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BIOACTIVE WOUND DRESSINGS AND IMPLANTABLE DEVICES
AND METHODS OF USE
FIELD OF THE INVENTION
[0001] The invention relates generally to compositions used in healing wounds
and
lesions, and in particular to biodegradable polymer dressings that promote
natural healing
processes at wound sites.
BACKGROUND INFORMATION
[0002] In general, wounds and lesions can be divided into two types: acute and
chronic.
In cases where a wound is not initially surgically closed (delayed primary
closure), the
wound is left open for a time sufficient to allow the inflammatory process and
angiogenesis
to begin before surgical closure. Wounds healing by secondary intention are
usually not
amenable to surgical closure. As a result, the wound is left to granulate and
epithelialize
from the wound bed and edges. Numerous dressing products were developed during
the past
few years to accelerate this type of healing process.
[0003] For these types of acute wounds, occlusive dressings increase re-
epithelialization
rates by 30% to 50% and collagen synthesis by 20% to 60% compared to wounds
exposed to
air by providing an optimal healing environment that exposes the wound
continuously to the
surrounding fluid of proteinases, chemotactic factors, complements, and growth
factors. An
electrical gradient that may stimulate fibroblast and epithelial cell
migration is maintained.
The use of non-adherent dressing prevents the stripping of the newly formed
epithelial layer.
[0004] An occlusive dressing is generally divided into a hydrating layer
(antibiotic
ointments or petrolatum jelly), a nonadherent contact layer, an absorbent and
cushioning
layer (gauze), and a securing layer (tape or wrap). Occlusive dressings are
commonly applied
within 2 hours of wounding and left on for at least 24 hours, rarely as long
as 48 hours, for
optimal healing of acute wounds. Initial wound hypoxia is important for
fibroblast
proliferation and angiogenesis; however, continued hypoxia at the wound site
delays wound
healing. As a result, if an occlusive dressing is applied to an ischemic
wound, healing is
severely impaired.
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[0005] Chronic wounds are defined as wounds that fail to heal after 3 months.
Venous
stasis ulcers, diabetic ulcers, pressure ulcers, and ischemic ulcers are the
most common
chronic wounds. Many of the dressing options that attempt to heal venous
stasis ulcers are a
variation on the classic paste compression bandage, Unna's boot. These wounds
can
sometimes have large amounts of exudates that require frequent debridement.
Alginates,
foams, and other absorptives can be used in this situation. Because chronic
wounds heal by
slightly different mechanisms than those of acute wounds, experimentation with
growth
factors is being investigated. Regranex and Procuren (Curative Health
Services, Inc.,
Hauppauge, NY) are the only medications approved by the US Food and Drug
Administration (FDA).
[0006] Despite these advances in the art, a need exists in the art for new and
better
methods and devices for restoring the natural process of wound healing at a
lesion, the repair
of which requires tissue remodeling and restoration.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the premise that multiple complex
processes,
involving the differential expression of dozens if not hundreds of genes, are
necessary for
optimal tissue repair and remodeling. Based on this concept, it follows that
optimal repair of
wounds and lesions cannot be achieved by the administration of single
proteins, or single
genes whose encoded products are known to be related to such processes nor,
because of the
complexity of tissue restoration processes, by the administration of a
combination of related
proteins or genes. This invention relies on the capacity of certain precursor
cells, such as
tissue-specific progenitor cells, to secrete the various factors, such as
growth factors and
cytokines, involved in tissue restoration in a time and concentration-
dependent coordinated
and appropriate sequence.
[0008] The present invention is based on the discovery that a biodegradable
polymer or
hydrogel can be loaded with allogenic or autologous precursor cells,
conditioned medium
obtained from such precursor cells, or a combination thereof, and used to
recruit and/or to
hold autologous natural healing cells at a lesion, the repair of which
requires tissue
remodeling and restoration. Tissue restoration is the process whereby multiple
damaged cell
types are replaced to restore the histoarchitecture and function to the
treated tissue. The
precursor cells are protected, nurtured in growth medium, and delivered by the
biodegradable
polymer(s) and/or hydrogels in the invention wound dressings and device
coatings.
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3
[0009] Accordingly, in one embodiment, the invention provides bioactive wound
dressings in which at least one precursor cell selected from stem cells,
tissue-specific
progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells,
conditioned
medium obtained from such cells, and combinations thereof is dispersed within
a
biodegradable polymer or hydrogel.
[0010] In another embodiment, the invention provides methods for promoting
restoration
of tissue at a lesion site in a mammalian subject by contacting the lesion
site with an
invention wound dressing under conditions suitable for promoting restoration
of the tissue at
the lesion site, wherein the wound dressing comprises at least one precursor
cell selected
from stem cells, tissue-specific progenitor cells, germ-layer lineage stem
cells, pluripotent
stem cells, conditioned medium obtained from such cells, and combinations
thereof,
dispersed within a biodegradable polymer or hydrogel.
[0011] In still anther embodiment, the invention provides polymer coatings for
coating at
least a portion of an implantable medical device. The invention polymer
coatings include a
biodegradable polymer having dispersed therein at least one precursor cell
selected from stem
cells, tissue-specific progenitor cells, germ-layer lineage stem cells, and
pluripotent stem
cells, conditioned medium obtained from such cells, and combinations thereof,
to enhance
tissue restoration at the site of implantation. The biodegradable polymer used
is a PEA
having a chemical structure described by stractural formula (1),
O O H O O H 0 O H
C-R-C-N-C-C-O-R4-O-CC-N C-R1-C-N-C-(CH2)4 N
H R3 R3 H m HI_O-R2 H tn'
~
O (I)
and whereiin n ranges from about 5 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 - C20)
alkylene or (C2-Cz0) alkenylene; R2 is hydrogen or (C6-Clo)aryl (C1-C6) alkyl
or t-butyl or
other protecting group; R3 is selected from the group consisting of hydrogen,
(C1-C6) alkyl,
(C2-C6) alkenyl, (C2-C6) alkynyl and (C6-Clo)aryl(C1-C6) alkyl; and R4 is
selected from the
group consisting of (C2-C20) alkylene, (C2-CZO) alkenylene or alkyloxy, and
bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of general formula (II):
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Formula ( II )
\
CH O
H2C' ~CH2
'i, CH
O
except that for unsaturated polymers having the chemical structure of
structural formula (I), Rl and R4 are selected from (C2-C20) alkylene and (CZ-
C20) alkenylene;
wherein at least one of Rl and R4 is (CZ-CZO) alkenylene; n is about 5 to
about 150; each R2 is
independently hydrogen, or (C6-Clo)aryl(C1-C6)alkyl; and each R3 is
independently lzydrogen,
(C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, or (C6-Clo)aryl(C1-C6)alkyl,
or a PEUR having a chemical formula described by structural formula (III),
lb O 0 H O 0 H 0 O H
C-O-R6-O-C-N-C-C-O-R4-O-C-C-N C-O-R6-O-C-N-C-(CH2)q-N
H R3 R3 m H~-O-R2 H p
O n
(III)
and wherein n ranges from about 5 to about 150, m ranges about 0.1 to about
0.9: p ranges
from about 0.9 to about 0.1; wherein RZ is hydrogen or (C6-Clo)aryl(Cl-C6)
alkyl or t-butyl or
other protecting group; R3 is selected from the group consisting of hydrogen,
(C1-C6) alkyl,
(C2-C6) alkenyl, (C2-C6) alkynyl and (C6-Clo) aryl(C1-C6) alkyl; and R4 is
selected from the
group consisting of (C2-C20) alkylene, (C2-C20) alkenylene or alkyloxy, and
bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of general formula (II); and R6 is
independently
selected from (C2-C20) alkylene, (CZ-CZO) alkenylene or alkyloxy, and bicyclic-
fragments of
1,4:3,6-dianhydrohexitols of general formula (II),
except that for unsaturated polymers having the structural formula (II) R~ and
R4 are
selected from (C2-C2o) alkylene and (C2_C2o) alkenylene; wherein at least one
of R6and R4 is
(C2-C20 alkenylene, and
wherein the wound dressing promotes in vivo tissue repair or remodeling at a
site at
which the device is implanted in a subject.
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A BRIEF DESCRIPTION OF THE FIGURE
[0012] Figure 1 is a graph showing the ATP generated in 48 hours by smooth
muscle cells
plated in Cambrex-complete-media (growth factors plus 5% fetal bovine serum
(FBS)),
Cambrex-minus-growth-factors (gf)-plus-5% FBS, or Cambrex-minus-gf-minus-FBS.
To
wells provided with the Cambrex-minus-gf-minus-FBS, inserts were added
containing alginic
acid (AA) hydrogel particles plus 0, 5 or 10% FBS. The AA particles released
the FBS into
the media leading to increased cell growth.
DETAILED DESCRIPTION OF THE INVENTION
[0013] In one aspect, the invention is based on the discovery that
biodegradable, bioactive
polymers or hydrogels can be used to create wound dressings, implantable
compositions, and
coatings for medical devices that promote endogenous tissue restoration
processes at a wound
site. Allogenic or autologous precursor cells dispersed in a polynzer or
hydrogel matrix
promote endogenous healing processes at the wound site by interaction with and
secretion
into the tissue surroundings of a mixture of factors and cytokines that
promote tissue
restoration. In addition, the polymers can be loaded with various bioactive
agents that either
attract or hold the precursor cells within the polymer matrix or promote the
natural healing
process in a wound, such as a chronic wound. As the hydrogels or polymers
biodegrade over
time, released bioactive agents can either be absorbed into a target cell in a
wound or lesion
site to act intracellularly, or the bioactive agent can bind to a cell surface
receptor molecule to
elicit a cellular response without entering the cell. Depending upon the rate
of
biodegradation of the polymer or hydrogel selected, the tissue rernodeling
properties of the
invention wound dressings, optionally implantable, will begin to take place
even before
biodegradation of the polymer.
[0014] Stem cells in embryonic tissues In general, human tissues have a very
limited
potential to regenerate. However, stem cells from embryos have the potential
to form all
adult tissues. Embryonic stem cells can be cultured to produce both a stable
pluripotent cell
population and its associated conditioned medium.
[0015] Stem cells in adult tissues Since the development of embryonic stein
cell
cultures, "stem cells" have been discovered to exist in many adult tissues. In
fact these
broadly labeled adult "stem cells" should more accurately be described as
"precursor cells",
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consisting of multiple populations of cells, including tissue-specific
progenitor cells, germ-
layer lineage stem cells, and pluripotent stem cells.
[0016] Tissue-specific progenitor cells display various capacities for
differentiation,
ranging from unipotency (forming a single cell type) to multipotency (forming
multiple cell
types).
[0017] Germ-layer lineage stem cells can form a wider range of cell types than
a
progenitor cell. An individual germ-layer lineage stem cell can form all cell
types within its
respective germ-layer lineage (i.e., ectodenn, mesoderm, or endoderm).
Pluripotent stem
cells can form a wider range of cell types than a single germ-layer lineage
stem cell. A single
pluripotent stem cell can form cells belonging to all three germ layer
lineages. Thus,
pluripotent stem cells are clonal cells that self-renew as well as
differentiate to regenerate
adult tissues.
[0018] Both germ-layer lineage stem cells and pluripotent stem cells have
extended
capabilities for self-renewal, far surpassing the limited life span of
progenitor cells.
[00191 A discussion follows of various categories of tissue wounds and lesions
and the
types of related precursor cells that have been identified as suitable for
promoting repair and
restoration of involved tissues at such sites. According to the invention,
wound dressings
containing the indicated type of precursor cells, or conditioned medium
thereof, can be used
for promoting tissue remodeling at the indicated tissue lesion sites.
Stem cells in adult tissues - from bone marrow
[0020] Adult bone marrow-derived progenitor cells have traditionally been
considered to
be tissue-specific cells with limited capacity for differentiation. However,
recent discoveries
have demonstrated that, under the influence of appropriate cytokine
molecules=such marrow-
derived progenitor cells actually possess the potential ability to regenerate
cells of various
different lineages. Thus the hypothesis that a common'interchangeable'
progenitor cell may
exist within the bone marrow capable of regenerating and repairing tissues
througliout the
body has been confirmed in a few examples to date. This result has created the
possibility of
using bone marrow-derived stem cells as a source of cells and conditioned
medium for
therapeutic tissue repair and regeneration in various body sites.
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ENDOTHELIA, AND ASSOCIATED TISSUE
Bone marrow-derived endothelial progenitor cells: adult neovascularization
[0021] Postnatal neovascularization is not restricted to angiogenesis, but
also includes
vasculogenesis. In the hematopoietic system the only long-term self-renewing
cells in the
stem and progenitor pool are the hematopoietic stem cells of the bone marrow.
In response to
vascular trauma and/or tissue ischemia, during adult vasculogenesis
endothelial progenitor
cells (EPCs), derived from the bone marrow stem cell pool, are recruited to
the systemic
circulation. From these progenitors, endothelial cell maturation and
differentiation into
arterial, venous, and lymphatic endothelium can occur, leading to either
vascular repair or
formation.
EPITHELIA
Stem cells in the eye
[0022] Limbal stem cells are essential for the maintenance of the comeal
epithelium, and
these cells are now used clinically for repair of severely damaged cornea.
Tooth development & repair
[0023] Future treatment could be based upon the discovery of dental epithelial
stem cells
in continuously growing teeth, such as impacted wisdom teeth in adults.
Lung morphogenesis and injury repair
[0024] Lung development, as well as epithelial injury repair in the lung, is
tightly
coordinated by a fine balance between stimulatory versus inhibitory genes that
appear to co-
regulate the function of stem/progenitor cells in the lung. Use of activated
lung
stem/progenitor cells in invention implantable compositions may aid in
ameliorating lung
injury, augment lung repair and/or induce lung regeneration in both children
and adults with
intractable pulmonary insufficiency.
Progenitor cells of glandular and myoepithelial lineages in the human adult
female
breast epithelium
[0025] Phenotypically and behaviourally, progenitor (committed adult stem)
cells of
human breast epithelium have the potential to differentiate into either
glandular or
myoepithelial cells through various intermediary cells and may be used in
invention
implantable compositions for tissue restoration at lesion sites in such
tissue.
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CONNECTIVE & SKELETAL TISSUES
Mesenchymal stem cells in osteobiology and applied bone regeneration.
[0026] Bone marrow contains a population of rare progenitor cells capable of
differentiating into bone, cartilage, muscle, tendon, and other connective
tissues. These cells
are referred to as mesenchymal stem cells (MSCs) and can be used in the
invention methods
and devices to enhance tissue reformation in bone, cartilage, muscle, tendon,
and other
connective tissues.
Muscle
Cardiac muscle
[0027] Cardiac progenitor cells have defeated the dogma that myocyte
regeneration
cannot occur in the adult heart. Most importantly, primitive and progenitor
cells have been
identified in the human heart. For cardiomyocyte proliferation in damaged
heart, the use of
bone marrow-derived stem cells provides a less invasive source for cells
useful in invention
devices and compositions associated with to cardiovascular tissue engineering,
such as heart
valves, blood vessels, and myocardium. Additionally, early embryonic cells can
be recruited
to the myocardial lineage and used for such purposes in cardiac tissue
restoration.
Bone
[0028] Ideally skeletal reconstruction depends on regeneration of normal
tissues that result
from initiation of progenitor cell activity. Basic requirements include a
scaffold conducive to
cell attachment and maintenance of cell function, together with a rich source
of progenitor
cells. In the latter respect, bone is a special case and there is a vast
potential for regeneration
from cells with stem cell characteristics. The development of osteoblasts,
chondroblasts,
adipoblasts, myoblasts, and fibroblasts has been shown to result from colonies
derived from
such single cells. In principle, these precursor cells may be used in the
invention wound
healing implantable compositions for regeneration of all tissues that these
varieties of cells
comprise: bone, cartilage, fat, muscle, tendons, and ligaments.
