessel to the
vessel wall of
blood borne progenitor endothelial cells and other blood factors that
participate in the
natural biological process of wound healing.
[0136] Alternatively, the polymer coating on the surface of the stmt structure
can be a
formed as a polymer sheath that is applied over the stmt structure. In this
embodiment
the sheath serves as a partial physical barner to macrophages so that a
relatively small
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number of smooth muscle cells are activated to cause neointimal proliferation.
To allow
for sufficient movement of bioactive material across the porous stmt
structure, such as
progenitor endothelial cells from the blood stream, the sheath can be laser
ablated to form
openings in the polymer coating. The stmt structure can be moved while the
laser is held
stationary to ablate the structure into a pattern, or alternatively, the laser
can be
programmed to move along a predetermined pattern by a method known to
artisans. A
combination of both, i.e. moving both the laser and the structure, is also
possible. In the
present invention, even a coated stmt having a complex stmt pattern can be
made with
high precision.
[0137] The stmt structure can be formed of any suitable substance, such as is
known
in the art, that can be processed (e.g., molded, stamped, woven, etc.) to
contain the porous
surface features described herein. For example, the stmt body can be formed
from a
biocompatible metal, such as stainless steel, tantalum, nitinol, elgiloy, and
the like, as
well as suitable combinations thereof.
[0138] For example, metal stmt structures can be formed of a material
comprising
metallic fibers uniformly laid to form a three-dimensional non-woven matrix
and sintered
to form a labyrinth structure exhibiting high porosity, typically in a range
from about 50
percent to about ~5 percent, preferably at least about 70 percent. The metal
fibers
typically have a diameter in the range from about 1 micron to 25 microns.
Pores in the
stmt structure can have an average diameter in the range from about 30 microns
to about
65 microns. For use in coronary arteries, the stmt structure should be made of
100%
stainless steel, with fully annealed stainless steel being a preferred metal.
The stmt
structure can be of the type that is balloon expandable, as is known in the
art.
[0139] In one embodiment, the stmt structure is itself entirely biodegradable,
being
made of cross-linkable "star structure polymers", or dendrimers, which are
well known to
those skilled in the art. In one aspect, the stmt structure is formed from
biodegradable
cross-linked polyester amide), polycaprolactone, or polyester urethane) as
described
herein. In invention multilayered biodegradable stems, the stmt structure
(i.e., the "stmt
struts") is preferably biodegradable and hence are made of such cross-linkable
polymers
or dendrimers.
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[0140] Residues of the polymers described herein can be formed employing any
suitable reagents and reaction conditions. Suitable reagents and reaction
conditions are
disclosed, e.g., in Advanced Organic Claemistfy, Part B: Reactiofas and
Synthesis,
Second Edition, Carey and Sundberg (1953); Advanced Organic Chenaistfy,
Reactions,
Mechanisms, and Structure, Second Edition, March (1977); and
Con2pf°elaensive Ofganic
Trafasfornaations, Second Edition, Larock (1999).
[0141] Additional bioactive agents As used herein, an "additional bioactive
agent"
refers to a therapeutic or diagnostic agent other than the "bioactive" agents
described
above that promote the natural wound healing process of re-endothelialization
of vessels
as disclosed herein. Such additional bioactive agents can also be attached
polymer
coatings on the surface of the invention stems or to polymers used for coating
other types
of insertable or implantable medical or therapeutic devices having different
treatment
aims as are known in the art, wherein contact of the polymer coating with a
treatment
surface or blood borne cell or factor or release from the polymer coating by
biodegradation is desirable. However, such additional bioactive agents are not
used in the
inner layer of the invention multilayered stems, which contain only the
bioactive agents
that promote the natural would healing process of re-endothelialization of
vessels.
[0142] Specifically, such additional bioactive agent can include, but is not
limited to,
one or more: polynucleotides, polypeptides, oligonucleotides, gene therapy
agents,
nucleotide analogs, nucleoside analogs, polynucleic acid decoys, therapeutic
antibodies,
abciximab, anti-inflammatory agents, blood modifiers, anti-platelet agents,
anti-
coagulation agents, immune suppressive agents, anti-neoplastic agents, anti-
cancer
agents, anti-cell proliferation agents, and nitric oxide releasing agents.
[0143] The polynucleotide can include deoxyribonucleic acid (DNA), ribonucleic
acid
(RNA), double stranded DNA, double stranded RNA, duplex DNA/RNA, antisense
polynucleotides, functional RNA or a combination thereof. In one embodiment,
the
polynucleotide can be RNA. In another embodiment, the polynucleotide can be
DNA. In
another embodiment, the polynucleotide can be an antisense polynucleotide. In
another
embodiment. the polynucleotide can be a sense polynucleotide. In another
embodiment,
the polynucleotide can include at least one nucleotide analog. In another
embodiment,
the polynucleotide can include a phosphodiester linked 3'-5' and 5'-3'
polynucleotide
backbone. Alternatively, the polynucleotide can include non-phosphodiester
linkages,
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such as phosphotioate type, phosphoramidate and peptide-nucleotide backbones.
In
another embodiment, moieties can be linked to the backbone sugars of the
polynucleotide.
Methods of creating such linkages are well known to those of skill in the art.
[0144] The polynucleotide can be a single-stranded polynucleotide or a double-
stranded polynucleotide. The polynucleotide can have any suitable length.
Specifically,
the polynucleotide can be about 2 to about 5,000 nucleotides in length,
inclusive; about 2
to about 1000 nucleotides in length, inclusive; about 2 to about 100
nucleotides in length,
inclusive; or about 2 to about 10 nucleotides in length, inclusive.
[0145] An antisense polynucleotide is typically a polynucleotide that is
complimentary
o an mRNA, which encodes a target protein. For example, the mRNA can encode a
cancer promoting protein i.e., the product of an oncogene. The antisense
polynucleotide
is complimentary to the single-stranded mRNA and will form a duplex and
thereby
inhibit expression of the target gene, i.e., will inhibit expression of the
oncogene. The ,
antisense polynucleotides of the invention can form a duplex with the mRNA
encoding a
target protein and will disallow expression of the target protein.
[0146] A "functional RNA" refers to a ribozyme or other RNA that is not
translated.
[0147] A "polynucleic acid decoy" is a polynucleic acid that inhibits the
activity of a
cellular factor upon binding of the cellular factor to the polynucleic acid
decoy. The
polynucleic acid decoy contains the binding site for the cellular factor.
Examples of
cellular factors include, but are not limited to, transcription factors,
polymerases and
ribosomes. An example of a polynucleic acid decoy for use as a transcription
factor
decoy will be a double-stranded polynucleic acid containing the binding site
for the
transcription factor. Alternatively, the polynucleic acid decoy for a
transcription factor
can be a single-stranded nucleic acid that hybridizes to itself to form a snap-
back duplex
containing the binding site for the target transcription factor. An example of
a
transcription factor decoy is the E2F decoy. E2F plays a role in transcription
of genes
that are involved with cell-cycle regulation and that cause cells to
proliferate. Controlling
E2F allows regulation of cellular proliferation. For example, after injury
(e.g.,
angioplasty, surgery, stenting) smooth muscle cells proliferate in response to
the injury.
