Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 4
CONTENANT LES PAGES 1 A 310
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 4
CONTAINING PAGES 1 TO 310
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
POLYMER COMPOSITIONS AND METHODS FOR THEIR USE
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates generally to polymer compositions that
include a therapeutic agent (e.g., a fibrosis-inhibiting agent or an anti-
infective
agent), and to methods of making and using such compositions.
Description of the Related Art
Polymeric compositions, particularly those that include synthetic
polymers or a combination of synthetic and naturally occurring polymers, have
been used in a variety of medical applications, such as the prevention of
surgical adhesions, tissue engineering, and as bioadhesive materials. U.S.
Patent No. 5,162,430 describes the use of collagen-synthetic polymer
conjugates prepared by covalently binding collagen to synthetic hydrophilic
polymers such as various derivatives of polyethylene glycol. In a related
patent, U.S. Patent No. 5,328,955, various activated forms of polyethylene
glycol and various linkages are described, which can be used to produce
collagen-synthetic polymer conjugates having a range of physical and chemical
properties. U.S. Patent No. 5,324,775 also describes synthetic hydrophilic
polyethylene glycol conjugates, but the conjugates involve naturally occurring
polymers such as polysaccharides. EP 0 732 109 A1 discloses a crosslinked
biomaterial composition that is prepared using a hydrophobic crosslinking
agent, or a mixture of hydrophilic and hydrophobic crosslinking agents. U.S.
Patent No. 5,614,587 describes bioadhesives that comprise collagen that is
crosslinked using a multifunctionally activated synthetic hydrophilic polymer.
U.S. application Ser. No. 08/403,360, filed Mar. 14, 1995, discloses a
composition useful in the prevention of surgical adhesions comprising a
substrate material and an anti-adhesion binding agent, where the substrate
1
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
material may comprise collagen and the binding agent may comprise at least
one tissue-reactive functional group and at least one substrate-reactive
functional group. U.S. application Ser. No. 08/476,825, filed Jun. 7, 1995,
discloses bioadhesive compositions comprising collagen crosslinked using a
multifunctionally activated synthetic hydrophilic polymer, as well as methods
of
using such compositions to effect adhesion between a first surface and a
second surface, wherein at least one of the first and second surfaces may be a
native tissue surface. U.S. Patent No. 5,874,500 describes a crosslinked
polymer composition that comprises one component having multiple
nucleophilic groups and another component having multiple electrophilic
groups. Covalently bonding of the nucleophilic and electrophilic groups forms
a
three dimensional matrix that has a variety of medical uses including tissue
adhesion, surface coatings for synthetic implants, and drug delivery. More
recent developments include the addition of a third component having either
nucleophilic or electrophilic groups, as is described in U.S. Patent No.
6,458,889 to Trollsas et al. US 5,874,500, US 6,051,648 and US 6,312,725
disclose the in situ crosslinking or crosslinked polymers, in particular
polyethylene glycol) based polymers, to produce a crosslinked composition.
West and Hubbell, Biomaterials (1995) 16:1153-1156, disclose the prevention
of post-operative adhesions using a photopolymerized polyethylene glycol-co-
lactic acid diacrylate hydrogel and a physically crosslinked polyethylene
glycol-
co-polypropylene glycol hydrogel, POLOXAMER 407 (BASF Corporation,
Mount Olive, NJ). Polymerizable cyanoacrylates have also been described for
use as tissue adhesives (Ellis, et al., J. Otolaryngol. 19:68-72 (1990)). Two-
part
synthetic polymer compositions have been described that, when mixed
together, form covalent bonds with one another, as well as with exposed tissue
surfaces (PCT WO 97/22371, which corresponds to U.S. application Ser. No.
08/769,806 U.S. Pat. No. 5,874,500).
2
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
BRIEF SUMMARY OF THE INVENTION
Briefly, in one aspect, the present invention provides compositions
that contain both an anti-fibrotic agent and either a polymer or a pre-
polymer,
i.e., a compound that forms a polymer. In one embodiment, these compositions
are formed in-situ when precursors thereof are delivered to a site in the
body, or
a site on an implant. For example, the compositions of the invention include
the
crosslinked reaction product that forms when two compounds (a multifunctional
polynucleophilic compound and a multi-functional polyelectrophilic compound)
are delivered to a site in a host (in other words, a patient) in the presence
of an
anti-fibrotic agent. However, the compositions of the invention also include a
mixture of anti-fibrotic agent and a polymer, where the composition can be
delivered to a site in a patient's body to achieve beneficial affects, e.g.,
the
beneficial affects described herein.
In some instances, the polymers themselves are useful in various
methods, including the prevention of surgical adhesions.
In another aspect, the present invention provides methods for
treating and/or preventing surgical adhesions. The surgical adhesions can be
the result of, for example, spinal or neurosurgical procedures, of
gynecological
procedures, of abdominal procedures, of cardiac procedures, of orthopedic
procedures, of reconstructive procedures, and cosmetic procedures.
In another aspect, the present invention provides methods for
treating or preventing inflammatory arthritis, such as osteoarthritis and
rheumatoid arthritis. The method includes delivering to patient in need
thereof
an anti-fibrotic agent, optionally with a polymer.
In another aspect, the present invention provides for the
prevention of cartilage loss as can occur, for example after a joint injury.
The
method includes delivering to the joint of the patient in need therof an anti-
fibrotic agent, optionally with a polymer.
In another aspect, the present invention provides for treating
hypertrophic scars and keloids. The method includes delivering to the scar or
3
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
keloid of the patient in need thereof an anti-fibrotic agent, optionally with
a
polymer.
In another aspect, the present invention provides a method for the
treatment of vascular disease, e.g., stenosis, restenosis or atherosclerosis.
The method includes the perivascular delivery of an anti-fibrotic agent.
In one aspect, the present invention provides a method for
implanting a medical device comprising: (a) infiltrating a tissue of a host
where
the medical device is to be, or has been, implanted with i) an anti-fibrotic
agent,
ii) an anti-infective agent, iii) a polymer; iv) a composition comprising an
anti-
fibrotic agent and a polymer, v) a composition comprising an anti-infective
agent and a polymer, or vi) a composition comprising an anti-fibrotic agent,
an
anti-infective agent and a polymer, and (b) implanting the medical device into
the host.
Optionally, in separate aspects, the invention provides: a method
for implanting a medical device comprising: (a) infiltrating a tissue of a
host
where the medical device is to be, or has been, implanted with an anti-
fibrotic
agent, and (b) implanting the medical device into the host; a method for
implanting a medical device comprising: (a) infiltrating a tissue of a host
where
the medical device is to be, or has been, implanted with an anti-infective
agent,
and (b) implanting the medical device into the host; a method for implanting a
medical device comprising: (a) infiltrating a tissue of a host where the
medical
device is to be, or has been, implanted with a polymer; and (b) implanting the
medical device into the host; a method for implanting a medical device
comprising: (a) infiltrating a tissue of a host where the medical device is to
be,
or has been, implanted with a composition comprising an anti-fibrotic agent
and
a polymer, and (b) implanting the medical device into the host; a method for
implanting a medical device comprising: (a) infiltrating a tissue of a host
where
the medical device is to be, or has been, implanted with a composition
comprising an anti-infective agent and a polymer, and (b) implanting the
medical device into the host; and a method for implanting a medical device
4
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
comprising: (a) infiltrating a tissue of a host where the medical device is to
be,
or has been, implanted with a composition comprising an anti-fibrotic agent,
an
anti-infective agent and a polymer, and (b) implanting the medical device into
the host.
These and other aspects of the present invention will become
evident upon reference to the following detailed description and attached
drawings. In addition, various references are set forth herein which describe
in
more detail certain procedures and/or compositions, and are therefore
incorporated by reference in the entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram showing how a cell cycle inhibitor acts at
one or more of the steps in the biological pathway.
Figure 2 is a graph showing the results for the screening assay for
assessing the effect of mitoxantrone on nitric oxide production by THP-1
macrophages.
Figure 3 is a graph showing the results for the screening assay for
assessing the effect of Bay 11-7082 on TNF-alpha production by THP-1
macrophages.
Figure 4 is a graph showing the results for the screening assay for
assessing the effect of rapamycin concentration for TNFa production by THP-1
macrophages.
Figure 5 is graph showing the results of a screening assay for
assessing the effect of mitoxantrone on proliferation of human fibroblasts.
Figure 6 is graph showing the results of a screening assay for
assessing the effect of rapamycin on proliferation of human fibroblasts.
Figure 7 is graph showing the results of a screening assay for
assessing the effect of paclitaxel on proliferation of human fibroblasts.
Figure 8 is a picture that shows an uninjured carotid artery from a
rat balloon injury model.
5
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Figure 9 is a picture that shows an injured carotid artery from a rat
balloon injury model.
Figure 10 is a picture that shows a paclitaxel/mesh treated carotid
artery in a rat balloon injury model.
Figure 11A schematically depicts the transcriptional regulation of
matrix metalloproteinases.
Figure 11 B is a blot which demonstrates that IL-1 stimulates AP-1
transcriptional activity.
Figure 11 C is a graph which shows that IL-1 induced binding
activity decreased in lysates from chondrocytes which were pretreated with
paclitaxel.
Figure 11 D is a blot which shows that IL-1 induction increases
collagenase and stromelysin in RNA levels in chondrocytes, and that this
induction can be inhibited by pretreatment with paclitaxel.
Figures 12A-H are blots that show the effect of various anti-
microtubule agents in inhibiting collagenase expression.
Figure 13 is a graph showing the results of a screening assay for
assessing the effect of paclitaxel on smooth muscle cell migration.
Figure 14 is a graph showing the results of a screening assay for
assessing the effect of geldanamycin on IL-1 ~i production by THP-1
macrophages.
Figure 15 is a graph showing the results of a screening assay for
assessing the effect of geldanamycin on IL-8 production by THP-1
macrophages.
Figure 16 is a graph showing the results of a screening assay for
assessing the effect of geldanamycin on MCP-1 production by THP-1
macrophages.
Figure 17 is graph showing the results of a screening assay for
assessing the effect of paclitaxel on proliferation of smooth muscle cells.
6
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Figure 18 is graph showing the results of a screening assay for
assessing the effect of paclitaxel for proliferation of the murine RAW 264.7
macrophage cell line.
Figure 19 is a graph showing the average rank of joint scores of
Hartley guinea pig knees with ACL damage treated with paclitaxel. A reduction
in score indicates an improvement in cartilage score. The dose response trend
is statistically significant (p < 0.02).
Figures 20A-C are examples of cross sections of Hartley guinea
pig knees of control and paclitaxel treated animals. Figure 20A. Control
speciment showing erosion of cartilage to the bone. Figure 20B. Paclitaxel
dose 1 (low dose) showing fraying of cartilage. Figure 20C. Paclitaxel dose 2
(medium dose) showing minor defects to cartilage.
Figures 21A-F are Safranin-O stained histological slides of
representative synovial tissues from naive (healthy) knees (Figures 21A and
21 D) and knees with arthritis induced by administration of albumin in
Freund's
complete adjuvant (Figures 21 B and 21 C) or carrageenan (Figures 21 E and
21 F). Arthritic knees received either control (Figures 21 B and 21 E) or 20%
paclitaxel-loaded microspheres (Figures 21 C and 21 F). The data illustrate
decreased proteoglycan red staining in arthritic knees treated with control
microspheres and the proteoglycan protection properties of the paclitaxel-
loaded formulation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Prior to setting forth the invention, it may be helpful to an
understanding thereof to first set forth definitions of certain terms that are
used
herein.
"Fibrosis," or "scarring," or "fibrotic response" refers to the
formation of fibrous (scar) tissue in response to injury or medical
intervention.
7
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Therapeutic agents which inhibit fibrosis or scarring are referred to herein
as
"fibrosis-inhibiting agents", "fibrosis-inhibitors", "anti-scarring agents",
and the
like, where these agents inhibit fibrosis through one or more mechanisms
including: inhibiting inflammation or the acute inflammatory response,
inhibiting
migration or proliferation of connective tissue cells (such as fibroblasts,
smooth
muscle cells, vascular smooth muscle cells), inhibiting angiogenesis, reducing
extracellular matrix (ECM) production or promoting ECM breakdown, and/or
inhibiting tissue remodeling. When scarring occurs in a confined space (e.g.,
within a lumen) following surgery or instrumentation (including implantation
of a
medical device or implant), such that a body passageway (e.g., a blood vessel,
the gastrointestinal tract, the respiratory tract, the urinary tract, the
female or
male reproductive tract, the eustacian tube etc.) is partially or completely
obstructed by scar tissue, this is referred to as "stenosis" (narrowing). When
scarring subsequently occurs to re-occlude a body passageway after it was
initially successfully opened by a surgical intervention (such as placement of
a
medical device or implant), this is referred to as "restenosis."
"Host", "person", "subject", "patient" and the like are used
synonymously to refer to the living being into which a device or implant of
the
present invention is implanted.
"Implanted" refers to having completely or partially placed a
device or implant within a host. A device is partially implanted when some of
the device reaches, or extends to the outside of, a host.
"Inhibit fibrosis", "reduce fibrosis", "inhibits scarring" and the like
are used synonymously to refer to the action of agents or compositions which
result in a statistically significant decrease in the formation of fibrous
tissue that
can be expected to occur in the absence of the agent or composition.
"Anti-infective agent" refers to an agent or composition which
prevents microrganisms from growing and/or slows the growth rate of
microorganisms and/or is directly toxic to microorganisms at or near the site
of
the agent. These processes would be expected to occur at a statistically
3
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
significant level at or near the site of the agent or composition relative to
the
effect in the absence of the agent or composition.
"Inhibit infection" refers to the ability of an agent or composition to
prevent microorganisms from accumulating andlor proliferating near or at the
site of the agent. These processes would be expected to occur at a
statistically
significant level at or near the site of the agent or composition relative to
the
effect in the absence of the agent or composition.
"Inhibitor" refers to an agent which prevents a biological process
from occurring or slows the rate or degree of occurrence of a biological
process. The process may be a general one such as scarring or refer to a
specific biological action such as, for example, a molecular process resulting
in
release of a cytokine.
"Antagonist" refers to an agent which prevents a biological
process from occurring or slows the rate or degree of occurrence of a
biological
process. While the process may be a general one, typically this refers to a
drug
mechanism where the drug competes with a molecule for an active molecular
site or prevents a molecule from interacting with the molecular site. In these
situations, the efFect is that the molecular process is inhibited.
"Agonist" refers to an agent which stimulates a biological process
or rate or degree of occurrence of a biological process. The process may be a
general one such as scarring or refer to a specific biological action such as,
for
example, a molecular process resulting in release of a cytokine.
"Anti-microtubule agents" should be understood to include any
protein, peptide, chemical, or other molecule which impairs the function of
microtubules, for example, through the prevention or stabilization of
polymerization. Compounds that stabilize polymerization of microtubules are
referred to herein as "microtubule stabilizing agents." A wide variety of
methods may be utilized to determine the anti-microtubule activity of a
particular compound, including for example, assays described by Smith et al.
9
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(Cancer Lett 79(2):213-219, 1994) and Mooberry et al., (Cancer Lett. 96(2):261-
266, 1995).
"Medical device", "implant", ""device", medical device", "medical
implant", "implant/device" and the like are used synonymously to refer to any
object that is designed to be placed partially or wholly within a patient's
body for
one or more therapeutic or prophylactic purposes such as for restoring
physiological function, alleviating symptoms associated with disease,
delivering
therapeutic agents, and/or repairing, replacing, or augmenting etc. damaged or
diseased organs and tissues. While normally composed of biologically
compatible synthetic materials (e.g., medical-grade stainless steel, titanium
and
other metals; polymers such as polyurethane, silicon, PLA, PLGA and other
materials) that are exogenous, some medical devices and implants include
materials derived from animals (e.g.', "xenografts" such as whole animal
organs;
animal tissues such as heart valves; naturally occurring or chemically-
modified
molecules such as collagen, hyaluronic acid, proteins, carbohydrates and
others), human donors (e.g., "allografts" such as whole organs; tissues such
as
bone grafts, skin grafts and others), or from the patients themselves (e.g.,
"autografts" such as saphenous vein grafts, skin grafts,
tendon/ligament/muscle
transplants). Representative examples of medical devices that are of
particular
utility in the present invention include, but are not restricted to, vascular
stents,
gastrointestinal stents, tracheal/bronchial stents, genital-urinary stents,
ENT
stents, intra-articular implants, intraocular lenses, implants for
hypertrophic
scars and keloids, vascular grafts, anastomotic connector devices, implantable
sensors, implantable pumps, soft tissue implants (e.g., cosmetic implants and
implants for reconstructive surgery), implantable electrical devices, such as
implantable neurostimulators and implantable electrical leads, surgical
adhesion barriers, glaucoma drainage devices, surgical films and meshes,
prosthetic heart valves, tympanostomy tubes, penile implants, endotracheal and
tracheostomy tubes, peritoneal dialysis catheters, intracranial pressure
monitors, vena cava fitters, central venous catheters (CVC's), ventricular
assist
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
devices (e.g., LVAD), spinal prostheses, urinary (Foley) catheters, prosthetic
bladder sphincters, orthopedic implants, and gastrointestinal drainage tubes.
"Chondroprotection" refers to the prevention of cartilage loss.
Cartilage is formed from chondrocytes, and chondroprotection is the protection
of the chrondrocytes so that they do not die.
"Release of an agent" refers to a statistically significant presence
of. the agent, or a subcomponent thereof, which has disassociated from the
implant/device and/or remains active on the surface of (or within) the
device/implant.
"Biodegradable" refers to materials for which the degradation
process is at least partially mediated by, and/or performed in, a biological
system. "Degradation" refers to a chain scission process by which a polymer
chain is cleaved into oligomers and monomers. Chain scission may occur
through various mechanisms, including, for example, by chemical reaction
(e.g.,
hydrolysis) or by a thermal or photolytic process. Polymer degradation may be
characterized, for example, using gel permeation chromatography (GPC),
which monitors the polymer molecular mass changes during erosion and drug
release. Biodegradable also refers to materials may be degraded by an erosion
process mediated by, and/or performed in, a biological system. "Erosion"
refers
to a process in which material is lost from the bulk. In the case of a
polymeric
system, the material may be a monomer, an oligomer, a part of a polymer
backbone, or a part of the polymer bulk. Erosion includes (i) surface erosion,
in
which erosion affects only the surface and not the inner parts of a matrix;
and
(ii) bulk erosion, in which the entire system is rapidly hydrated and polymer
chains are cleaved throughout the matrix. Depending on the type of polymer,
erosion generally occurs by one of three basic mechanisms (see, e.g., Heller,
J., CRC Critical Review in Therapeutic Drug Carrier Systems (1984), 1 (1 ), 39-
90); Siepmann, J. et al., Adv. Drug Del. Rev. (2001 ), 48, 229-247): (1 )
water-
soluble polymers that have been insolubilized by covalent cross-links and that
solubilize as the cross-links or the backbone undergo a hydrolytic cleavage;
(2)
11
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polymers that are initially water insoluble are solubilized by hydrolysis,
ionization, or pronation of a pendant group; and (3) hydrophobic polymers are
converted to small water-soluble molecule's by backbone cleavage.
Techniques for characterizing erosion include thermal analysis (e.g., DSC), X-
ray diffraction, scanning electron microscopy (SEM), electron paramagnetic
resonance spectroscopy (EPR), NMR imaging, and recording mass loss during
an erosion experiment. For microspheres, photon correlation spectroscopy
(PCS) and other particles size measurement techniques may be applied to
monitor the size evolution of erodible devices versus time.
As used herein, "analogue" refers to a chemical compound that is
structurally similar to a parent compound, but differs slightly in composition
(e.g., one atom or functional group is different, added, or removed). The
analogue may or may not have different chemical or physical properties than
the original compound and may or may not have improved biological and/or
chemical activity. For example, the analogue may be more hydrophilic or it may
have altered reactivity as compared to the parent compound. The analogue
may mimic the chemical and/or biologically activity of the parent compound
(i.e., it may have similar or identical activity), or, in some cases, may have
increased or decreased activity. The analogue may be a naturally or non-
naturally occurring (e.g., recombinant) variant of the original compound. An
example of an analogue is a mutein (i.e., a protein analogue in which at least
one amino acid is deleted, added, or substituted with another amino acid).