[0029] In particular, the repair of a fracture necessarily entails synthesis
of osseous tissue
requiring the transformation of undifferentiated osteochondral progenitor
cells to mature
osteoblasts and chondrocytes. It has been proposed that there are stem cells
for all
mesenchymal tissues, resident in bone marrow throughout life, that have a
lineage
comparable to that described for hematopoiesis. Marrow derived and periosteal
derived
progenitor cells have been shown to produce bone and cartilage in numerous in
vivo and in
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vitro studies. For example, in an in vitro study of chondrogenesis, the marrow
derived
progenitor cells were shown to differentiate into their terminal phenotype,
the hypertrophic
chondrocyte.
Repair of articular cartilage
[0030] There is no natural repair mechanism to heal damaged or diseased
cartilage.
However, full-thickness defects involving the subchondral bone can be repaired
with the use
of pluripotent progenitor cells from bone marrow or from transplanted
perichondrium or
periosteum. The mechanism of fibrocartilaginous repair appears to be mediated
by
proliferation and differentiation of mesenchymal cells obtained from the bone
marrow.
Biologic grafts such as perichondrium have been successfully used to repair
full-thickness
defects, presumably because grafts of perichondrium contain progenitor cells
that can
differentiate into chondroblasts, and the like.
Stem cells and tissue engineering: prospects for regenerating tissues in
dental practice
[0031] Specific signal molecules have been identified that regulate the
development of
teeth and bones from progenitor cells. This information is already being used
for generation
of dentoalveolar tissues in vitro and in vivo. Future developments will be to
grow new
enamel, dentine, periodontal ligament, bone, or even whole new teeth in
patients. It has been
demonstrated that the potential for regeneration of the periodontium is highly
dependent upon
defect morphology and the availability of "progenitor cells."
Tissue engineering of ligament and tendon healing
[0032] Ligaments and tendons are bands of dense connective tissue that mediate
nonnal
joint movement and stability. Introduction of mesenchymal progenitor cells as
a pluripotent
cell source into the healing environment can be used to restore damage to
ligaments and
tendons.
Hair cell regeneration in the inner ear
[0033] Hearing and balance disorders caused by the loss of inner ear hair
cells are a
common problem encountered in otolaryngology-head and neck surgery. Current
work is
focused on the cellular progenitor source of new hair cells and the trigger
mechanism
responsible for inducing hair cell regeneration.
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NEURONAL
Brain repair: neurogenesis and gliogenesis
[0034] Neural stem cells (NSCs) are subscribed extraordinary potential in
repair of the
damaged nervous system. However, the molecular identity of NSCs has not been
fully
established. Most NSC cultures contain large numbers of multipotent, bipotent,
and lineage
restricted neural progenitors - the majority of which appear to lose
neurogenic potential after
expansion. Thus, a single NSC is capable of generating various kinds of cells
witllin the
central nervous system (CNS), including neurons, astrocytes, and
oligodendrocytes. Because
of these characteristics, there is increasing interest in NSCs and neural
progenitor cells for
therapeutic applications in the damaged brain.
[0035] Major advances in the study of neural progenitor cells have resulted in
rational
approaches to the repair of the damaged nervous system using transplanted
progenitor cells.
Role of Muller glia in neuroprotection and regeneration in the retina
[0036] The vertebrate retina is derived from paired evaginations from the
neural tube in
embryonic development and is initially produced by progenitor cells similar to
those that
generate the neurons and glia of other areas of the central nervous system.
Glial cells are tliought to protect neurons from various neurological insults.
When there is
injury to retina, Muller cells, which are the predominant glial element in the
retina, undergo
significant morphological, cellular and molecular changes. Muller cells
contact to
endothelial cells to facilitate the neovascularization process during hypoxic
conditions.
Recent studies have pointed to a role of Muller cells in retinal regeneration
after damage,
dedifferentiating to progenitor cells and then giving rise to different
neuronal cell types.
Enhanced neurogenesis following stroke
[0037] Proliferation, migration, and maturation of neural precursors are
affected by
ischemia. There exists compelling evidence that neural precursors resident in
the brain
initiate a compensatory response to stroke, resulting in the production of new
neurons.
Evidence for neuronal self-repair following insults to the adult brain has
been scarce until
very recently. Ischaemic insults have now been shown to trigger neurogenesis
from neural
stem cells or progenitor cells located in the dentate subgranular zone, the
subventricular zone
lining the lateral ventricle, and the posterior periventricle adjacent to the
hippocampus (See
K.S Aboody et al. PNAS (2002) 97(23):12846-12851 and R-L. Zhao, Experinzetatal
Neurology (2002)174(1):11-20)
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Olfactory ensheathing cells: their potential use for repairing the injured
spinal cord
[0038] Intraspinal transplants (e.g., fetal neuronal cells, progenitor stem
cells, and
olfactory ensheathing cells) have been used to restore intraspinal circuitry
or to serve as a
"bridge" for damaged axons. Olfactory ensheathing cells (EC)from olfactory
la.mina propria
in the nose are among the best transplants for "bridging" descending and
ascending pathways
in damaged spinal cord. In particular, assays of growth factor expression in
cultured ECs
have shown that ECs expressed nerve growth factor (NGF), brain derived
neurotrophic factor
(BDNF) and glial cell-line derived neurotrophic factor (GDNF). ELISA
confirined the
intracellular presence of NGF and BDNF and showed that, compared to BDNF,
about seven
times as much NGF was secreted by ECs. RT-PCR analysis demonstrated expression
of
inRNA for NGF, BDNF, GDNF and neurturin (NTN). In addition, ECs also expressed
the
receptors trkB, GFRalpha-1 and GFRalpha-2. Thus, ECs express a number of
growth factors
and that BDNF in particular can act both in a paracrine and autocrine manner.
CE. Woodhall
et al, Brain Res Mol Brain Res. (2001) 88(1-2):203-13).
Neural stem/progenitor cells during demyelination: repair mechanisms in
multiple
sclerosis
[0039] In recent years, it has become evident that the adult mammalian central
nervous
system (CNS) contains a population of neural stem cells (NSCs) described as
iinmature,
undifferentiated, multipotent cells, which may be recmited for repair in
neurodegenerative
and demyelinating diseases. Theses NSCs may give rise to oligodendrocyte
progenitor cells
(OPCs) and other myelinating cells.
[0040] As myelinating OPCs fail to differentiate in multiple sclerosis,
emerging
knowledge of the molecules involved in this maturation may help in the design
of future stem
cell-based treatment of demyelinating diseases, such as multiple sclerosis.
Experimental
studies indicate that transplanted neonatal OPCs can repopulate the large
areas of
demyelination characteristic of MS with much greater efficiency than
endogenous OPCs.
Promotion of recovery from spinal cord injury
[0041] The implantation of embryonic stem cells for remyelinating damaged
axons is
proposed and in clinical trials in certain countries. Human neural stem cells
can replace
damaged cells and improve function in a mouse model of spinal cord injury when
human
neural stem cells were injected into the site of spinal cord contusion injury
in inice, the
human cells survived and engrafted extensively within the injured mouse
spirzal cord, with
cells persisting 17 weeks after transplantation. The injected neural stem
cells differentiated
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into neurons and formed synapses between neurons. The mice so treated showed
evidence of
recovering coordinated locomotor function and stepping ability 16 weeks after
engraftmeut
(B.J. Cummings, PNAS (2005) Online edition September 19, 2005).
VISCERAL ORGANS
Liver regeneration and repair
[00421 Stem and progenitor cells for hepatocytes have been known for some
years Other,
non-physiologic sources of cells for therapeutic needs are not limited to
those that participate
in physiological repair processes. Alternates include stem cells from other
adult populations
such as bone marrow stromal cells, from fetal liver tissue, or from ex vivo
differentiation of
embryonic stem cells. Isolated, cultured, and expanded ex vivo, fetal
hepatoblasts and fetal
hepatocytes were the first to be studied. It was presumed that these cells
were already
"comrnitted" to an hepatic lineage, bidirectional in the case of hepatoblasts
(i.e. toward both
cholangiocytes and hepatocytes) and hepatocyte-committed in the case of fetal
hepatocytes.
Differentiation of mouse embryonic stem (ES) cells into mature hepatocytes has
now been
readily demonstrated by a number of groups.
[0043] The diseases which are potentially most likely to show real benefit
fiom such a
procedure include primary liver diseases or diseases where extra-hepatic
manifestations arise
from abnormal gene expression or defective protein production by the liver
(Wilson's
disease, alpha-l-antitrypsin deficiency, tyrosenemia type I, hyperlipidoses,
and porphyria,
metabolic deficiencies eg. Crigler-Najjar syndrome, familial
hypercholesterolemia and
amyloidosis, oxalosis and coagulation defects like hemophilia A, Factor IX
deficiency.
Acquired liver diseases, particularly acute failure secondary to toxic or
viral injury, have been
treated in limited clinical trials with fetal and adult hepatocytes. The
efficacy of these
treatments in helping patients to survive until a donor organ became
available, with
improvement of clinical measures such as hepatic encephalopathy and cerebral
perfusion
pressures, is promising (S. Sell, Cancer Research (1990), 50(13):3811-3815).
Kidney regeneration and repair
[0044] There are renal stem cells and progenitor cells.
Progenitor cells in the adult pancreas: certain diabetics
[0045] The beta-cell mass in the adult pancreas possesses the ability to
undergo limited
regeneration following injury. Pancreatic beta-cell replacement represents an
attractive
approach for treatment of type 1 and insulin requiring type 2 diabetic
patients. Identifying
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the progenitor cells involved in this process and understanding the mechanisms
leading to
their maturation will produce new treatment opportunities.
[0046] This prospect is currently restricted by the limited availability of
donor cells.
Recent developments, including beta-cell expansion by reversible
immortalization and
generation of beta cells by differentiation from embryonic and adult tissue
progenitor cells,
may provide abundant sources of cultured human beta cells. Such cells could be
genetically
modified, as well as encapsulated in semi-permeable membranes, to increase
their resistance
to beta-cell degenerative agents (in type 2 diabetics) and to recurring
autoimmunity (in type 1
diabetics)
AUTOIMMUNE DISEASE
High dose chemotherapy and autologous hematopoietic stem cell transplantation
for
rheumatoid arthritis
[0047] A new treatment approach, involving intense immunosuppression and
autologous
hematopoietic stem cell transplantation (SCT), has emerged in recent years for
the treatment
of severe, refractory rheumatic autoimmune diseases including rlieumatoid
arthritis (RA).
The rationale of this strategy is based on the concept of immunoablation by
intense
immunosuppression with subsequent regeneration of naive T lymphocytes derived
from
reinfused hematopoietic progenitor cells.
[0048] In one aspect, the invention describes wound dressings and implants
that comprise
(a) a bioabsorbable hydrogel or polymeric (polymer, copolymer or polymer
alloy) carrier into
which is dispersed, mixed, dissolved, homogenized, and/or covalently bound
("dispersed")
(b) at least one allogenic or autologous precursor cells, conditioned medium
produced by
such cells, or a combination thereof, effective for promoting natural wound
healing processes
over days, weeks, or months. The invention wound dressings and implants of the
present
invention can be in any appropriate form into which the polymer matrix,
including precursor
cells or conditioned medium and other bioactive agents, can be formed with
polymer
technological processing methods.
[0049] Experimental evidence suggests that tissue remodeling is impaired in
the elderly,
who represent the largest cohort of patients in need of tissue remodeling
therapy. Therefore,
the age-related factors that impair tissue remodeling would also impair the
activity of
autologous precursor cells, such as various types of progenitor cells,
retrieved from older
patients and delivered to their wound or lesion.
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14
[0050] These drawbacks can be overcome, according to the invention methods, by
administering to such patients cell-free conditioned medium prepared by
culturing isolated
allogenic precursor cells obtained from young healthy individuals, and
processing the
conditioned medium to remove cells therefrom to yield a cell-free conditioned
medium.
[0051] For example, when cell-free conditioned medium is prepared from either
autologous or allogenic bone marrow, the bone marrow can optionally be
filtered prior to
placement in the growth medium to remove particles larger than about 300 to
about 200 .
Bone marrow cells can also be separated from the filtered ABM for growth
leading to
production of precursor cells. Usually the growth time required to move from
bone marrow
to a composition comprising only a few cells among which are one or a few
precursor cells is
about 7 to 10 days. The bone marrow-derived precursor cells can be isolated,
and
additionally grown in a suitable growth medium for a suitable period of time,
for example,
about 24 hours, for the cells to secrete into the growth medium a mixture of
cytokines and
other factors. The conditioned medium containing the mixture of cytokines,
factors, and the
like, can be collected through a filter selected to remove cells or processed
to substantially
remove cells to produce a cell-free medium. Suitable culture conditions for
both cell growth
steps are well known in the art. Similar (but not necessarily identical)
methods can be used if
the precursor cells are derived from other tissues.
[0052] Cell-free medium derived from growth in vitro of autologous or
allogenic
precursor cells, such as, but not limited to, those obtained from bone marrow,
can be used in
the place of conditioned mediuni derived from growth in culture of cells
obtained from
autologous bone inarrow to deliver to the tissue of a patient the many
angiogenic factors
secreted by precursor cells that participate in tissue restoration. The
invention cell-free
medium is produced by culturing isolated allogenic or autologous progenitor
cells under
suitable conditions and for a time sufficient for the progenitor cells to
secrete mixed secretion
products into the conditioned medium. The conditioned medium is then processed
to yield a
cell-free medium containing the mixed secretion products. As with preparation
of donor
blood for transfusion, in which only the red cells are typed and cross-
matched, the other cells
administered, as well as the plasma, are administered without any typing. The
incidence of
serious allergic responses to any of these products is very low. The cell-free
conditioned
medium derived from cells of an allogenic or autologous source can be
considered as free of
allergens as serum or plasma obtained from various donors.
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[00531 The cytokines remaining in the cell-free medium are relatively small
molecules as
compared with the size of proteins and, therefore, lack features that the
mammalian body
recognizes as non-self, leading to immune response. The size differential
between cells and
cytokines makes it convenient to remove the cells from the growth medium to
yield the cell-
free medium by filtering the growth medium or by centrifugation, for example
for five
minutes at 10k x g. The cell-free medium may be further processed, such as by
freezing or
lyophilization and placed into small containers, to make handling, storage and
distribution
convenient. Those of skill in the art would understand that the frozen or
lyophilized cell-free
medium would be readily reconstituted for use by addition of such fluids as
sterilized water,
physiological saline, and the like, using the techniques know in the art as
suitable for
preparing other types of blood cells and blood products for administration to
a patient.
[0054] The bone marrow (BM) is a natural source of a broad spectrum of
cytokines (e.g.,
growth factors), various factors and cells that are involved in the control of
angiogenic
processes, which are referred to herein collectively as "mixed secretion
products" for
convenience. It is therefore believed that the intramyocardial injection of
autologous (A) BM
or bone marrow cells derived therefrom, by taking advantage of the natural
ability of these
cells to secrete many angiogenic factors in a time-appropriate manner,
provides an optimal
intervention for achieving therapeutic collateral development in ischemic
myocardium.
[0055] When the conditioned medium is not prepared to be cell-free, for
example when
the precursor cells are autologous, the filtering step is omitted.