Proliferation may cause restenosis of the treated area (closure of an artery
through cellular
proliferation). Therefore, modulation of E2F activity allows control of cell
proliferation
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and can be used to decrease proliferation and avoid closure of an artery.
Examples of
other such polynucleic acid decoys and target proteins include, but are not
limited to,
promoter sequences for inhibiting polymerases and ribosome binding sequences
for
inhibiting ribosomes. It is understood that the invention includes polynucleic
acid decoys
constructed to inhibit any target cellular factor.
[0148] A "gene therapy agent" refers to an agent that causes expression of a
gene
product in a target cell through introduction of a gene into the target cell
followed by
expression. An example of such a gene therapy agent would be a genetic
construct that
causes expression of a protein, such as insulin, when introduced into a cell.
Alternatively,
a gene therapy agent can decrease expression of a gene in a target cell. An
example of
such a gene therapy agent~would be the introduction of a polynucleic acid
segment into a
cell that would integrate into a target gene and disrupt expression of the
gene. Examples
of such agents include viruses and polynucleotides that are able to disrupt a
gene through
homologous recombination. Methods of introducing and disrupting genes with
cells are
well known to those of skill in the art.
[0149] An oligonucleotide of the invention can have any suitable length.
Specifically,
the oligonucleotide can be about 2 to about 100 nucleotides in length,
inclusive; up to
about 20 nucleotides in length, inclusive; or about 15 to about 30 nucleotides
in length, ,
inclusive. The oligonucleotide can be single-stranded or double-stranded. In
one
embodiment, the oligonucleotide can'be single-stranded. The oligonucleotide
can be
DNA or RNA. In one embodiment, the oligonucleotide can be DNA. In one
embodiment, the oligonucleotide can be synthesized according to commonly known
chemical methods. In another embodiment, the oligonucleotide can be obtained
from a
commercial supplier. The oligonucleotide can include, but is not limited to,
at least one
nucleotide analog, such as bromo derivatives, azido derivatives, fluorescent
derivatives or
a combination thereof. Nucleotide analogs are well known to those of skill in
the art.
The oligonucleotide can include a chain terminator. The oligonucleotide can
also be
used, e.g., as a cross-linking reagent or a fluorescent tag. Many common
linkages can be
employed to couple an oligonucleotide to another moiety, e.g., phosphate,
hydroxyl, etc.
Additionally, a moiety may be linked to the oligonucleotide through a
nucleotide analog
incorporated into the oligonucleotide. In another embodiment, the
oligonucleotide can
include a phosphodiester linked 3'-5' and 5'-3' oligonucleotide backbone.
Alternatively,
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the oligonucleotide can include non-phosphodiester linkages, such as
phosphotioate type,
phosphoramidate and peptide-nucleotide backbones. In another embodiment,
moieties
can be linked to the backbone sugars of the oligonucleotide. Methods of
creating such
linkages are well known to those of skill in the art.
[0150] Nucleotide and nucleoside analogues are well known on the art. Examples
of
such nucleoside analogs include, but are not limited to, Cytovene~ (Roche
Laboratories),
Epivir~ (Glaxo Wellcome), Gemzar~ (Lilly), Hivid~ (Roche Laboratories),
Rebetron~
(Schering), Videx~ (Bristol-Myers Squibb), Zerit~ (Bristol-Myers Squibb), and
Zovirax~ (Glaxo Wellcome). See, Physician's Desk Reference, 2005 Edition.
[0151] Polypeptides acting as additional bioactive agents attached to the
polymers in
the invention stmt coverings and other medical devices can have any suitable
length.
Specifically, the polypeptides can be about 2 to about 5,000 amino acids in
length,
inclusive; about 2 to about 2,000 amino acids in length, inclusive; about 2 to
about 1,000
amino acids in length, inclusive; or about 2 to about 100 amino acids in
length, inclusive.
[0152] In one embodiment, the additional bioactive agent polypeptide attached
to the
polymer coatings for the invention medical devices can be an antibody. In one
embodiment, the antibody can bind to a cell adhesion molecule, such as a
cadherin,
integrin or selectin. In another embodiment, the antibody can bind to an
extracellular
matrix molecule, such as collagen, elastin, fibronectin or laminin. In still
another
embodiment, the antibody can bind to a receptor, such as an adrenergic
receptor, B-cell
receptor, complement receptor, cholinergic receptor, estrogen receptor,
insulin receptor,
low-density lipoprotein receptor, growth factor receptor or T-cell receptor.
Antibodies
attached to polymers (either directly or by a linker) in the invention medical
devices can
also bind to platelet aggregation factors (e.g., fibrinogen), cell
proliferation factors (e.g.,
growth factors and cytokines), and blood clotting factors (e.g., fibrinogen).
In another
embodiment, an antibody can be conjugated to an active agent, such as a toxin.
In
another embodiment, the antibody can be Abciximab (ReoProR)). Abciximab is an
Fab
fragment of a chimeric antibody that binds to beta(3) integrins. Abciximab is
specific for
platelet glycoprotein IIb/IIIa receptors, e.g., on blood cells. Human aortic
smooth muscle
cells express alpha(v)beta(3) integrins on their surface. Treating beta(3)
expressing
smooth muscle cells may prohibit adhesion of other cells and decrease cellular
migration
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or proliferation, thus reducing restenosis following percutaneous coronary
interventions
(CPI) e.g., stenosis, angioplasty, stenting. Abciximab also inhibits
aggregation of blood
platelets.
[0153] In one embodiment, the peptide can be a glycopeptide. "Glycopeptide"
refers
to oligopeptide (e.g. heptapeptide) antibiotics, characterized by a mufti-ring
peptide core
optionally substituted with saccharide groups, such as vancomycin. Examples of
glycopeptides included in this definition may be found in "Glycopeptides
Classification,
Occurrence, and Discovery," by Raymond C. Rao and Louise W. Crandall,
("Bioactive
agents and the Pharmaceutical Sciences" Volume 63, edited by Ramakrishnan
Nagarajan,
published by Marcal Dekker, Inc.). Additional examples of glycopeptides are
disclosed
in U.S. Patent Nos. 4,639,433; 4,643,987; 4,497,802; 4,698,327, 5,591,714;
5,840,684;
and 5,843,889; in EP 0 802 199; EP 0 801 075; EP 0 667 353; WO 97/28812; WO
97/38702; WO 98/52589; WO 98/52592; and in J. Amef~. Cl2em. Soc., 1996, 118,
13107-
13108; .I. An2er. Chem. Soc., 1997, 119, 12041-12047; and J. Amen°.