Other types of analogues include isomers (enantiomers, diasteromers, and the
like) and other types of chiral variants of a compound, as well as structural
isomers. The analogue may be a branched or cyclic variant of a linear
compound. For example, a linear compound may have an analogue that is
branched or otherwise substituted to impart certain desirable properties
(e.g.,
improve hydrophilicity or bioavailability).
As used herein, "derivative" refers to a chemically or biologically
modified version of a chemical compound that is structurally similar to a
parent
12
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
compound and (actually or theoretically) derivable from that parent compound.
A "derivative" differs from an "analogue" in that a parent compound may be the
starting material to generate a "derivative," whereas the parent compound may
not necessarily be used as the starting material to generate an "analogue." A
derivative may or may not have different chemical or physical properties of
the
parent compound. For example, the derivative may be more hydrophilic or it
may have altered reactivity as compared to the parent compound.
Derivatization (i.e., modification) may involve substitution of one or more
moieties within the molecule (e.g., a change in functional group). For
example,
a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or
a
hydroxyl group (-OH) may be replaced with a carboxylic acid moiety (-COOH).
The term "derivative" also includes conjugates, and prodrugs of a parent
compound (i.e., chemically modified derivatives which can be converted into
the
original compound under physiological conditions). For example, the prodrug
may be an inactive form of an active agent. Under physiological conditions,
the
prodrug may be converted into the active form of the compound. Prodrugs may
be formed, for example, by replacing one or two hydrogen atoms on nitrogen
atoms by an acyl group (acyl prodrugs) or a carbamate group (carbamate
prodrugs). More detailed information relating to prodrugs is found, for
example,
in Fleisher et al., Advanced Drug Delivery Reviews 19 (1996) 115; Design of
Prodrugs, H. Bundgaard (ed.), Elsevier, 1985; or H. Bundgaard, Drugs of the
Future 16 (1991 ) 443. The term "derivative" is also used to describe all
solvates, for example hydrates or adducts (e.g., adducts with alcohols),
active
metabolites, and salts of the parent compound. The type of salt that may be
prepared depends on the nature of the moieties within the compound. For
example, acidic groups, for example carboxylic acid groups, can form, for
example, alkali metal salts or alkaline earth metal salts (e.g., sodium salts,
potassium salts, magnesium salts and calcium salts, and also salts with
physiologically tolerable quaternary ammonium ions and acid addition salts
with
ammonia and physiologically tolerable organic amines such as, for example,
13
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
triethylamine, ethanolamine or tris-(2-hydroxyethyl)amine). Basic groups can
form acid addition salts, for example with inorganic acids such as
hydrochloric
acid, sulfuric acid or phosphoric acid, or with organic carboxylic acids and
sulfonic acids such as acetic acid, citric acid, benzoic acid, malefic acid,
fumaric
acid, tartaric acid, methanesulfonic acid or p-toluenesulfonic acid. Compounds
which simultaneously contain a basic group and an acidic group, for example a
carboxyl group in addition to basic nitrogen atoms, can be present as
zwitterions. Salts can be obtained by customary methods known to those
skilled in the art, for example by combining a compound with an inorganic or
organic acid or base in a solvent or diluent, or from other salts by cation
exchange or anion exchange.
"Hyaluronic acid" or "HA" as used herein refers to all forms of
hyaluronic acid that are described or referenced herein, including those that
have been processed or chemically or physically modified, as well as
hyaluronic
acid that has been crosslinked (for example, covalently, ionically, thermally
or
physically). HA is a glycosaminoglycan composed of a' linear chain of about
2500 repeating disaccharide units. Each disaccharide unit is composed of an
N-acetylglucosamine residue linked to a glucuronic acid. Hyaluronic acid is a
natural substance that is found in the extracellular matrix of many tissues
including synovial joint fluid, the vitreous humor of the eye, cartilage,
blood
vessels, skin and the umbilical cord. Commercial forms of hyaluronic acid
having a molecular weight of approximately 1.2 to 1.5 million Daltons (Da) are
extracted from rooster combs and other animal sources. Other sources of HA
include HA that is isolated from cell culture / fermentation processes. Lower
molecular weight HA formulations are also available from a variety of
commercial sources. The molecule can be of variable lengths (i.e., different
numbers of repeating disaccharide units and different chain branching
patterns)
and can be modified at several sites (through the addition or subtraction of
different functional groups) without deviating from the scope of the present
invention.
14
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
The term "inter-react" refers to the formulation of covalent bonds,
noncovalent bonds, or both. The term thus includes crosslinking, which
involves both intermolecular crosslinks and optionally intramolecular
crosslinks
as well, arising from the formation of covalent bonds. Covalent bonding
between two reactive groups may be direct, in which case an atom in reactive
group is directly bound to an atom in the other reactive group, or it may be
indirect, through a linking group. Noncovalent bonds include ionic
(electrostatic) bonds, hydrogen bonds, or the association of hydrophobic
molecular segments, which may be the same or different. A crosslinked matrix
may, in addition to covalent bonds, also include such intermolecular and/or
intramolecular noncovalent bonds.
When referring to polymers, the terms "hydrophilic" and
"hydrophobic" are generally defined in terms of an HLB value, i.e., a
hydrophilic
lipophilic balance. A high HLB value indicates a hydrophilic compound, while a
low HLB value characterizes a hydrophobic compound. HLB values are well
known in the art, and generally range from 1 to 18. Preferred multifunctional
compound cores are hydrophilic, although as long as the multifunctional
compound as a whole contains at least one hydrophilic component,
crosslinkable hydrophobic components may also be present.
The term "synthetic" is used to refer to polymers, compounds and
other such materials that are "chemically synthesized." For example, a
synthetic material in the present compositions may have a molecular structure
that is identical to a naturally occurring material, but the material per se,
as
incorporated in the compositions of the invention, has been chemically
synthesized in the laboratory or industrially. "Synthetic" materials also
include
semi-synthetic materials, i.e., naturally occurring materials, obtained from a
natural source, that have been chemically modified in some way. Generally,
however, the synthetic materials herein are purely synthetic, i.e., they are
neither semi-synthetic nor have a structure that is identical to that of a
naturally
occurring material.
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
The term "effective amount" refers to the amount of composition
required in order to obtain the effect desired. For example, a "tissue growth-
promoting amount" of a composition refers to the amount needed in order to
stimulate tissue growth to a detectable degree. Tissue, in this context,
includes
connective tissue, bone, cartilage, epidermis and dermis, blood, and other
tissues. The actual amount that is determined to be an effective amount will
vary depending on factors such as the size, condition, sex and age of the
patient and can be more readily determined by the caregiver.
The term "in situ" as used herein means at the site of
administration. Thus, compositions of the invention can be injected or
otherwise applied to a specific site within a patient's body, e.g., a site in
need of
augmentation, and allowed to crosslink at the site of injection. Suitable
sites
will generally be intradermal or subcutaneous regions for augmenting dermal
support, at a bone fracture site for bone repair, within sphincter tissue for
sphincter augmentation (e.g., for restoration of continence), within a wound
or
suture, to promote tissue regrowth; and within or adjacent to vessel
anastomoses, to promote vessel regrowth.
The term "aqueous medium" includes solutions, suspensions,
dispersions, colloids, and the like containing water. The term "aqueous
environment" means an environment containing an aqueous medium.
Similarly, the term "dry environment" means an environment that does not
contain an aqueous medium.
With regard to nomenclature pertinent to molecular structures, the
following definitions apply:
The term "alkyl" as used herein refers to a branched or
unbranched saturated hydrocarbon group typically although not necessarily
containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t butyl, octyl, decyl, and the like, as well as
cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally,
although again not necessarily, alkyl groups herein contain 1 to about 12
16
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
carbon atoms. The term "lower alkyl" intends an alkyl group of one to six
carbon atoms, preferably one to four carbon atoms. "Substituted alkyl" refers
to
alkyl substituted with one or more substituent groups. "Alkylene," "lower
alkylene" and "substituted alkylene" refer to divalent alkyl, lower alkyl, and
substituted alkyl groups, respectively.
The term "aryl" as used herein, and unless otherwise specified,
refers to an aromatic substituent containing a single aromatic ring
(monocyclic)
or multiple aromatic rings that are fused together, linked covalently, or
linked to
a common group such as a methylene or ethylene moiety. The common linking
group may also be a carbonyl as in benzophenone, an oxygen atom as in
diphenylether, or a nitrogen atom as in diphenylamine. Preferred aryl groups
contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl,
naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like.
"Substituted aryl" refers to an aryl moiety substituted with one or more
substituent groups, and the terms "heteroatom-containing aryl" and
"heteroaryl"
refer to aryl in which at least one carbon atom is replaced with a heteroatom.
The terms "arylene" and "substituted arylene" refer to divalent aryl and
substituted aryl groups as just defined.
The term "heteroatom-containing" as in a "heteroatom-containing
hydrocarbyl group" refers to a molecule or molecular fragment in which one or
more carbon atoms is replaced with an atom other than carbon, e.g., nitrogen,
oxygen, sulfur, phosphorus or silicon.
"Hydrocarbyl" refers to univalent hydrocarbyl radicals containing 1
to about 30 carbon atoms, preferably 1 to about 24 carbon atoms, most
preferably 1 to about 12 carbon atoms, including branched or unbranched,
saturated or unsaturated species, such as alkyl groups, alkenyl groups, aryl
groups, and the like. The term "lower hydrocarbyl" intends a hydrocarbyl group
of one to six carbon atoms, preferably one to four carbon atoms. The term
"hydrocarbylene" intends a divalent hydrocarbyl moiety containing 1 to about
30
carbon atoms, preferably 1 to about 24 carbon atoms, most preferably 1 to
17
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
about 12 carbon atoms, including branched or unbranched, saturated or
unsaturated species, or the like. The term "lower hydrocarbylene" intends a
hydrocarbylene group of one to six carbon atoms, preferably one to four carbon
atoms. "Substituted hydrocarbyl" refers to hydrocarbyl substituted with one or
more substituent groups, and the terms "heteroatom-containing hydrocarbyl"
and "heterohydrocarbyl" refer to hydrocarbyl in which at least one carbon atom
is replaced with a heteroatom. Similarly, "substituted hydrocarbylene" refers
to
hydrocarbylene substituted with one or more substituent groups, and the terms
"heteroatom-containing hydrocarbylene" and "heterohydrocarbylene" refer to
hydrocarbylene in which at least one carbon atom is replaced with a
heteroatom. If not otherwise indicated, "hydrocarbyl" indicates both
unsubstituted and substituted hydrocarbyls, "heteroatom-containing
hydrocarbyl" indicates both unsubstituted and substituted heteroatom-
containing hydrocarbyls and so forth.
By "substituted" as in "substituted hydrocarbyl," "substituted alkyl,"
and the like, as alluded to in some of the aforementioned definitions, is
meant
that in the hydrocarbyl, alkyl, or other moiety, at least one hydrogen atom
bound to a carbon atom is replaced with one or more substituents that are
functional groups such as alkoxy, hydroxy, halo, vitro, and the like. Unless
otherwise indicated, it is to be understood that specified molecular segments
can be substituted with one or more substituents that do not compromise a
compound's utility. For example, "succinimidyl" is intended to include
unsubstituted succinimidyl as well as sulfosuccinimidyl and other succinimidyl
groups substituted on a ring carbon atom, e.g., with alkoxy substituents,
polyether substituents, or the like.
Any concentration ranges, percentage range, or ratio range
recited herein are to be understood to include concentrations, percentages or
ratios of any integer within that range and fractions thereof, such as one
tenth
and one hundredth of an integer, unless otherwise indicated. Also, any number
range recited herein relating to any physical feature, such as polymer
subunits,
18
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
size or thickness, are to be understood to include any integer within the
recited
range, unless otherwise indicated. As used herein, the term "about" refers to
~
15% of any indicated structure, value, or range.
"A" and "an" refer to one or more of the indicated items. For
example, "a" polymer refers to both one polymer or a mixture comprising two or
more polymers; "a multifunctional compound " refers not only to a single
multifunctional compound but also to a combination of two or more of the same
or different multifunctional compounds; "a reactive group" refers to a
combination of reactive groups as well as to a single reactive group, and the
like.
As discussed above, the present invention provides polymeric
compositions which greatly increase the ability to inhibit the formation of
reactive scar tissue on, or around, the surface of a device or implant or at a
treatment site. Numerous polymeric compositions and therapeutic agents are
described herein.
The present invention provides for the combination of
compositions (e.g., polymers) which include one or more therapeutic agents,
described below. Also described in more detail below are methods for making
and methods for utilizing such compositions.
A. Therapeutic Agents
In one aspect, the present invention discloses pharmaceutical
agents which inhibit one or more aspects of the production of excessive
fibrous
(scar) tissue. Suitable fibrosis-inhibiting or stenosis-inhibiting agents may
be
readily determined based upon the in vitro and in vivo (animal) models such as
those provided in Examples 20-33. Agents which inhibit fibrosis may be
identified through in viv~ models including inhibition of intimal hyperplasia
development in the rat balloon carotid artery model (Examples 25 and 33). The
assays set forth in Examples 24 and 32 may be used to determine whether an
agent is able to inhibit cell proliferation in fibroblasts and/or smooth
muscle
19
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
cells. In one aspect of the invention, the agent has an IC5° for
inhibition of cell
proliferation within a range of about 10-6 to about 10-x° M. The assay
set forth in
Example 28 may be used to determine whether an agent may inhibit migration
of fibroblasts and/or smooth muscle cells. In one aspect of the invention, the
agent has an IC5° for inhibition of cell migration within a range of
about 10-6 to
about 10-9M. Assays set forth herein may be used to determine whether an
agent is able to inhibit inflammatory processes, including nitric oxide
production
in macrophages (Example 20), and/or TNF-alpha production by macrophages
(Example 21 ), and/or IL-1 beta production by macrophages (Example 29),
and/or IL-8 production by macrophages (Example 30), and/or inhibition of MCP-
1 by macrophages (Example 31 ). In one aspect of the invention, the agent has
an IC5° for inhibition of any one of these inflammatory processes
within a range
of about 10-6 to about 10-~°M. The assay set forth in Example 26 may be
used
to determine whether an agent is able to inhibit MMP production. In one aspect
of the invention, the agent has an IC5° for inhibition of MMP
production within a
range of about 10-4 to about 10-$M. The assay set forth in Example 27 (al'so
known as the CAM assay) may be used to determine whether an agent is able
to inhibit angiogenesis. In one aspect of the invention, the agent has an
IC5° for
inhibition of angiogenesis within a range of about 10-6 to about 10-
~°M. Agents
which reduce the formation of surgical adhesions may be identified through in
vivo models including the rabbit surgical adhesions model (Examples 23, 42
and 43) and the rat caecal sidewall model (Example 22). These
pharmacologically active agents (described below) can then be delivered at
appropriate dosages into to the tissue either alone, or via carriers
(described
herein), to treat the clinical problems described herein.
Numerous therapeutic compounds capable of inhibiting fibrosis
have been identified that are of utility in the invention including:
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
1 ) Anaiogenesis Inhibitors
In one embodiment, the pharmacologically active fibrosis-
inhibiting compound is an angiogenesis inhibitor (e.g., 2-ME (NSC-659853), PI-
88 (D-mannose, O-6-O-phosphono-alpha-D-mannopyranosyl-(1-3)-O-alpha-D-
mannopyranosyl-(1-3)-O-alpha-D-mannopyranosyl-(1-3)-O-alpha-D-
mannopyranosyl-(1-2)- hydrogen sulfate), thalidomide (1H-isoindole-1,3(2H)-
dione, 2-(2,6-dioxo-3-piperidinyl)-), CDC-394, CC-5079, ENMD-0995 (S-3-
amino-phthalidoglutarimide), AVE-8062A, vatalanib, SH-268, halofuginone
hydrobromide, atiprimod dimaleate (2-azaspivo(4.5)decane-2-propanamine,
N,N-diethyl-8,8-dipropyl, dimaleate), ATN-224, CHIR-258, combretastatin A-4
(phenol, 2-methoxy-5-(2-(3,4,5-trimethoxyphenyl)ethenyl)-, (Z)-), GCS-100LE,
or an analogue or derivative thereof).
2) 5-Lipoxygenase Inhibitors and Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a 5-lipoxygenase inhibitor or antagonist (e.g., Wy-
50295
(2-naphthaleneacetic acid, alpha-methyl-6-(2-quinolinylmethoxy)-, (S)-), ONO-
LP-269 (2,11,14-eicosatrienamide, N-(4-hydroxy-2-(1H-tetrazol-5-yl)-8-
quinolinyl)-, (E,Z,Z)-), licofelone (1 H-pyrrolizine-5-acetic acid, 6-(4-
chlorophenyl)-2,3-dihydro-2,2-dimethyl-7-phenyl-), CMI-568 (urea, N-butyl-N-
hydroxy-N'-(4-(3-(methylsulfonyl)-2-propoxy-5-(tetrahydro-5-(3,4,5-
trimethoxyphenyl)-2-furanyl)phenoxy)butyl)-,trans-), IP-751 ((3R,4R)-(delta 6)-
THC-DMH-11-oic acid), PF-5901 (benzenemethanol, alpha-pentyl-3-(2-
quinolinylmethoxy)-), LY-293111 (benzoic acid, 2-(3-(3-((5-ethyl-4'-fluoro-2-
hydroxy(1,1'-biphenyl)-4-yl)oxy)propoxy)-2-propylphenoxy)-), RG-5901-A
(benzenemethanol, alpha-pentyl-3-(2-quinolinylmethoxy)-, hydrochloride),
rilopirox (2(1 H)-pyridinone, 6-((4-(4-chlorophenoxy)phenoxy)methyl)-1-hydroxy-
4-methyl-), L-674636 (acetic acid, ((4-(4-chlorophenyl)-1-(4-(2-
quinolinylmethoxy)phenyl)butyl)thio)-AS)), 7-((3-(4-methoxy-tetrahydro-2H-
pyran-4-yl)phenyl)methoxy)-4-phenylnaphtho(2,3-c)furan-1 (3H)-one, MK-886
21
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(1H-indole-2-propanoic acid, 1-((4-chlorophenyl)methyl)-3-((1,1-
dimethylethyl)thio)-alpha, alpha-dimethyl-5-(1-methylethyl)-), quiflapon (1 H-
indole-2-propanoic acid, 1-((4-chlorophenyl)methyl)-3-((1,1-
dimethylethyl)thio)-
alpha, alpha-dimethyl-5-(2-quinolinylmethoxy)-), quiflapon (1 H-Indole-2-
propanoic acid, 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,
alpha-dimethyl-5-(2-quinolinylmethoxy)-), docebenone (2,5-cyclohexadiene-1,4-
dione, 2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-), zileuton (urea, N-
(1-
benzo(b)thien-2-ylethyl)-N-hydroxy-), or an analogue or derivative thereof).
3) Chemokine Receptor Antagonists CCR (1, 3, and 5)
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a chemokine receptor antagonist which inhibits one or
more subtypes of CCR (1, 3, and 5) (e.g., ONO-4128 (1,4,9-
triazaspiro(5.5)undecane-2,5-dione, 1-butyl-3-(cyclohexylmethyl)-9-((2,3-
dihydro-1,4-benzodioxin-6-yl)methyl-), L-381, CT-112 (L-arginine, L-threonyl-L-
threonyl-L-seryl-L-glutaminyl-L-valyl-L-arginyl-L-prolyl-), AS-900004, SCH-C,
ZK-811752, PD-172084, UK-427857, SB-380732, vMIP II, SB-265610, DPC-
168, TAK-779 (N, N-dimethyl-N-(4-(2-(4-methylphenyl)-6,7-dihydro-5H-
benzocyclohepten-8-ylcarboxamido)benyl)tetrahydro-2H-pyran-4-aminium
chloride), TAK-220, KRH-1120), GSK766994, SSR-150106, or an analogue or
derivative thereof). Other examples of chemokine receptor antagonists include
a-Immunokine-NNS03, BX-471, CCX-282, Sch-350634; Sch-351125; Sch-
417690; SCH-C, and analogues and derivatives thereof.