Alternatively still, the isolated
precursor cells can be loaded live into the hydrogel or polymer matrix in a
suitable growth
medium and the invention wound dressing can be placed into a site requiring
tissue
restoration to allow production in situ of mixed secretion products by the
cells while the cells
are sequestered within the polymer or hydrogel matrix. In any event, the
precursor cells, if
present, need to remain alive only transiently after placement of the wound
dressing or coated
device, only long enough for interaction with the surrounding tissue to call
forth appropriate
secretion products and begin the endogenous processes tissue remodeling. The
exact period
of time required for this therapeutic effect to take place will differ
according to the type of
precursor cell employed and the type of surrounding tissue into which the
invention wound
dressing or coated device is placed. However, in general, the in situ life
span of the precursor
cells will be in the range from 10 hours to 10 days.
[0056] An "effective amount" of precursor cells or conditioned medium
containing mixed
precursor cell-secretion products, as the term is used herein, means an amount
sufficient to
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16
stimulate development of tissue remodeling in a wound or lesion site. The
effective amount
for any particular patient will be determined by a physician taking into
account such factors
as patient general health and age, severity of the condition being treated,
body weigh-t, and the
like.
[0057] The discovery that the mixture of secretion products secreted by
precursor cells
into growth medium can influence and promote tissue restoration has lead to
the coriclusion
that conditioned medium obtained from isolated autologous or allogenic
precursor cells, as
described herein, can be substituted for autologous precursor cells to produce
tissue
restoration effects. Moreover, the advantages of using allogenic donor-
provided precursor
cells to produce the therapeutic conditioned medium are several. First, an
ischemic or older
patient does not have to undergo anesthesia to obtain autologous precursor
cells, such as can
be obtained from bone marrow. Young healthy donors produce more vigorous
precursor
cells and, hence, use of allogenic cells or especially cell-free conditioned
medium produced
by allogenic precursor cells reduces the trauma to a patient of obtaining the
cells used in the
invention wound dressings and compositions. In addition, the wound healing
composition
can be produced in advance and stored for immediate use by a recipient
patient. For
example, the wound dressing composition containing cell-free conditioned
medium from
allogenic cells can be frozen to accommodate storage. Alternatively, the cell-
free
conditioned medium can be prepared as described herein and frozen or
lyophilized for
storage and then reconstituted for infusion or "loading" into the invention
polymer or
hydrogel dressings at the point of use.
[0058] In one embodiment, the invention provides bioactive wound dressings or
device
coatings are designed for implantation into an internal body site and comprise
at least one
layer of a bioabsorbable polymer that releases the dispersed precursor cells,
conditioned
medium, wound healing drug or bioactive agent over a considerable period of
time, for
example, over a period of twenty-four hours, about seven days, about thirty
days, about
ninety days, and about one hundred twenty days. A cross-linked poly (ester
amide),
polycaprolactone, or poly (ester urethane) as described herein can be used for
this purpose so
that the wound dressing is completely bioabsorbable. In this case, over time,
the w~ound
dressing will be re-absorbed by the body through natural enzymatic action and
at a controlled
rate dependent upon the selection of the polymer, allowing the re-established
cell axchitecture
to resume its natural function. Thus, the precursor cells or conditioned
medium obta.ined
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17
from the precursor cells is released in situ as a result of biodegradation of
the polymer carrier
by enzymes found in mammalian subjects, such as humans.
[0059] The invention wound dressings and implantable compositions are also
intended for
use in veterinary treatment of wounds and lesions in of a variety of mammalian
patients, such
as pets (for example, cats, dogs, rabbits, ferrets), farm animals (for
example, swine, horses,
mules, dairy and meat cattle) and race horses.
[0060] Preferred additional bioactive agents for dispersion into and release
from the
biodegradable polymers used in the invention wound dressings and implantable
compositions
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 -limus
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.
[0061] As used herein, "biodegradable" as used to describe the polymers and
hydrogels
used in the invention wound dressings, implantable compositions and device
coating is
capable of being broken down into innocuous and bioactive products in the
normal
functioning of the body. In one embodiment, the entire wound dressing or
device coating is
biodegradable. The preferred biodegradable, bioactive polymers have
hydrolyzable ester
linkages that provide the biodegradability, and are typically chain terminated
predonlinantly
with amino groups.
[0062] As used herein "dispersed" means a wound healing drug or mixture of
drugs ancl/or
one or more other bioactive agents as disclosed herein is dispersed, mixed,
dissolved,
homogenized, and/or covalently bound ("dispersed") in a polymer or hydrogel.
[0063] Polymers suitable for use in the practice of the invention can 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 arrxide
group. As will be described herein, the biodegradable, bioactive polymer and
the bioactive
agent can contain numerous complementary functional groups that can be used to
covalently
attach the bioactive agent to the biodegradable, bioactive polymer.
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18
[0064] A polymer used in making an invention wound dressing or device coating,
whether
or not present in a formulation as described herein, whether or not linked to
a bioactive agent
as described herein, and whetlzer or not intermixed with a bioactive agent as
described herein,
can also be used in medical therapy. For example, the polymer can be used in
the
manufacture of a medical device or a coating for at least a portion of an
implantable medical
or drug delivery device. Such implantable medical devices include, for
example, orthopedic
implants, such as artificial joints, artificial bones or intravertebral
implants; bone pins and
plates, surgical implants and wraps, implantable drug delivery devices,
cardiovascular
medical devices, stents, shunts, medical devices useful in angioplastic
therapy, artificial heart
valves, artificial by-passes, sutures, artificial arteries, vascular delivery,
monitoring and
treatment catheters, and adhesion barriers for local bioactive agent delivery
systems.
[0065] As used herein, "bioactive" means the wound dressing or device coating
contains a
polyiner having dispersed precursor cells and/or conditioned medium from such
cells that
play an active role in the endogenous healing processes at a wound site or
site of device
implant by holding the precursor cells or conditioned medium at the site of
the wound or
lesion for a period of time sufficient to allow the precursor cells and/or
conditioned medium
from growth of such cells to interact with surrounding tissue to affect tissue
remodeling
processes, while slowly releasing the precursor cells or drug or bioactive
agent during
biodegradation of the polymer and/or hydrogel contained therein. In addition,
in certain
embodiments the polymers disclosed herein (i.e., those having stru.ctural
formulae (I-VIII and
XI) may be bioactive upon enzymatic degradation, providing essential amino
acids that
nurture cells while the other breakdown products can be metabolized in the way
that fatty
acids and sugars are metabolized.
[0066] Bioactive agents contemplated for dispersion within the polymers and
lzydrogels
used in the invention wound dressings and device coatings include agents that,
when freed or
eluted from the polymer or hydrogel during its degradation, prornote
endogenous production
of a therapeutic natural wound healing agent, such as nitric oxide, which is
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. These bioactive agents can be any agent that donates,
transfers, or releases
nitric oxide, 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,
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19
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-l-
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.
[0067] Small proteinaceous motifs, such as the B domain of bacterial Protein A
and the
functionally equivalent region of Protein G, that are known to bind to, and
thereby capture,
such antibody molecules can be covalently attached by the Fc region to the
polymers and will
act as ligands to attach antibodies for use as capture antibodies to hold
precursor cells or
capture cells out of the patient's blood stream. Therefore, the antibody types
that can be
attached to polymer coatings using a Protein A or Protein G functional region
are those that
contain an Fc region. The capture antibodies will in turn bind to ancl hold
precursor cells,
such as progenitor cells, near the polymer surface while the precursor cells,
which are
preferably bathed in a growth medium within the polymer or hydrogel, secrete
various factors
and interact with other cells of the subject at the lesion site. In addition,
one or more active
agents contained in the wound dressing, such as the bradykinins, may activate
the precursor
cells.
[0068] For example, bioactive agents for attaching precursor cells or for
capturing PECs
from the subject's blood are monoclonal antibodies directed against a known
precursor cell
surface marker. For example, complementary determinants (CDs) that have been
reported to
decorate the surface of endothelial cells include CD31, CD34+, CD34-, CD102,
CD105,
CD106, CD109, CDwl30, CD141, CD142, CD143, CD144, CDwl45, CD146, CD147, and
CD 166. These cell surface markers can be of varying specificity and the
degree of specificity
for a particular cell/developmental type/stage is in many cases not fully
characterized. In
addition these cell marker molecules against which antibodies have been raised
will overlap
(in tenns of antibody recognition) especially with CDs on cells of the same
lineage:
inonocytes in the case of endothelial cells. Circulating endothelial
progenitor cells are some
way along the developmental pathway from (bone marrow) monocytes to mature
endothelial
cells. CDs 106, 142 and 144 have been reported to mark mature endothelial
cells witli some
specificity. CD34 is presently known to be specific for progenitor endothelial
cells and
therefore is currently preferred for capturing progenitor endothelial cells
out of blood in the
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site into which the wound dressing is implanted. Examples of such antibodies
include single-
chain antibodies, chimeric antibodies, monoclonal antibodies, polyclonal
antibodies, antibody
fragments, Fab fragments, IgA, IgG, IgM, IgD, IgE and humanized antibodies.
However, it
should be noted that access of the wound dressing to circulating blood may be
minimal,
especially in treatment of chronic wounds.
Y
[0069] The following drugs and bioactive agents will be particularly effective
for
dispersion within the polymers used in making invention wound dressings,
whether dispersed
within a time release biodegradable hydrogel, as described herein, or a
biodegradable
polymer, for example, one having a chemical structure described by formulae I -
XI herein.
The bioactive agents that are incorporated into the invention wound dressings,
and device
coatings, like the type of precursor cells, may differ depending upon the
particular tissue type
or tissue site under treatment.
[0070] In general, the suitable bioactive agents are not limited to, but
include, various
classes of compounds that facilitate or contribute to wound healing when
presented in a time-
release fashion to the wound or lesion surface. Such bioactive agents include
wound-healing
cells in addition to precursor cells, which can be protected, nurtured and
delivered by the
biodegradable polymer(s) and/or hydrogels in the invention wound dressings.
Such
additional wound healing cells include, for example, pericytes and endothelial
cells, as well
as inflammatory healing cells. To recruit such cells to the wound bed or
lesion site, the
wound dressings can include ligands for such cells, such as antibodies and
smaller molecule
ligands, that specifically bind to "cellular adhesion molecules" (CAMs).
Exemplary ligands
for wound healing cells include those that specifically bind to Intercellular
adhesion
molecules (ICAMs), such as ICAM-1 (CD54 antigen); ICAM-2 (CD102 antigen); ICAM-
3
(CD50 antigen); ICAM-4 (CD242 antigen); and ICAM-5; Vascular cell adhesion
molecules
(VCAMs), such as VCAM-1 (CD106 antigen)]; Neural cell adhesion molecules
(NCAMs),
such as NCAM-1 (CD56 antigen); or NCAM-2; Platelet endothelial cell adhesion
molecules
PECAMs, such as PECAM-1 (CD31 antigen); Leukocyte-endothelial cell adhesion
molecules
(ELAMs), such as LECAM-1; or LECAM-2 (CD62E antigen), and the like.].
[0071] These wound healing cells, for example, can be dispersed within a
hydrogel loaded
with a suitable growth medium for the cells. Synthetic tissue grafts, such as
Apligraf0
(Novartis), which is specifically formulated for healing of diabetic chronic
wounds, can be
supported by attachment to polymer layers in invention wound dressings.
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21
[0072] In another aspect, the wound-healing bioactive agents include extra
cellular matrix
proteins, macromolecules that can be dispersed into the invention wound
dressings or
implants, e.g., attached either covalently or non covalently. Examples of
useful extra-cellular
matrix proteins include, for example, glycosaminoglycans, usually linked to
proteins
(proteoglycans), and fibrous proteins (e.g., collagen; elastin; fibronectins
and laminin). Bio-
mimics of extra-cellular proteins can also be used. These are usually non-
human, but
biocompatible, glycoproteins, such as alginates and chitin derivatives. Wound
healing
peptides that are specific fragments of such extra-cellular matrix proteins
and/or their bio-
mimics can also be used.
[0073] Proteinaceous growth factors are an additional category of wound
healing
bioactive agents suitable for incorporation into the various invention wound
dressings
described herein. For example, Platelet Derived Growth Factor-BB (PDGF-BB),
Tumor
Necrosis Factor-alpha (TNF-alpha), Epidermal Growth Factor (EGF), Keratinocyte
Growth
Factor (KGF), Thymosin B4; and, various angiogenic factors such as vascular
Endothelial
Growth Factors (VEGFs), Fibroblast Growth Factors (FGFs), Tumor Necrosis
Factor-beta
(TNF-beta), and Insulin-like Growth Factor-1 (IGF-1). Many of these
proteinaceous growth
factors are available commercially or can be produced reconibinantly using
techniques well
known in the art.
[0074] Alternatively, expression systems comprising vectors, particularly
adenovirus
vectors, incorporating genes encoding such proteinaceous growth factors can be
dispersed
into the invention wound dressings for administration of the growth factors to
the wound bed.
For care of chronic wounds, the growth factors such as VEGFs, PDGFs, FGF, NGF,
and
evolutionary and functionally related biologics, and angiogenic enzymes, such
as thrombin
are preferred. For case of acute wounds, the cell recruitment biologics, such
as therapeutic
antibodies and cell receptor or receptor ligand molecules, and active
fragments thereof, are
preferred.
[0075] Drugs that enable healing are an additional category of wound healing
bioactive
agents suitable for dispersion into the various invention wound dressings,
polymer implants
and device coatings described herein. Such healing enabler drugs include, for
example,
antimicrobials and anti-inflammatory agents as well as certain healing
promoters, such as, for
example, vitamin A and synthetic inhibitors of lipid peroxidation.
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22
[0076] A variety of antibiotics can be dispersed in the invention wound
dressings,
implants and implantable device coatings to indirectly promote natural healing
processes by
preventing or controlling infection. Suitable antibiotics include many
classes, such as
aminoglycoside antibiotics or quinolones or beta-lactams, such as
cefalosporines, e.g.,
ciprofloxacin, gentamycin, tobramycin, erythromycin, vancomycin, oxacillin,
cloxacillin,
methicillin, lincomycin, ampicillin, and colistin. Suitable antibiotics have
been described in
the literature.
[0077] Suitable antimicrobials include, for example, Adriamycin PFS/RDFO
(Pharmacia
and Upjohn), Blenoxane (Bristol-Myers Squibb Oncology/Immunology), Cerubidine
(Bedford), Cosmegen (Merck), DaunoXomeO (NeXstar), DoxilO (Sequus),
Doxorubicin
Hydrochloride (Astra), Idamycin0 PFS (Pharmacia and Upjohn), Mithracin0
(Bayer),
Mitamycin (Bristol-Myers Squibb Oncology/Immunology), NipenO (SuperGen),
Novantrone0 (hnmunex) and RubexO (Bristol-Myers Squibb Oncology/Immunology).
In one embodiment, the peptide can be a glycopeptide. "Glycopeptide" refers to
oligopeptide
(e.g. heptapeptide) antibiotics, characterized by a multi-ring peptide core
optionally
substituted with saccharide groups, such as vancomycin.
[0078] Examples of glycopeptides included in this category of antimicrobials
may be
found in "Glycopeptides Classification, Occurrence, and Discovery," by Raymond
C. Rao
and Louise W. Crandall, ("Bioactive agents and the Pharmaceutical Sciences"
Voluine 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. Amer. Chem.
Soc., 1996, 118, 13107-13108; J. Amer. Chem. Soc., 1997, 119, 12041-12047; and
J. Amer.
Chem. 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-7205 1, Vancomycin, and the
like. The
term "glycopeptide" or "glycopeptide antibiotic" as used herein is also
intended to include
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23
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 vancosainine.
[0079] 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 phospliorous.
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.