Chena. Soc., 1994,
116, 4573-4590. Representative glycopeptides include those identified as A477,
A35512,
A40926, A41030, A42867, A47934, A80407, A82846, A83850, A84575, AB-65,
Actaplanin, Actinoidin, Ardacin, Avoparcin, Azureomycin, Balhimyein,
Chloroorientiein,
Chloropolysporin, Decaplanin, -demethylvancomycin, Eremomycin, Galacardin,
Helvecardin, Izupeptin, Kibdelin, LL-AM374, Mannopeptin, MM45289, MM47756,
MM47761, MM49721, MM47766, MM55260, MM55266, MM55270, MM56597,
MM56598, OA-7653, Orenticin, Parvodicin, Ristocetin, Ristomycin, Synmonicin,
Teicoplanin, UK-68597, UD-69542, UK-72051, Vancomycin, and the like. The term
"glycopeptide" or "glycopeptide antibiotic" as used herein is also intended to
include the
general class of glycopeptides disclosed above on which the sugar moiety is
absent, i.e.
the aglycone series of glycopeptides. For example, removal of the disaccharide
moiety
appended to the phenol on vancomycin by mild hydrolysis gives vancomycin
aglycone.
Also included within the scope of the term "glycopeptide antibiotics" are
synthetic
derivatives of the general class of glycopeptides disclosed above, included
alkylated and
acylated derivatives. Additionally, within the scope of this term are
glycopeptides that
have been further appended with additional saccharide residues, especially
aminoglycosides, in a manner similar to vancosamine.
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[0154] The term "lipidated glycopeptide" refers specifically to those
glycopeptide
antibiotics that have been synthetically modified to contain a lipid
substituent. As used
herein, the term "lipid substituent" refers to any substituent contains 5 or
more carbon
atoms, preferably, 10 to 40 carbon atoms. The lipid substituent may optionally
contain
from 1 to 6 heteroatoms selected from halo, oxygen, nitrogen, sulfur, and
phosphorous.
Lipidated glycopeptide antibiotics are well known in the art. See, for
example, in U.S.
Patent Nos. 5,840,684, 5,843,889, 5,916,873, 5,919,756, 5,952,310, 5,977,062,
5,977,063,
EP 667, 353, WO 98/52589, WO 99/56760, WO 00/04044, WO 00/39156, the
disclosures
of which are incorporated herein by reference in their entirety.
[0155] Anti-inflammatory agents useful for attachment to polymer coatings of
the
invention stems and other medical devices, or for loading into the outer layer
of the
invention multilayered stems include, e.g. analgesics (e.g., NSAIDS and
salicyclates),
antirheumatic agents, gastrointestinal agents, gout preparations, hormones
(glucocorticoids), nasal preparations, ophthalmic preparations, otic
preparations (e.g.,
antibiotic and steroid combinations), respiratory agents, and skin & mucous
membrane
agents. See, Physician's Desk Reference, 2005 Edition: Specifically, the anti-
inflammatory agent can include dexamethasone, which is chemically designated
as (119,
16I)-9-fluro-11,17,21-trihydroxy-16-methylpregna-1,4-dime-3,20-dione.
Alternatively,
the anti-inflammatory agent can include sirolimus (rapamycin), which is a
triene
macrolide antibiotic isolated from Steptomyces laygf°oscopicus.
[0156] Anti-platelet or anti-coagulation agents include, e.g., Coumadin~
(DuPont),
Fragmin~ (Pharmacia & Upjohn), Heparin~ (Wyeth-Ayerst), Lovenox~, Normiflo~,
Orgaran ~ (Organon), Aggrastat~ (Merck), Agrylin~ (Roberts), Ecotrin~
(Smithkline
Beecham), Flolan~ (Glaxo Wellcome), Halfprin~ (Kramer), Integrillin~ (COR
Therapeutics), Integrillin~ (Key), Persantine~ (Boehringer Ingelheim), Plavix~
(Bristol-
Myers Squibb), ReoPro~ (Centecor), Ticlid~ (Roche), Abbokinase~ (Abbott),
Activase~ (Genentech), Eminase~ (Roberts), and Strepase~ (Astray. See,
Physician's
Des7~Reference, 2005 Edition. Specifcally, the anti-platelet or anti-
coagulation agent can
include trapidil (avantrin), cilostazol, heparin, hirudin, or ilprost.
[0157] Trapidil is chemically designated as N,N-dimethyl-5-methyl-
[1,2,4]triazolo[1,-
5-a]pyrimidin-7-amine.
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[0158] Cilostazol is chemically designated as 6-[4-(1-cyclohexyl-1H-tetrazol-5-
yl)-
butoxy]-3,4-dihydro-2( 1 H)-quinolinone.
[0159] Heparin is a glycosaminoglycan with anticoagulant activity; a
heterogeneous
mixture of variably sulfonated polysaccharide chains composed of repeating
units of D-
glucosamine and either L-iduronic or D-glucuronic acids.
[0160] Hirudin is an anticoagulant protein extracted from leeches, e.g.,
Flirudo
mediciyaalis.
[0161] Iloprost is chemically designated as 5-[Hexahydro-5-hydroxy-4-(3-
hydroxy-4-
methyl-1-octen-6-ynyl)-2(1H)-pentalenylidene]pentanoic acid.
[0162] The immune suppressive agent can include, e.g., Azathioprine~ (Roxane),
BayRho-D~ (Bayer Biological), CellCept~ (Roche Laboratories), Imuran~ (Glaxo
Wellcome), MiCRhoGAM~ (Ortho-Clinical Diagnostics), Neoran~ (Novartis),
Orthoclone OKT3~ (Ortho Biotech), Prograf~ (Fujisawa), PhoGAM~ (Ortho-Clinical
Diagnostics), Sandimmune~ (Novartis), Simulect~ (Novartis), and Zenapax~
(Roche
Laboratories).
[0163] Specifically, the immune suppressive agent can include rapamycin or
thalidomide. Rapamycin is a triene macrolide isolated from Streptofnyces
laygroscopiczis.
[0164] Thalidomide is chemically designated as 2-(2,6-dioxo-3-piperidinyl)-1H-
iso-
indole-1,3(2H)-dione.
[0165] Anti-cancer or anti-cell proliferation agents that can be used as an
additional
bioactive agent, for example, in the outer layer of the invention multilayered
stems
include, e.g., nucleotide and nucleoside analogs, such as 2-chloro-
deoxyadenosine,
adjunct antineoplastic agents, alkylating agents, nitrogen mustards,
nitrosoureas,
antibiotics, antimetabolites, hormonal agonists/antagonists, androgens,
antiandrogens,
antiestrogens, estrogen ~ nitrogen mustard combinations, gonadotropin
releasing
hormone (GNRH) analogues, progestrins, immunomodulators, miscellaneous
antineoplastics, photosensitizing agents, and skin and mucous membrane agents.
See,
Physicia~z's DeskReferehce, 2005 Edition.
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[0166] Suitable adjunct antineoplastic agents include Anzemet~ (Hoeschst
Marion
Roussel), Aredia~ (Novartis), Didronel~ (MGI), Diflucan~ (Pfizer), Epogen~
(Amgen), Ergamisol~ (Janssen), Ethyol~ (Alza), Kytril~ (SmithKline Beecham),
Leucovorin~ (Immunex), Leucovorin~ (Glaxo Wellcome), Leucovorin~ (Astray,
Leukine~ (Immunex), Marinol~ (Roxane), Mesnex~ (Bristol-Myers Squibb
Oncology/Immunology), Neupogen (Amgen), Procrit~ (Ortho Biotech), Salagen~
(MGI), Sandostatin~ (Novartis), Zinecard~ (Pharmacia and Upjohn), Zofran~
(Glaxo
Wellcome) and Zyloprim~ (Glaxo Wellcome).