4) Cell Cycle Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a cell cycle inhibitor. Representative examples of such
agents include taxanes (e.g., paclitaxel (discussed in more detail below) and
docetaxel) (Schiff et al., Nature 277:665-667, 1979; Long and Fairchild,
Cancer
Research 54:4355-4361, 1994; Ringel and Horwitz, J. Nat'I Cancer Inst.
22
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
83(4):288-291, 1991; Pazdur et al., Cancer Treat. Rev. 79(40):351-386, 1993),
etanidazole, nimorazole (B.A. Chabner and D.L. Longo. Cancer Chemotherapy
and Biotherapy - Principles and Practice. Lippincott-Raven Publishers, New
York,.1996, p.554), perfluorochemicals with hyperbaric oxygen, transfusion,
erythropoietin,~BW12C, nicotinamide, hydralazine, BSO, WR-2721, IudR,
DUdR, etanidazole, WR-2721, BSO, mono-substituted keto-aldehyde
compounds (L.G. Egyud. Keto-aldehyde-amine addition products and method
of making same. U.S. Patent No. 4,066,650, Jan 3, 1978), nitroimidazole (K.C.
Agrawal and M. Sakaguchi. Nitroimidazole radiosensitizers for Hypoxic tumor
cells and compositions thereof. U.S. Patent No. 4,462,992, Jul. 31, 1984), 5-
substituted-4-nitroimidazoles (Adams et al., Int. J. Radiat. 8i~1. Relat.
Stud.
Phys., Chem. Med. 40(2):153-61, 1981 ), SR-2508 (Brown et al., Int. J. Radiat.
Oncol., Biol. Phys. 7(6):695-703, 1981 ), 2H-isoindolediones (J.A. Myers, 2H-
Isoindolediones, the synthesis and use as radiosensitizers. Patent 4,494,547,
Jan. 22, 1985), chiral (((2-bromoethyl)-amino)methyl)-nitro-1 H-imidazole-1-
ethanol (V.G. Beylin, et al., Process for preparing chiral (((2-bromoethyl)-
amino)methyl)-nitro-1 H-imidazole-1-ethanol and related compounds. U.S.
Patent No. 5,543,527, Aug. 6, 1996; U.S. Patent No. 4,797,397; Jan. 10, 1989;
U.S. Patent No. 5,342,959, Aug. 30, 1994), nitroaniline derivatives (W.A.
Denny, et al. Nitroaniline derivatives and the use as anti-tumor agents. U.S.
Patent No. 5,571,845, Nov. 5, 1996), DNA-affinic hypoxia selective cytotoxins
(M.V. Papadopoulou-Rosenzweig. DNA-affinic hypoxia selective cytotoxins.
U.S. Patent No. 5,602,142, Feb. 11, 1997), halogenated DNA ligand (R.F.
Martin. Halogenated DNA ligand radiosensitizers for cancer therapy. U.S.
Patent No. 5,641,764, Jun 24, 1997), 1,2,4 benzotriazine oxides (W.W. Lee et
al. 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic
agents. U.S. Patent No. 5,616,584, Apr. 1, 1997; U.S. Patent No. 5,624,925,
Apr. 29, 1997; Process for Preparing 1,2,4 Benzotriazine oxides. U.S. Patent
No. 5,175,287, Dec. 29, 1992), nitric oxide (J.B. Mitchell et al., Use of
Nitric
oxide releasing compounds as hypoxic cell radiation sensitizers. U.S. Patent
23
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
No. 5,650,442, Jul. 22, 1997), 2-nitroimidazole derivatives (M.J. Suto et al.
2-
Nitroimidazole derivatives useful as radiosensitizers for hypoxic tumor cells.
U.S. Patent No. 4,797,397, Jan. 10, 1989; T. Suzuki. 2-Nitroimidazole
derivative, production thereof, and radiosensitizer containing the same as
active
ingredient. U.S. Patent No. 5,270,330, Dec. 14, 1993; T. Suzuki et al. 2-
Nitroimidazole derivative, production thereof, and radiosensitizer containing
the
same as active ingredient. U.S. Patent No. 5,270,330, Dec 14, 1993; T.
Suzuki. 2-Nitroimidazole derivative, production thereof and radiosensitizer
containing the same as active ingredient; Patent EP 0 513 351 B1, Jan. 24,
1991 ), fluorine-containing nitroazole derivatives (T. Kagiya. Fluorine-
containing
nitroazole derivatives and radiosensitizer comprising the same. U.S. Patent
No. 4,927,941, May 22, 1990), copper (M.J. Abrams. Copper Radiosensitizers.
U.S. Patent No. 5,100,885, Mar. 31, 1992), combination modality cancer
therapy (D.H. Picker et al. Combination modality cancer therapy. U.S. Patent
No. 4,681,091, Jul. 21, 1987). 5-CIdC or (d)H4U or 5-halo-2'-halo-2'-deoxy-
cytidine or -uridine derivatives (S.B. Greer. Method and Materials for
sensitizing neoplastic tissue to radiation. U.S. Patent No. 4,894,364 Jan. 16,
1990), platinum complexes (K.A. Skov. Platinum Complexes with one
radiosensitizing ligand. U.S. Patent No. 4,921,963. May 1, 1990; K.A. Skov.
Platinum Complexes with one radiosensitizing ligand. Patent EP 0 287 317
A3), fluorine-containing nitroazole (T. Kagiya, et al. Fluorine-containing
nitroazole derivatives and radiosensitizer comprising the same. U.S. Patent
No. 4,927,941. May 22,1990), benzamide (W.W. Lee. Substituted Benzamide
Radiosensitizers. U.S. Patent No. 5,032,617, Jul. 16, 1991 ), autobiotics
(L.G.
Egyud. Autobiotics and the use in eliminating nonself cells in vivo. U.S.
Patent
No. 5,147,652. Sep. 15,1992), benzamide and nicotinamide (W.W. Lee et al.
Benzamide and Nictoinamide Radiosensitizers. U.S. Patent No. 5,215,738, Jun
1 1993), acridine-intercalator (M. Papadopoulou-Rosenzweig. Acridine
Intercalator based hypoxia selective cytotoxins. U.S. Patent No. 5,294,715,
Mar. 15,1994), fluorine-containing nitroimidazole (T. Kagiya et al. Fluorine
24
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
containing nitroimidazole compounds. U.S. Patent No. 5,304,654, Apr. 19,
1994), hydroxylated texaphyrins (J.L. Sessler et al. Hydroxylated texaphrins.
U.S. Patent No. 5,457,183, Oct. 10, 1995), hydroxylated compound derivative
(T. Suzuki et al. Heterocyclic compound derivative, production thereof and
radiosensitizer and antiviral agent containing said derivative as active
ingredient. Publication Number 011106775 A (Japan), Oct. 22,1987; T. Suzuki
et al. Heterocyclic compound derivative, production thereof and
radiosensitizer,
antiviral agent and anti cancer agent containing said derivative as active
ingredient. Publication Number 01139596 A (Japan), Nov. 25, 1987; S.
Sakaguchi et al. Heterocyclic compound derivative, its production and
radiosensitizer containing said derivative as active ingredient; Publication
Number 63170375 A (Japan), Jan. 7, 1987), fluorine containing 3-vitro-1,2,4-
triazole (T. Kagitani et al. Novel fluorine-containing 3-vitro-1,2,4-triazole
and
radiosensitizer containing same compound. Publication Number 02076861 A
(Japan), Mar. 31, 1988), 5-thiotretrazole derivative or its salt (E. Kano et
al.
Radiosensitizer for Hypoxic cell. Publication Number 61010511 A (Japan), Jun.
26, 1984), Nitrothiazole (T. Kagitani et al. Radiation-sensitizing agent.
Publication Number 61167616 A (Japan) Jan. 22, 1985), imidazole derivatives
(S. Inayma et al. Imidazole derivative. Publication Number 6203767 A (Japan)
Aug. 1,1985; Publication Number 62030768 A (Japan) Aug. 1, 1985;
Publication Number 62030777 A (Japan) Aug. 1, 1985), 4-vitro-1,2,3-triazole
(T. Kagitani et al. Radiosensitizer. Publication Number 62039525 A (Japan),
Aug. 15,1985), 3-vitro-1,2,4-triazole (T. Kagitani et al. Radiosensitizer.
Publication Number 62138427 A (Japan), Dec. 12, 1985), Carcinostatic action
regulator (H. Amagase. Carcinostatic action regulator. Publication Number
63099017 A (Japan), Nov. 21, 1986), 4,5-dinitroimidazole derivative (S.
Inayama. 4,5-Dinitroimidazole derivative. Publication Number 63310873 A
(Japan) Jun. 9, 1987), nitrotriazole Compound (T. Kagitanil Nitrotriazole
Compound. Publication Number 07149737 A (Japan) Jun. 22, 1993), cisplatin,
doxorubin, misonidazole, mitomycin, tiripazamine, nitrosourea, mercaptopurine,
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
methotrexate, flurouracil, bleomycin, vincristine, carboplatin, epirubicin,
doxorubicin, cyclophosphamide, vindesine, etoposide (1.F. Tannock. Review
Article: Treatment of Cancer with Radiation and Drugs. Journal of Clinical
Oncology 14(12):3156-3174, 1996), camptothecin (Ewend M.G. et al. Local
delivery of chemotherapy and concurrent external beam radiotherapy prolongs
survival in metastatic brain tumor models. Cancer Research 56(22):5217-5223,
1996) and paclitaxel (Tishler R.B. et al. Taxol: a novel radiation sensitizer.
International Journal of Radiation Oncology and Biological Physics 22(3):613-
617, 1992).
A number of the above-mentioned cell cycle inhibitors also have a
wide variety of analogues and derivatives, including, but not limited to,
cisplatin,
cyclophosphamide, misonidazole, tiripazamine, nitrosourea, mercaptopurine,
methotrexate, flurouracil, epirubicin, doxorubicin, vindesine and etoposide.
Analogues and derivatives include (CPA)2Pt(DOLYM) and (DACH)Pt(DOLYM)
cisplatin (Choi et al., Areh. Pharmacal Res. 22(2):151-156, 1999), Cis-
(PtCl2(4,7-H-5-methyl-7-oxo)1,2,4(triazolo(1,5-a)pyrimidine)2) (Navarro et
al., J.
Med. Chem. 41(3):332-338, 1998), (Pt(cis-1,4-DACH)(trans-
C12)(CBDCA)) ~'/~MeOH cisplatin (Shamsuddin et al., Inorg. Chem.
36(25):5969-5971, 1997), 4-pyridoxate diammine hydroxy platinum (Tokunaga
et al., Pharm. Sci. 3(7):353-356, 1997), Pt(II) ~ ~ ~ Pt(II)
(Pt2(NHCHN(C(CH2)(CH3)))4) (Navarro et al., Inorg. Chem. 35(26):7829-7835,
1996), 254-S cisplatin analogue (Koga et al., Neurol. Res. 13(3):244-247,
1996), o-phenylenediamine ligand bearing cisplatin analogues (Koeckerbauer &
Bednarski, J. Inorg. Biochem. 62(4):281-298, 1996), trans,cis-(Pt(OAc)~I~(en))
(Kratochwil et al., J. Med. Chem. 39(13):2499-2507, 1996), estrogenic 1,2-
diarylethylenediamine ligand (with sulfur-containing amino acids and
glutathione) bearing cisplatin analogues (Bednarski, J. Inorg. Biochem.
62(1):75, 1996), cis-1,4-diaminocyclohexane cisplatin analogues (Shamsuddin
et al., J. Inorg. Biochem. 61(4):291-301, 1996), 5' orientational isomer of
cis-
(Pt(NH3)(4-aminoTEMP-O){d(GpG)}) (Dunham & Lippard, J. Am. Chem. Soc.
26
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
7 7 7(43):10702-12, 1995), chelating diamine-bearing cisplatin analogues
(Koeckerbauer & Bednarski, J. Pharm. Sci. 84(7):819-23, 1995), 1,2-
diarylethyleneamine ligand-bearing cisplatin analogues (Otto et al., J. Cancer
Res. Clin. Oncol. 727(1):31-8, 1995), (ethylenediamine)platinum(II) complexes
(Pasini et al., J. Chem. Soc., Dalton Trans. 4:579-85, 1995), CI-973 cisplatin
analogue (Yang et al., Int. J. Oncol. 5(3):597-602, 1994), cis-
diamminedichloroplatinum(11) and its analogues cis-1,1-
cyclobutanedicarbosylato(2R)-2-methyl-1,4-butanediam-mineplatinum(II) and
cis-diammine(glycolato)platinum (Claycamp & Zimbrick, J. Inorg. Biochem.,
26(4):257-67, 1986; Fan et al., Cancer Res. 48(11 ):3135-9, 1988; Heiger-
Bernays et al., Biochemistry 29(36):8461-6, 1990; Kikkawa et al., J. Exp.
Clin.
Cancer Res. 72(4):233-40, 1993; Murray et al., Biochemistry 37(47):11812-17,
1992; Takahashi et al., Cancer Chemother. Pharmacol. 33(1 ):31-5, 1993), cis-
amine-cyclohexylamine-dichloroplatinum(II) (Yoshida et al., Biochem.
Pharmacol. 48(4):793-9, 1994), gem-diphosphonate cisplatin analogues (FR
2683529), (meso-1,2-bis(2,6-dichloro-4-hydroxyplenyl)ethylenediamine)
dichloroplatinum(II) (Bednarski et al., J. Med. Chem. 35(23):4479-85, 1992),
cisplatin analogues containing a tethered dansyl group (Hartwig et al., J. Am.
Chem. Soc. 7 74(21 ):8292-3, 1992), platinum(II) polyamines (Siegmann et al.,
Inorg. Met.-Containing Polym. Mater., (Pros. Am. Chem. Soc. Int. Symp.), 335-
61, 1990), cis-(3H)dichloro(ethylenediamine)platinum(II) (Eastman, Anal.
Biochem. 797(2):311-15, 1991 ), trans-diamminedichloroplatinum(II) and cis-
(Pt(NH3)2(N3-cytosine)CI) (Bellon & Lippard, Biophys. Chem. 35(2-3):179-88,
1990), 3H-cis-1,2-diaminocyclohexanedichloroplatinum(II) and 3H-cis-1,2-
diaminocyclohexanemalonatoplatinum (II) (Oswald et al., Res. Commun. Chem.
Pathol. Pharmacol. 64(1 ):41-58, 1989), diaminocarboxylatoplatinum (EPA
296321 ), trans-(D,1 )-1,2-diaminocyclohexane carrier ligand-bearing platinum
analogues (Wyrick & Chaney, J. Labelled Compd. Radiopharm. 25(4):349-57,
1988), aminoalkylaminoanthraquinone-derived cisplatin analogues (Kitov et al.,
Eur. J. Med. Chem. 23(4):381-3, 1988), spiroplatin, carboplatin, iproplatin
and
27
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
JM40 platinum analogues (Schroyen et al., Eur. J. Cancer Clin. Oncol.
24(8):1309-12, 1988), bidentate tertiary diamine-containing cisplatinum
derivatives (Orbell et al., Inorg. Chim. Acta 752(2):125-34, 1988),
platinum(II),
platinum(IV) (Liu & Wang, Shandong Yike Daxue Xuebao 24(1):35-41, 1986),
cis-diammine(1,1-cyclobutanedicarboxylato-)platinum(II) (carboplatin, JM8) and
ethylenediammine-malonatoplatinum(II) (JM40) (Begg et al., Radiother. Oncol.
9(2):157-65, 1987), JM8 and JM9 cisplatin analogues (Harstrick et al., Int. J.
Androl. 70(1); 139-45, 1987), (NPr4)2((PtCL4).cis-(PtCl2-(NH2Me)2))
(Brammer et al., J. Chem. Soc., Chem. Commun. 6:443-5, 1987), aliphatic
tricarboxylic acid platinum complexes (EPA 185225), cis-dichloro(amino
acid)(tert-butylamine)platinum(II) complexes (Pasini & Bersanetti, Inorg.
Chim.
Acta 707(4):259-67, 1985); 4-hydroperoxycylcophosphamide (Ballard et al.,
Cancer Chemother. Pharmacol. 26(6):397-402, 1990), acyclouridine
cyclophosphamide derivatives (Zakerinia et al., Helv. Chim. Acta 73(4):912-15,
1990), 1,3,2-dioxa- and -oxazaphosphorinane cyclophosphamide analogues
(Yang et al., Tetrahedron 44(20):6305-14, 1988), C5-substituted
cyclophosphamide analogues (Spada, University of Rhode Island Dissertation,
1987), tetrahydrooxazine cyclophosphamide analogues (Valente, University of
Rochester Dissertation, 1988), phenyl ketone cyclophosphamide analogues
(Hales et al., Teratology 39(1):31-7, 1989), phenylketophosphamide
cyclophosphamide analogues (Ludeman et al., J. Med. Chem. 29(5):716-27,
1986), ASTA Z-7557 cyclophosphamide analogues (Evans et al., Int. J. Cancer
34(6):883-90, 1984), 3-(1-oxy-2,2,6,6-tetramethyl-4-
piperidinyl)cyclophosphamide (Tsui et al., J. Med. Chem. 25(9):1106-10, 1982),
2-oxobis(2-(3-chloroethylamino)-4-,6-dimethyl-1,3,2-oxazaphosphorinane
cyclophosphamide (Carpenter et al., Phosphorus Sulfur 72(3):287-93, 1982), 5-
fluoro- and 5-chlorocyclophosphamide (Foster et al., J. Med. Chem.
24(12):1399-403, 1981 ), cis- and trans-4-phenylcyclophosphamide (Boyd et al.,
J. Med. Chem. 23(4):372-5, 1980), 5-bromocyclophosphamide, 3,5-
dehydrocyclophosphamide (Ludeman et al., J. Med. Chem. 22(2):151-8, 1979),
28
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
4-ethoxycarbonyl cyclophosphamide analogues (Foster, J. Pharm. Sci.
67(5):709-10, 1978), arylaminotetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide
cyclophosphamide analogues (Hamacher, Arch. Pharm. (Weinheim, Ger.)
310(5):J,428-34, 1977), NSC-26271 cyclophosphamide analogues
(Montgomery & Struck, Cancer Treat. Rep. 60(4):J381-93, 1976), benzo
annulated cyclophosphamide analogues (Ludeman & Zon, J. Med. Chem.
18(12):J1251-3, 1975), 6-trifluoromethylcyclophosphamide (Farmer & Cox, J.
Med. Chem. 18(11 ):J1106-10, 1975), 4-methylcyclophosphamide and 6-
methycyclophosphamide analogues (Cox et al., Biochem. Pharmacol.
24(5):J599-606, 1975); FCE 23762 doxorubicin derivative (Quaglia et al., J.
Liq.
Chromatogr. 17(18):3911-3923, 1994), annamycin (Zoo et al., J. Pharm. Sci.
82(11 ):1151-1154, 1993), ruboxyl (Rapoport et al., J. Controlled Release
58(2):153-162, 1999), anthracycline disaccharide doxorubicin analogue (Pratesi
et al., Clin. Cancer Res. 4(11 ):2833-2839, 1998), N-
(trifluoroacetyl)doxorubicin
and 4'-O-acetyl-N-(trifluoroacetyl)doxorubicin (Berube & Lepage, Synth.
Common. 28(6):1109-1116, 1998), 2-pyrrol~inodoxorubicin (Nagy et al., Proc.