[0080] Anti-inflamniatory agents useful for dispersion in polymers and/or
hydrogels used
in invention wound dressings, implants and device coatings, depending on the
body site to be
treated, include, e.g. analgesics (e.g., NSAIDS and salicyclates), steroids,
antirheumatic
agents, gastrointestinal agents, gout preparations, hormones
(glucocorticoids), nasal
preparations, ophthalmic preparations, oticpreparations (e.g., antibiotic and
steroid
combinations), respiratory agents, and skin & mucous membrane agents. See,
Physician's
Desk Reference, 2004 Edition. Specifically, the anti-inflaminatory agent can
include
dexamethasone, which is cheniically designated as (11a, 16I)-9-fluro-11,17,21-
trihydroxy-
16-methylpregna-1,4-diene-3,20-dione. Alternatively, the anti-inflammatory
agent can
include sirolimus (rapamycin), which is a triene macrolide antibiotic isolated
from
Steptomyces hygroscopicus.
[0081] In certain embodiments of the invention, the bioactive agents are
covalently
bonded to the polymers used in the invention wound dressings, iniplants and
device coatings.
The following examples illustrate the ease with which certain categories of
bioactive agents
can be dispersed into the invention polymers. Aminoxyls contemplated for use
as bioactive
agents have the structure:
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24
eN 0 [0082] Exemplary aminoxyls include the following compounds:
NJ N-O' N-O' - O'
> >
2 3,
2,2,6,6-tetramethylpiperidine-l-oxy (1); 2,2,5,5-tetramethylpyrrolidine-l-oxy
(2); and
2,2,5,5-tetramethylpyrroline-l-oxy-3-carbonyl (3). Further aminoxyls
contemplated for use
include 4-amino-2,2,6,6-tetramethylpiperidine-l-oxy (TEMPAMINE); 4-(N,N-
dimethyl-N-
hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-l-oxy, iodide (CAT16); 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-l-
oxy; N-(4-(iodoacetyl)amino-2,2,6,6-tetramethylpiperidine-l-oxy(TEMPO 1A); N-
(2,2,6,6-
tetramethylpiperidine-l-oxy-4-yl)maleimide(TEMPO maleimide, MAL-6); and 4-
trimethylammonium-2,2,6,6-tetramethylpiperidine-l-oxy, iodide (CAT 1); 3-amino-
2,2,5,5-
tetramethylpyrrolidine-l-oxy; and N-(3-(iodoacetyl)amino)-2,2,5,5-
tetramethylpyrrolidine-l-
oxy(PROXYL lA); succinimidy12,2,5,5-tetramethyl-3-pyrroline-l-oxy-3-
carboxylate and
2,2,5,5 -tetramethyl-3 -pyrroline- 1 -oxy-3 -carboxylic acid, and the like.
[0083] Furoxans contemplated for use as bioactive agents have the structure:
/ \+
N N~0
\0/
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[0084] An exemplary furoxan is 4-phenyl-3-f-uroxancarbonitrile, as set forth
below:
C/
11N
N /N~,-
0
O
[0085] Nitrosothiols include compounds bearing the -S-N=O moiety, such as the
exemplary nitrosothiol set forth below:
COOH
0=N-S
NHCOCH3.
[0086] Anthocyanins are also contemplated for use as bioactive agents.
Anthocyanins are
glycosylated anthocyanidins and have the structure:
3'
O I
HO J",-- 51
3
5 OH
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.
[0087] In further embodiments, the bioactive agent dispersed in the polymer is
a ligand for
attaching to or capturing progenitor endothelial cells floating within the
blood stream within a
blood vessel. In one example, the ligand is a"sticky" peptide or polypeptide,
such as Protein
A and Protein G. Protein A is a constituent of staphylococcus A bacteria that
binds the Fc
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26
region of particular antibody or immunoglobulin molecules, and is used
extensively to
identify and isolate these molecules. For example the Protein A ligand can be
or contain the
amino acid sequence:
MTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGV
WTYDDATKTFTVTE (SEQ ID NO: 1)
or a functionally equivalent peptidic derivative thereof, such as, by way of
an example, the
functionally equivalent peptide having the amino acid sequence:
TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDAT
KTFTVTE (SEQ ID NO:2)
[0088] 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 ligand can be, or contain Protein G
having an amino
acid sequence:
MTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVW
TYDDATKTFTVTE (SEQ ID NO:3)
or a functionally equivalent peptidic derivative thereof, such as, by way of
an example, the
functionally equivalent peptide having the amino acid sequence:
TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDAT
KTFTVTE (SEQ ID NO:4)
[0089] Other bioactive peptides contemplated for dispersion in the polymers
and/or
hydrogels used in the invention wound dressings and device coatings include
the
bradykinins. Bradykinins are vasoactive nonapeptides formed by the action of
proteases on
kininogens, to produce the decapeptide kallidin (KRPPGFSPFR) (SEQ ID NO:5),
which can
undergo further C-terminal proteolytic cleavage to yield the bradykinin 1
nonapeptide:
(KRPPGFSPF) (SEQ ID NO: 6), or N-terminal proteolytic cleavage to yield the
bradykinin 2
nonapeptide: (RPPGFSPFR) (SEQ ID NO: 7). Bradykinins 1 and 2 are functionally
distinct
as agonists of specific bradykinin cell surface receptors B1 and B2
respectively: both kallidin
and bradykinin 2 are natural ligands for the B2 receptor whereas their C-
terminal metabolites
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27
(bradykinin 1 and the octapeptide RPPGFSPF (SEQ ID N0:8) respectively) are
ligands for
the Bl 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
pootent
vasodilators, increasing penneability of post-capillary venules, and acting on
endothelial cells
to activate calmodulin and thereby nitric oxide synthase.
[0090] Bradykinin peptides are dispersed into the polymers used in the
invention wound
dressings by attachment at one end of the peptide. In general, the unattached
end of the
bradykinin extends freely from the polymer to contact endothelial cells at the
lesion site,
thereby activating the endothelial cells with which contact is made.
Endothelial cells
activated in this way activate further progenitor endothelial cells with which
they come into
contact, thereby causing a cascade of endothelial cell activation at the site
of the injury that
results in endogenous production of nitric oxide.
[0091] 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.
[0092] Polymers contemplated for use in forming the blood-compatible,
hydrophilic
polymer layer or coating in the invention wound dressings include polyesters,
poly(amino
acids), polyester amides, polyurethanes, or copolymers thereof. In particular,
examples of
biodegradable polyesters include poly(a-hydroxy Cl -C5 alkyl carboxylic
aacids), e.g.,
polyglycolic acids, poly-L-lactides, and poly-D,L-lactides; poly-3-hydroxy
butyrate;
polyhydroxyvalerate; polycaprolactones, e.g., poly(s-caprolactone); and
rnodified poly(a-
hydroxyacid)homopolymers, 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 Kimura, Y., "Biocompatible Polymers" in Biomedical
Applications
of Polymeric Materials, Tsuruta, T., et al, eds., CRC Press, 1993, page 179-
[0093] Examples of biodegradable copolymer polyesters useful in forming the
blood-
compatible, hydrophilic layer or coating 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
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28
ratio of glycolic acid to L-lactic acid ranging from 5:95 to 95:5 and
preferably a monomex
mole ratio of glycolic acid to L-lactic acid ranging from 45:65 to 95:5. The
glycolide-
caprolactone copolymers include glycolide and s-caprolactone block copolymer,
e.g.,
Monocryl or Poliglecaprone.
[0094] Further examples of polymers contemplated for use in the practice of
the invention
include polyester amides that have built-in functional groups on PEA
backbones, 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 further.
Therefore, the PEAs
used in the invention methods are ready for reaction with other chemicals
having a
hydrophilic structure to increase water solubility and with drugs and other
bioactive agernts,
without the necessity of prior modification. In addition, the PEAs preferred
for use in the
invention wound dressings and device coating display no llydrolytic
degradation when t(-- ; sted
in a saline (PBS) medium, but in an enzymatic solution, such as chymotrypsin
or CT) a
uniform and linear erosive behavior has been observed.
[0095] In one embodiment, the biodegradable polymer used is a PEA having a
chernical
structure described by structural formula (I),
O 0 H O O H 0 0 H
C-R1 -C-N-C-C-O-R4-OC-C-N C-R1-C-N-C-(CH2)4 N
H R3 R3 m H C-0-R2 H
~ j
0 (I)
and wherein n ranges from about 5 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 - C2o)
alkylene or (C2-C2o) alkenylene; R2 is hydrogen or (C6-Cio)aryl (C1-C6) alkyl
or a protecting
group; R3 is selected from the group consisting of hydrogen, (C1-C6) alkyl,
(C2-C6) alkenyl,
(C2-C6) alkynyl and (C6-Clo)aryl(C1-C6) alkyl; and R4 is selected from the
group consisting of
(C2-C20) alkylene, (C2-C20) alkenylene or alkyloxy, and bicyclic-fragments of
1,4:3,6-
dianhydrohexitols of general formula (II):
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29
Formula ( II )
\
CH 0
H2 ; ~ H2
0 CH
except that for unsaturated polymers having the chemical structure of
structural fonnula (I), Rl and R4 are selected from (C2-C20) alkylene and (CZ-
CZo) alkenylene;
wherein at least one of Ri and R4 is (C2-C20) alkenylene; n is about 5 to
about 150; each R2 is
independently hydrogen, or (C6-CIo)aryl(C1-C6)alkyl; and each R3 is
independently hydrogen,
(C1-C6)alkyl, (C2-C6)alkenyl, (CZ-C6)alkynyl, or (C6-Clo)aryl(C1-C6)alkyl,
or a PEUR having a chemical formula described by stiuctural formula (III),
H
11O 0 H O O H 0 0
C-O-R6-0-C-N-C-C-O-R4-0-C-C-N C-O-R6-0-C-N-C-(CH2)4-N
H R3 R3 m H C-O-R~ H p
O n
and wherein n ranges from about 5 to about 150, m ranges about 0.1 to about
0.9: p ranges
from about 0.9 to about 0.1; wherein R2 is lhydrogen or (C6-Cio)aryl(C1-C6)
alkyl or t-butyl or
other protecting group; R3 is independently selected from the group consisting
of hydrogen,
(C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl and (C6-C10) aryl(Cl-C6)
alkyl; and R4 is
selected from the group consisting of (C2-C20) alkylene, (C2-C20) alkenylene
or alkyloxy, and
bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II); and
R6is
independently selected from (C2-C20) alkylene, (C2-C20) alkenylene or
alkyloxy, and bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of general formula (II),
except that for unsaturated polymers having the structural formula (II) R6 and
R4 are selected from (C2-C20) alkylene and (C2_C20) alkenylene; wherein at
least one of R~and
R4 is (C2-C20 alkenylene.
[0096] The bicyclic-fragments of 1,4:3,6-dianhydrohexitols can be derived from
"sugar
alcohols," such as D-glucitol, D-mannitol, and L-iditol. Useful protecting
groups include t-
butyl and others as is known in the art.
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[0097] In one alternative, at least one of the a-amino acids used in
fabrication of the
invention polymers is a biological a-amino acid. For example, when the R3s are
CH2Ph, the
biological a amino acid used in synthesis is L-phenylalanine. In alternatives
wherein the R3s
are CH2-CH(CH3)2, the polymer contains the biological a amino acid, leucine.
By varying
the R3s, other biological a-amino acids can also be used, e.g., glycine (when
the R3s are H),
alanine (when the R3s are CH3), valine (when the R3s are CH(CH3)2), isoleucine
(when the
R3s are CH(CH3)-CHZ-CH3), phenylalanine (when the R3s are CH2-C6H5), lysine
(when the
R3s are (CH2)4-NH2); or methionine (when the R3s or R4s are -(CHZ)2SCH3), and
mixtures
thereof. In yet anotlier embodiment, all of the various a-amino acids
contained in the
invention PEA and PEUR polymers are such biological a-amino acids, as
described herein.
[0098] As used herein, the terms "amino acid", and "a-amino acid" mean a
chemical
compound containing an amino group, a carboxyl group and R3 groups as defined
herein. As
used herein, the terms "biological amino acid" and "biological a-amino acid"
mean the
amino acid(s) used in synthesis is L-phenylalanine, leucine, glycine, alanine,
valine,
isoleucine, lysine, or methionine, or a mixture thereof.
[0099] 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 and
naphthyl, and
nitrophenyl.
[0100] 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.
[0101] The molecular weights and polydisperities herein are determined by gel
permeation chromatograph using polystyrene standards. More particularly,
number and
weight average molecular weights (Mõ and M ,) are determined using a Model 510
gel
permeation chromatograph (Water Associates, Inc., Milford, MA) equipped with a
high-
pressure liquid chromatographic pump, a Waters 486 LN detector and a Waters
2410
differential refractive index detector. Tetrahydrofuran (THF) is used as the
eluent (1.0
mL/min). The polystyrene standards have a narrow molecular weight
distribution.
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31
[0102] Methods for making the polymers of structural formulas (I) and (III),
containing an
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-
ainino 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 R2-OH. As a result,
ester
fragments are formed. Then, tlie 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, e.g., di-
para-nitrophenoxy
di-acid, can also be used.
[0103] More particularly, synthesis of the unsaturated poly(ester-amide)s
(UPEAs) useful
as biodegradable polymers of the structural formula (I) as described above
will be described,
O
O O II
C
(a) _R 1 ~I C is C
C
H 11
wherein 0
and/or (b) R4 is -CH2-CH=CH-CH2- . In cases where (a) is present and (b) is
not present, R4
in (I) is -C4H$- or -C6H12-. In cases where (a) is not present and (b) is
present, Rl in (I) is -
C4Hg- or -CgH]6-.
[0104] The UPEAs can be prepared by solution polycondensation of either (1) di-
p-
toluene sulfonic acid salt of diester of alpha-amino acid and unsaturated diol
and di-p-
nitrophenyl ester of saturated dicarboxylic acid or (2) di-p-toluene sulfonic
acid salt of alpha-
amino acid and saturated diol and di-nitrophenyl ester of unsaturated
dicarboxylic acid or (3)
di-p-toluene sulfonic acid salt of diester of alpha-amino acid and unsaturated
diol and di-
nitrophenyl ester of unsaturated dicarboxylic acid.
[0105] The aryl sulfonic acid salts are used instead of the free base because
the aryl
sulfonic acid group is a very good leaving group which can promote the
condensation
reaction to move to the right of the reaction equation so product is obtained
in high yield and
because the p-toluene sulfonic acid salts are known for use in synthesizing
polymers
containing amino acid residues.
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32
[0106] The di-p-nitrophenyl esters of unsaturated dicarboxylic acid can be
synthesized
from p-nitrophenyl and unsaturated dicarboxylic acid chloride, e.g., by
dissolving
triethylamine and p-nitrophenyl in acetone and adding unsaturated dicarboxylic
acid chloride
dropwise with stirring at -78 C and pouring into water to precipitate product.
Suitable acid
chlorides include fumaric, maleic, mesaconic, citraconic, glutaconic,
itaconic, ethenyl-butane
dioic and 2-propenyl-butanedioic acid chlorides. Additiona.l compounds that
can be used in
the place of di-p-nitrophenyl esters of unsaturated dicarboxylic acid include
those having
structural formula (IV):
0 0
~~ 11 RS-0-C-0-R6-0-C-0-RS
(IV)
wherein each RS is independently (C1 -Clo)aryl optionally substituted with one
or more nitro,
cyano, halo, trifluoromethyl, or trifluoromethoxy; and R6 is independently (C2
-C20)alkylene
or (C2 -C$)alkyloxy(CZ -C20)alkylene.