[0167] Suitable miscellaneous alkylating agents include Myleran~ (Glaxo
Wellcome),
Paraplatin~ (Bristol-Myers Squibb OncologylImmunology), Platinol~ (Bristol-
Myers
Squibb Oncology/Immunology) and Thioplex~ (Immunex).
[0168] Suitable nitrogen mustards include Alkeran~ (Glaxo Wellcome), Cytoxan~
(Bristol-Myers Squibb Oncology/Immunology), Ifex~ (Bristol-Myers Squibb
Oncology/Immunology), Leukeran~ (Glaxo Wellcome) and Mustargen~ (Merck).
[0169] Suitable nitrosoureas include BiCNU~ (Bristol-Myers Squibb
Oncology/Immunology), CeeNU~ (Bristol-Myers Squibb OncologylImmunology),
Gliadel~ (Rhone-Poulenc Rover) and Zanosar~ (Pharmacia and Upjohn).
(0170] Suitable antibiotics include Adriamycin PFS/RDF~ (Pharmacia and
Upjohn),
Blenoxane~ (Bristol-Myers Squibb Oncology/Immunology), Cerubidine~ (Bedford),
Cosmegen~ (Merck), DaunoXome~ (NeXstar), Doxil~ (Sequus), Doxorubicin
Hydrochloride~ (Astray, Idamycin~ PFS (Pharmacia and Upjohn), Mithracin~
(Bayer),
Mitamycin~ (Bristol-Myers Squibb Oncology/Iminunology), Nipen~ (SuperGen),
Novantrone~ (Immunex) and Rubex~ (Bristol-Myers Squibb Oncology/Immunology).
[0171] Suitable antimetabolites include Cytostar-U~ (Pharmacia and Upjohn),
Fludara~ (Berlex), Sterile FUDR~ (Ruche Laboratories), Leustatin~ (Ortho
Biotech),
Methotrexate~ (hnmunex), Parinethol~ (Glaxo Wellcome), Thioguanine~ (Glaxo
Wellcome) and Xeloda~ (Ruche Laboratories).
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[0172] Suitable androgens include Nilandron~ (Hoechst Marion Roussel) and
Teslac~ (Bristol-Myers Squibb Oncology/Immunology).
[0173] Suitable antiandrogens include Casodex~ (Zeneca) and Eulexin~
(Schering).
[0174] Suitable antiestrogens include Arimidex~ (Zeneca), Fareston~
(Schering),
Femara~ (Novartis) and Nolvadex~ (Zeneca).
[0175] Suitable estrogen and nitrogen mustard. combinations include Emcyt~
(Pharmacia and Upjohn).
[0176] Suitable estrogens include Estrace~ (Bristol-Myers Squibb) and Estrab~
(Solway).
[0177] Suitable gonadotropin releasing hormone (GNRH) analogues include
Leupron
Depot~ (TAP) and Zoladex~ (Zeneca).
[0178] Suitable progestins include Depo-Provera~ (Phannacia and Upjohn) and
Megace~ (Bristol-Myers Squibb Oncology/Immunology).
[0179] Suitable immunomodulators include Erganisol~ (Janssen) and Proleukin~
(Chiron Corporation).
[0180] Suitable miscellaneous antineoplastics include Camptosar~ (Pharmacia
and
Upjohn), Celestone~ (Schering), DTIC-Dome~ (Bayer), Elspar~ (Merck),
Etopophos~
(Bristol-Myers Squibb Oncology/Immunology), Etopoxide~ (Astray, Gemzar~
(Lilly),
Hexalen~ (U.S. Bioscience), Hycantin~ (SmithKline Beecham), Hydrea~ (Bristol-
Myers Squibb Oncology/Immunology), Hydroxyurea~ (Roxane), Intron A~
(Schering),
Lysodren~ (Bristol-Myers Squibb Oncology/Immunology), Navelbine~ (Glaxo
Wellcome), Oncaspar~ (Rhone-Poulenc Rover), Oncovin~ (Lilly), Proleukin~
(Chiron
Corporation), Rituxan~ (IDEC), Rituxan~ (Genentech), Roferon-A~ (Roche
Laboratories), Taxol~ (paclitaxol/paclitaxel, Bristol-Myers Squibb
Oncology/Immunology), Taxotere~ (Rhone-Poulenc Rover), TheraCys~ (Pasteur
Merieux Connaught), Tice BCG~ (Organon), Velban~ (Lilly), VePesid~ (Bristol-
Myers
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Squibb Oncology/Immunology), Vesanoid~ (Roche Laboratories) and Vumon~
(Bristol-
Myers Squibb Oncology/Immunology).
[0181] Suitable photosensitizing agents include Photofrin~ (Sanofi).
[0182] Specifically, the anti-cancer or anti-cell proliferation agent can
include Taxol~
(paclitaxol), a nitric oxide-like compound, or NicOX (NCX-4016). Taxol~
(paclitaxol) is
chemically designated as 5(3,20-Epoxy-1,2a4,7(3,10(3,13a-hexahydroxytax-11-en-
9-one
4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine.
[0183] A nitric oxide-like agent includes any bioactive agent that contains a
nitric
oxide releasing functional group. Suitable nitric oxide-like compounds are S-
nitrosothiol
derivative (adduct) of bovine or human serum albumin and as disclosed, e.g.,
in LT.S.
Patent No. 5,650,447. See, e.g., David Marks et al., "Inhibition of neointimal
proliferation in rabbits after vascular injury by a single treatment with a
protein adduct of
nitric oxide," .I Glin. Ifavest.(1995) 96:2630-2638. NCX-4016 is chemically
designated as
2-acetoxy-benzoate 2-(nitroxymethyl)-phenyl ester, and is an antithrombotic
agent.
[0184] It is appreciated that those skilled in the art understand that the
bioactive agent
or additional bioactive agent useful in the present invention is the bioactive
substance
present in any of the bioactive agents or agents disclosed above. For example,
Taxol~ is
typically available as an injectable, slightly yellow viscous solution. The
bioactive agent,
however, is a crystalline powder with the chemical name 5(3,20-Epoxy-
1,2a,4,7(3,10(3,13a-hexahydroxytax-11-en-9-one 4,10-diacetate 2-benzoate 13-
ester with
(2R,3S)-N-benzoyl-3-phenylisoserine. Physician's Des7~Refereface (PIER),
Medical
Economics Company (Montvale, NJ), (53rd Ed.), pp. 1059-1067.
[0185] As used herein a "residue of a bioactive agent" or "residue of an
additional
bioactive agent" is a radical of such bioactive agent as disclosed herein
having one or
more open valences. Any synthetically feasible atom or atoms of the bioactive
agent can
be removed to provide the open valence, provided bioactivity is substantially
retained
when the radical is attached to a residue of a polymer described herein. Based
on the
linkage that is desired, those skilled in the art can select suitably
functionalized starting
materials that can be derived from a bioactive agent using procedures that are
known in
the art.