Nat'1 Acad. Sci. U.S.A. 95(4):1794-1799, 1998), disaccharide doxorubicin
analogues (Arcamone et al., J. Nat'I Cancer Inst. 89(16):1217-1223, 1997), 4-
demethoxy-7-O-(2,6-dideoxy-4-O-(2,3,6-trideoxy-3-amino-a-L-lyxo-
hexopyranosyl)-cc-L-lyxo-hexopyranosyl)adriamicinone doxorubicin
disaccharide analogue (Monteagudo et al., Carbohydr. Res. 300(1 ):11-16,
1997), 2-pyrrolinodoxorubicin (Nagy et al., Proc. Nat'I Acad. Sci. U.S.A.
94(2):652-656, 1997), morpholinyl doxorubicin analogues (Duran et al., Cancer
Chemother. Pharmacol. 38(3):210-216, 1996), enaminomalonyl-~-alanine
doxorubicin derivatives (Seitz et al., Tetrahedron Lett. 36(9):1413-16, 1995),
cephalosporin doxorubicin derivatives (Vrudhula et al., J. Med. Chem.
38(8):1380-5, 1995), hydroxyrubicin (Solary et al., Int. J. Cancer 58(1 ):85-
94,
1994), methoxymorpholino doxorubicin derivative (Kohl et al., Cancer
Chemother. Pharmacol. 33(1 ):10-16, 1993), (6-maleimidocaproyl)hydrazone
doxorubicin derivative (Willner et al., Bioconjugate Chem. 4(6):521-7, 1993),
N-
29
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(5,5-diacetoxypent-1-yl) doxorubicin (Cherif & Farquhar, J. Med. Chem.
35(17):3208-14, 1992), FCE 23762 methoxymorpholinyl doxorubicin derivative
(Ripamonti et al., Br. J. Cancer 65(5):703-7, 1992), N-hydroxysuccinimide
ester
doxorubicin derivatives (Demant et al., Biochim. Biophys. Acta 7778(1):83-90,
1991 ), polydeoxynucleotide doxorubicin derivatives (Ruggiero et al., Biochim.
Biophys. Acta 7729(3):294-302, 1991), morpholinyl doxorubicin derivatives
(EPA 434960), mitoxantrone doxorubicin analogue (Krapcho et al., J. Med.
Chem. 34(8):2373-80. 1991 ), AD198 doxorubicin analogue (Traganos et al.,
Cancer Res. 57(14):3682-9, 1991 ), 4-demethoxy-3'-N-trifluoroacetyldoxorubicin
(Horton et al., Drug Des. Delivery 6(2):123-9, 1990), 4'-epidoxorubicin
(Drzewoski et al., Pol. J. Pharmacol. Pharm. 40(2):159-65, 1988; Weenen et
al., Eur. J. Cancer Clin. Oncol. 20(7):919-26, 1984), alkylating
cyanomorpholino
doxorubicin derivative (Scudder et al., J. Nat'I Cancer Inst. 80(16):1294-8,
1988), deoxydihydroiodooxorubicin (EPA 275966), adriblastin (Kalishevskaya et
al., Vestn. Mosk. Univ., 16(Biol. 1 ):21-7, 1988), 4'-deoxydoxorubicin
(Schoelzel
et al., Leuk. Res. 70(12):1455-9, 1986), 4-demethyoxy-4'-o-methyldoxorubicin
(Giuliani et al., Proc. Int. Congr. Chemother. 76:285-70-285-77, 1983), 3'-
deamino-3'-hydroxydoxorubicin (Horton et al., J. Antibiot. 37(8):853-8, 1984),
4-
demethyoxy doxorubicin analogues (Barbieri et al., Drugs Exp. Clin. Res.
10(2):85-90, 1984), N-L-leucyl doxorubicin derivatives (Trouet et al.,
Anthracyclines (Proc. Int. Symp. Tumor Pharmacother.), 179-81, 1983), 3'-
deamino-3'-(4-methoxy-1-piperidinyl) doxorubicin derivatives (4,314,054), 3'-
deamino-3'-(4-mortholinyl) doxorubicin derivatives (4,301,277), 4'-
deoxydoxorubicin and 4'-o-methyldoxorubicin (Giuliani et al., Int. J. Cancer
27(1 ):5-13, 1981 ), aglycone doxorubicin derivatives (Chan & Watson, J.
Pharm.
Sci. 67(12):1748-52, 1978), SM 5887 (Pharma Japan 7468:20, 1995), MX-2
(Pharma Japan 7420:19, 1994), 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin (EP
275966), morpholinyl doxorubicin derivatives (EPA 434960), 3'-deamino-3'-(4-
methoxy-1-piperidinyl) doxorubicin derivatives (4,314,054), doxorubicin-14-
valerate, morpholinodoxorubicin (5,004,606), 3'-deamino-3'-(3"-cyano-4"-
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
morpholinyl doxorubicin; 3'-deamino-3'-(3"-cyano-4"-morpholinyl)-13-
dihydoxorubicin; (3'-deamino-3'-(3"-cyano-4"-morpholinyl) daunorubicin; 3'-
deamino-3'-(3"-cyano-4"-morpholinyl)-3-dihydrodaunorubicin; and 3'-deamino-
3'-(4"-morpholinyl-5-iminodoxorubicin and derivatives (4,585,859), 3'-deamino-
3'-(4-methoxy-1-piperidinyl) doxorubicin derivatives (4,314,054) and 3-deamino-
3-(4-morpholinyl) doxorubicin derivatives (4,301,277); 4,5-
dimethylmisonidazole
(Born et al., Biochem. Pharmacol. 43(6):1337-44, 1992), azo and azoxy
misonidazole derivatives (Gattavecchia & Tonelli, Int. J. Radiat. BioL Relat.
Stud. Phys., Chem. Med. 45(5):469-77, 1984); RB90740 (Wardman et al., Br. J.
Cancer, 74 Suppl. (27):S70-S74, 1996); 6-bromo and 6-chloro-2,3-dihydro-1,4-
benzothiazines nitrosourea derivatives (Rai et al., Heterocyel. Common.
2(6):587-592, 1996), diamino acid nitrosourea derivatives (Dulude et al.,
Bioorg.
Med. Chem. Lett. 4(22):2697-700, 1994; Dulude et al., Bioorg. Med. Chem.
3(2):151-60, 1995), amino acid nitrosourea derivatives (Zheleva et al.,
Pharmazie 50(1 ):25-6, 1995), 3',4'-didemethoxy-3',4'-dioxo-4-
deoxypodophyllotoxin nitrosourea derivatives (Miyahara et al., Heterocycles
39(1 ):361-9, 1994), ACNU (Matsunaga et al., Immunopharmacology 23(3):199-
204, 1992), tertiary phosphine oxide nitrosourea derivatives (Guguva et al.,
Pharmazie 46(8):603, 1991 ), sulfamerizine and sulfamethizole nitrosourea
derivatives (Chiang et al., Zhonghua Yaozue Zazhi 43(5):401-6, 1991 ),
thymidine nitrosourea analogues (Zhang et al., Cancer Common. 3(4):119-26,
1991 ), 1,3-bis(2-chloroethyl)-1-nitrosourea (August et al., Cancer Res.
57(6):1586-90, 1991 ), 2,2,6,6-tetramethyl-1-oxopiperidiunium nitrosourea
derivatives (U.S.S.R. 1261253), 2- and 4-deoxy sugar nitrosourea derivatives
(4,902,791 ), nitroxyl nitrosourea derivatives (U.S.S.R. 1336489), fotemustine
(Boutin et al., Eur. J. Cancer Clin. Oncol. 25(9):1311-16, 1989), pyrimidine
(II)
nitrosourea derivatives (Wei et al., Chung-hua Yao Hsueh Tsa Chih 41(1 ):19-
26, 1989), CGP 6809 (Schieweck et al., Cancer Chemother. Pharmacol.
23(6):341-7, 1989), B-3839 (Prajda et al., In Vivo 2(2):151-4, 1988), 5-
halogenocytosine nitrosourea derivatives (Chiang & Tseng, T'ai-wan Yao
31
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Hsueh Tsa Chih 38(1 ):37-43, 1986), 1-(2-chloroethyl)-3-isobutyl-3-(~i-
maltosyl)-
1-nitrosourea (Fujimoto & Ogawa, J. Pharmacobio-Dyn. 10(7):341-5, 1987),
sulfur-containing nitrosoureas (Tang et al., Yaoxue Xuebao 21(7):502-9, 1986),
sucrose, 6-((((2-chloroethyl)nitrosoamino-)carbonyl)amino)-6-deoxysucrose
(NS-1 C) and 6'-((((2-chloroethyl)nitrosoamino)carbonyl)amino)-6'-deoxysucrose
(NS-1 D) nitrosourea derivatives (Tanoh et al., Chemotherapy (Tokyo)
33(11):969-77, 1985), CNCC, RFCNU and chlorozotocin (Mena et al.,
Chemotherapy (Basel) 32(2):131-7, 1986), CNUA (Edanami et al.,
Chemotherapy (Tokyo) 33(5):455-61, 1985), 1-(2-chloroethyl)-3-isobutyl-3-(~-
maltosyl)-1-nitrosourea (Fujimoto & Ogawa, Jpn. J. Cancer Res. (Gann)
76(7):651-6, 1985), choline-like nitrosoalkylureas (Belyaev et al., Izv. Akad.
NAUK SSSR, Ser. Khim. 3:553-7, 1985), sucrose nitrosourea derivatives (JP
84219300), sulfa drug nitrosourea analogues (Chiang et al., Proc. Nat'I Sci.
Counc., Repub. China, Part A 8(1 ):18-22, 1984), DONU (Asanuma et al., J.
Jpn. Soc. Cancer Ther. 17(8):2035-43, 1982), N,N'-bis (N-(2-chloroethyl)-N-
nitrosocarbamoyl)cystamine (CNCC) (Blazsek et al., Toxicol. Appl. Pharmacol.
74(2):250-7, 1984), dimethylnitrosourea (4Crutova et al., Izv. Akad. NAUK
SSSR, Ser. Biol. 3:439-45, 1984), GANU (Sava & Giraldi, Cancer Chemother.
Pharmacol. 10(3):167-9, 1983), CCNU (Capelli et al., Med., Biol., Environ.
11(1 ):111-16, 1983), 5-aminomethyl-2'-deoxyuridine nitrosourea analogues
(Shiau, Shih Ta Hsueh Pao (Taipei) 27:681-9, 1982), TA-077 (Fujimoto ~
Ogawa, Cancer Chemother. Pharmacol. 9(3):134-9, 1982), gentianose
nitrosourea derivatives (JP 82 80396), CNCC, RFCNU, RPCNU AND
chlorozotocin (CZT) (Marzin et al., INSERM Symp., 19(Nitrosoureas Cancer
Treat.):165-74, 1981 ), thiocolchicine nitrosourea analogues (George, Shih Ta
Hsueh Pao (Taipei) 25:355-62, 1980), 2-chloroethyl-nitrosourea (teller &
Eisenbrand, Oncology 38(1 ):39-42, 1981 ), ACNU, (1-(4-amino-2-methyl-5-
pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride) (Shibuya et
al., Gan To Kagaku Ryoho 7(8):1393-401, 1980), N-deacetylmethyl
thiocolchicine nitrosourea analogues (Lin et al., J. Med. Chem. 23(12):1440-2,
32
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
1980), pyridine and piperidine nitrosourea derivatives (Crider et al., J. Med.
Chem. 23(8):848-51, 1980), methyl-CCNU (Zimber & Perk, Refu. Vet. 35(1 ):28,
1978), phensuzimide nitrosourea derivatives (Crider et al., J. Med. Chem.
23(3):324-6, 1980), ergoline nitrosourea derivatives (Crider et al., J. Med.
Chem. 22(1 ):32-5, 1979), glucopyranose nitrosourea derivatives (JP 78 95917),
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (Farmer et al., J. Med. Chem.
27(6):514-20, 1978), 4-(3-(2-chloroethyl)-3-nitrosoureid-o)-cis-
cyclohexanecarboxylic acid (Drewinko et al., Cancer Treat. Rep. 61(8):J1513-
18, 1977), RPCNU (ICIG 1163) (Larnicol et al., Biomedicine 26(3):J176-81,
1977), IOB-252 (Sorodoc et al., Rev. Rouen. Med., Virol. 28(1 ):J 55-61,
1977),
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (Siebert & Eisenbrand, Mutat. Res.
42(1 ):J45-50, 1977), 1-tetrahydroxycyclopentyl-3-nitroso-3-(2-chloroethyl)-
urea
(4,039,578), d-1-1-(~-chloroethyl)-3-(2-oxo-3-hexahydroazepinyl)-1-nitrosourea
(3,859,277) and gentianose nitrosourea derivatives (JP 57080396); 6-S-
aminoacyloxymethyl mercaptopurine derivatives (Harada et al., Chem. Pharm.
Bull. 43(10):793-6, 1995), 6-mercaptopurine (6-MP) (Kashida et al., Biol.
Pharm. Bull. 18(11 ):1492-7, 1995), 7,8-polymethyleneimidazo-1,3,2-
diazaphosphorines (Nilov et al., Mendeleev Commun. 2:67, 1995), azathioprine
(Chifotides et al., J. Inorg. Biochem. 56(4):249-64, 1994), methyl-D-
glucopyranoside mercaptopurine derivatives (Da Silva et al., Eur. J. Med.
Chem. 29(2):149-52, 1994) and s-alkynyl mercaptopurine derivatives (Ratsino
et al., Khim.-Farm. Zh. 75(8):65-7, 1981 ); indoline ring and a modified
ornithine
or glutamic acid-bearing methotrexate derivatives (Matsuoka et al., Chem.
Pharm. Bull. 45(7):1146-1150, 1997), alkyl-substituted benzene ring C bearing
methotrexate derivatives (Matsuoka et al., Chem. Pharm. Bull. 44(12):2287-
2293, 1996), benzoxazine or benzothiazine moiety-bearing methotrexate
derivatives (Matsuoka et al., J. Med. Chem. 40(1 ):105-111, 1997), 10-
deazaaminopterin analogues (DeGraw et al., J. Med. Chem. 40(3):370-376,
1997), 5-deazaaminopterin and 5,10-dideazaaminopterin methotrexate
analogues (Piper et al., J. Med. Chem. 40(3):377-384, 1997), indoline moiety-
33
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
bearing methotrexate derivatives (Matsuoka et al., Chem. Pharm. Bull.
44(7):1332-1337, 1996), lipophilic amide methotrexate derivatives (Pignatello
et
al., World Meet. Pharm., Biopharm. Pharm. Technol., 563-4, 1995), L-threo-
(2S, 4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamic acid-containing
methotrexate analogues (Hart et al., J. Med. Chem. 39(1 ):56-65, 1996),
methotrexate tetrahydroquinazoline analogue (Gangjee, et al., J. Heterocycl.
Chem. 32(1 ):243-8, 1995), N-(a-aminoacyl) methotrexate derivatives (Cheung
et al., Pteridines 3(1-2):101-2, 1992), biotin methotrexate derivatives (Fan
et al.,
Pteridines 3(1-2):131-2, 1992), D-glutamic acid or D-erythrou, threo-4-
fluoroglutamic acid methotrexate analogues (McGuire et al., Biochem.
Pharmacol. 42(12):2400-3, 1991 ), ~,y-methano methotrexate analogues
(Rosowsky et al., Pteridines 2(3):133-9, 1991 ), 10-deazaaminopterin (10-
EDAM) analogue (Braakhuis et al., Chem. Biol. Pteridines, Proc. Int Symp.
Pteridines Folic Acid Deriv., 1027-30, 1989), y-tetrazole methotrexate
analogue
(Kalman et al., Chem. Biol. Pteridines, Proc. Int. Symp. Pteridines Folic Acid
Deriv., 1154-7, 1989), N-(L-a-aminoacyl) methotrexate derivatives (Cheung et
al., Heterocycles 28(2):751-8, 1989), meta and ortho isomers of aminopterin
(Rosowsky et al., J. Med. Chem. 32(12):2582, 1989),
hydroxymethylmethotrexate (DE 267495), y-fluoromethotrexate (McGuire et al.,
Cancer Res. 49(16):4517-25, 1989), polyglutamyl methotrexate derivatives
(Kumar et al., Cancer Res. 46(10):5020-3, 1986), gem-diphosphonate
methotrexate analogues (WO 88/06158), a- and y-substituted methotrexate
analogues (Tsushima et al., Tetrahedron 44(17):5375-87, 1988), 5-methyl-5-
deaza methotrexate analogues (4,725,687), N5-acyl-Na-(4-amino-4-
deoxypteroyl)-L-ornithine derivatives (Rosowsky et al., J. Med. Chem.
37(7):1332-7, 1988), 8-deaza methotrexate analogues (Kuehl et al., Caneer
Res. 48(6):1481-8, 1988), acivicin methotrexate analogue (Rosowsky et al., J.
Med. Chem. 30(8):1463-9, 1987), polymeric platinol methotrexate derivative
(Carraher et al., Polym. Sci. Technol. (Plenum), 35(Adv. Biomed. Polym.):311-
24, 1987), methotrexate-y-dimyristoylphophatidylethanolamine (Kinsky et al.,
34
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Biochim. Biophys. Acta 917(2):211-18, 1987), methotrexate polyglutamate
analogues (Rosowsky et al., Chem. Biol. Pteridines, Pteridines Folic Acid
Deriv., Proc. Int. Symp. Pteridines Folic Acid Deriv.: Chem., Biol. Clin.
Aspects:
985-8, 1986), poly-y-glutamyl methotrexate derivatives (Kisliuk et al., Chem.
Biol. Pteridines, Pteridines Folic Acid Deriv., Proc. Int. Symp. Pteridines
Folic
Acid Deriv.: Chem., Biol. Clin. Aspects: 989-92, 1986), deoxyuridylate
methotrexate derivatives (Webber et al., Chem. Biol. Pteridines, Pteridines
Folic
Acid Deriv., Proc. Int. Symp. Pteridines Folic Acid Deriv.: Chem., Biol. Clin.
Aspects: 659-62, 1986), iodoacetyl lysine methotrexate analogue (Delcamp et
al., Chem. Biol. Pteridines, Pteridines Folic Acid Deriv., Proc. Int. Symp.
Pteridines Folic Acid Deriv.: Chem., Biol. Clin. Aspects: 807-9, 1986),
2,.omega.-diaminoalkanoid acid-containing methotrexate analogues (McGuire
et al., Biochem. Pharmacol. 35(15):2607-13, 1986), polyglutamate
methotrexate derivatives (Kaman & Winick, Methods Enzymol. 122 (Vitam.
Coenzymes, Pt. G):339-46, 1986), 5-methyl-5-deaza analogues (Piper et al., J.
Med. Chem. 29(6):1080-7, 1986), quinazoline methotrexate analogue
(Mastropaolo et al., J. Med. Chem. 29(1 ):155-8, 1986), pyrazine methotrexate
analogue (Lever & Vestal, J. Heterocycl. Chem. 22(1 ):5-6, 1985), cysteic acid
and homocysteic acid methotrexate analogues (4,490,529), y-tart-butyl
methotrexate esters (Rosowsky et al., J. Med. Chem. 28(5):660-7, 1985),
fluorinated methotrexate analogues (Tsushima et al., Heterocycles 23(1 ):45-9,
1985), folate methotrexate analogue (Trombe, J. Bacteriol. 160(3):849-53,
1984), phosphonoglutamic acid analogues (Sturtz & Guillamot, Eur. J. Med.
Chem.--Chim. Ther. 19(3):267-73, 1984), poly (L-lysine) methotrexate
conjugates (Rosowsky et al., J. Med. Chem. 27(7):888-93, 1984), dilysine and
trilysine methotrexate derivates (Forsch & Rosowsky, J. Org. Chem.
' 49(7):1305-9, 1984), 7-hydroxymethotrexate (Fabre et al., Cancer Res.