[0107] The di-aryl sulfonic acid salts of diesters of alpha-anzino 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-
l,4-diol and
1, 1 8-octadec-9-en-diol.
[0108] 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.
[0109] Synthesis the unsaturated poly(ester-amide)s (T.TPEAs) useful as
biodegradable
polymers of the structural formula (I) as described above will now be
described.
Unsaturated compounds having the structural formula (I) can be made in similar
fashion to
the compound (VII) of U. S. Patent No. 6,503,538 Bl, except that R4 of (III)
of 6,503,535
and/or Rl of (V) of 6,503,538 is CZ-C20 alkenylene as described above. The
reaction is
carried out, for example, by adding dry triethylamine to a znixture 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
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33
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 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 unsaturated product (I) with dilute acid.
[0110] A working example of a saturated compound having structural formula (I)
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-1,4-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.
[0111] In unsaturated compounds having structural formula (I), the following
hold:
Aminoxyl radical e.g., 4-amino TEMPO can be attached using carbonyldiimidazol
as a
condensing agent. Bioactive agents, additional bioactive agents and wound-
healing drugs,
and the like, as described herein, can be attached via the double bond
functionality.
Hydrophilicity can be imparted by bonding to poly(ethylene glycol) diacrylate.
[0112] The biodegradable polymers and copolyniers preferably have weight
average
molecular weights ranging from 10,000 to 300,000; 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.
[0113] In yet another aspect, polymers contemplated for use in forming the
invention
wound healing dressings, implants and 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.
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34
[0114] In this embodiment, the biodegradable polymers and copolymers may
contain up
to two amino acids, such as biological amino acids, 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
fiom 0.3 to 4.0, preferably ranging from 0.5 to 3.5.
[0115] Such poly(caprolactones) contemplated for use have an exemplary
structural
formula (V) as follows:
0
O
j
~=
[0116] Poly(glycolides) contemplated for use have an exemplary stnxctural
formula (VI)
as follows:
O
O
(VI)=
[0117] Poly(lactides) contemplated for use have an exemplary structural
formula (VII) as
follows:
O
O
H Me
(VIn=
[0118] 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
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lactide and s-caprolactone in the presence of benzyl alcohol using stannous
octoate as the
catalyst to form a polymer of structural formula (VIII).
0
O
O
O
CH2OH + +
O
O
/ \ O
p 0H
n 0 m
(VHD
[0119] The hydroxy terminated polymer chains can then be capped with maleic
anhydride
to foniz polymer chains having structural formula (IX):
O
p O OH
~M~-~
n 0
(LX)
[0120] At this point, 4-amino-2,2,6,6-tetramethylpiperidine-l-oxy can be
reacted with the
carboxylic end group to covalently attach the aminoxyl moiety to the copolymer
via the
amide bond which results from the reaction between the 4-amino group and the
carboxylic
acid end group. Alternatively, the maleic acid capped copolymer can be grafted
with
polyacrylic acid to provide additional carboxylic acid moieties for subsequent
attaclunent of
further aminoxyl groups.
[0121] 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.
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36
[0122] The precursor cells are dispersed within the polymer matrix without
direct
chemical linkage to the polymer carrier, although it is contemplated that the
precursor cells
may be held within the polymer by a capture ligarid, such as an antibody that
binds to a cell
surface marker, as described herein. However one or more bioactive agent can
be covalently
bound to the biodegradable, bioactive polymers via a wide variety of suitable
functional
groups. For example, when the biodegradable, bioactive polymer is a polyester,
the carboxyl
group chain end can be used to react with a complimentary moiety on the
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 Organic ClzemistT-y, Reactions,
Mechanisms, and
Structure, Fifth Edition, (2001); and Comprehensive Organic TYansformations,
Second
Edition, Larock (1999).
[0123] In other embodiments, a bioactive agent can be linked to any of the
polymers of
structures (I) and (III) through an amide, ester, ether, amino, ketone,
thioether, sulfinyl,
sulfonyl, disulfide, and the like, or a direct linkage. Such a linkage can be
formed from
suitably functionalized starting materials using synthetic procedures that are
known in the art.
[0124] In one embodiment of the present invention, a polymer can be linked to
the
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 of a
bioactive agent or a
hydroxyl functional group of a bioactive agent to provide a biodegradable,
bioactive polyiner
having a bioactive agent attached via an amide linkage or carboxylic ester
linkage,
respectively. In another embodiment, the carboxyl group of the polymer can be
transformed
into an acyl halide, acyl anhydride/"mixed" anhydride, or active ester.
[0125] Alternatively, the bioactive agent may be attached to the polymer via a
linker.
Indeed, to improve surface hydrophobicity of the biodegradable, bioactive
polymer, to
improve accessibility of the biodegradable, bioactive polymer towards enzyme
activation,
and to improve the release profile of the biodegradable, bioactive polymer, a
linker may be
utilized to indirectly attach the bioactive agent to the biodegradable,
bioactive polymer. In
certain embodiments, the linker compounds include poly(ethylene glycol) having
a molecular
weight (MW) of about 44 to about 10,000, preferably 44 to 2000; amino acids,
such as serine;
polypeptides with repeat units from 1 to 100; and any other suitable low
molecular weiglit
polymers. The linker typically separates the bioactive agent from the polymer
by about 5
angstroms up to about 200 angstroms.
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[0126] In still further embodiments, the linker is a divalent radical of
formula W-A-Q,
wherein A is (C1-CZ4)alkyl, (C2-C24)alkenyl, (C2-C24)alkynyl, (C3-
C8)cycloalkyl, or (C6-C1o)
aryl, and W and Q are each independently N(R)C(=0)-, -C(=0)N(R)-, -OC(=0)-, -
C(=O)O,
-0-, -S-, -S(O), -S(0)2-, -S-S-, -N(R)-, -C(=0)-, wherein each R is
independently H or (C1-
C6)alkyl.
[0127] 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 uiiderstood that a wide variety of catalysts can
be used to form
polymers suitable for use in the practice of the invention.
[0128] In certain embodiments, the bioactive agent can be covalently bound to
the
biodegradable, bioactive polymers via a wide variety of suitable functional
groups. For
example, when the biodegradable, bioactive polymer is a polyester, the:
carboxyl group chain
end can be used to react witli a complimentary moiety on the bioactive agent,
such as
hydroxy, amino, thio, and the like. A wide variety of suitable reagents and
reaction
conditions are disclosed, e.g., inAdvanced Organic Cheniistr.y, Reacti ns,
Mechanisms, and
Structure, Fifth Edition, (2001); and Con2prehensive OrgaTiic Transfoy-
mations, Second
Edition, Larock (1999).
[0129] In other embodiments, precursor cells and bioactive agents can be
dispersed into
the polymer by "loading" onto the polymer without formation of a cheinical
bond or the
bioactive agent can be linked to any of functional group in the polymeYs, such
as an amide,
ester, ether, amino, ketone, thioether, sulfmyl, sulfonyl, disulfide, and the
like, to form a
direct linkage. Such a linkage can be formed from suitably fixnctionalized
starting materials
using synthetic procedures that are known in the art.
[0130] Alternatively still, the bioactive agent may be attached to th-e
polymer via a linker.
Indeed, to improve surface hydrophobicity of the biodegradable, bioactive
polymer, to
improve accessibility of the biodegradable, bioactive polymer towards enzyme
activation,
and to improve the release profile of the biodegradable, bioactive polymer, a
linker may be
utilized to indirectly attach the bioactive agent to the biodegradable,
bioactive polymer. In
certain embodiments, the linker compounds include poly(ethylene glycol) having
a molecular
weight (MW) of about 44 to about 10,000, preferably 44 to 2000; amino acids,
such as serine;
polypeptides with repeat units from 1 to 100; and any other suitable low
molecular weight
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polymers. The linker typically separates the bioactive agent from the polymer
by about 5
angstroms up to about 200 angstroms.
[0131] 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-
C$)cycloalkyl, or (C6-C1O)
aryl, and W and Q are each independently N(R)C(=O)-, -C(=O)N(R)-, -OC(=O)-, -
C(=0)O,
-0-, -S-, -S(O), -S(0)2-, -S-S-, -N(R)-, -C(=0)-, wherein each R is
independently H or (C1-
C6)alkyl.
[0132] As used herein, 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.
[0133] As used herein, "alkenyl" refers to straight or branched chain
hydrocarbyl groups
having one or more carbon-carbon double bonds.
[0134] As used herein, "alkynyl" refers to straight or branched chain
hydrocarbyl groups
having at least one carbon-carbon triple bond.
[0135] As used herein, "aryl" refers to aromatic groups having in the range of
6 up to 14
carbon atoms.
[0136] 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-
lysine, poly-L-
glutamic acid, poly-L-aspartic acid, poly-L-histidine, poly-L-omithine, 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.
[0137] The linker can be attached first to the polymer or to the bioactive
agent. During
synthesis of polymers containing bioactive agents indirectly attached via a
linker, the linker
can be either in unprotected form or protected from, using a variety of
protecting groups well
known to those skilled in the art.
[0138] In the case of a protected linker, the unprotected end of the linker
can first be
attached to the polymer or the bioactive agent. The protecting group can then
be de-protected
using Pd/H2 hydrogen lysis, mild acid or base hydrolysis, or any other common
de-protection
method that are known in the art. The de-protected linker can then be attached
to the
bioactive agent. An example using poly(ethylene glycol) as the linker is shown
in Scheme 1.
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Scheme 1
[0139] Poly(ethylene glycol) employed as the linker between polymer and
drug/biologic.
O
R
n
O
R
n
O
R
n
wherein =nnnr represents the polymer;
R can be either a drug or bioactive agent; and
n can range from 1 to 200; preferable from 1 to 50.
[0140] An exemplary synthesis of a biodegradable, bioactive polymer according
to the
invention (wherein the bioactive agent is an aminoxyl) is set forth as
follows.
[0141] A polyester can be reacted with an aminoxyl, e.g., 4-amino-2,2,6,6-
tetrainetliylpiperidine- 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 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.
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[0142] 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, methylene
chloride and chloroform at room temperature to 50 C suitably dissolve the
polyester.
[0143] In anotlier aspect, the biodegradable polymers or hydrogels can be
coated onto the
surface of a medical device, in many ways, such as dip-coating, spray-coating,
ionic
deposition, and the like, as is well known in the art, prior to loading with
the precursor cells.
In coating a porous surface of a medical device, care must be taken not to
occlude the pores,
which are needed to allow access and migration of cells, factors, and the
like, from the
surface of the device to the interior of the device, for example endothelial
cells and other
blood factors that participate in the natural biological process of wound
healing and tissue
reconstruction.
[0144] The medical device can be formed of any suitable substance, such as is
known in
the art. For example, the medical device can be formed from a bioceramic, such
as a porous
calcium phosphate cement or implantable object made tlierefrom, or a
biocompatible metal,
such as stainless steel, tantalum, iiitinol, elgiloy, and the like, and
suitable combinations
thereof.
[0145] In another embodiment, the medical device can itself be substantially
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 medical device
is formed from
biodegradable cross-linked poly(ester amide), polycaprolactone, or poly(ester
urethane) as
described herein.
Polymer / Bioactive agent Linkage
[0146] In one embodiment, the polymers used to make the wound dressings and
device
coverings as described herein have one or more bioactive agents that promote
natural wound
healing directly linked to the polymer. The residues of the polymer can be
linked to the
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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. Alternatively, more than one bioactive agent, or a
mixture of
bioactive agents, that promote natural re-endothelialization of vessels 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.
[0147] 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., carboxyl) 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.
[0148] As used herein, a "residue of a compound of structural formula (*)"
refers to a
radical of a compound of formula s(I, III - VII) and (X) having one or more
open valences.
Any synthetically feasible atom, atoms, or functional group of the compound of
formulas (I-
X) (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 fiinctional
group (e.g., carboxyl)
can be created on the compound of formulas (I, II - VII) and (X) (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, III - VII) and (X) using
procedures that are
kllown in the art.
[0149] For example, the residue of a bioactive agent can be linked to the
residue of a
compound of structural formula (I, III - VII) and (X) through an amide (e.g., -
N(R)C(=0)- or
-C(=0)N(R)-), ester (e.g., -OC(=O)- or -C(=O)O-), ether (e.g., -0-), amino
(e.g., -N(R)-),
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ketone (e.g., -C(=O)-), thioether (e.g., -S-), sulfinyl (e.g., -S(O)-),
sulfonyl (e.g., -S(O)Z-),
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, III - VII) and
(X) and from a
given residue of a bioactive agent using procedures that are known in the art.
The residue of
the bioactive agent can be linked to any synthetically feasible position on
the residue of a
compound of structural formula (I, III - VII) and (X). Additionally, the
invention also
provides compounds having more than one residue of a bioactive agent or
bioactive agents
directly linked to a compound of structural formula (I, III - VII) and (X).
[0150] The number of bioactive agents that can be linked to the polyiner can
typically
depend upon the molecular weight of the polymer. For example, for a compound
of
structural formula (I), wherein n is about 50 to about 150, up to about 300
bioactive agents
(i.e., residues thereof) can be directly linked to the polymer (i.e., residue
thereof) by reacting
the bioactive agent with end groups of the polymer. In unsaturated polymers,
the bioactive
agents can also be reacted with double (or triple) bonds in the polymer.
Hydrogels for use in wound dressings
[0151] Non-stick wound healing dressings and non-stick layers used in the
invention
wound-healing dressings and implantable cell or conditioned medium delivery
compositions
comprise a biodegradable hydrogel. Although any biodegradable hydrogel known
in the art
that can be loaded with precursor cells as described herein, wound healing
drugs or bioactive
agents for in situ delivery can be used for this purpose, preferred hydrogels
have both
hydrophobic and hydrophilic components and form a one-phase crosslinked
polymer network
structure by free radical polymeriza.tion. Such hydrogels effectively
accommodate precursor
cells as well as hydrophobic drugs (as well as hydrophilic drugs) and
hydrogels with
hydrophobic and hydrophilic components have the advantage of maintaining
structural
integrity for relatively longer periods of time and having increased
mechanical strength
compared to totally hydropllilic-based hydrogels. Due to its non-stick nature,
the hydrogel
layer can be placed directly into the wound bed or lesion to deliver its load
of precursor cells
or conditioned medium in situ and can be removed without damage to the
developing cell
architecture in the wound bed.
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[0152] In one aspect, such a hydrogel is formed from a hydrogel-forming system
that
comprises from 0.01 to 99.99% by weight, for example, from 95% to 5%, by
weight of (A),
wherein (A) is a hydrophobic macromer with unsaturated group terminated ends,
and from
99.99 to 0.01% by weight, for example, from 5% to 95%, by weight of (B),
wherein (B) is a
hydrophilic polysaccharide containing hydroxyl groups that are reacted with
the unsaturated
groups of the hydrophobic macromer. The total of the percentages of (A) and
(B) is 100%.
The hydrophobic macromer is biodegradable and is readily prepared by reacting
diol--
obtained by converting hydroxyls of terminal carboxylic acid groups of
poly(lactic acid) to
aminoethanol groups-- with the unsaturated group-introducing compound.
[0153] Preferably, the hydrophilic polymer is dextran wherein one or more
hydroxyls in a
glucose unit of the dextran are reacted with the unsaturated group-introducing
compound. In
one case, the hydrophilic polymer can be dextran-maleic acid monoester as
described in
PCT/US99/18818.