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[0186] The residue of a bioactive agent or additional bioactive agent, as
described
herein, can be formed employing any suitable reagents and reaction conditions.
Suitable
reagents and reaction conditions are disclosed, e.g., in Advanced Organic
Claenaistry, Paf~t
B: Reactions and Synthesis, Second Edition, Carey and Sundberg (1983);
Advanced
Ofganic Chemistry, Reactions, Mechanisms and Structuf°e, Second
Edition, March
(1977); and Compf°ehensive Organie T'ransfof°mations, Second
Edition, Larock (1999).
[0187] In certain embodiments, the polymer-bioactive agent linkage can degrade
to
provide a suitable and effective amount of free bioactive agent. As will be
appreciated by
those of skill in the art, depending upon the chemical and therapeutic
properties of the
biological agent, in certain other embodiments, the bioactive agent attached
to the
polymer performs its therapeutic effect while still attached to the polymer,
such as is the
case with the "sticky" polypeptides Protein A and Protein G, known herein as
"bioligands", which function while attached to the polymer to hold a target
molecule
close to the polymer, and the bradykinins and antibodies, which function by
contacting
(e.g., bumping into) a receptor on a target molecule. Any suitable and
effective amount
of bioactive agent can be released and will typically depend, e.g., on the
specific polymer,
bioactive agent, and polymer/bioactive agent linkage chosen. Typically, up to
about
100% of the bioactive agent can be released from the polymer by degradation of
the
polymer/bioactive agent linkage. Specifically, up to about 90°/~, up to
75%, up to 50%, or
up to 25% of the bioactive agent can be released from the polymer. Factors
that typically
affect the amount of the bioactive agent that is released from the polymer is
the type of
polymer/bioactive agent linkage, and the nature and amount of additional
substances
present in the formulation.
[0188] The polymer-bioactive agent linkage can degrade over a period of time
to
provide time release of a suitable and effective amount of bioactive agent.
Any suitable
and effective period of time can be chosen. Typically, the suitable and
effective amount
of bioactive agent can be released in about twenty-four hours, in about seven
days, in
about thirty days, in about ninety days, or in about one hundred and twenty
days. Factors
that typically affect the length of time in which the bioactive agent is
released from the
polymer-bioactive agent include, e.g., the nature and amount of polymer, the
nature and
amount of bioactive agent, the nature of the polymer/bioactive agent linkage,
and the
nature and amount of additional substances present in the formulation.
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[0189] Polyfne~ Intel°mixed with Bioactive Agent or Additional
Bioactive Agent In
addition to being linked to one or more bioactive agents, either directly or
through a
linker, a polymer used for coating a medical device or making a sheath for a
stmt
structure as described herein can be physically intermixed with one or more
bioactive
agents or additional bioactive agents to provide a polymer formulation that is
used for
coating a medical device or a stmt structure.
(0190] As used herein, "intermixed" refers to a polymer of the present
invention
physically mixed with a bioactive agent or a polymer as described herein that
is
physically in contact with a bioactive agent.
[0191] As used herein, a "formulation" refers to a polymer as described herein
that is
intermixed with one or more bioactive agents or additional bioactive agents.
The
formulation includes such a polymer having one or more bioactive agents
present on the
surface of the polymer, partially embedded in the polymer, or completely
embedded in
the polymer. Additionally, the formulation includes a polymer as described
herein and a
bioactive agent forming a homogeneous composition (i.e., a homogeneous
formulation).
(0192] By contrast, in the invention multilayered stems, in the outer layer
non-
covalently bound bioactive agents and/or additional bioactive agents can be
intermingled
with or "loaded into" any biocompatible biodegradable polymer as is known in
the art
since the outer layer in this embodiment of the invention does not come into
contact with
blood. However, the inner layer has only bioactive agents covalently attached
to a
hydrophilic, blood-compatible polymer as described herein.
[0193] Any suitable amount of polymer and bioactive agent can be employed to
provide the formulation. The polymer can be present in about 0.1 wt.% to about
99.9
wt.% of the formulation. Typically, the polymer can be present above about 25
wt.% of
the formulation; above about 50 wt.% of the formulation; above about 75 wt.% %
of the
formulation; or above about 90 wt.% of the formulation. Likewise, the
bioactive agent
can be present in about 0.1 wt.% to about 99.9 wt.% of the formulation.
Typically, the
bioactive agent can be present above about 5 wt.% of the formulation; above
about 10
wt.% of the formulation; above about 15 wt.% of the formulation; or above
about 20
wt.% of the formulation.
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[0194] In yet another embodiment of the invention the polymer coating having a
bioactive agent dispersed therein can be applied as a polymeric film onto at
least a portion
of the surface of any medical device to be implanted into a diabetic that is
exposed to
blood and upon which it is desirable to establish an endothelial layer (e.g.,
a heart valve,
or a synthetic bypass artery). The polymeric film can have any suitable
thickness on the
medical device. For example, the thickness of the polymeric film on the
medical device
can be about 1 to about 50 microns thick or about 5 to about 20 microns thick.
In the
invention stems and multilayered stems, each of the layers can be from 0.1
micron to 50
microns thick, for example from 0.5 micron to 5 microns in thickness.
[0195] The polymeric film can effectively serve as a bioactive agent-eluting
polymeric
coating on a medical device, such as a stmt structure. This bioactive agent
eluting
polymeric coating can be created on the medical device by any suitable coating
process,
e.g., dip coating, vacuum depositing, or spray coating the polymeric film, on
the medical
device. Additionally, the bioactive agent eluting polymer coating system can
be applied
onto the surface of a stmt, a vascular delivery catheter, a delivery balloon,
a separate stmt
cover sheet configuration, or a stmt bioactive agent delivery sheath, as
described herein
to create a type of local bioactive agent delivery system. When the polymer is
used as a
cover sheet for a stmt, the polymer can be processed, for example by extrusion
or
spiraling as is known in the art, to form a woven sheet or mat of fine polymer
fibers to
which the bioligand is covalently attached, either directly or by means of a
linker, as
described herein.
[0196] The bioactive agent-eluting polymer coated stems and other medical
devices
cari be used in conjunction with, e.g., hydrogel-based bioactive agent
delivery systems.
For example, in one embodiment, the above-described polymer coated stems and
medical
devices, can be coated with an additional formulation layer applied over the
polymer
coated stmt surface as a sandwich type of configuration to deliver to the
blood vessels
bioactive agents that promote natural re-endothelialization processes and
prevent or
reduce in-stmt restenosis. Such an additional layer of hydrogel-based drug
release
formulation can comprise various bioactive agents mixed with hydrogels (see,
U.S. Patent
No. 5,610,241, which is incorporated by reference herein in its entirety) to
provide an
elution rate different than that of the polymer-active agent coating on the
stmt structure or
medical device surface.
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[0197] Any suitable size of polymer and bioactive agent can be employed to
provide
such a formulation. For example, the polymer can have a size of less than
about 1 x 10~
meters, less than about 1 x 10-5 meters, less than about 1 x 10-6 meters, less
than about 1 x
10-~ meters, less than about 1 x 10-8 meters, or less than about 1 x 10-9
meters.