43(10):4648-52, 1983), poly-y-glutamyl methotrexate analogues (Piper &
Montgomery, Adv. Exp. Med. Biol., 163(Folyl Antifolyl Polyglutamates):95-100,
1983), 3',5'-dichloromethotrexate (Rosowsky & Yu, J. Med. Chem. 26(10):1448-
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
52, 1983), diazoketone and chloromethylketone methotrexate analogues
(Gangjee et al., J. Pharm. Sci. 71(6):717-19, 1982), 10-propargylaminopterin
and alkyl methotrexate homologs (Piper et al., J. Med. Chem. 25(7):877-80,
1982), lectin derivatives of methotrexate (Lin et al., JNCI 66(3):523-8, 1981
),
polyglutamate methotrexate derivatives (Galivan, Mol. Pharmacol. 17(1 ):105-
10, 1980), halogentated methotrexate derivatives (Fox, JNCI 58(4):J955-8,
1977), 8-alkyl-7,8-dihydro analogues (Chaykovsky et al., J. Med. Chem.
20(10):J1323-7, 1977), 7-methyl methotrexate derivatives and
dichloromethotrexate (Rosowsky & Chen, J. Med. Chem. 17(12):J1308-11,
1974), lipophilic methotrexate derivatives and 3',5'-dichloromethotrexate
(Rosowsky, J. Med. Chem. 16(10):J1190-3, 1973), deaza amethopterin
analogues (Montgomery et al., Ann. N. Y. Acad. Sci. 186:J227-34, 1971 ),
MX068 (Pharma Japan, 1658:18, 1999) and cysteic acid and homocysteic acid
methotrexate analogues (EPA 0142220); N3-alkylated analogues of 5-
fluorouracil (Kozai et al., J. Chem. Soc., Perlein Trans. 1(19):3145-3146,
1998),
5-fluorouracil derivatives with 1,4-oxaheteroepane moieties (Gomez et al.,
Tetrahedron 54(43):13295-13312, 1998), 5-fluorouracil and nucleoside
analogues (Li, Anticancer Res. 17(1A):21-27, 1997), cis- and trans-5-fluoro-
5,6-
dihydro-6-alkoxyuracil (Van der Wilt et al., Br. J. Cancer 68(4):702-7, 1993),
cyclopentane 5-fluorouracil analogues (Hronowski & Szarek, Can. J. Chem.
70(4):1162-9, 1992), A-OT-fluorouracil (Zhang et al., Zongguo Yiyao Gongye
Zazhi 20(11 ):513-15, 1989), N4-trimethoxybenzoyl-5'-deoxy-5-fluorocytidine
and 5'-deoxy-5-fluorouridine (Miwa et al., Chem. Pharm. Bull. 38(4):998-1003,
1990), 1-hexylcarbamoyl-5-fluorouracil (Hoshi et al., J. Pharmacobio-Dun.
3(9):478-81, 1980; Maehara et al., Chemotherapy (Basel) 34(6):484-9, 1988),
B-3839 (Prajda et al., In Vivo 2(2):151-4, 1988), uracil-1-(2-tetrahydrofuryl)-
5-
fluorouracil (Anai et al., Oncology 45(3):144-7, 1988), 1-(2'-deoxy-2'-fluoro-
(i-D-
arabinofuranosyl)-5-fluorouracil (Suzuko et al., Mol. Pharmacol. 31(3):301-6,
1987), doxifluridine (Matuura et al., Oyo Yakuri 29(5):803-31, 1985), 5'-deoxy-
5-
fluorouridine (Bollag & Hartmann, Eur. J. Cancer 16(4):427-32, 1980), 1-acetyl-
36
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
3-O-toluyl-5-fluorouracil (Okada, Hiroshima J. Med. Sci. 28(1 ):49-66, 1979),
5-
fluorouracil-m-formylbenzene-sulfonate (JP 55059173), N'-(2-furanidyl)-5-
fluorouracil (JP 53149985) and 1-(2-tetrahydrofuryl)-5-fluorouracil (JP
52089680); 4'-epidoxorubicin (Lanius, Adv. Chemother. Gastrointest. Cancer,
(Int. Symp.), 159-67, 1984); N-substituted deacetylvinblastine amide
(vindesine)
sulfates (Conrad et al., J. Med. Chem. 22(4):391-400, 1979); and Cu(II)-VP-16
(etoposide) complex (Tawa et al., Bioorg. Med. Chem. 6(7):1003-1008, 1998),
pyrrolecarboxamidino-bearing etoposide analogues (Ji et al., Bioorg. Med.
Chem. Lett. 7(5):607-612, 1997), 4~i-amino etoposide analogues (Hu,
University of North Carolina Dissertation, 1992), y-lactone ring-modified
arylamino etoposide analogues (Zhou et al., J. Med. Chem. 37(2):287-92,
1994), N-glucosyl etoposide analogue (Allevi et al., Tetrahedron Lett.
34(45):7313-16, 1993), etoposide A-ring analogues (Kadow et al., Bioorg. Med.
Chem. Lett. 2(1 ):17-22, 1992), 4'-deshydroxy-4'-methyl etoposide (Saulnier et
al., Bioorg. Med. Chem. Lett. 2(10):1213-18, 1992), pendulum ring etoposide
analogues (Sinha et al., Eur. J. Cancer 26(5):590-3, 1990) and E-ring desoxy
etoposide analogues (Saulnier et al., J. Med. Chem. 32(7):1418-20, 1989).
Within one embodiment of the invention, the cell cycle inhibitor is
paclitaxel, a compound which disrupts mitosis (M-phase) by binding to tubulin
to form abnormal mitotic spindles or an analogue or derivative thereof.
Briefly,
paclitaxel is a highly derivatized diterpenoid (Wani et al., J. Am. Chem. Soc.
93:2325, 1971 ) which has been obtained from the harvested and dried bark of
Taxus brevifolia (Pacific Yew) and Taxomyces Andreanae and Endophytic
Fungus of the Pacific Yew (Stierle et al., Science 60:214-216, 1993).
"Paclitaxel" (which should be understood herein to include formulations,
prodrugs, analogues and derivatives such as, for example, TAXOL (Bristol
Myers Squibb, New York, NY, TAXOTERE (Aventis Pharmaceuticals, France),
docetaxel, 10-desacetyl analogues of paclitaxel and 3'N-desbenzoyl-3'N-t-
butoxy carbonyl analogues of paclitaxel) may be readily prepared utilizing
techniques known to those skilled in the art (see, e.g., Schiff et al., Nature
37
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
277:665-667, 1979; Long and Fairchild, Cancer Research 54:4355-4361, 1994;
Ringel and Horwitz, J. Nat'1 Cancer Inst. 83(4):288-291, 1991; Pazdur et al.,
Cancer Treat. Rev. 79(4):351-386, 1993; WO 94/07882; WO 94/07881; WO
94/07880; WO 94/07876; WO 93/23555; WO 93/10076; W094/00156;
WO 93/24476; EP 590267; WO 94/20089; U.S. Patent Nos. 5,294,637;
5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; 5,254,580;
5,412,092; 5,395,850; 5,380,751; 5,350,866; 4,857,653; 5,272,171; 5,411,984;
5,248,796; 5,248,796; 5,422,364; 5,300,638; 5,294,637; 5,362,831; 5,440,056;
4,814,470; 5,278,324; 5,352,805; 5,411,984; 5,059,699; 4,942,184;
Tetrahedron Letters 35(52):9709-9712, 1994; J. Med. Chem. 35:4230-4237,
1992; J. Med. Chem. 34:992-998, 1991; J. Natural Prod. 57(10):1404-1410,
1994; J. Natural Prod. 57(11 ):1580-1583, 1994; J. Am. Chem. Soc. 110:6558-
6560, 1988), or obtained from a variety of commercial sources, including for
example, Sigma Chemical Co., St. Louis, Missouri (T7402 - from Taxus
brevifolia).
Representative examples of paclitaxel derivatives or analogues
include 7-deoxy-docetaxol, 7,8-cyclopropataxanes, N-substituted 2-azetidones,
6,7-epoxy paclitaxels, 6,7-modified paclitaxels, 10-desacetoxytaxol, 10-
deacetyltaxol (from 10-deacetylbaccatin III), phosphonooxy and carbonate
derivatives of taxol, taxol 2',7-di(sodium 1,2-benzenedicarboxylate, 10-
desacetoxy-11,12-dihydrotaxol-10,12(18)-diene derivatives, 10-
desacetoxytaxol, Protaxol (2'-and/or 7-O-ester derivatives), (2'-and/or 7-O-
carbonate derivatives), asymmetric synthesis of taxol side chain, fluoro
taxols,
9-deoxotaxane, (13-acetyl-9-deoxobaccatine III, 9-deoxotaxol, 7-deoxy-9-
deoxotaxol, 10-desacetoxy-7-deoxy-9-deoxotaxol, Derivatives containing
hydrogen or acetyl group and a hydroxy and tert-butoxycarbonylamino,
sulfonated 2'-acryloyltaxol and sulfonated 2'-O-acyl acid taxol derivatives,
succinyltaxol, 2'-y-aminobutyryltaxol formate, 2'-acetyl taxol, 7-acetyl
taxol, 7-
glycine carbamate taxol, 2'-OH-7-PEG(5000) carbamate taxol, 2'-benzoyl and
2',7-dibenzoyl taxol derivatives, other prodrugs (2'-acetyltaxol; 2',7-
38
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
diacetyltaxol; 2'succinyltaxol; 2'-(beta-alanyl)-taxol); 2'gamma-
aminobutyryltaxol formate; ethylene glycol derivatives of 2'-succinyltaxol; 2'-
glutaryltaxol; 2'-(N,N-dimethylglycyl) taxol; 2'-(2-(N,N-
dimethylamino)propionyl)taxol; 2'orthocarboxybenzoyl taxol; 2'aliphatic
carboxylic acid derivatives of taxol, Prodrugs (2'(N,N-
diethylaminopropionyl)taxol, 2'(N,N-dimethylglycyl)taxol, 7(N,N-
dimethylglycyl)taxol, 2',7-di-(N,N-dimethylglycyl)taxol, 7(N,N-
diethylaminopropionyl)taxol, 2',7-di(N,N-diethylaminopropionyl)taxol, 2'-(L-
glycyl)taxol, 7-(L-glycyl)taxol, 2',7-di(L-glycyl)taxol, 2'-(L-alanyl)taxol, 7-
(L-
alanyl)taxol, 2',7-di(L-alanyl)taxol, 2'-(L-leucyl)taxol, 7-(L-leucyl)taxol,
2',7-di(L-
leucyl)taxol, 2'-(L-isoleucyl)taxol, 7-(L-isoleucyl)taxol, 2',7-di(L-
isoleucyl)taxol,
2'-(L-valyl)taxol, 7-(L-valyl)taxol, 2'7-di(L-valyl)taxol, 2'-(L-
phenylalanyl) taxol, 7-
(L-phenylalanyl)taxol, 2',7-di(L-phenylalanyl)taxol, 2'-(L-prolyl)taxol, 7-(L-
prolyl)taxol, 2',7-di(L-prolyl)taxol, 2'-(L-lysyl)taxol, 7-(L-lysyl)taxol,
2',7-di(L-
lysyl)taxol, 2'-(L-glutamyl)taxol, 7-(L-glutamyl)taxol, 2',7-di(L-
glutamyl)taxol, 2'-
(L-arginyl)taxol, 7-(L-arginyl)taxol, 2',7-di(L-arginyl)taxol}, taxol
analogues with
modified phenylisoserine side chains, TAXOTERE, (N-debenzoyl-N-tert-
(butoxycaronyl)-10-deacetyltaxol, and taxanes (e.g., baccatin III,
cephalomannine, 10-deacetylbaccatin III, brevifoliol, yunantaxusin and
taxusin);
and other taxane analogues and derivatives, including 14-beta-hydroxy-10
deacetybaccatin III, debenzoyl-2-acyl paclitaxel derivatives, benzoate
paclitaxel
derivatives, phosphonooxy and carbonate paclitaxel derivatives, sulfonated 2'-
acryloyltaxol; sulfonated 2'-O-aryl acid paclitaxel derivatives, 18-site-
substituted
paclitaxel derivatives, chlorinated paclitaxel analogues, C4 methoxy ether
paclitaxel derivatives, sulfenamide taxane derivatives, brominated paclitaxel
analogues, Girard taxane derivatives, nitrophenyl paclitaxel, 10-deacetylated
substituted paclitaxel derivatives, 14- beta -hydroxy-10 deacetylbaccatin III
taxane derivatives, C7 taxane derivatives, C10 taxane derivatives, 2-debenzoyl-
2-acyl taxane derivatives, 2-debenzoyl and -2-acyl paclitaxel derivatives,
taxane
and baccatin III analogues bearing new C2 and C4 functional groups, n-acyl
39
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
paclitaxel analogues, 10-deacetylbaccatin III and 7-protected-10-
deacetylbaccatin III derivatives from 10-deacetyl taxol A, 10-deacetyl taxol
B,
and 10-deacetyl taxol, benzoate derivatives of taxol, 2-aroyl-4-acyl
paclitaxel
analogues, orthro-ester paclitaxel analogues, 2-aroyl-4-acyl paclitaxel
analogues and 1-deoxy paclitaxel and 1-d'eoxy paclitaxel analogues.
In one aspect, the cell cycle inhibitor is a taxane having the
formula (C1 ):
(C1 ),
where the gray-highlighted portions may be substituted and the non-highlighted
portion is the taxane core. A side-chain (labeled "A" in the diagram) is
desirably
present in order for the compound to have good activity as a cell cycle
inhibitor.
Examples of compounds having this structure include paclitaxel (Merck Index
entry 7117), docetaxol (TAXOTERE, Merck Index entry 3458), and 3'-
desphenyl-3'-(4-ntirophenyl)-N-debenzoyl-N-(t-butoxycarbonyl)-10-
deacetyltaxol.
In one aspect, suitable taxanes such as paclitaxel and its
analogues and derivatives are disclosed in U.S. Patent No. 5,440,056 as
having the structure (C2):
v
HgC~~
R2
CH3
R~O'~ '_ ~ O
Rb =
Rs0 Ra0
(C2)
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
wherein X may be oxygen (paclitaxel), hydrogen (9-deoxy derivatives),
thioacyl,
or dihydroxyl precursors; R~ is selected from paclitaxel or TAXOTERE side
chains or alkanoyl of the formula (C3)
O
R~
' (C3)
wherein R~ is selected from hydrogen, alkyl, phenyl, alkoxy, amino, phenoxy
(substituted or unsubstituted); R$ is selected from hydrogen, alkyl,
hydroxyalkyl,
alkoxyalkyl, aminoalkyl, phenyl (substituted or unsubstituted), alpha or beta-
naphthyl; and R9 is selected from hydrogen, alkanoyl, substituted alkanoyl,
and
aminoalkanoyl; where substitutions refer to hydroxyl, sulfhydryl, allalkoxyl,
carboxyl, halogen, thioalkoxyl, N,N-dimethylamino, alkylamino, dialkylamino,
nitro, and -OS03H, and/or may refer to groups containing such substitutions;
R2
is selected from hydrogen or oxygen-containing groups, such as hydrogen,
hydroxyl, alkoyl, alkanoyloxy, aminoalkanoyloxy, and peptidyalkanoyloxy; R3 is
selected from hydrogen or oxygen-containing groups, such as hydrogen,
hydroxyl, alkoyl, alkanoyloxy, aminoalkanoyloxy, and peptidyalkanoyloxy, and
may further be a silyl containing group or a sulphur containing group; R4 is
selected from acyl, alkyl, alkanoyl, aminoalkanoyl, peptidylalkanoyl and
aroyl;
R5 is selected from acyl, alkyl, alkanoyl, aminoalkanoyl, peptidylalkanoyl and
aroyl; R6 is selected from hydrogen or oxygen-containing groups, such as
hydrogen, hydroxyl alkoyl, alkanoyloxy, aminoalkanoyloxy, and
peptidyalkanoyloxy.
In one aspect, the paclitaxel analogues and derivatives useful as
cell cycle inhibitors are disclosed in PCT International Patent Application
No.
WO 93/10076. As disclosed in this publication, the analogue or derivative
41
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
should have a side chain attached to the taxane nucleus at C~3, as shown in
the
structure below (formula C4), in order to confer antitumor activity to the
taxane.
9
13
5
(C4)
WO 93/10076 discloses that the taxane nucleus may be
5 substituted at any position with the exception of the existing methyl
groups.
The substitutions may include, for example, hydrogen, alkanoyloxy,
alkenoyloxy, aryloyloxy. In addition, oxo groups may be attached to carbons
labeled 2, 4, 9, and/or 10. As well, an oxetane ring may be attached at
carbons
4 and 5. As well, an oxirane ring may be attached to the carbon labeled 4.
10 In one aspect, the taxane-based cell cycle inhibitor useful in the
present invention is disclosed in U.S. Patent 5,440,056, which discloses 9-
deoxo taxanes. These are compounds lacking an oxo group at the carbon
labeled 9 in the taxane structure shown above (formula C4). The taxane ring
may be substituted at the carbons labeled 1, 7 and 10 (independently) with H,
OH, O-R, or O-CO-R where R is an alkyl or an aminoalkyl. As well, it may be
substituted at carbons labeled 2 and 4 (independently) with aryol, alkanoyl,
aminoalkanoyl or alkyl groups. The side chain of formula (C3) may be
substituted at R7 and R$ (independently) with phenyl rings, substituted phenyl
rings, linear alkanes/alkenes, and groups containing H, O or N. R9 may be
substituted with H, or a substituted or unsubstituted alkanoyl group.
Taxanes in general, and paclitaxel is particular, is considered to
function as a cell cycle inhibitor by acting as an anti-microtubule agent, and
more specifically as a stabilizer. These compounds have been shown useful in
42
4
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
the treatment of proliferative disorders, including: non-small cell (NSC)
lung;
small cell lung; breast; prostate; cervical; endometrial; head and neck
cancers.
In another aspect, the anti-microtuble agent (microtubule inhibitor)
is albendazole (carbamic acid, (5-(propylthio)-1 H-benzimidazol-2-yl)-, methyl
ester), LY-355703 (1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone,
10-((3-chloro-4-methoxyphenyl)methyl)-6,6-dimethyl-3-(2-methylpropyl)-16-
((1S)-1-((2S,3R)-3-phenyloxiranyl)ethyl)-, (3S,10R,13E,16S)-), vindesine
(vincaleukoblastine, 3-(aminocarbonyl)-04-deacetyl-3-de(methoxycarbonyl)-),
or WAY-174286
In another aspect, the cell cycle inhibitor is a vinca alkaloid. Vinca
alkaloids have the following general structure. They are indole-dihydroindole
dimers.
ale
dihydroindole
U-Kp
As disclosed in U.S. Patent Nos. 4,841,045 and 5,030,620, R~ can
be a formyl or methyl group or alternately H. R~ can also be an alkyl group or
an aldehyde-substituted alkyl (e.g., CH2CH0). R~ is typically a CH3 or NH2
group. However it can be alternately substituted with a lower alkyl ester or
the
ester linking to the dihydroindole core may be substituted with C(O)-R where R
is NH2, an amino acid ester or a peptide ester. R3 is typically C(O)CH3, CH3
or
H. Alternately, a protein fragment may be linked by a bifunctional group, such
as maleoyl amino acid. R3 can also be substituted to form an alkyl ester which
may be further substituted. R4 may be -CH2- or a single bond. R5 and R6 may
43
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
be H, OH or a lower alkyl, typically -CH2CH3. Alternatively R6 and R~ may
together form an oxetane ring. R7 may alternately be H. Further substitutions
include molecules wherein methyl groups are substituted with other alkyl
groups, and whereby unsaturated rings may be derivatized by the addition of a
side group such as an alkane, alkene, alkyne, halogen, ester, amide or amino
group.
Exemplary vinca alkaloids are vinblastine, vincristine, vincristine
sulfate, vindesine, and vinorelbine, having the structures:
R1 Rz R3 Ra Rs
Vinblastine:CH3 C(O)CH3 CHZ
CH3 OH
Vincristine:CH3 C(O)CH3 CHZ
CH20 OH
Vindesine: NHZ H OH CH2
CH3
Vinorelbine:CH3 CH3 H single
CH3 bond
Analogues typically require the side group (shaded area) in order
to have activity. These compounds are thought to act as cell cycle inhibitors
by
functioning as anti-microtubule agents, and more specifically to inhibit
polymerization. These compounds have been shown useful in treating
proliferative disorders, including NSC lung; small cell lung; breast;
prostate;
brain; head and neck; retinoblastoma; bladder; and penile cancers; and soft
tissue sarcoma.