[0154] A precursor cell, conditioned medium, wound-healing bioactive agent or
drug, as
described herein, can be loaded into (i.e., dispersed in) the hydrogel by a
number of means
depending on the molecular weight of the cells, agents or drug. For example, a
drug of
weight average molecular weight ranging from 200 to 1,000, as exemplified by
indomethacin, can be entrapped in the three dimensional crosslinked polymer
network for
controlled release therefrom. Alternatively, a water-soluble macromolecule of
weight
average molecular weight ranging from 1,000 to 10,000, e.g., a polypeptide, as
exemplified
by insulin, can be entrapped in the three dimensional crosslinked polymer
network for
controlled release therefrom. In still another example, a precursor cell,
e.g., weighing in the
femtogram range, can be entrapped in the three dimensional crosslinked polymer
network for
controlled release therefrom.
[0155] The term "hydrogel" is used herein to mean a polymeric material that
exhibits the
ability to swell in water or other aqueous solution and to retain a
significant portion of the
aqueous solution within its structure without dissolving. Thus the hydrogels
described herein
are particularly suitable for loading of precursor cells in growth medium or
for loading with
conditioned medium, such as cell-free conditioned medium.
[0156] In certain embodiments, the biodegradable hydrogel used in the
invention methods
and devices is a hydrogel formed from a hydrogel forming system containing at
least one
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biodegradable component, i.e., component that is degraded by water and/or by
enzymes
found in wounds and lesions of mammalian patients, such as humans and other
animals.
[0157] The term "crosslinked polymer network structure" is used herein to mean
an
interconnected structure where crosslinks are formed between hydrophobic
molecules,
between hydrophilic molecules and between hydrophobic molecules and
hyd.rophilic
molecules.
[0158] The term "photocrosslinking" is used herein to mean causing vinyl bonds
in the
initiator to break and other vinyl bonds to form crosslinks by the application
of radiant
energy.
[0159] The term "macromer" is used herein to mean a monomer having a_ weight
average
molecular weight ranging from 500 to 80,000.
[0160] The term "unsaturated group-introducing compound" as used herein with
respect to
hydrogels means a compound that reacts with an hydroxyl group and provicles a
pendant or
end group containing an unsaturated group, e.g., a pendant group with a vinyl
group at its
end.
[0161] The weight average molecular weights and number average molecular
weights
herein are determined by gel permeation chromatography.
[0162] A detailed description of such biodegradable hydrogels and their
znethods of
preparation are described in U.S. Patent Nos. 6,388,047 and 6,583,219.
[0163] Suitable compounds for use as the hydrophobic macromer (A) in the
preparation of
biodegradable hydrogels are readily obtained by converting the end groups f a
starting
material macromer to groups with terminal hydroxyl group if such are not
already present as
end groups, i.e., to provide a diol, and reacting the terminal hydroxyls with
an unsaturated
group-introducing compound to provide terminal unsaturated groups, e.g., vinyl
groups, on
the macromer. The starting material macromer preferably has a weight average
molecular
weight ranging up from 500 to 20,000, such as the aliphatic polyester
poly(Iactic acid) having
a weight average molecular weight ranging from 600 to 8,000, e.g., 600 to
1,000 or 6,500 to
8,000, e.g., poly-D-,L-lactic acid (sometimes denoted PDLLA). Poly-D,L-Iactic
acid has
widely been used as a biodegradable hydrophobic polymeric material due to its
combination
of biodegradability, biocompatibility, and adequate mechanical strength. Tlle
degradation of
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poly-D,L-lactic acid in vivo is well understood and the degradation products
are natural
metabolites that can be readily eliminated by the human body. Other starting
material
macromers that can be used include, for exainple, other aliphatic polyesters,
such as
poly(glycolic acid), poly(epsilon-caprolactone), poly(glycolide-co-lactide),
poly(lactide-
epsilon-caprolactone), polycaprolactone diols (e.g., with Mn equal to 530,
1250 or 2000),
polycaprolactone triols (e.g., with Mõ equal to 300 or 900), or any synthetic
biodegradable
macromer having one carboxyl end group and one hydroxyl end group, carboxyl
groups at
both ends, or hydroxyl groups at both ends.
[0164] Reaction of a diol with the unsaturated group-introducing compound
provides a
hydrophobic polymer with unsaturated end groups. The unsaturated group-
introducing
compound can be, for example, acryloyl chloride, methacryloyl chloride,
acrylic acid,
methacrylic acid, or isocyanate having unsaturated, e.g., vinyl, group at one
end of the
molecule, e.g., allyl isocyanate or isocyanatoethyl methacrylate. Vinyl
terminated
hydrophobic macromer A can be prepared from poly-D,L-lactic acid with mers
ranging from
8 to 120.
[0165] The hydrophilic polymer (B) is a polysaccharide derivative. Suitable
polysaccharides useful for preparing (B) have hydroxy functional pendant
groups and
include, for example, dextran, inulin, starch, cellulose, pullan, levan,
mannan, chitin, xylan,
pectin, glucuronan, laminarin, galactomannan, amylose, amylopectin, and
phytoglucans.
These polysaccharides have multiple hydroxy functional groups that permit the
production of
a three-dimensional network. The named polysaccharides are inexpensive.
Dextran, which is
the preferred polysaccharide starting material, is one of the most abundant
naturally occurring
biodegradable polymers. It is susceptible to enzymatic digestion in the body
and consists
mainly of (1--->6) alpha-D-glucoside linkages with about 5-10% of (1->3) alpha-
linked
branching. It contains three hydroxyl groups per glucose repeating unit and
therefore
mediates formation of a crosslinked polymer network. Preferably, the dextran
starting
material has a weight average molecular weight ranging from 40,000 to 80,000.
[0166] The polysaccharide hydroxy groups are reacted with an unsaturated group-
introducing compound. Suitable unsaturated group-introducing compounds for use
in
making biodegradable hydrogels include, for example, acryloyl chloride,
methacryloyl
chloride, acrylic acid, methacrylic acid, or isocyanate having an unsaturated,
e.g., vinyl,
group at one end of the molecule, e.g., allyl isocyanate or isocyanatoethyl
methacrylata.
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[0167] The percentages of (A) and (B), the molecular weight of the hydrophobic
macromer, the molecular weight of the hydrophilic polymer, and the degree of
substitution in
the hydrophilic polymer, are variables affecting
hydrophobicity/hydrophilicity, mechanical,
swelling ratio and biodegradation properties of the hydrogel prepared from the
hydrogel-
forming systems described herein. The "swelling ratio" is obtained by
immersing a known
weight of dry hydrogel in a vial containing 15 ml liquid, removing swollen
hydrogel from the
liquid at regular time intervals wiping off surface water and weighing, until
equilibrium is
obtained.
[0168] Decreasing the percentage of (B) and increasing the percentage of (A)
increases
hydrophobicity (and compatibility with hydrophobic agents and milieus) and
decreases
swelling ratio (with the largest percentage decrease in swelling ratio being
found in
decreasing the percentage of (B) from 80% to 60% and increasing the percentage
of (A) from
20% to 40%). Increasing the percentage of (B) and decreasing the percentage of
(A) increases
hydrophilicity and compatibility of hydrogel with hydrophilic agents and
milieus. Increasing
the percentage of (A) improved mechanical properties in the hydrogels forined
from the
hydrogel-forming systems. Increasing the molecular weight of (A) increases
hydrophobicity
and mechanical properties, increases swelling ratio where the percentage of A
or B is high
and causes increase in biodegradation time for formed hydrogel. Increase in
the molecular
weight of (B) decreases hydrophobicity, decreases swelling ratio, causes
increase in
mechanical properties, and where (B) is a dextran derivative increases time
for degradation
by dextranase, in formed hydrogel. Increase in degree of substitution in
hydrophilic polymer
decreases hydrophilicity and swelling ratio (in higher weight percentage
dextran derivative
compositions), increases mechanical property and increases degradation time,
in formed
hydrogel.
[0169] The hydrogel formed herein can chemically incorporate a wound-healing
bioactive
agent which reacts with either or both of the components of the hydrogel-
forming system;
this can be accomplished by reacting the bioactive agent witli one or both of
the components
of the hydrogel-forming system herein.
[0170] Wound-healing agents which are not reactive with components of the
hydrogel-
forming system herein can be physically entrapped within the hydrogel or
physically
encapsulated within the hydrogel by including them in the reaction mixture
subjected to
photocrosslinking so that the photocrosslinking causes formation of hydrogel
with bioactive
agent entrapped therein or encapsulated thereby.
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[0171] By varying the parameters as discussed above, to vary mechanical
properties,
hydrophobicity/hydrophilicity, swelling ratio and biodegradation properties,
the hydrogel-
forming system described herein can be tailored to produce hydrogels that
control the release
rate from the invention wound dressings and device coatings of the precursor
cells and/or
conditioned medium as well as bioactive agents dispersed therein. As described
above,
higher swelling ratios give faster release rates and are connected with high
hydrophilicity,
which is important for wound cleaning utilities, and provide better absorption
for sanitary
purposes. In one embodiment, the invention wound dressings utilize the
hydrogels
containing precursor cells as scaffolds for tissue engineering.
[0172] The synthetic or natural polymers that can be incorporated into
biodegradable
hydrogels include, for example, proteins, peptides, polysaccharides, and
polymucosaccharides. Proteins for this alternative include, for example,
lysozyme,
interleukin-1, and basic fibroblast growth factor. This alternative provides a
good approach
for controlled release administration of syntlietic or natural polymer drugs.
[0173] Entrapped precursor cells, conditioned medium and wound-healing
bioactive
agents are readily incorporated into the biodegradable hydrogel by forming a
solution of
components (A) and (B) to provide a concentration of 30 to 50% (w/v) of total
of (A) and (B)
in the solution, adding photo initiator and then adding, for example, from 0.5
to 3% (w/w
based on the total weight of (A) and (B)) of cells and molecules to be
entrapped, and then
effecting free radical polymerization. The solvent should be one in which (A)
and (B), and
agent to be entrapped are soluble. Such solvents in which (A) and (B) are
soluble typically
include, for example, dimethyl fluoride (DMF) and dimethyl sulfoxide (DMSO),
and
selection is made from among the solvents in which (A) and (B) are soluble, to
obtain solvent
that also dissolves the cells and bioactive agent to be entrapped.
Additional bioactive agents
[0174] As used herein in connection with wound-healing dressings and implants,
an
"additional bioactive agent" refers to a therapeutic or diagnostic agents
other than the
"wound-healing" 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 dispersed within a polymer matrix or coating on the surface of insertable
or implantable
medical or therapeutic devices having different treatment aims as are known in
the art,
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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.
[0175] Specifically, such additional bioactive agent can include, but are not
limited to, one
or more: polynucleotides, polypeptides, oligonucleotides, nucleotide analogs,
nucleoside
analogs, polynucleic acid decoys, tlierapeutic antibodies, abciximab, 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.
[0176] 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,
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.
[0177] 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.
[0178] An antisense polynucleotide is typically a polynucleotide that is
complinientary to
an mRNA, which encodes a target protein. For example, the mRNA can encode a
cance:x
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.
[0179] A "functional RNA" refers to a ribozyme or other RNA that is not
translated.
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49
[0180] 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 of
the gene product. 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 within
cells are
well known to those of skill in the art.
[0181] 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 coinmonly 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, 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.
[0182] Nucleotide and nucleoside analogues are well known in 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),
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Videx (Bristol-Myers Squibb), ZeritO (Bristol-Myers Squibb), and ZoviraxO
(Glaxo
Wellcome). See, Physician's Desk Reference, 2004 Edition.
[0183] Polypeptides acting as additional bioactive agents dispersed within the
polymers in
the invention wound dressings and coatings on implantable 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.
[0184] The 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 conlpound
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 a1.
(1987) J. Med.
Chem., 30:1229; and are usually developed with the aid of computerized
molecular modeling.
Generally, peptidomimetics are structarally similar to a paradigm polypeptid.e
(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.=
- -CH2NH--, --CH2S--, CH2-CHZ--, --CH=CH-- (cis and trans), --COCH2--, --
CH(OH)CH2--
and --CH2SO--, 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., Vega
Data (March 1983), 1(3), "Peptide Backbone Modifications" (general review);
Morley, J.S.,
Ti-ends. Pharfn. Sci., (1980) pp. 463-468 (general review); Hudson, D. et aL,
Int. J Pept.
Prot. Res., (1979) 14:177-185 (--CH2NH--, CH2CH2--); Spatola, A.F. et al.,
Life Sci., (1986)
38:1243-1249 (--CH2-S--); Harm, M. M., J. Chein. Soc. Perkin Trans I(1982) 307-
314 (--
CH=CH--, cis and trans); Almquist, R.G. et al., J. Med. Chem., (1980) 23:2533
(--COCH2--);
Jennings-Whie, C. et al., Tetrahedron Lett., (1982) 23:2533 (--COCH2--);
Szelke, M. et al.,
European Applnõ EP 45665 (1982) Ck 97:39405 (1982) (--CH(OH)CH2--); Holladay,
M.
W. et al., Tetrahedron Lett., (1983) 24:4401-4404 (--C(OH)CH2--); and Hru.by,
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,
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51
efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological
activities), reduced
antigenicity, and others.
[0185] Additionally, substitution of one or more amino acids within a
polypeptide (e.g,
with a D-Lysine in place of L-Lysine) may be used to generate more stable
polypeptides and
polypeptides resistant to endogenous proteases.
[0186] In one embodiment, the additional bioactive agent polypeptide dispersed
in the
polymers or hydrogels used in the invention wound dressings, implants and
coatings of
medical devices can be an antibody. In one aspect, the antibody can bind to a
cell adhesion
molecule, such as a cadherin, integrin or selectin. In another aspect, the
antibody can bind to
an extracellular matrix molecule, such as collagen, elastin, fibronectin or
laminin. In still
another aspect, 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) 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 a Fab fragment of a cllimeric 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 or proliferation. Abciximab also inhibits aggregation of
blood platelets.
[0187] Useful anti-platelet or anti-coagulation agents that may be used
include, e.g.,
Coumadin0 (DuPont), Fragmin0 (Pharmacia & Upjohn), Heparin0 (Wyeth-Ayerst),
Lovenox0, Normiflo0, Orgaran O(Organon), Aggrastat0 (Merck), Agrylin0
(Roberts),
Ecotrin0 (Smithkline Beecham), Flolan0 (Glaxo Wellcome), Halfprin0 (Kramer),
Integrillin0 (COR Therapeutics), Integrillin0 (Key), Persantine0 (Boebringer
Ingelheim),
Plavix0 (Bristol-Myers Squibb), ReoProO (Centecor), Ticlid0 (Roche),
Abbokinase0
(Abbott), Activase0 (Genentech), Eminase0 (Roberts), and Strepase0 (Astra).
See,
Physician's Desk Reference, 2001 Edition. Specifically, the anti-platelet or
anti-coagulation
agent can include trapidil (avantrin), cilostazol, heparin, hirudin, or
ilprost. Trapidil is
chemically designated as N,N-dimethyl-5-methyl-[1,2,4]triazolo[1,-5-
a]pyrimidin-7-amine.
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52
Cilostazol is chemically designated as 6-[4-(1-cyclohexyl-lH-tetrazol-5-yl)-
butoxy]-3,4-
dihydro-2(1H)-quinolinone. 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. Hirudin is
an
anticoagulant protein extracted from leeches, e.g., Hirudo inedicinalis.
Iloprost is chemically
designated as 5-[Hexahydro-5-hydroxy-4-(3-hydroxy-4-methyl-l-octen-6-ynyl)-
2(lH)-
pentalenylidene] pentanoic acid.