[0198] The formulation can degrade to provide a suitable and effective amount
of the
bioactive agents. Any suitable and effective amount of bioactive agent can be
released
and will typically depend, e.g., on the specific formulation chosen.
Typically, up to about
100% of the bioactive agent can be released from the formulation.
Specifically, up to
about 90%, up to 75%, up to 50%, or up to 25% of the bioactive agent can be
released
from the formulation. Factors that typically affect the amount of the
bioactive agent that
is released from the formulation include, e.g., the nature and amount of
polymer, the
nature and amount of bioactive agent, and the nature and amount of additional
substances
present in the formulation.
[0199] The formulation can degrade over a period of time to provide the
suitable and
effective amount of bioactive agent. Any suitable and effective period of time
can be
chosen. Typically, the suitable and effective amount of bioactive agent can be
released in
about twenty-four hours, in about seven days, in about thirty days, in about
ninety days,
or in about one hundred and twenty days. Factors that typically affect the
length of time
in which the bioactive agent is released from the formulation include, e.g.,
the nature and
amount of polymer, the nature and amount of bioactive agent, and the nature
and amount
of additional substances present in the formulation.
[0200] The present invention also provides for an invention stmt coated with a
formulation that includes a polymer as described herein physically intermixed
with one or
more bioactive agents. The polymer that is present in the formulation can also
be linked,
either directly or through a linker, to one or more (e.g., 1, 2, 3, or 4)
bioactive agents. As
such, the polymer can be intermixed with one or more (e.g., l, 2, 3, or 4)
bioactive agents
and can be linked, either directly or through a linker, to one or more (e.g.,
1, 2, 3, or 4)
bioactive agents.
(0201] A polymer used in making an invention stmt can include one or more
bioactive
agents. In one embodiment, the polymer is physically intermixed with one or
more
bioactive agents. In another embodiment, the polymer is linked to one or more
bioactive
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agents, either directly or through a linker. In another embodiment, the
polymer is linked
to one or more bioactive agents, either directly or through a linker, and the
resulting
polymer can also be physically intermixed with one or more bioactive agents.
[0202] A polymer used in making an invention stmt, whether or not present in a
formulation as described herein, whether or not linked to a bioactive agent as
described
herein, and whether or not intermixed with a bioactive agent as described
herein, can also
be used in medical therapy or medical diagnosis. For example, the polymer can
be used
in the manufacture of a medical device. Suitable medical devices include,
e.g., artificial
joints, artificial bones, cardiovascular medical devices, stems, shunts,
medical devices
useful in angioplastic therapy, artificial heart valves, artificial by-passes,
sutures, artificial
arteries, vascular delivery catheters, drug delivery balloons, separate
tubular stmt cover
sheet configurations (referred to herein as "sheaths"), and stmt bioactive
agent delivery
sleeve types for local bioactive agent delivery systems.
[0203] In still another embodiment, the invention provides methods for
treating a
patient suffering from diabetes having a vessel with a damaged endothelium by
implanting an invention stmt in the vessel at the locus of damage and allowing
the stmt
to interact with blood components within the vessel. The invention may further
comprise
testing a blood sample from the diabetic patient to determine the amount of
therapeutic
PECs in the sample as compared with a parallel sample of blood from a healthy
non-
diabetic individual to detect a decrease in the amount of therapeutic PECs in
the blood
from the diabetic patient. Such testing may be conducted prior to implantation
of an
invention stem to determine whether the diabetic patient has a decreased
amount or
concentration of therapeutic PECs as compared with the normal concentration in
healthy
non-diabetic patients.
[0204] In another embodiment, the invention methods for treating diabetics
having
damaged vasculature may further comprise obtaining therapeutic PECs from the
circulating blood of the diabetic, expanding the patient's therapeutic PECs ex
vivo, and
transfusing the autologous PECs into the circulating blood of the diabetic
patient either
before or contemporaneously with implantation of the invention stems.
[0205] All publications, patents, and patent documents are incorporated by
reference
herein, as though individually incorporated by reference. The invention has
been
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described with reference to various specific, and preferred embodiments and
techniques.
However, it should be understood that many variations and modifications may be
made
while remaining within the spirit and scope of the invention.
[0206] The invention will be further understood with reference to the
following
examples, which are purely exemplary, and should not be taken as limiting the
true scope
of the present invention as described in the claims.
EXAMPLE 1
[0207] Amide Bond Formation-This example illustrates the coupling of a
carboxyl
group of a polymer with an amino functional group of the bioactive agent, or
equally, the
coupling of a carboxyl group of the bioactive agent with an amino functional
group of a
polymer.
[0208] Coupling Tlzt°oztgh Pt°e-Foamed Active Esters;
Caf°bodiimide Mediated
Couplings-Conjztgation of 4-Amino-Ternpo to Polyme~° The free
carboxylic acid form
of the PEA polymer is converted first to its active succinimidyl ester (PEA-
OSu) or
benzotriazolyl ester (PEA-OBt). This conversion can be achieved by reacting
dried PEA-
R polymer with N Hydroxysuccinimide (NHS) or 1-Hydroxybenzotriazole (HOBt) and
a
suitable dehydrating agent, such as dicyclohexylcarbodiimide (DCC), in
anhydrous
CH2C12 at room temperature for 16 hrs. After filtering away the precipitated
dicyclohexylurea (DCU), the PEA-OSu product may be isolated by precipitation,
or used
without further purification, in which case the PEA-OSu solution is
transferred to a round
bottom flask, diluted to the desired concentration, and cooled to 0°C.
Next, a solution of
the free amine-containing bioactive agent-the nucleophile, specifically, 4-
Amino-
Tempo--in CH2Cl2 is added in a single shot at 0°C. (Equally, the
nucleophile may be
revealed in situ by treating the ammonium salt of the bioactive agent with a
hindered
base, preferably a tertiary amine, such as like triethylamine or,
diisopropylethylamine, in
a suitable aprotic solvent, such as dichloromethane (DCM)). The reaction is
monitored
by tracking consumption of the free amine by TLC, as indicated by ninhydrin
staining.
Work-up for the polymer involves customary precipitation of the reaction
solution into a
mixture of non-solvent, such as hexanelethyl acetate. Solvent is then
decanted, polymer
residue is resuspended in a suitable solvent, filtered, concentrated by roto-
evaporation,
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cast onto a clean teflon tray, and dried under vacuum to furnish the PEA-
bioactive agent
conjugate, specifically, PEA-4-Amino-Tempo.
[0209] Amii2iumlUf°onium Salt and Phosphonium Salt Mediated Couplifags
Two
effective catalysts for this type of coupling include: HBTU, O-(benzotriazol-1-
yl)-1,1,3,3-
teramethyluronium hexafluorophosphate, and BOP, 1-
benzotriazolyoxytris(dimethyl-
amino)phosphonium hexafluorophosphate (Castro's Reagent). These reagents are
employed in the presence of equimolar amounts of the carboxyl group of the
polymer and
the amino functional group of the bioactive agent (neutral or as the ammonium
salt), with
a tertiary amine such as diisopropylethylamine, N methylmorpholine, or
dimethyl-
substituted pyridines (DMAP), in solvents such as DMF, THF, or acetonitrile.