In another aspect, the cell cycle inhibitor is a camptothecin, or an
analog or derivative thereof. Camptothecins have the following general
structure.
44
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In this structure, X is typically O, but can be other groups, e.g., NH
in the case of 21-lactam derivatives. R1 is typically H or OH, but may be
other
groups, e.g., a terminally hydroxylated C1_3 alkane. R2 is typically H or an
amino containing group such as (CH3)2NHCH~, but may be other groups e.g.,
NO~, NHS, halogen (as disclosed in, e.g., U.S. Patent 5,552,156) or a short
alkane containing these groups. R3 is typically H or a short alkyl such as
C2H5.
R4 is typically H but may be other groups, e.g., a methylenedioxy group with
R1.
Exemplary camptothecin compounds include topotecan,
irinotecan (CPT-11 ), 9-aminocamptothecin, 21-lactam-20(S)-camptothecin,
10,11-methylenedioxycamptothecin, SN-38, 9-nitrocamptothecin, 10-
hydroxycamptothecin. Exemplary compounds have the structures:
R~ R3 O
R~
\ ~N ~ ~X
E
/ N \ o
H3C- ' OH
Ri R2 R3
Camptothecin: H H H
Topotecan: OH (CH3)2NHCHz H
SN-3S: OH H CZHS
x: O for most analogs, NH for 21-lactam analogs
Camptothecins have the five rings shown here. The ring labeled
E must be intact (the lactone rather than carboxylate form) for maximum
activity
and minimum toxicity. These compounds are useful to as cell cycle inhibitors,
where they can function as topoisomerase I inhibitors and/or DNA cleavage
agents. They have been shown useful in the treatment of proliferative
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
disorders, including, for example, NSC lung; small cell lung; and cervical
cancers.
In another aspect, the cell cycle inhibitor is a podophyllotoxin, or a
derivative or an analogue thereof. Exemplary compounds of this type are
etoposide or teniposide, which have the following structures:
Etoposide C'N3
Teniposide s
HgC~
OH
These compounds are thought to function as cell cycle inhibitors
by being topoisomerase II inhibitors and/or by DNA cleaving agents. They have
been shown useful as antiproliferative agents in, e.g., small cell lung,
prostate,
and brain cancers, and in retinoblastoma.
Another example of a DNA topoisomerase inhibitor is lurtotecan
dihydrochloride (11H-1,4-dioxino(2,3-g)pyrano(3',4':6,7)indolizino(1,2-
b)quinoline-9,12(8H,14H)-dione, 8-ethyl-2,3-dihydro-8-hydroxy-15-((4-methyl-1-
piperazinyl)methyl)-, dihydrochloride, (S)-).
In another aspect, the cell cycle inhibitor is an anthracycline.
Anthracyclines have the following general structure, where the R groups may
be a variety of organic groups:
46
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
According to U.S. Patent 5,594,158, suitable R groups are: R~ is
CH3 or CH20H; R2 is daunosamine or H; R3 and R4 are independently one of
OH, N02, NH2, F, Cl, Br, I, CN, H or groups derived from these; R5_~ are all H
or
R5 and R6 are H and R~ and R$ are alkyl or halogen, or vice versa: R~ and R$
are H and R5 and R6 are alkyl or halogen.
According to U.S. Patent 5,843,903, R2 may be a conjugated
peptide. According to U.S. Patent Nos. 4,215,062 and 4,296,105, R5 may be
OH or an ether linked alkyl group. R~ may also be linked to the anthracycline
ring by a group other than C(O), such as an alkyl or branched alkyl group
having the C(O) linking moiety at its end, such as -CH2CH(CH2-X)C(O)-R~,
wherein X is H or an alkyl group (see, e.g., U.S. Patent 4,215,062). R2 may
alternately be a group linked by the functional group =N-NHC(O)-Y, where Y is
a group such as a phenyl or substituted phenyl ring. Alternately R3 may have
the following structure:
HsC O
NH
F29
R,o
in which R9 is OH either in or out of the plane of the ring, or is a second
sugar
moiety such as R3. Ran may be H or form a secondary amine with a group such
as an aromatic group, saturated or partially saturated 5 or 6 membered
heterocyclic having at least one ring nitrogen (see U.S. Patent 5,843,903).
Alternately, R~o may be derived from an amino acid, having the structure -
C(O)CH(NHR~~)(R~2), in which R~~ is H, or forms a C3_4 membered alkylene with
R~2. R~2 may be H, alkyl, aminoalkyl, amino, hydroxy, mercapto, phenyl, benzyl
or methylthio (see U.S. Patent 4,296,105).
Exemplary anthracyclines are doxorubicin, daunorubicin,
idarubicin, epirubicin, pirarubicin, zorubicin, and carubicin. Suitable
compounds
have the structures:
47
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
0
O OH
R,
....OH
R, o OH O
H,C O
NHz
R7
R, R. R3
Doxorubicin:OCH,CHzOHOH out
of ring
plane
Epirubicin:OCH,CHiOHOH in
dng
plane
(4' oxoruhicin)
epimer
of
d
Daunorubicin: CHe - OH
OCH3 out
of ring
plane
Idarubicin:H CH, OH out
of ring
plane
PirarubicinOCH,OH A
ZorubicinOCH3=N-NHC(0)CeHe
B
CarubicinOH CH3 B
A: ~ / 3: O /
O CHI O
OH
NH,
Other suitable anthracyclines are anthramycin, mitoxantrone,
menogaril, nogalamycin, aclacinomycin A, olivomycin A, chromomycin A3, and
plicamycin having the structures:
OH OH
H
HaC ~ N Anthramycin
N
NHS
O
O
R, Rz R3
Menogaril H OCH3 H
off O HN~NH~OH Nogalamycin O-sugar H COOCH3
cH3
sugar: H3C O
O
OH O HN~ ~ 'OH H3C0 CH3 OCHa
N' VH
Mitoxantrone i
O OCHa
O
CH3
W~ OH
HO - HO~ ~ ~
O O ~/
CH3
HaC O
R,O
HO
R, RZ R3 RQ
Olivomycin A COCH(CH3)zCOCHeH
CHI
Chromomycin A, COCH~ COCH,CH3
CH3
Plicamycin H H H CH3
48
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
These compounds are thought to function as cell cycle inhibitors
by being topoisomerase inhibitors andlor by DNA cleaving agents. They have
been shown useful in the treatment of proliferative disorders, including small
cell lung; breast; endometrial; head and neck; retinoblastoma; liver; bile
duct;
islet cell; and bladder cancers; and soft tissue sarcoma.
In another aspect, the cell cycle inhibitor is a platinum compound.
In general, suitable platinum complexes may be of Pt(II) or Pt(IV) and have
this
basic structure:
Z1
X
R1 ~/Pt~
R/
2
22
wherein X and Y are anionic leaving groups such as sulfate, phosphate,
carboxylate, and halogen; R1 and R2 are alkyl, amine, amino alkyl any may be
further substituted, and are basically inert or bridging groups. For Pt(II)
complexes Z1 and Z~ are non-existent. For Pt(IV) Z1 and Z2 may be anionic
groups such as halogen, hydroxy, carboxylate, ester, sulfate or phosphate.
See, e.g., U.S. Patent Nos. 4,588,831 and 4,250,189.
Suitable platinum complexes may contain multiple Pt atoms. See,
e.g., U.S. Patent Nos. 5,409,915 and 5,380,897. For example bisplatinum and
triplatinum complexes of the type:
49
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Z~ Zt
X~ I / Rt X~ I ~ R2
Y/ It\A~ It\Y
Zz Zz
X Zt R X Zt A Zt X
\Pt/ \ ~t~ ~ It/
Y/ I ~A~ I \Y Rz~ I \Y
Z2 Z2 Z2
Zt Zt
X\ I / Rz Rz\ / X
Y/ It\A~ It\Y
Z2 Zz
Zz~ / R3
Pt
Y/I\Zt
X
Exemplary platinum compounds are cisplatin, carboplatin,
oxaliplatin, and miboplatin having the structures:
N3
NH3 O O~
Pt
CI~ t-NH3 I ~NH3
O
CI
O
Cisplatin Carboplatin
O O
NHZ O NHZ
~Pt
~ H
NHZ~'1~ O HN
O// O
Oxaliplatin Miboplatin
These compounds are thought to function as cell cycle inhibitors
by binding to DNA, i.e., acting as alkylating agents of DNA. These compounds
have been shown useful in the treatment of cell proliferative disorders,
including, e.g., NSC lung; small cell lung; breast; cervical; brain; head and
neck;
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
esophageal; retinoblastom; liver; bile duct; bladder; penile; and vulvar
cancers;
and soft tissue sarcoma.
In another aspect, the cell cycle inhibitor is a nitrosourea.
Nitrosourease have the following general structure (C5), where typical R
groups
are shown below.
0
R'~ ~R
N NH
N~
~O
(C5)
R Group:
H2C
O
'CI OH
Carmu~stine OH OH O-CHs
Ranimustine Lomustine
CH3 ~ NHS OH
\CH3 O
OH
O /~
~CHa ~ ~CH3 OH OH
Fotemustine Nimustine Chlorozotocin Streptozocin
Other suitable R groups include cyclic alkanes, alkanes, halogen
substituted groups, sugars, aryl and heteroaryl groups, phosphonyl and
sulfonyl
groups. As disclosed in U.S. Patent No. 4,367,239, R may suitably be CH2-
C(X)(Y)(Z), wherein X and Y may be the same or different members of the
following groups: phenyl, cyclyhexyl, or a phenyl or cyclohexyl group
substituted with groups such as halogen, lower alkyl (C~_4), trifluore methyl,
cyano, phenyl, cyclohexyl, lower alkyloxy (C~_4). Z has the following
structure:
-alkylene-N-R~ R2, where R~ and R2 may be the same or different members of
the following group: lower alkyl (C~_4) and benzyl, or together R~ and R2 may
form a saturated 5 or 6 membered heterocyclic such as pyrrolidine, piperidine,
51
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
morfoline, thiomorfoline, N-lower alkyl piperazine, where the heterocyclic may
be optionally substituted with lower alkyl groups.
As disclosed in U.S. Patent No. 6,096,923, R and R' of formula
(C5) may be the same or different, where each may be a substituted or
unsubstituted hydrocarbon having 1-10 carbons. Substitutions may include
hydrocarbyl, halo, ester, amide, carboxylic acid, ether, thioether and alcohol
groups. As disclosed in U.S. Patent No. 4,472,379, R of formula (C5) may be
an amide bond and a pyranose structure (e.g., methyl 2'-(N-(N-(2-chloroethyl)-
N-nitroso-carbamoyl)-glycyl)amino-2'-deoxy-a-D-glucopyranoside). As
disclosed in U.S. Patent No. 4,150,146, R of formula (C5) may be an alkyl
group of 2 to 6 carbons and may be substituted with an ester, sulfonyl, or
hydroxyl group. It may also be substituted with a carboxylic acid or CONH~
group.
Exemplary nitrosoureas are BCNU (carmustine), methyl-CCNU
(semustine), CCNU (lomustine), ranimustine, ~nimustine, chlorozotocin,
fotemustine, and streptozocin, having the structures:
0
CI~ ~ ~R
NH R Group:
N
O
'CI
Carmustine
HZC OH
OH O OH O
OH OH O-CH OH OH
Ranimustine Lomustine O
H3C~ ~
NHZ OH N"NH
N
OH O \O
N CH3 OH ~ pH
Nimustine Chlorozotocin
\CH3
~~ \O~CH3
O
Fotemustine
52
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
These nitrosourea compounds are thought to function as cell
cycle inhibitors by binding to DNA, that is, by functioning as DNA alkylating
agents. These cell cycle inhibitors have been shown useful in treating cell
proliferative disorders such as, for example, islet cell; small cell lung;
melanoma; and brain cancers.
In another aspect, the cell cycle inhibitor is a nitroimidazole,
where exemplary nitroimidazoles are metronidazole, benznidazole, etanidazole,
and misonidazole, having the structures:
R~
N R2
R3~
Ri Rz R3
Metronidazole OH CH3 NOz
Benznidazole C(O)NHCHz benzyl NOz H
Etanidazole CONHCHZCH20H NOz H
Suitable nitroimidazole compounds are disclosed in, e.g., U.S.
Patent Nos. 4,371,540 and 4,462,992.
In another aspect, the cell cycle inhibitor is a folic acid antagonist,
such as methotrexate or derivatives or analogues thereof, including
edatrexate,
trimetrexate, raltitrexed, piritrexim, denopterin, tomudex, and pteropterin.
Methotrexate analogues have the following general structure:
The identity of the R group may be selected from organic groups,
particularly those groups set forth in U.S. Patent Nos. 5,166,149 and
5,382,582.
53
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
For example, R~ may be N, R2 may be N or C(CH3), R3 and R3' may H or alkyl,
e.g., CH3, R4 may be a single bond or NR, where R is H or alkyl group. 85,6,8
may be H, OCH3, or alternately they can be halogens or hydro groups. R7 is a
side chain of the general structure:
HO
wherein n = 1 for methotrexate, n = 3 for pteropterin. The carboxyl groups in
the side chain may be esterified or form a salt such as a Zn~+ salt. R9 and
Rio
can be NH2 or may be alkyl substituted.
Exemplary folic acid antagonist compounds have the structures:
N NHp
~N
2
RO
RaRi R: Rs Ra RsRa Re
Rn
MelholrexateNH,N N H N(CHa)H H A(n.t)H
EdalrexaieNH,N N H N(CH,CHa)H H A(n=t)H
TrimetrexateNHzN C(CHa)H NH H OCH, OCHa
OCH,
PteroplennNH,N N H N(CH,)H H A H
(n=3)
DenoplerinOHN N CH,N(CH,)H H A(n=t)H
PiritreximNH,N C(CH,)singleOCH~H H OCHaH
H
bond
A: 0
,,yy NH
H0~
O
0 off n
N CHa
HOOC~ O ~ H3
5 N ~ ~ NH
HOOC NH
O
Tomudex
These compounds are thought to function as cell cycle inhibitors
by serving as antimetabolites of folic acid. They have been shown useful in
the
treatment of cell proliferative disorders including, for example, soft tissue
54
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
sarcoma, small cell lung, breast, brain, head and neck, bladder, and penile
cancers.
In another aspect, the cell cycle inhibitor is a cytidine analogue,
such as cytarabine or derivatives or analogues thereof, including enocitabine,
FMdC ((E(-2'-deoxy-2'-(fluoromethylene)cytidine), gemcitabine, 5-azacitidine,
ancitabine, and 6-azauridine. Exemplary compounds have the structures:
R, RZ R3 RQ
Cytarabine OH H CH
H
Enocitabine OH H CH
C(O)(CHZ)ZOCH3
Gemcitabine F F CH
H
Azacitidine H OH N
H
FMdC H CHzFH CH
Ancitabine 6-Azauridine
These compounds are thought to function as cell cycle inhibitors
as acting as antimetabolites of pyrimidine. These compounds have been
shown useful in the treatment of cell proliferative disorders including, for
example, pancreatic, breast, cervical, NSC lung, and bile duct cancers.
In another aspect, the cell cycle inhibitor is a pyrimidine analogue.
In one aspect, the pyrimidine analogues have the general structure:
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
wherein positions 2', 3' and 5' on the sugar ring (R2, R3 and R4,
respectively)
can be H, hydroxyl, phosphoryl (see, e.g., U.S. Patent 4,086,417) or ester
(see,
e.g., U.S. Patent 3,894,000). Esters can be of alkyl, cycloalkyl, aryl or
heterocyclo/aryl types. The 2' carbon can be hydroxylated at either R2 or R~',
the other group is H. Alternately, the 2' carbon can be substituted with
halogens e.g., fluoro or difluoro cytidines such as Gemcytabine. Alternately,
the sugar can be substituted for another heterocyclic group such as a furyl
group or for an alkane, an alkyl ether or an amide linked alkane such as
C(O)NH(CH2)5CH3. The 2° amine can be substituted with an aliphatic
acyl (R~)
linked with an amide (see, e.g., U.S. Patent 3,991,045) or urethane (see,
e.g.,
U.S. Patent 3,894,000) bond. It can also be further substituted to form a
quaternary ammonium salt. R5 in the pyrimidine ring may be N or CR, where R
is H, halogen containing groups, or alkyl (see, e.g., U.S. Patent No.
4,086,417).
R6 and R~ can together can form an oxo group or R6 = -NH-R~ and R~ = H. R$
is H or R~ and R$ together can form a double bond or R$ can be X, where X is:
CN
O ~ ~ O O
0 N O
Specific pyrimidine analogues are disclosed in U.S. Patent No.
3,894,000 (see, e.g., 2'-O-palmityl-ara-cytidine, 3'-O-benzoyl-ara-cytidine,
and
more than 10 other examples); U.S. Patent No. 3,991,045 (see, e.g., N4-acyl-1-
(3-D-arabinofuranosylcytosine, and numerous acyl groups derivatives as listed
therein, such as palmitoyl.
56
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another aspect, the cell cycle inhibitor is a fluoropyrimidine
analogue, such as 5-fluorouracil, or an analogue or derivative thereof,
including
carmofur, doxifluridine, emitefur, tegafur, and floxuridine. Exemplary
compounds have the structures:
0
Rz F
O '~N~
R~
R1 Rz
5-FluorouracilH H
CarmofurC(O)NH(CHZ)5CH3
H
DoxifluridineA, H
FloxuridineA2 H
EmitefurCH20CHZCH3
B
TegafurH
A, HO Hz HO
p O CH3
off off off
B CN
O ~ ~ O O
O N O
C
O
Other suitable fluoropyrimidine analogues include 5-FudR (5-
fluoro-deoxyuridine), or an analogue or derivative thereof, including 5-
iododeoxyuridine (5-IudR), 5-bromodeoxyuridine (5-BudR), fluorouridine
triphosphate (5-FUTP), and fluorodeoxyuridine monophosphate (5-dFUMP).
Exemplary compounds have the structures:
57
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
5-Fluoro-2'-deoxyuridine: R = F
5-Bromo-2'-deoxyuridine: R = Br
5-lodoo-2'-deoxyuridine: R = I
These compounds are thought to function as cell cycle inhibitors
by serving as antimetabolites of pyrimidine. These compounds have been
shown useful in the treatment of cell proliferative disorders such as breast,
cervical, non-melanoma skin, head and neck, esophageal, bile duct, pancreatic,
islet cell, penile, and vulvar cancers.
In another aspect, the cell cycle inhibitor is a purine analogue.
Purine analogues have the following general structure.
R2
N
N
R~ N
R3
wherein X is typically carbon; R~ is H, halogen, amine or a substituted
phenyl;
R2 is H, a primary, secondary or tertiary amine, a sulfur containing group,
typically -SH, an alkane, a cyclic alkane, a heterocyclic or a sugar; R3 is H,
a
sugar (typically a furanose or pyranose structure), a substituted sugar or a
cyclic or heterocyclic alkane or aryl group. See, e.g., U.S. Patent No.
5,602,140 for compounds of this type.
In the case of pentostatin, X-R2 is -CH2CH(OH)-. In this case a
second carbon atom is inserted in the ring between X and the adjacent nitrogen
atom. The X-N double bond becomes a single bond.
58
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
U.S. Patent No. 5,446,139 describes suitable purine analogues of
the type shown in the formula.
R~
X~
\Y
wherein N signifies nitrogen and V, W, X, Z can be either carbon or nitrogen
with the following provisos. Ring A may have 0 to 3 nitrogen atoms in its
structure. If two nitrogens are present in ring A, one must be in the W
position.