[0188] The immune suppressive agent can include, e.g., Azathioprine0 (Roxane),
BayRho-DO (Bayer Biological), CellCeptO (Roche Laboratories), Imuran0 (Glaxo
Wellcome), MiCRhoGAMO (Ortho-Clinical Diagnostics), Neoran0 (Novartis),
Orthoclone
OKT30 (Ortho Biotech), Prograf0 (Fujisawa), PhoGAMO (Ortho-Clinical
Diagnostics),
Sandimmune0 (Novartis), Simulect0 (Novartis), and Zenapax0 (Roche
Laboratories).
[0189] Specifically, the immune suppressive agent can include rapamycin or
thalidomide.
Rapamycin is a triene macrolide isolated from Streptomyces liygroscopicus..
Thalidomide is
chemically designated as 2-(2,6-dioxo-3-piperidinyl)-1H-iso-indole-1,3(2H)-
dione.
[0190] Anti-cancer or anti-cell proliferation agents that can be incorporated
as an
additional bioactive agent in the invention wound dressings, implants and
device coatings
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 and nitrogen mustard combinations, gonadotropin releasing hormone
(GNRH)
analogues, progestrins, immunomodulators, miscellaneous antineoplastics,
photosensitizing
agents, and skin and mucous membrane agents. See, Physician's Desk Reference,
2005
Edition.
[0191] Suitable adjunct antineoplastic agents include Anzemet0 (Hoeschst
Marion
Roussel), Aredia0 (Novartis), Didronel0 (MGI), Diflucan0 (Pfizer), Epogen0
(Amgen),
Ergamisol0 (Janssen), Ethyol0 (Alza), KytrilO (SmithKline Beecham),
Leucovorin0
(Immunex), Leucovorin0 (Glaxo Wellcome), Leucovorin0 (Astra), Leukine0
(Immunex),
Marinol0 (Roxane), Mesnex0 (Bristol-Myers Squibb Oncology/Immunology),
Neupogen
(Amgen), Procrit0 (Ortho Biotech), Salagen0 (MGI), Sandostatin0 (Novartis),
Zinecard0
(Pharmacia and Upjohn), ZofranO (Glaxo Wellcome) and Zyloprim0 (Glaxo
Wellcome).
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53
[0192] Suitable miscellaneous alkylating agents include Myleran0 (Glaxo
Wellcome),
Paraplatin0 (Bristol-Myers Squibb Oncology/.Immunology), Platinol0 (Bristol-
Myers
Squibb Oncology/Immunology) and Thioplex0 (Immunex).
[0193] Suitable nitrogen mustards include Alkeran0 (Glaxo Wellcome), Cytoxan0
(Bristol-Myers Squibb Oncology/Immunology), IfexO (Bristol-Myers Squibb
Oncology/Immunology), Leukeran0 (Glaxo Wellcome) and Mustargen0 (Merck).
[0194] Suitable nitrosoureas include BiCNUO (Bristol-Myers Squibb
Oncology/Immunology), CeeNUO (Bristol-Myers Squibb Oncology/Immunology),
Gliadel0
(Rhone-Poulenc Rover) and Zanosar0 (Phannacia and Upjohn).
[0195] Suitable antimetabolites include Cytostar-UO (Phamiacia and Upjohn),
Fludara0
(Berlex), Sterile FUDRO (Roche Laboratories), Leustatin0 (Ortho Biotech),
Methotrexate0
(Immunex), Parinethol0 (Glaxo Wellcome), Thioguanine0 (Glaxo Wellcome) and
Xeloda0
(Roche Laboratories).
[0196] Suitable androgens include Nilandron0 (Hoechst Marion Roussel) and
Teslac0
(Bristol-Myers Squibb Oncology/Immunology).
[0197] Suitable antiandrogens include Casodex0 (Zeneca) and Eulexin0
(Schering).
[0198] Suitable antiestrogens include Arimidex0 (Zeneca), Fareston0
(Schering),
Femara0 (Novartis) and Nolvadex0 (Zeneca).
[0199] Suitable estrogen and nitrogen mustard combinations include EmcytO
(Pharmacia
and Upjohn).
[0200] Suitable estrogens include Estrace0 (Bristol-Myers Squibb) and Estrab0
(Solvay).
[0201] Suitable gonadotropin releasing hormone (GNRH) analogues include
Leupron
DepotO (TAP) and Zoladex0 (Zeneca).
[0202] Suitable progestins include Depo-Provera0 (Pharmacia and Upjohn) and
Megace0 (Bristol-Myers Squibb Oncology/Immunology).
[0203] Suitable immunomodulators include Erganisol0 (Janssen) and Proleukin0
(Chiron
Corporation).
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54
[0204] Suitable miscellaneous antineoplastics include Camptosar0 (Pharmacia
and
Upjohn), Celestone0 (Schering), DTIC-Dome (Bayer), Elspar0 (Merck), Etopophos
(Bristol-Myers Squibb Oncology/Immunology), Etopoxide0 (Astra), Gemzar0
(Lilly),
Hexalen0 (U. S. Bioscience), Hycantin0 (SmithKline Beecham), Hydrea0 (Bristol-
Myers
Squibb Oncology/Immunology), Hydroxyurea0 (Roxane), Intron AO (Schering),
Lysodren0
(Bristol-Myers Squibb Oncology/Immunology), Navelbine0 (Glaxo Wellcome),
Oncaspar0
(Rhone-Poulenc Rover), Oncovin0 (Lilly), Proleukin0 (Chiron Corporation),
Rituxan0
(IDEC), Rituxan0 (Genentech), Roferon-AO (Roche Laboratories), TaxolO
(paclitaxol/paclitaxel, Bristol-Myers Squibb Oncology/Immunology), Taxotere0
(Rhone-
Poulenc Rover), TheraCysO (Pasteur Merieux Connaught), Tice BCGO (Organon),
Velban0
(Lilly), VePesidO (Bristol-Myers Squibb Oncology/Immunology), Vesanoid0 (Roche
Laboratories) and Vuinon0 (Bristol-Myers Squibb Oncology/Immunology).
[0205] Suitable photosensitizing agents include Photofrin0 (Sanofi).
[0206] Specifically, useful anti-cancer or anti-cell proliferation agents can
include TaxolO
(paclitaxol), a nitric oxide-like compound, or NicOX (NCX-4016). TaxolO
(paclitaxol) is
chemically designated as 5(3,20-Epoxy-1,2a4,7(3,10P,13a-hexahydroxytax-11-en-9-
one 4,10-
diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine,
rapamycin,
sirolimus, everolimus, paclitaxel and its taxene analogs, 17AAG and other
geldanamycins,
Epothilone D and other epothilones, Estradiol and related steroid derivatives;
Lantrunculin D,
Cytochalasin A, nitric oxide, dexamethasone, and Angiopeptin.. Anti-
proliferant drugs can
be used to treat a wide range of abnormal growth related indications resulting
from excessive
cell proliferation, including, but not limited to restenosis, hemangiomas
(vascular
malformations); inflammatory conditions, malignant or benign neoplasia,
endometriosis,
(congenital or endocrine/honnonal abnormalities), adhesions (abdominal or
plural), keloid
formation, bone overgrowth and infections.
[0207] A nitric oxide-like compound includes any compound (e.g., polymer) to
which is
bound 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
U.S. Patent No. 5,650,447. See, e.g., "Inhibition of neointimal proliferation
in rabbits after
vascular injury by a single treatment with a protein adduct of nitric oxide";
David Marks et
al. JClin. Invest. (1995) 96:2630-2638. NCX-4016 is chemically designated as 2-
acetoxy-
benzoate 2-(nitroxymethyl)-phenyl ester, and is an antithrombotic agent.
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[0208] It is appreciated that those skilled in the art understand that the
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.
Plzysician's Desk
Reference (PDR), Medical Economics Company (Montvale, NJ), (53rd Ed.), pp.
1059-1067.
[0209] Preferred bioactive agents and drugs for inclusion in the invention
wound dressings
and device coatings include, rapamycin and any of its analogs or derivatives,
paclitaxel or
any of its taxene analogs or derivatives, everolimus, Sirolimus, tacrolimus,
or any of its -
limus named family of drugs, and statins such as simvastatin, atorvastatin,
fluvastatin,
pravastatin, lovastatin, rosuvastatin, geldanamycins, such as 17AAG (17-
allylamino-17-
demethoxygeldanamycin); Kosan KOS-862, 17-dimethylaminoethylamino-17-demethoxy-
geldanamycin and other polyketide inhibitors of heat shock protein 90 (Hsp90)
and
Cilostazol, and the like.
[0210] 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 t11e
radical is attached to a residue of compound of structural formulas s(I, III -
VII) or (X).
Based on the linkage that is desired, those slcilled 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.
[0211] The residue of a bioactive agent can be formed employing any suitable
reagents
and reaction conditions. Suitable reagents and reaction conditions are
disclosed, e.g., in
Advanced Organic Chemistfy, Part B: Reactions and Synthesis, Second Edition,
Carey and
Sundberg (1983); Advanced Organic Claemistiy, Reactions, Mechanisms and
Structure,
Second Edition, March (1977); and Comprehensive ONganic Transformations,
Second
Edition, Larock (1999).
[0212] 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
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56
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 "ligands",
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.
[0213] The polymer/bioactive agent linkage can be selected to degrade over a
desired
period of time to provide time release of a suitable and effective amount of
bioactive agent
according to the type of wound being treated. Any suitable and effective
period of time can
be chosen by judicious choice of the chemical properties of the linkage of the
bioactive agent
to the polymer. Typically, the suitable and effective amount of bioactive
agent can be
released over a time selected from about twenty-four hours, about seven days,
about thirty
days, about ninety days, and about one hundred and twenty days. Longer time
spans are
particularly suitable for implantable wound dressings and device coatings.
Additional factors
that typically affect the length of time over which the bioactive agent is
released from the
polymer 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.
Polymer/Linker/Bioactive agent Linkage
[0214] In addition to being directly linked (e.g., covalently) to the residue
of a compound
of structural formulas (I, III - VII) and (X), the residue of a bioactive
agent can also be linked
to the residue of a compound of structural formulas (I, III - VII) and (X) by
a suitable linker.
The stnicture of the linker is not crucial, provided the resulting compound of
the invention
has an effective therapeutic index as a bioactive agent.
[0215] Suitable linkers include linkers that separate the residue of a
compound of
structural formulas (I, III - VII) and (X) from the residue of a bioactive
agent by a distance of
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57
about 5 angstroms to about 200 angstroms, inclusive. Other suitable linkers
include linkers
that separate the residue of a compound of structural formulas s (I, III -
VII) and (X) and the
residue of a bioactive agent by a distance of about 5 angstroms to about 100
angstroms,
inclusive, as well as linkers that separate the residue of a compound of
structural formulas (I,
III - VII) and (X) from the residue of a bioactive agent by a distance of
about 5 angstrorns to
about 50 angstroms, or by about 5 angstroms to about 25 angstroms, inclusive.
[0216] The linker can be linked to any synthetically feasible position on the
residue of a
compound of structural formulas(I, III - VII) and (X). Based on the linkage
that is desired,
those skilled in the art carn select suitably functionalized starting
materials that can be derived
from a compound of structural formulas (I, III - VII) and (X) and a bioactive
agent using
procedures that are known in the art.
[0217] The linker can conveniently be linked to the residue of a coinpound of
structural
formulas (I, III - VII) and (X) or to the residue of a bioactive agent 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., -
0-), 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-), amino (e.g., -N(R)-) or a direct (e.g., C-C) linkage, wherein
each R is
independently H or (C1-Cg)alkyl. The linkage can be formed from suitably
functionalized
starting materials using synthetic procedures that are known in the art. Based
on the lirnkage
that is desired, those skilled in the art can select suitably functionalized
starting materials that
can be derived from a residue of a compound of structural formulas (I, III -
VII) and (X), a
residue of a bioactive agcnt, and from a given linker using procedures that
are known in the
art.
[0218] Specifically, the linker can be a divalent radical of the formula W-A-Q
wherein A
is (C1-C24)alkyl, (C2-C24)alkenyl, (C2-C24)alkynyl, (C3-C$)cycloalkyl, or (C6-
Clo)aryl,
wherein W and Q are each independently -N(R)C(=O)-, -C(=O)N(R)-, OC(=O)-, -
C(=O)O-,
-0-, -S-, -S(O)-, -S(0)2-, -S-S-, -N(R)-, -C(=0)-, or a direct bond (i.e., W
and/or Q is absent);
wherein each R is indepe;ndently H or (C1-C6)alkyl.
[0219] Specifically, tbe linker can be a divalent radical of the formula W-
(CH2)n-Q,
wherein n is from about 1 to about 20, from about 1 to about 15, from about 2
to about 10,
from about 2 to about 6, or from about 4 to about 6; wherein W and Q are each
independently
-N(R)C(=O)-, -C(=0)N(R)-, -OC(=O)-, -C(=O)O-, -0-, -S-, -S(O)-, -S(O)2-, -S-S-
, -C(=O)-, -
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58
N(R)-, or a direct bond (i.e., W and/or Q is absent); wherein each R is
independently IEI or
(C1-C6)alkyl.
[0220] Specifically, W and Q can each independently be -N(R)C(=O)-, -C(=O)N(R)-
, -
OC(=O)-, -N(R)-, -C(=O)O-, -0-, or a direct bond (i.e., W and/or Q is absent).
Specifically,
the linker can be a divalent radical formed from a saccharide. Specifically,
the linker can be
a divalent radical formed from a cyclodextrin. Specifically, the linker can be
a divalent
radical, i.e., divalent radicals formed from a peptide or an amino acid. The
peptide can
comprise 2 to about 25 amino acids, 2 to about 15 amino acids, or 2 to about
12 amino acids.
[0221] Specifically, the peptide can be poly-L-lysine (i.e., [-
NHCH[(CH2)4NH2]CO-]m Q
wherein Q is H, (C1-C14)alkyl, or a suitable carboxy protecting group; and
wherein ni is about
2 to about 25. The poly-L-lysine can contain about 5 to about 15 residues
(i.e., m is from
about 5 to about 15). For example, the poly-L-lysine can contain from about 8
to about 11
residues (i.e., m is from about 8 to about 11).
[0222] Specifically, the peptide can also be 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, or poly-L-lysine-L-
tyrosine.
[0223] Specifically, the linker can be prepared from 1,6-diaminohexane
H2N(CH2)6NH2,
1,5-diaminopentane H2N(CH2)5NH2, 1,4-diaminobutane H2N(CHZ)4NH2, or 1,3-
diaminopropane H2N(CH2)3NH2.
[0224] One or more bioactive agents can be linked to the polymer through a
linker.
Specifically, the residue of each of the bioactive agents can each be linked
to the residue of
the polymer through a linker. Any suitable number of bioactive agents (i.e.,
residues thereof)
can be linked to the polymer (i.e., residue tliereof) through a linker. The
number of bioactive
agents that can be linked to the polymer through a linker can typically depend
upon the
molecular weight of the polymer and whether the polymer is saturated or
unsaturated. For
example, up to about 450 bioactive agents (i.e., residues thereof) can be
linked to the polymer
(i.e., residue thereof) through a linker, up to about 300 bioactive agents
(i.e., residues thereof)
can be linked to the polymer (i.e., residue thereof) through a linker, or up
to about 150
bioactive agents (i.e., residues thereof) can be linked to the polymer (i.e.,
residue thoreof)
through a linker.
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[0225] In one embodiment of the present invention, a polymer (i.e., residue
thereof) as
disclosed herein can be linked to the linker via a carboxyl group (e.g., COOR)
of the
polymer. For example, a polymer residue containing a free hydrogen, or (C6-
Clo)aryl(CI-
C6)alkyl, can react with an amino functional group of the linker or a hydroxyl
functional
group of the linker, to provide a Polymer/Linker having an amide linkage or a
Polymer/Linker having a carboxyl ester linkage, respectively. In another
embodiment, the
carboxyl group can be transformed into an acyl halide or ari acyl anhydride.