EXAMPLE 2
[0210] Ester Bond Formation - This example illustrates coupling of a carboxyl
group
of a polymer with a hydroxyl functional group of the bioactive agent, or
equally, coupling
of a carboxyl group of the bioactive agent with a hydroxyl functional group of
a polymer.
[0211] Caf°b~diimide Mediated Esterification For the conjugation, a
sample of the
carboxyl-group-containing polymer was dissolved in DCM. To this slightly
viscous
solution was added a solution of the hydroxyl-containing-drug/biologic and
DMAP in
DCM. The flask was then placed in an ice bath and cooled to 0 °C. Next,
a solution of
1,3-diisopropylcarbodiimide (DIPC) in DCM was added, the ice bath removed, and
the
reaction warmed to room temperature. The conjugation reaction was stirred at
room
temperature for 16 hours during which time TLC was periodically performed to
monitor
consumption of the hydroxyl functional group of the bioactive agent. After the
allotted
time, the reaction mixture was precipitated, and the polymer-bioactive agent
conjugate
was isolated as described above in Example 1.
EXAMPLE 3
[0212] PEC Isolations To establish the protocol for isolating the progenitor
endothelial cells (PECs) from peripheral blood, blood from healthy, normal
donors was
used. A literature review generated multiple PEC isolation protocols (J. C.I.
(2000)
105:71-77 ; Cif°c. (2003) 107:143-149; Cif°c. (2003) 107:1164-
1169; Plast. Reconstf-uc.
SZif g~°. (2004) 113:284; and Am. J. Physiol. Heaf-t Circ. Playsiol.
(2004) 286:H1985-
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H 1993). Surprisingly, however, preliminary attempts required modification of
the known
protocols to ensure successful isolations. The flow chart in Fig. 2 presents a
modified
protocol followed in isolation of PECs.
[0213] From a trial PEC isolation, it was determined that cells would attach
and grow
better on fibronectin-coated plates than on gelatin-coated plates. Cells were
isolated from
120 milliliters of peripheral blood and then single aliquots of cells were
plated in
Endothelial Basal Medium and 5% FBS (Cambrex). The media was changed every 4-5
days. The total cell number obtained from the isolations was donor-dependent
and ranged
from 40 million to 200 million cells.
[0214] Table 1 below indicates the isolation methods and the PEC isolation
outcome
for PEC isolation from various donors. Initially, both a mononuclear cell
Ficoll gradient
protocol (designed to isolate human mononuclear cells from peripheral blood)
and a
CD 133+ magnetic bead purification step were used to ensure the isolation of
PECs. It did
not appear that the CD133+ purification step was increasing the isolation of
PECs, so this
step was omitted from the last two donors.
TABLE 1
Donor IdentifierGradientCD133+ PEC
Donor 1 Yes Yes Yes
Donor 2 Yes Yes No
Donor 3 Yes Yes No
Donor 1 Yes Yes Yes
Donor 4 Yes No No
Donor 5 Yes No Yes
Cells were plated either in 12-well or 6-well fibronectin-coated plates and
monitored
daily, over a span of about 28-30 days. The culture media used in the PEC
isolations was
Endothelial Basal Medium plus SingleQuot Kit (Cambrex Corporation, East
Rutherford,
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New Jersey), a mixture of hydrocortisone, hEGF, FBS, VEGF, hFGF-B, R3-IGF-1,
ascorbic acid and heparin. Generally from 10-15 days in culture after the
isolation were
required before a monolayer became apparent.
[0215] Once a monolayer was identified, cells were further characterized with
DiI-
acetylated-Low Density Lipoprotein (LDL). The human LDL complex delivers
cholesterol to cells via receptor-mediated endocytosis. However, the
acetylated form of
LDL is not taken up by the LDL receptor, but is taken up by macrophages and
endothelial
cells via a "scavenger" receptor specific for the modified LDL. Decreased
uptake by
endothelial cells as compared with macrophages was determined by microscope
and
photographed (100x magnification). The monolayer remains actively growing for
a few
months. Cells were replated and reformed the monolayer for several passages
(about 30
days in culture) before becoming senescent.
[0216] The number of circulating PECs is known to be extremely low, below 0.1
%;
accordingly, the success rate of PEC isolations was found to be about 40%.
(Herz (2002)
27: 579-SS).
EXAMPLE 4
[0217] Cell Recruitment to Bioactive Agents To select appropriate bioligands
for
use as recruitment factors in stmt applications, an in vitro adhesion assay
was developed.
This assay can distinguish between endothelial cells (ECs) and smooth muscle
cells
(SMCs) to aid in selecting potential attachment factors. Both the ECs and SMCs
used in
these assays were purchased from Cambrex (Baltimore, MD) (HASMC = Human Aortic
Smooth Muscle Cells and HCAEC = Human Coronary Artery Endothelial Cells).
[0218] Fig. 3 shows the flow chart of the protocol followed for this assay.
The
attachment factor, in a phosphate buffered saline (PBS) solution, was coated
onto a non-
tissue culture dish and allowed to adsorb overnight at 4°C. The
following day the plate
was blocked for 1 hour at room temperature with heat-inactivated, 0.2% bovine
serum
albumin (BSA) solution (in PBS) to prevent non-specific attachment. A timed
adhesion
assay was then conducted. The assay includes negative control wells coated
only with
PBS and positive control wells coated with fibronectin. So far, none of the
adhesion
factors tested has surpassed the cell adhesion and cell spreading induced by
fibronectin.
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61
In addition to adhesion, spreading is also an important consideration in
determining the
suitability of a substrate. If the cells are not able to spread, it is
unlikely that the cells will
proliferate on that surface.
[0219] Initial efforts focused on potential recruitment factors with low
affinity but
present in high density. A variety of potential recruitment factors were
tested, including:
1. Sialyl Lewis X, a ligand for Selectin receptors found on endothelium;
2. CSS, whose amino acid sequence is Gly-Glu-Glu-Ile-Gln-Ile-Gly-His-Ile-Pro-
Afg-Glu-Asp- Teal-Asp-Tyj°-His-Leu-Tyr-Pro (SEQ ID NO:1). CSS is found
in the Type
III connecting segment of fibronectin, an extracellular matrix protein known
to bind many
different cells, including ECs. The sequence for the CSS peptide contains the
amino acid
sequence REDVDY (underlined) (SEQ ID N0:2); and
3. GREDVDY (SEQ ID NO:l 1), which includes a G linker placed on the
REDVDY sequence.)
[0220] Of the bioligands tested to date, CSS and GREDVDY gave the most
promising
adhesion data with the best sites for conjugation to the polymers used in
making the
invention stems. Even though neither of these peptide sequences equaled the
large
molecule fibronectin in cell adhesion or spreading, surprisingly both peptide
sequences
showed specificity for ECs over SMCs and these small peptide sequences can be
readily
synthesized and bound to the polymers used in the polymers used in manufacture
of the
invention stems and implantable medical device coverings.