If only one is present, it must not be in the Q position. V and Q must not be
simultaneously nitrogen. Z and Q must not be simultaneously nitrogen. If Z is
nitrogen, R3 is not present. Furthermore, R~_3 are independently one of H,
halogen, C~_~ alkyl, C~_~ alkenyl, hydroxyl, mercapto, C~_~ alkylthio, C~_~
alkoxy,
C~_7 alkenyloxy, aryl oxy, nitro, primary, secondary or tertiary amine
containing
group. R5_$ are H or up to two of the positions may contain independently one
of OH, halogen, cyano, azido, substituted amino, R5 and R~ can together form a
double bond. Y is H, a C~_7 alkylcarbonyl, or a mono- di or tri phosphate.
Exemplary suitable purine analogues include 6-mercaptopurine,
thiguanosine, thiamiprine, cladribine, fludaribine, tubercidin, puromycin,
pentoxyfilline; where these compounds may optionally be phosphorylated.
Exemplary compounds have the structures:
59
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Ra
R~~N~ ~~
R~
Ft, FtzRs A: o~ B,'Ho
6-Mercaptopurine SH H
H
ThioguanosineNHzSH B~ j
OH OH
ThiamiprineNHzA H
Ba:
CladribineCINHzBz Ho Ho
FludarabineF NH,Ba H o"
PuromycinH N(CH,)z ON ON
Bq
Tubercidin H NHz B, Be'
H
NH OH
CH3
O N
HaC N
N
~Hg
Pentoxyfilline
These compounds are thought to function as cell cycle inhibitors
by serving as antimetabolites of purine.
In another aspect, the cell cycle inhibitor is a nitrogen mustard.
Many suitable nitrogen mustards are known and are suitably used as a cell
cycle inhibitor in the present invention. Suitable nitrogen mustards are also
known as cyclophosphamides.
A preferred nitrogen mustard has the general structure:
R1
N
A~ ~CI
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
where A is:
P\O
N~
Rz
R3
or -CH3 or other alkane, or chloronated alkane, typically CH2CH(CH3)CI, or a
polycyclic group such as B, or a substituted phenyl such as C or a
heterocyclic
group such as D.
HO
o 1l
O
H3~ H ~~''H
~~''H
HOOC
NHZ
(iii)
H
N
O
H
(IV)
Examples of suitable nitrogen mustards are disclosed in U.S.
Patent No. 3,808,297, wherein A is:
61
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
O~P
\O
N~
R2
R3
R~_2 are H or CH2CH2C1; R3 is H or oxygen-containing groups such
as hydroperoxy; and R4 can be alkyl, aryl, heterocyclic.
The cyclic moiety need not be intact. See, e.g., U.S. Patent Nos.
5,472,956, 4,908,356, 4,841,085 that describe the following type of structure:
F2
R ~\P~N~CI
O
R Rs N F22
wherein R~ is H or CH~CH2CI, and R2_6 are various substituent groups.
Exemplary nitrogen mustards include methylchloroethamine, and
analogues or derivatives thereof, including methylchloroethamine oxide
10' hydrohchloride, novembichin, and mannomustine (a halogenated sugar).
Exemplary compounds have the structures:
CI
N I CI
R
\ ~ I HCI
R \
CH3
Mechlorethanime CH3 Mechlorethanime Oxide HCI
Novembichin CH~CH(CH3)CI
The nitrogen mustard may be cyclophosphamide, ifosfamide,
perfosfamide, or torofosfamide, where these compounds have the structures:
62
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Ri RZ R3
Cyclophosphamide CHZCH2C1H
H
Ifosfamide CH~CHzCIH H
PerfOSfamide CHZCH~CIH OOH
Torofosfamide CH~CHZCICH2CHZCIH
The nitrogen mustard may be estramustine, or an analogue or
derivative thereof, including phenesterine, prednimustine, and estramustine
P04. Thus, suitable nitrogen mustard type cell cycle inhibitors of the present
invention have the structures:
The nitrogen mustard may be chlorambucil, or an analogue or
derivative thereof, including melphalan and chlormaphazine. Thus, suitable
nitrogen mustard type cell cycle inhibitors of the present invention have the
structures:
63
R
Estramustine OH
Phenesterine C(CH3)(CHZ)3CH(CH3)z
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
N I
R~
R~ R3 I
R~ RZ R3
Chlorambucil H H
CH2COOH
Melphalan COOH NHZ H
Chlornaphazine together
H forms
a
benzene
ring
The nitrogen mustard may be uracil mustard, which has the
structure:
H
O V ~ ~ I
H
CI
The nitrogen mustards are thought to function as cell cycle
inhibitors by serving as alkylating agents for DNA. Nitrogen mustards have
been shown useful in the treatment of cell proliferative disorders including,
for
example, small cell lung, breast, cervical, head and neck, prostate,
retinoblastoma, and soft tissue sarcoma.
The cell cycle inhibitor of the present invention may be a
hydroxyurea. Hydroxyureas have the following general structure:
0
R3 O-X
~N N~
R2 R~
Suitable hydroxyureas are disclosed in, for example, U.S. Patent
No. 6,080,874, wherein R~ is:
S
R2
Rs
64
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
and R2 is an alkyl group having 1-4 carbons and R3 is one of H, acyl, methyl,
ethyl, and mixtures thereof, such as a methylether.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 5,665,768, wherein R~ is a cycloalkenyl group, for example N-(3-(5-(4-
fluorophenylthio)-furyl)-2-cyclopenten-1-yl)N-hydroxyurea; R2 is H or an alkyl
group having 1 to 4 carbons and R3 is H; X is H or a cation.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 4,299,778, wherein R~ is a phenyl group substituted with on or more
fluorine atoms; R2 is a cyclopropyl group; and R3 and X is H.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 5,066,658, wherein R2 and R3 together with the adjacent nitrogen form:
c~~Zm
Y . N-
(CHZ)m
wherein m is 1 or 2, n is 0-2 and Y is an alkyl group.
In one aspect, the hydroxy urea has the structure:
0
,oH
H2N NH
Hydroxyurea
Hydroxyureas are thought to function as cell cycle inhibitors by
serving to inhibit DNA synthesis.
In another aspect, the cell cycle inhibitor is a mytomicin, such as
mitomycin C, or an analogue or derivative thereof, such as porphyromycin.
Exemplary compounds have the structures:
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
R
Mitomycin C H
Porphyromycin CH3
(N-methyl Mitomycin C)
These compounds are thought to function as cell cycle inhibitors
by serving as DNA alkylating agents. Mitomycins have been shown useful in
the treatment of cell proliferative disorders such as, for example,
esophageal,
liver, bladder, and breast cancers.
In another aspect, the cell cycle inhibitor is an alkyl sulfonate,
such as busulfan, or an analogue or derivative thereof, such as treosulfan,
improsulfan, piposulfan, and pipobroman. Exemplary compounds have the
structures:
0
H ~~ ~R~/O ~~ CH3
O p
R
Busulfan single bond
Improsulfan -CHz NH-GHZ
Piposulfan ~ p
~--N~i --~
0
B N Br
O
Pipobroman
These compounds are thought to function as cell cycle inhibitors
by serving as DNA alkylating agents.
66
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another aspect, the cell cycle inhibitor is a benzamide. In yet
another aspect, the cell cycle inhibitor is a nicotinamide. These compounds
have the basic structure:
A
wherein X is either O or S; A is commonly NH2 or it can be OH or an alkoxy
group; B is N or C-R4, where R4 is H or an ether-linked hydroxylated alkane
such as OCH2CH20H, the alkane may be linear or branched and may contain
one or more hydroxyl groups. Alternately, B may be N-R5 in which case the
double bond in the ring involving B is a single bond. R5 may be H, and alkyl
or
an aryl group (see, e.g., U.S. Patent No. 4,258,052); R2 is H, OR6, SR6 or
NHR6, where R6 is an alkyl group; and R3 is H, a lower alkyl, an ether linked
lower alkyl such as -O-Me or-O-ethyl (see, e.g., U.S. Patent No. 5,215,738).
Suitable benzamide compounds have the structures:
x
z
~NHZ
Y
N
Benzamides
X=OorS
Y = H, OR, CH3, or acetoxy
Z = H, OR, SR, or NHR
R = alkyl group
where additional compounds are disclosed in U.S. Patent No. 5,215,738,
(listing some 32 compounds).
Suitable nicotinamide compounds have the structures:
67
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
x
z
\~ ~NHZ
N
Nicotinamides
X=OorS
Z = H, OR, SR, NHR
R = alkyl group
where additional compounds are disclosed in U.S. Patent No.
5,215,738,
R O
z O
Rz /I'I~
N-~~-NH~O~R~
Rz~ I O
N
Rz ~ H3C N
O
Rz Ra
Rz Rz ~II
N O"NHz
R' Ra
Benzodepa phenyl H
CH3
Meturedepa CH3 CH3 Carboquone
Uredepa CH3 H
In another aspect, the cell cycle inhibitor is a halogenated sugar,
such as mitolactol, or an analogue or derivative thereof, including
mitobronitol
and mannomustine. Examplary compounds have the structures:
CH~Br CHpBr CHzNHZ~CH2CHzCl
H OH HO H HO H
HO H NO H HO H
HO H H OH H OH
H OH H OH H OH
CHaBr CHaBr CHaNHp+CHZCH~CI
Mitolactol Mitobronitol Mannomustine
In another aspect, the cell cycle inhibitor is a diazo compound,
such as azaserine, or an analogue or derivative thereof, including 6-diazo-5-
oxo-L-norleucine and 5-diazouracil (also a pyrimidine analog). Examplary
compounds have the structures:
68
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
O
N=N~ Rt~RZ
OH
O N H~
R~ Rz
Azaserine O single bond
6-diazo-5-oxo-
L-norleucine single bond GHz
Other compounds that may serve as cell cycle inhibitors
according to the present invention are pazelliptine; wortmannin;
metoclopramide; RSU; buthionine sulfoxime; tumeric; curcumin; AG337, a
thymidylate synthase inhibitor; levamisole; lentinan, a polysaccharide;
razoxane, an EDTA analogue; indomethacin; chlorpromazine; a and ~3
interferon; MnBOPP; gadolinium texaphyrin; 4-amino-1,8-naphthalimide;
staurosporine derivative of CGP; and SR-2508.
Thus, in one aspect, the cell cycle inhibitor is a DNA alylating
agent. In another aspect, the cell cycle inhibitor is an anti-microtubule
agent.
In another aspect, the cell cycle inhibitor is a topoisomerase inhibitor. In
another aspect, the cell cycle inhibitor is a DNA cleaving agent. In another
aspect, the cell cycle inhibitor is an antimetabolite. In another aspect, the
cell
cycle inhibitor functions by inhibiting adenosine deaminase (e.g., as a purine
analogue). In another aspect, the cell cycle inhibitor functions by inhibiting
purine ring synthesis and/or as a nucleotide interconversion inhibitor (e.g.,
as a
purine analogue such as mercaptopurine). In another aspect, the cell cycle
inhibitor functions by inhibiting dihydrofolate reduction and/or as a
thymidine
monophosphate block (e.g., methotrexate). In another aspect, the cell cycle
inhibitor functions by causing DNA damage (e.g., bleomycin). In another
aspect, the cell cycle inhibitor functions as a DNA intercalation agent and/or
RNA synthesis inhibition (e.g., doxorubicin, aclarubicin, or detorubicin
(acetic
acid, diethoxy-, 2-(4-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-
1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-
naphthacenyl)-2-oxoethyl ester, (2S-cis)-)). In another aspect, the cell cycle
69
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
inhibitor functions by inhibiting pyrimidine synthesis (e.g., N-
phosphonoacetyl-L-
aspartate). In another aspect, the cell cycle inhibitor functions by
inhibiting
ribonucleotides (e.g., hydroxyurea). In another aspect, the cell cycle
inhibitor
functions by inhibiting thymidine monophosphate (e.g., 5-fluorouracil). In
another aspect, the cell cycle inhibitor functions by inhibiting DNA synthesis
(e.g., cytarabine). In another aspect, the cell cycle inhibitor functions by
causing DNA adduct formation (e.g., platinum compounds). In another aspect,
the cell cycle inhibitor functions by inhibiting protein synthesis (e.g., L-
asparginase). In another aspect, the cell cycle inhibitor functions by
inhibiting
microtubule function (e.g., taxanes). In another aspect, the cell cycle
inhibitor
acts at one or more of the steps in the biological pathway shown in FIG. 1.
Additional cell cycle inhibitor s useful in the present invention, as
well as a discussion of the mechanisms of action, may be found in Hardman
J.G., Limbird L.E. Molinoff R.B., Ruddon R W., Gilman A.G. editors,
Chemotherapy of Neoplastic Diseases in Goodman and Gilman's The
Pharmacological Basis of Therapeutics Ninth Edition, McGraw-Hill Health
Professions Division, New York, 1996, pages 1225-1287. See also U.S. Patent
Nos. 3,387,001; 3,808,297; 3,894,000; 3,991,045; 4,012,390; 4,057,548;
4,086,417; 4,144,237; 4,150,146; 4,210,584; 4,215,062; 4,250,189; 4,258,052;
4,259,242; 4,296,105; 4,299,778; 4,367,239; 4,374,414; 4,375,432; 4,472,379;
4,588,831; 4,639,456; 4,767,855; 4,828,831; 4,841,045; 4,841,085; 4,908,356;
4,923,876; 5,030,620; 5,034,320; 5,047,528; 5,066,658; 5,166,149; 5,190,929;
5,215,738; 5,292,731; 5,380,897; 5,382,582; 5,409,915; 5,440,056; 5,446,139;
5,472,956; 5,527,905; 5,552,156; 5,594,158; 5,602,140; 5,665,768; 5,843,903;
6,080,874; 6,096,923; and RE030561.
In another embodiment, the cell-cycle inhibitor is camptothecin,
mitoxantrone, etoposide, 5-fluorouracil, doxorubicin, methotrexate, peloruside
A, mitomycin C, or a CDK-2 inhibitor or an analogue or derivative of any
member of the class of listed compounds.
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another embodiment, the cell-cycle inhibitor is HTI-286,
plicamycin; or mithramycin, or an analogue or derivative thereof.
Other examples of cell cycle inhibitors also include, e.g., 7-
hexanoyltaxol (QP-2), cytochalasin A, lantrunculin D, actinomycin-D, Ro-31
7453 (3-(6-nitro-1-methyl-3-indolyl)-4-(1-methyl-3-indolyl}pyrrole-2,5-dione),
PNU-151807, brostallicin, C2-ceramide, cytarabine ocfosfate (2(1 H)
pyrimidinone, 4-amino-1-(5-O-(hydroxy(octadecyloxy)phosphinyl)-f3-D-
arabinofuranosyl)-, monosodium salt), paclitaxel (5f3,20-epoxy-1,2
alpha,4,7f3,10f3,13 alpha-hexahydroxytax-11-en-9-one-4,10-diacetate-2-
benzoate-13-(alpha-phenylhippurate)), doxorubicin (5,12-naphthacenedione,
10-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-
tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-, (8S)-cis-),
daunorubicin (5,12-naphthacenedione, 8-acetyl-10-((3-amino-2,3,6-trideoxy-
alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihyd roxy-1-
methoxy-, (8S-cis)-), gemcitabine hydrochloride (cytidine, 2'-deoxy-2', 2'-
difluoro-,monohydrochloride), nitacrine (1,3-propanediamine, N,N-dimethyl-N'-
(1-nitro-9-acridinyl)-), carbopiatin (platinum, diammine(1,1-
cyclobutanedicarboxylato(2-))-, (SP-4-2)-), altretamine (1,3,5-triazine-2,4,6-
triamine, N,N,N',N',N",N"-hexamethyl-), teniposide (furo(3',4':6,7)naphtho(2,3-
d)-1,3-dioxol-6(5aH)-one, 5,8,8a,9-tetrahydro-5-(4-hydroxy-3,5-
dimethoxyphenyl)-9-((4,6-O-(2-thienylmethylene)-f3-D-glucopyranosyl)oxy)-,
(5R-(5alpha,5a(3,8aAlpha,9(3(R*)))-), eptaplatin (platinum, ((4R,5R)-2-(1-
methylethyl)-1,3-dioxolane-4,5-dimethanamine-kappa N4,kappa
N5)(propanedioato(2-)-kappa 01, kappa 03)-, (SP-4-2)-), amrubicin
hydrochloride (5,12-naphthacenedione, 9-acetyl-9-amino-7-((2-deoxy-f3-D-
erythro-pentopyranosyl)oxy)-7,8,9,10-tetrahydro-6,11-dihydroxy-,
hydrochloride,
(7S-cis)-), ifosfamide (2H-1,3,2-oxazaphosphorin-2-amine, N,3-bis(2-
chloroethyl)tetrahydro-,2-oxide), cladribine (adenosine, 2-chloro-2'-deoxy-),
mitobronitol (D-mannitol, 1,6-dibromo-1,6-dideoxy-), fludaribine phosphate (9H-
purin-6-amine, 2-fluoro-9-(5-O-phosphono-f3-D-arabinofuranosyl)-), enocitabine
71
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(docosanamide, N-(1-f3-D-arabinofuranosyl-1,2-dihydro-2-oxo-4-pyrimidinyl)-),
vindesine (vincaleukoblastine, 3-(aminocarbonyl)-O4-deacetyl-3-
de(methoxycarbonyl)-), idarubicin (5,12-naphthacenedione, 9-acetyl-7-((3-
amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-
6,9,11-trihydroxy-, (7S-cis)-), zinostatin (neocarzinostatin), vincristine
(vincaleukoblastine, 22-oxo-), tegafur (2,4(1 H,3H)-pyrimidinedione, 5-fluoro-
1-
(tetrahydro-2-furanyl)-), razoxane (2,6-piperazinedione, 4,4'-(1-methyl-1,2-
ethanediyl)bis-), methotrexate (L-glutamic acid, N-(4-(((2,4-diamino-6-
pteridinyl)methyl)methylamino)benzoyl)-), raltitrexed (L-glutamic acid, N-((5-
(((1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-
thienyl)carbonyl)-), oxaliplatin (platinum, (1,2-cyclohexanediamine-
N,N')(ethanedioato(2-)-O,O')-, (SP-4-2-(1 R-trans))-), doxifluridine (uridine,
5'-
deoxy-5-fluoro-), mitolactol (galactitol, 1,6-dibromo-1,6-dideoxy-),
piraubicin
(5,12-naphthacenedione, 10-((3-amino-2,3,6-trideoxy-4-O-(tetrahydro-2H-
pyran-2-yl)-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-
trihydroxy-8-(hydroxyacetyl)-1-methoxy-, (8S-(8 alpha, 10 alpha(S*)))-),
docetaxel ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester
with 5(3,20-epoxy-1,2 alpha,4,7f3,10(3,13 alpha-hexahydroxytax-11-en-9-one 4-
acetate 2-benzoate-), capecitabine (cytidine, 5-deoxy-5-fluoro-N-
((pentyloxy)carbonyl)-), cytarabine (2(1 H)-pyrimidone, 4-amino-1-f3-D-arabino
furanosyl-), valrubicin (pentanoic acid, 2-(1,2,3,4,6,11-hexahydro-2,5,12-
trihydroxy-7-methoxy-6,11-dioxo-4-((2,3,6-trideoxy-3-((trifluoroacetyl)amino)-
alpha-L-lyxo-hexopyranosyl)oxy)-2-naphthacenyl)-2-oxoethyl ester (2S-cis)-),
trofosfamide (3-2-(chloroethyl)-2-(bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-
oxazaphosphorin 2-oxide), prednimustine (pregna-1,4-diene-3,20-dione, 21-(4-
(4-(bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy)-11,17-dihydroxy-, (11(3)-),
lomustine (Urea, N-(2-chloroethyl)-N'-cyclohexyl-N-nitroso-), epirubicin (5,12-
naphthacenedione, 10-((3-amino-2,3,6-trideoxy-alpha-L-arabino-
hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-
methoxy-, (8S-cis)-), or an analogue or derivative thereof).