[0226] In one embodiment of the invention, a bioactive agent (i.e., residue
thereof) can be
linked to the linker via a carboxyl group (e.g., COOR, wherein R is hydrogen,
(C6-
Clo)aryl(C1-C6)alkyl or (C1-Q)alkyl) of the linker. Specifically, an amino
functional group
of the bioactive agent or a hydroxyl functional group of the bioactive agent
can react with the
carboxyl group of the linker, to provide a Linker/Bioactive agent having an
amide linkage or
a Linker/Bioactive agent having a carboxylic ester linkage, respectively. In
another
embodiment, the carboxyl group of the linker can be transformed into an acyl
halide or an
acyl anhydride.
[0227] The bioactive agent is released as the polymer degrades, whether the
bioactive
agent is linked to the polymer, dissolved within the polymer, or intermixed
with the polymer.
Any suitable and effective amount of bioactive agent can be released and will
typically
depend, e.g., on the specific polymer, bioactive agent, linker, and
polymer/linker/bioactive
agent linkage chosen. Typically, up to about 100% of the bioactive agent can
be released
from the polymer. Specifically, up to about 90%, up 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 released from the polymer/linker/bioactive agent include,
e.g., the nature
and amount of polymer, the nature and amount of bioactive agent, the nature
and amount of
linker, the nature of any polymer/linker/bioactive agent linkage, and the
nature and amount of
additional substances present in the formulation.
[0228] The polymer degrades 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
precursor cells and/or bioactive agent is released from the polymer include,
e.g., the nature
and amount of polymer, the nature and amount of precursor cells, conditioned
medium or
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growth medium for the cells, the nature and amount of bioactive agent, the
nature of any
linker used, the nature of the polymer/linker/bioactive agent linkage, and the
nature and
amount of additional substances present in the formulation.
Polynaer Intermixed with Bioactive Agent or Additional Bioactive Agent
[0229] 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 structure as
described herein
can be physically intermixed with one or more bioactive agents or additional
bioactive agents
to provide a formulation.
[0230] 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.
[0231] As used herein, a"foimulation" 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 conlposition (i.e., a homogeneous formulation).
[0232] 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.
[0233] In yet another embodiment of the invention the polymer/bioactive agent,
polymer/linker/bioactive agent, formulation, or combination thereof as
described herein, can
be applied, as a polymeric film onto the surface of a medical device. At least
a portion of the
surface of the medical device can be coated with a film of polymer or hydrogel
having any
suitable thickness. For example, the thickness of the film on the medical
device can be about
1 to about 50 microns thick or about 5 to about 20 microns thick.
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[0234] The polymer and/or hydrogel film can effectively serve to sequester
precursor cells
or conditioned medium thereof as well as a bioactive agent-eluting coating on
a medical
device, such as an orthopedic implant. This bioactive coating can be createcl
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 to create a type of local bioactive
agent delivery
system that enhances restoration of a tissue lesion at the site of implant.
[0235] In one embodiment, the wound dressing is a single or multilayered wound
dressing
wherein the above-described polymer is in the form of a membrane or mat of
fine polymer
threads, for example NanoskinTM (MediVas, LLP, San Diego, CA). Such a polymer
dressing
can be used as a surgical wrap, for example, for restoration of tissue for
burn victims when
seeded with epithelial precursor cells or infused with conditioned medium
obtained from
allogenic epithelial precursor cells, wherein a surface barrier enhances
tissue restoration and
yet the polymer membrane is bioabsorbed. In other embodiments, the polyrner
threads can be
woven, webbed, braided, and the like. For example, electrospinning of the
polymer can
produce a random webbing or mat of microfibers of the polymer.
[0236] At least one surface of the polymer membrane may be coated with an
additional
formulation layer in a sandwich type of configuration to deliver to the
tissue, bioactive agents
that promote natural tissue restoration processes. Such an additional layer of
hydrogel-based
drug release formulation can comprise various bioactive agents dispersed in a
hydrogel to
provide an elution rate different than that of the polymer component of the
wound dressing.
Optionally the multilayered wound dressing may further include an occlusive
layer lying atop
the hydrogel layer.
[0237] 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
10-4 meters,
less than about 1 x 10-5 meters, less than about 1 x 10"6 meters, less than
about 1 x 10-7
meters, less than about 1 x 10"8 meters, or less than about 1 x 10-9 meters.
[0238] The formulation can degrade to provide a suitable and effective amount
of the at
least one progenitor precursor cell, which can be autologous or allogenic. Any
suitable and
effective amount of the precursor cells can be released and will typically
depend, e.g., on the
specific formulation chosen. Typically, up to about 100% of the precursor
cells or
conditioned medium can be released from the formulation. Specifically, up to
about 90%, up
to 75%, up to 50%, or up to 25% of the precursor cells or conditioned medium
can be
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released from the formulation at a substantially linear rate. Factors that
typically affect the
amount or rate of the precursor cells or conditioned medium that is released
from the
formulation include, e.g., the type of tissue in contact with the wound
dressing, the nature and
ainount of polymer or hydrogel, the type of dispersal of the precursor cells
in the polymer or
hydrogel, the nature and amount of the precursor cells, and the nature and
amount of any
additional wound healing bioactive agent(s) present in the formulation.
[0239] 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.
For example, the polymer can be selected to release the bioactive agent over
about twenty-
four hours, over about two days, over about seven days, over about ninety
days, or over about
one hundred and twenty days, the latter being particularly useful when an
iinplantable wound
dressing is desired. Factors that typically affect the length of time over
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.
[0240] In one embodiment, a polymer used in making an invention wound dressing
is
physically intermixed with at least one bioactive agent. In another
embodirrient, the polymer
is linked to at least one bioactive agent, either directly or through a
linker. ]En another
embodiment, the precursor cells disbursed in the polymer are held within the
polymer during
polymer biodegradation, either directly or tlirough a ligand or linker, and
the polymer or
hydrogel can also be physically intermixed with one or more precursor cells in
growth
medium or with conditioned medium, such as cell-free conditioned mediuna.
[0241] The invention polymer/hydrogel coatings for medical devices containing
precursor
cells and/or conditioned medium, 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 be used in the manufacture of
a medical
device. Suitable medical devices include, e.g., artificial joints, artificial
bones and
intravertebral implants, cardiovascular medical devices, stents, shunts,
sutuxes, artificial
arteries, teeth and other body implants.
[0242] In yet another embodiment, the invention provides methods for the
invention
provides methods for promoting restoration of tissue at a lesion site in a
mammalian subject
by using a biodegradable wound dressing comprising at least one precursor cell
selected from
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stem cells, tissue-specific progenitor cells, germ-layer lineage stern cells,
and pluripotent
stem cells, and/or conditioned medium obtained from such cells, dispersed
within a
biodegradable polymer or hydrogel and contacting the lesion site with the
wound dressing
under conditions suitable for promoting restoration of the tissue at the
lesion site.
[02431 To this end, in treating a chronic wound, the polymer of the wound
dressing can be
placed in contact with the wound bed and the precursor cells and conditioned
medium can be
allowed to interact with cells and factors in surrounding tissue while the
polymer biodegrades
over a suitable period time, releasing bioactive agents therein into the wound
bed while the
polymer is absorbed therein. Alternatively, the wound dressing used in
treatment of a chronic
wound will include a biodegradable hydrogel layer (i.e., non-stick: layer),
which can be
placed in contact with the wound bed. The formulations of the polymer layer
and the
hydrogel layer can be selected to release their respective cells, conditioned
medium and
bioactive agents at different rates. The invention methods are beneficially
used in treatment
of such chronic wounds as venous stasis ulcer, diabetic ulcer, pressure ulcer,
or ischemic
ulcer.
[0244] The following examples are meant to illustrate and not to limit the
invention.
EXAMPLES
EXAMPLE 1
Delivery of Growth/Survival Components by Alginic Acid
[0245] This example illustrates experiments that were conducted to test the
ability of
hydrogel particles to deliver components required for cell growtlz and
survival. The hydrogel
particles were formed using low viscosity alginic acid (AA) (Sigrna Chemicals,
A2158)
prepared using an adaptation of a published protocol (J Ra.ymond et al., Aa
JNeuroradzol
(2003) 24:1214-1221).
[0246] Preliminary experiments demonstrated that 4% low viscosity alginate
made up in
0.5% sodium chloride can absorb about one third of its volume of the desired
media, such as
conditioned medium from progenitor cell cultures that had been isolated from
human blood,
and then be crosslinked by adding 5% calcium chloride. Polyrnerization occurs
very rapidly
and AA. particles form, encapsulating the media within the alginate. The rate
of release of
media from the particles during the first 24 hours is very rapid, with about
half of the media
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being released. The release rate then slows over the next 48 hours until the
majority of the
media is released by 72 hours.
[0247] To illustrate the release rate from AA particles, trypan blue was
incorporated into
the AA as an indicator compound. The trypan blue-containing beads were placed
into wells
without PBS, as controls to demonstrate the starting color of the beads, and
into wells plus
PBS to observe the release of the trypan blue into the colorless PBS. At each
time point (24,
48 and 72 hours), the trypan-blue containing beads were removed from the PBS
and placed
into an empty well to observe any change in color when compared to control
beads.
Observations were recorded and photographed at each time point.
[0248] At 24 hours, about 50% of the blue color had been released fr~ om the
hydrogel
particles, by 48 hours there was still a small amount of color left in the
particles, but by 72
hours the AA particles no longer had any color left, indicating that all the
trypan blue had
been released.
Cell Rescue Experiment
[0249] This example illustrates the efficiency of AA particles for delivering
a survival
component to cells. An experiment was designed using fetal bovine serum (FBS)
encapsulated in AA particles, which were provided to cells growing without
other access to
FBS. Endothelial or smooth muscle cells were plated into 12-well tissue
culture plates with
FBS-containing particles placed in adjoining cell inserts in the plates (BD 35-
3180, pore size
0.4 microns).
[0250] The experimental protocol included the following conditions for both
endothelial
cells and smooth muscle cells:
[0251] 1. Cells were plated at 15,000 cells/well in 12-well plates in the
following media
(the basal medium for SMC is SmGM-2 BulletKit (Cambrex, #CC-3182) and for
EC'is
EGM-2 BulletKit (Cambrex #CC-3162)).
2. Complete growth factors (for SMC this included hEGF, insulin, hFGF-B; and
for EC this included hEGF, VEGF, hFGF-B, R3-IGF-1) and 5% FBS.
3. Minus growth factors, plus 5% FBS.
4. Minus growth factors, minus serum.
5. Minus growth factors, plus AA particle containing 0% FBS.
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6. Minus growth factors, plus AA particle containing 5% FBS.
7. Minus growth factors, plus AA particle containing 10% FBS
[0252] AA particles were made as follows: Sterile 4% low viscosity alginic
acid (AA)
was added to 1/3 (v/v) FBS and then crosslinked by addition of 5% calciuni
chloride in a
volume equal to AA. Particles were rinsed twice in PBS and then placed into
cell inserts.
[0253] Microscopic observations were made at 24 and 48 hours, and standard ATP
assays
were conducted at 48 hours to measure cell growth. This same experiment was
repeated
several times, demonstrating that the AA particles were able to deliver FBS to
both
endothelial and smooth muscle cells to enhance cell growth (Fig. 1).
EXAMPLE 2
Encapsulation of Cells in Hydrogel
[0254] A highly purified and well-characterized sodium alginate with very low
levels of
endotoxins and proteins, optimal for in vitro and in vivo applications, was
used to encapsulate
cells (NovaMatrix, Pronova UltraPure (UP) LVM alginate). This alginate is made
up in 2%
HEPES with some sonication, to help the AA go into complete suspension, and
then filter
sterilized. Cell-containing-particles are made by mixing cells plus fetal
bovine serum (FBS)
with the alginate, drawing the mixture up into a Icc syringe and forming
particles drop wise
into a 5% calcium chloride solution. This process produces about 100
particles/ml with about
2000 cells/particle. The cells used for this experiment were SMCs, wliich are
a robust,
anchorage-dependent cell line nonnally not grown in suspension.
[0255] To determine cell viability within the AA particles, two viability dyes
were used:
(1) Calcein AM, which is retained in cells that have intact membranes. Calcein
AM does not
label dead cells and is rapidly lost under conditions that cause cell lysis.
Once inside the cell,
the colorless, nonfluorescent AM ester is cleaved by nonspecific esterases
causing the
compound to fluoresce. (2) Propidium oodide, which binds to DNA by
intercalating between
the bases. Propidium Iodide is membrane impermeant and does not easily enter
viable cells.
Control particles encapsulated SMCs in the AA particles, but were exposed to
no dyes.
Microscopic observations were recorded and photographed (20x mag Bar = 50
microns)
using a Nikon Eclipse TE2000-S phase contrast microscope equipped with an epi-
fluorescence attachment
~
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[0256] After up to 6 h the cells were photographed in color. Microscopic
observations
were recorded and photographed using a Nikon Eclipse TE2000-S phase contrast
microscope
equipped with an epi-fluorescence attachment and the photographs showed that
the Calcein
AM was taken up by viable cells while the Propidium Iodide (PI) was not. This
result
indicated that the cells in the particles were still alive following several
hours inside the AA
particles.
[0257] A series of time course experiments using SMC encapsulated in AA
particles
exposed to the two viability dyes, indicated that SMCs remained viable for up
to 6 hours
because the Calcein AM was taken up by encapsulated cells while the propidium
iodide was
not.
EXAMPLE 3
Polymer Coating of Hydrogel Encapsulated Cells
[0258] PEA conjugated to fluorescent dansyl-lysine was used to coat particles
to visualize
the polymer coating for consistency and completeness of coating. The results
of this
experiment indicated that a reliable method to coat AA particles to achieve
consistency and
completeness of coating was to suspend them in the polymer and allow the
polymer to flow
through a mesh that captures the particles. The particles were then rapidly
removed from the
mesh by resuspending in the appropriate buffer.
[0259] Then, to determine if the SMCs contained in AA particles can survive
coating with
PEA.H polymer, AA particles were formed containing 2000 smooth muscle
cells/particle.
Test AA particles were coated with a 10% (w/v ethanol) polymer solution and
incubated in
the presence of the two viability dyes shown in Example 2 above as able to
enter the
encapsulated cells. Control particles encapsulated the SMCs, but were also
exposed to the
dyes, but were not coated with the PEA polymer. Microscopic observations were
recorded
and photographed (200x mag; Bar = 50 microns) using a Nikon Eclipse TE2000-S
phase
contrast microscope equipped with an epi-fluorescence attachment.
[0260] It had been demonstrated in Example 2 above, that SMCs in AA particles
remained
viable for up to 6 hours as measured by Calcein AM uptake and propidium iodide
exclusion.
Visual examination of these polymer-coated particles after 4 four hours
indicated that the
overall cell population was less viable than the cells in the control
particles. Cells nearest the
surface were most affected by the polymer coating; whereas some of the more
interior cells
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67
were able to survive for a few hours. Therefore, polymer coating of the AA-
encapsulated
SMCs reduced the length of time that the cells were viable, but the principle
of polymer
encapsulation of cells was demonstrated.
[0261] All publications, patents, and patent documents are incorporated by
reference
herein, as though individually incorporated by reference. The invention has
been described
with reference to various specific and preferred embodiments and techniques.
However, it
should be understood that many variations and modifications might be made
while remaining
within the spirit and scope of the invention.
[0262] 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 herein is limited only by
the following
claims.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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