[0221] In addition to microscopic observations, cell adhesion was quantitated
using an
ATP assay. Data of a representative adhesion assay quantitation by ATP
standard curve
is shown in the graph in Fig. 4, which illustrates the comparative results
obtained at 2, 4
and 6 hours into the assay. The assay can identify the number of cells that
are adhered to
a specific substrate; however, it does not take into consideration cell
spreading. The cell
spreading determined in microscopic observations may indicate that cell
spreading can
increase the overall degree of cell adhesion since more space is occupied by a
well spread
cell than by an adhered cell that has not spread on the surface, due to timing
of data points
or appropriateness of the substrate used. The ATP data are useful to support
the
observational findings of the adhesion assay but cannot replace the adhesion
assay.
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EXAMPLE 5
[0222] Cell Recruitment to Bioactive Agent-Polymer Conjugates Based upon the
promising results from the adhesion assays, the next step was to conjugate the
most
effective of the identified recruitment factors to the stmt polymer to assess
the increased
adhesion to the polymer induced with these potential recruitment factors. The
first
conjugation was done to the PEA-H version of the polymer (acid) since this
polymer has
suitable sites for conjugation. The peptides can be covalently bound to this
polymer via a
wide variety of suitable functional groups. For example, when the
biodegradable polymer
is a polyester amide) (PEA) containing Lysine residues, the carboxyl groups
from the
Lysine residues can be used to react with a complementary moiety on the
peptide, such as
an hydroxy, amino, thio moiety, and the like(5). Specifically, the PEA-H
polymer with
free COOH reacts with water soluble carbodiimide (WSC) and N-
Hydroxysuccinimide
(HOSu) to produce an activated ester, which, in turn, reacts with an amino
functional
group of a peptide to provide an amide linkage (Fig. 6B). By using a
fluorescent dansyl-
lysine (Fig. 5), the optimal reaction conditions for activation and
conjugation were
determined (Fig. 6A).
[0223] The conjugation of CSS and GREDVDY peptides to the polymer was then
performed using the same protocol (Fig. 6B). The adhesion assay showed that
the
conjugation of the peptides did not alter their ability to bind to cells; and,
further, that the
ECs when compared to the SMCs adhered significantly better to the conjugated
peptides
than on the unconjugated PEA-H polymer.
[0224] A similar protocol (see flow chart Fig. 6B) was used to conjugate
combinations
of the acid polymer with PEA polymer of structure (I) containing acetylated
ends and
benzylated COOH groups, (PEA-AcBz) and PEA-TEMPO (50/50 and 10/90),
respectively. By combining the conjugatable acid form with the other polymers,
a
determination could be made whether the presence of the recruitment peptide on
the
polymer conferred an advantage in EC recruitment.
[0225] Microscopic observations taken at 2h, 4h and 6h from duplicate wells
from two
representative adhesion assays are summarized in Table 2 below.
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Table 2
Summary of Assays with Conjugated Peptides on Polymer
50/50 HBz 50/50 HBz 10/90 HBz 10/90
Coating/Conj2a 2a 2a HBz
& 2b & 2b & 2b 2a & 2b
50/50 H/T 50/50 H/T 10/90 H/T 10/90 lasticlastic
3a 3a 3a H/T
& 3b & 3b & 3b 3a & 3b
2h Assay 1 Assay 2 Assay 1 Assay Assay Assay
2 1 2
2A
PBS 20% r 20-30%r/s 30% r/s 30% r/s 20% 20-30%
r/s r/s/sp
Conj CSS 20% r 20-30% 30% r/s 30% r/s
r
2B
PBS 20% 30% r/s 30% r 30% r/s 20-30%30% r/s
r
Conj REDV 20-30% 30-40% 30% r/s 30% r/s/sp
r r/s/sp
3A
PBS 30%r 20-30%r/s 30% r/s 30% r/s 20-30%20-30%
r r/s
Conj CSS 20-30% 30% r/s 30% r/s 30% r/s/sp
r/s
3B
PBS 20-30% 30% r/s/sp20-30% 30% r/s/sp20-30%20-30%
r r/s r r/s
Conj REDV 20-30%r 30% r/s/sp30% r/s 30% r/s/sp
4h
2A
PBS 30% r 20-30% 40% r/s 30% r/s/sp30%
r r/s
Conj CSS 30% r 30% rls/sp40% r/sp 30% s/sp
2B
PBS 30% r 30-40% 30% r/s 30-40% 20% 30% s/sp
r/s r/s/sp r
Conj REDV 30% s/sp 30-40% 30-40% 30-40%
s/sp r/s/sp s/sp
3A
PBS 30% r 30% r/s 30% r/s 30% s/sp 30% 30% r/s
r
Conj CSS 30% r 30-40% 30% r/s/sp30-40%
r/s/sp s/sp
3B
PBS 30% r 30%r/s/sp 30% r/s/sp30% s/sp 30% 20-30%
r r/s/sp
Conj REDV 30% 30% r/s/sp30-40% 40% s/sp
s/sp
6h
2A
PBS 20% r 20% r/s 30% r/s 30% r/s/sp20% 30% r/s/sp
r/s
Conj CSS 20% r 30% r/s 30-40% 30% r/s/sp
s/sp
2B
PBS 20% r 30% r/s/sp30% rls/sp30-40% 20% 30% r/s/sp
r/s/sp r
Conj REDV 30% r/s 30-40% 30% s/sp 30-40%
r/s/sp r/s/sp
3A
PBS 20% r 30% r/s 30% r/s/sp30-40% 20% 30-40%
r/s/sp r rls
Conj CSS 20% r 30% r/s 30-40% 30% r/s/sp
r/s/sp
3B
PBS 20% r 30% r/s 30% r 30-40% 20% 30% r/s
s/sp r
Conj REDV 20%r 30-40% 30-40% 40% s/sp
s/sp r/s/sp
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r=round, s=spindly, sp=spread; 50/50 HBz = 50% PEA-H and 50% PEA-Ac-Bz; 10190
HBz = 10% PEA-
H and 90% PEA-Ac-Bz; 50/50 H/T = 50% PEA-H and 50% PEA-Ac-TEMPO; 10/90 H/T =
10% PEA-H
and 90% PEA-Ac-TEMPO.
[0226] A complete evaluation of the assays with conjugated peptides on the
polymer
(Table 2), showed a benefit to the presence of the recruitment peptides on the
polymer.
The following combinations of polymer conjugated to the GREDVDY peptide
resulted in
an increased adhesion over basal levels in both assays 1 and 2 (early and late
time points).
50/50 PEA-H/PEA-Ac-Bz (H/Bz) and 10/90 PEA-H/PEA-TEMPO (H/T) conjugated to
GREDVDY - at middle and late time points. Surprisingly, the shorter peptide (7
mer)
proved more robust in cell recruitment than the longer (20 mer) CSS peptide.
[0227] Although the invention has been described with reference to the above
examples, it will be understood that modifications and variations are
encompassed within
the spirit and scope of the invention. Accordingly, the invention is limited
only by the
following claims.
DEMANDES OU BREVETS VOLUMINEUX
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CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
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