72
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
5) Cyclin Dependent Protein Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a cyclin dependent protein kinase inhibitor (e.g., R-
roscovitine, CYC-101, CYC-103, CYC-400, MX-7065, alvocidib (4H-1-
Benzopyran-4-one, 2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-
piperidinyl)-, cis-(-)-), SU-9516, AG-12275, PD-0166285, CGP-79807,
fascaplysin, GW-8510 (benzenesulfonamide, 4-(((Z)-(6,7-dihydro-7-oxo-8H-
pyrrolo(2,3-g)benzothiazol-8-ylidene)methyl)amino)-N-(3-hydroxy-2,2-
dimethylpropyl)-), GW-491619, Indirubin 3' monoxime, GW8510, AZD-5438,
ZK-CDK or an analogue or derivative thereof).
6) EGF (Epidermal Growth Factor) Receptor Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an EGF (epidermal growth factor) kinase inhibitor
(e.g.,
erlotinib (4-quinazolinamine, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-,
monohydrochloride), erbstatin, BIB?C-1382, gefitinib (4-quinazolinamine, N-(3-
chloro-4-fluorophenyl)-7-methoxy-6-(3-(4-morpholinyl)propoxy)), or an analogue
or derivative thereof).
7) Elastase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an elastase inhibitor (e.g., ONO-6818, sivelestat
sodium
hydrate (glycine, N-(2-(((4-(2,2-dimethyl-1-
oxopropoxy)phenyl)sulfonyl)amino)benzoyl)-), erdosteine (acetic acid, ((2-oxo-
2-((tetrahydro-2-oxo-3-thienyl)amino)ethyl)thio)-), MDL-100948A, MDL-104238
(N-(4-(4-morpholinylcarbonyl)benzoyl)-L-valyl-N'-(3,3,4,4,4-pentafluoro-1-(1-
methylethyl)-2-oxobutyl)-L-2-azetamide), MDL-27324 (L-prolinamide, N-((5-
(dimethylamino)-1-naphthalenyl)sulfonyl)-L-alanyl-L-alanyl-N-(3,3,3-trifluoro-
1-
(1-methylethyl)-2-oxopropyl)-, (S)-), SR-26831 (thieno(3,2-c)pyridinium, 5-((2-
chlorophenyl)methyl)-2-(2,2-dimethyl-1-oxopropoxy)-4,5,6,7-tetrahydro-5-
73
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
hydroxy-), Win-68794, Win-63110, SSR-69071 (2-(9(2-piperidinoethoxy)-4-oxo-
4H-pyrido(1,2-a)pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methyoxy-1,2-
benzisothiazol-3(2H)-one-1,1-dioxide), (N(Alpha)-(1-
adamantylsulfonyl)N(epsilon)-succinyl-L-lysyl-L-prolyl-L-valinal), Ro-31-3537
(N
alpha-(1-adamantanesulphonyl)-N-(4-carboxybenzoyl)-L-lysyl-alanyl-L-valinal),
R-665, FCE-28204, ((6R,7R)-2-(benzoyloxy)-7-methoxy-3-methyl-4-pivaloyl-3-
cephem 1,1-dioxide), 1,2-benzisothiazol-3(2H)-one, 2-(2,4-dinitrophenyl)-, 1,1-
dioxide, L-658758 (L-proline, 1-((3-((acetyloxy)methyl)-7-methoxy-8-oxo-5-thia-
1-azabicyclo(4.2.0)oct-2-en-2-yl)carbonyl)-, S,S-dioxide, (6R-cis)-), L-659286
(pyrrolidine, 1-((7-methoxy-8-oxo-3-(((1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-
1,2,4-triazin-3-yl)thio)methyl)-5-this-1-azabicyclo(4.2.0)oct-2-en-2-
yl)carbonyl)-,
S,S-dioxide, (6R-cis)-), L-680833 (benzeneacetic acid, 4-((3,3-diethyl-1-(((1-
(4-
methylphenyl)butyl)amino)carbonyl)-4-oxo-2-azetidinyl)oxy)-, (S-(R*,S*))-), FK-
706 (L-prolinamide, N-(4-(((carboxymethyl)amino)carbonyl)benzoyl)-L-valyl-N-
(3,3,3-trifluoro-1-(1-methylethyl)-2-oxopropyl)-, monosodium salt), Roche 8-
665, or an analogue or derivative thereof).
8) Factor Xa Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a factor Xa inhibitor (e.g., CY-222, fondaparinux
sodium
(alpha-D-glucopyranoside, methyl O-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-
glucopyranosyl-( 1-4)-O-f3-D-glucopyranuronosyl-( 1-4)-O-2-deoxy-3,6-di-O-
sulfo-2-(sulfoamino)-alpha-D-glucopyranosyl-( 1-4)-O-2-O-sulfo-alpha-L-
idopyranuronosyl-(1-4)-2-deoxy-2-(sulfoamino)-, 6-(hydrogen sulfate)),
danaparoid sodium, or an analogue or derivative thereof).
9) Farnesyltransferase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a farnesyltransferase inhibitor (e.g.,
dichlorobenzoprim
(2,4-diamino-5-(4-(3,4-dichlorobenzylamino)-3-nitrophenyl)-6-ethylpyrimidine),
74
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
B-581, B-956 (N-(8(R)-amino-2(S)-benzyl-5(S)-isopropyl-9-sulfanyl-3(Z),6(E)-
nonadienoyl)-L-methionine), OSI-754, perillyl alcohol (1-cyclohexene-1-
methanol, 4-(1-methylethenyl)-, RPR-114334, lonafarnib (1-
piperidinecarboxamide, 4-(2-(4-((11 R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-
benzo(5,6)cyclohepta(1,2-b)pyridin-11-yl)-1-piperidinyl)-2-oxoethyl)-), Sch-
48755, Sch-226374, (7,8-dichloro-5H-dibenzo(b,e)(1,4)diazepin-11-y1)-pyridin-
3-ylmethylamine, J-104126, L-639749, L-731734 (pentanamide, 2-((2-((2-
amino-3-mercaptopropyl)amino)-3-methylpentyl)amino)-3-methyl-N-(tetrahydro-
2-oxo-3-furanyl)-, (3S-(3R*(2R*(2R*(S*),3S*),3R*)))-), L-744832 (butanoic
acid,
2-((2-((2-((2-amino-3-mercaptopropyl)amino)-3-methylpentyl)oxy)-1-oxo-3-
phenylpropyl)amino)-4-(methylsulfonyl)-, 1-methylethyl ester, (2S-
(1(R*(R*)),2R*(S*),3R*))-), L-745631 (1-piperazinepropanethiol, f3-amino-2-(2-
methoxyethyl)-4-(1-naphthalenylcarbonyl)-, (f3R,2S)-), N-acetyl-N-
naphthylmethyl-2(S)-((1-(4-cyanobenzyl)-1 H-imidazol-5-yl)acetyl)amino-3(S)-
methylpentamine, (2alpha)-2-hydroxy-24,25-dihydroxylanost-8-en-3-one, BMS-
316810, UCF-1-C (2,4-decadienamide, N-(5-hydroxy-5-(7-((2-hydroxy-5-oxo-1-
cyclopenten-I-yl)amino-oxo-1,3,5-heptatrienyl)-2-oxo-7-oxabicyclo(4.1.0)hept-3-
en-3-yl)-2,4,6-trimethyl-, (1S-(1alpha,3(2E,4E,6S*),5 alpha, 5(1E,3E,5E), 6
alpha))-), UCF-116-B, ARGLABIN (3H-oxireno(8,8a)azuleno(4,5-b)furan-
8(4aH)-one, 5,6,6a,7,9a,9b-hexahydro-1,4a-dimethyl-7-methylene-,
(3aR,4aS,6aS,9aS,9bR)-) from ARGLABIN - Paracure, Inc. (Virginia Beach,
VA), or an analogue or derivative thereof).
10) Fibrinogen Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a fibrinogen antagonist (e.g., 2(S)-((p-
toluenesulfonyl)amino)-3-(((5,6,7,8,-tetrahydro-4-oxo-5-(2-(piperidin-4-
yl)ethyl)-
4H-pyrazolo-(1,5-a)(1,4)diazepin-2-yl)carbonyl)-amino)propionic acid,
streptokinase (kinase (enzyme-activating), strepto-), urokinase (kinase
(enzyme-activating), uro-), plasminogen activator, pamiteplase, monteplase,
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
heberkinase, anistreplase, alteplase, pro-urokinase, picotamide (1,3-
benzenedicarboxamide, 4-methoxy-N,N'-bis(3-pyridinylmethyl)-), or an
analogue or derivative thereof).
11 ) Guanylate Cyclase Stimulants
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a guanylate cyclase stimulant (e.g., isosorbide-5-
mononitrate (D-glucitol, 1,4:3,6-dianhydro-, 5-nitrate), or an analogue or
derivative thereof).
12) Heat Shock Protein 90 Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a heat shock protein 90 antagonist (e.g., geldanamycin;
NSC-33050 (17-allylaminogeldanamycin), rifabutin (rifamycin XIV, 1',4-
didehydro-1-deoxy-1,4-dihydro-5'-(2-methylpropyl)-1-oxo-), 17AAG, or an
analogue or derivative thereof).
13) HMGCoA Reductase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an HMGCoA reductase inhibitor (e.g., BCP-671, BB-
476, fluvastatin (6-heptenoic acid, 7-(3-(4-fluorophenyl)-1-(1-methylethyl)-1
H-
indol-2-yl)-3,5-dihydroxy-, monosodium salt, (R*,S*-(E))-(~)-), dalvastatin
(2H-
pyran-2-one, 6-(2-(2-(2-(4-fluoro-3-methylphenyl)-4,4,6,6-tetramethyl-1-
cyclohexen-1-yl)ethenyl)tetrahydro)-4-hydroxy-, (4alpha,6f3(E))-(+/-)-),
glenvastatin (2H-pyran-2-one, 6-(2-(4-(4-fluorophenyl)-2-(1-methylethyl)-6-
phenyl-3-pyridinyl)ethenyl)tetrahydro-4-hydroxy-, (4R-(4alpha,6(3(E)))-), S-
2468,
N-(1-oxododecyl)-4Alpha,10-dimethyl-8-aza-trans-decal-3(3-0l, atorvastatin
calcium (1 H-Pyrrole-1-heptanoic acid, 2-(4-fluorophenyl)-(3,delta-dihydroxy-5-
(1-methylethyl)-3-phenyl-4-((phenylamino)carbonyl)-, calcium salt (R-(R*,R*))-
),
CP-83101 (6,8-nonadienoic acid, 3,5-dihydroxy-9,9-diphenyl-, methyl ester,
76
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(R*,S*-(E))-(+/-)-), pravastatin (1-naphthaleneheptanoic acid, 1,2,6,7,8,8a-
hexahydro-(3,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-,
monosodium salt, (1 S-(1 alpha(f3S*,deltaS*),2 alpha,6 alpha,8f3(R*),8a
alpha))-
), U-20685, pitavastatin (6-heptenoic acid, 7-(2-cyclopropyl-4-(4-
fluorophenyl)-
3-quinolinyl)-3,5-dihydroxy-, calcium salt (2:1), (S-(R*,S*-(E)))-), N-((1-
methylpropyl)carbonyl)-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-
perhydro-isoquinoline, dihydromevinolin (butanoic acid, 2-methyl-,
1,2,3,4,4a,7,8,8a-octahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-
pyran-2-yl)ethyl)-1-naphthalenyl ester(1 alpha(R*), 3 alpha, 4a
alpha,7f~,8f3(2S*,4S*),8af3))-), HBS-107, dihydromevinolin (butanoic acid, 2-
methyl-, 1,2,3,4,4a,7,8,8a-octahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-
oxo-2H-pyran-2-yl)ethyl)-1-naphthalenyl ester(1 alpha(R*), 3 alpha,4a
alpha,7f3,8f3(2S*,4S*),8af~))-), L-669262 (butanoic acid, 2,2-dimethyl-,
1,2,6,7,8,8a-hexahydro-3,7-dimethyl-6-oxo-8-(2-(tetrahydro-4-hydroxy-6-oxo-
2H-pyran-2-yl)ethyl)-1-naphthalenyl(1S-(1Alpha,7(3,8(3(2S*,4S*),8af3))-),
simvastatin (butanoic acid, 2,2-dimethyl-, 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-
8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenyl ester,
(1S-(1alpha, 3alpha,7f3,8(3(2S*,4S*),8af3))-), rosuvastatin calcium (6-
heptenoic
acid, 7-(4-(4-fluorophenyl)-6-(1-methylethyl)-2-(methyl(methylsulfonyl)amino)-
5-
pyrimdinyl)-3,5-dihydroxy- calcium salt (2:1 ) (S-(R*, S*-(E)))), meglutol (2-
hydroxy-2-methyl-1,3-propandicarboxylic acid), lovastatin (butanoic acid, 2-
methyl-, 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-
2H-pyran-2-yl)ethyl)-1-naphthalenyl ester, (1S-(1 alpha.(R*),3
alpha,7f~,8f3(2S*,4S*),8af3))-), or an analogue or derivative thereof).
14) Hydroorotate Dehydrogenase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a hydroorotate dehydrogenase inhibitor (e.g.,
leflunomide (4-isoxazolecarboxamide, 5-methyl-N-(4-(trifluoromethyl)phenyl)-),
laflunimus (2-propenamide, 2-cyano-3-cyclopropyl-3-hydroxy-N-(3-methyl-
77
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
4(trifluoromethyl)phenyl)-, (Z)-), or atovaquone (1,4-naphthalenedione, 2-(4-
(4-
chlorophenyl)cyclohexyl)-3-hydroxy-, traps-, or an analogue or derivative
thereof).
15) IKK2lnhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an IKK2 inhibitor (e.g., MLN-120B, SPC-839, or an
analogue or derivative thereof).
16) IL-1, ICE and IRAK Antagonists
In another embodiri~ent, the pharmacologically active fibrosis-
inhibiting compound is an IL-1, ICE or an IRAK antagonist (e.g., E-5090 (2-
propenoic acid, 3-(5-ethyl-4-hydroxy-3-methoxy-1-naphthalenyl)-2-methyl-, (Z)-
), CH-164, CH-172, CH-490, AMG-719, iguratimod (N-(3-(formylamino)-4-oxo-
6-phenoxy-4H-chromen-7-yl) methanesulfonamide), AV94-88, pralnacasan
(6H-pyridazino(1,2-a)(1,2)diazepine-1-carboxamide, N-((2R,3S)-2-
ethoxytetrahydro-5-oxo-3-furanyl)octahydro-9-((1-isoquinolinylcarbonyl)amino)-
6,10-dioxo-, (1S,9S)-), (2S-cis)-5-(benzyloxycarbonylamino-1,2,4,5,6,7-
hexahydro-4-(oxoazepino(3,2,1-hi)indole-2-carbonyl)-amino)-4-oxobutanoic
acid, AVE-9488, esonarimod (benzenebutanoic acid, alpha-((acetylthio)methyl)-
4-methyl-gamma-oxo-), pralnacasan (6H-pyridazino(1,2-a)(1,2)diazepine-1-
carboxamide, N-((2R,3S)-2-ethoxytetrahydro-5-oxo-3-furanyl)octahydro-9-((1-
isoquinolinylcarbonyl)amino)-6,10-dioxo-, (1S,9S)-), tranexamic acid
(cyclohexanecarboxylic acid, 4-(aminomethyl)-, traps-), Win-72052, romazarit
(Ro-31-3948) (propanoic acid, 2-((2-(4-chlorophenyl)-4-methyl-5-
oxazolyl)methoxy)-2-methyl-), PD-163594, SDZ-224-015 (L-alaninamide N-
((phenylmethoxy)carbonyl)-L-valyl-N-((1S)-3-((2,6-dichlorobenzoyl)oxy)-1-(2-
ethoxy-2-oxoethyl)-2-oxopropyl)-), L-709049 (L-alaninamide, N-acetyl-L-tyrosyl-
L-valyl-N-(2-carboxy-1-formylethyl)-, (S)-), TA-383 (1 H-imidazole, 2-(4-
chlorophenyl)-4,5-dihydro-4,5-diphenyl-, monohydrochloride, cis-), EI-1507-1
78
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(6a,12a-epoxybenz(a)anthracen-1,12(2H,7H)-dione, 3,4-dihydro-3,7-dihydroxy-
8-methoxy-3-methyl-), ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-
triazol-1-yl methyl)quinoline-3-carboxylate, EI-1941-1, TJ-114, anakinra
(interleukin 1 receptor antagonist (human isoform x reduced), N2-L-methionyl-
),
IX-207-887 (acetic acid, (10-methoxy-4H-benzo(4,5)cyclohepta(1,2-b)thien-4-
ylidene)-), K-832, or an analogue or derivative thereof).
17) IL-4 Aaonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an IL-4 agonist (e.g., glatiramir acetate (L-glutamic
acid,
polymer with L-alanine, L-lysine and L-tyrosine, acetate (salt)), or an
analogue
or derivative thereof).
18) Immunomodulatory Agents
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an immunomodulatory agent (e.g., biolimus, ABT-578,
methylsulfamic acid 3-(2-methoxyphenoxy)-2-
(((methylamino)sulfonyl)oxy)propyl ester, sirolimus (also referred to as
rapamycin or RAPAMUNE (American Home Products, Inc., Madison, NJ)), CCI-
779 (rapamycin 42-(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate)), LF-15-
0195, NPC15669 (L-leucine, N-(((2,7-dimethyl-9H-fluoren-9-
yl)methoxy)carbonyl)-), NPC-15670 (L-leucine, N-(((4,5-dimethyl-9H-fluoren-9-
yl)methoxy)carbonyl)-), NPC-16570 (4-(2-(fluoren-9-yl)ethyloxy-
carbonyl)aminobenzoic acid), sufosfamide (ethanol, 2-((3-(2-
chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-yl)amino)-,
methanesulfonate (ester), P-oxide), tresperimus (2-(N-(4-(3-
aminopropylamino)butyl)carbamoyloxy)-N-(6-guanidinohexyl)acetamide), 4-(2-
(fluoren-9-yl)ethoxycarbonylamino)-benzo-hydroxamic acid, iaquinimod, PBI-
1411, azathioprine (6-((1-Methyl-4-nitro-1 H-imidazol-5-yl)thio)-1 H-purine),
PB10032, beclometasone, MDL-28842 (9H-purin-6-amine, 9-(5-deoxy-5-fluoro-
79
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(3-D-threo-pent-4-enofuranosyl)-, (Z)-), FK-788, AVE-1726, ZK-90695, ZK-
90695, Ro-54864, didemnin-B, Illinois (didemnin A, N-(1-(2-hydroxy-1-
oxopropyl)-L-prolyl)-, (S)-), SDZ-62-826 (ethanaminium, 2-((hydroxy((1-
((octadecyloxy)carbonyl)-3-piperidinyl)methoxy)phosphinyl)oxy)-N, N, N-
trimethyl-, inner salt), argyrin B ((4S,7S,13R,22R)-13-Ethyl-4-(1 H-indol-3-
ylmethyl)-7-(4-methoxy-1 H-indol-3-ylmethyl)18,22-dimethyl-16-methyl-ene-24-
thia-3,6, 9,12,15,18,21,26-octaazabicyclo(21.2.1 )-hexacosa-1 (25),23(26)-
diene-
2,5,8,11,14,17,20-heptaone), everolimus (rapamycin, 42-O-(2-hydroxyethyl)-),
SAR-943, L-687795, 6-((4-chlorophenyl)sulfinyl)-2,3-dihydro-2-(4-methoxy-
phenyl)-5-methyl-3-oxo-4-pyridazinecarbonitrile, 91Y78 (1 H-imidazo(4,5-
c)pyridin-4-amine, 1-f3-D-ribofuranosyl-), auranofin (gold, (1-thio-f3-D-
glucopyranose 2,3,4,6-tetraacetato-S)(triethylphosphine)-), 27-0-
demethylrapamycin, tipredane (androsta-1,4-dien-3-one, 17-(ethylthio)-9-fluoro-
11-hydroxy-17-(methylthio)-, (11 (3,17 alpha)-), AI-402, LY-178002 (4-
thiazolidinone, 5-((3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl)methylene)-),
SM-8849 (2-thiazolamine, 4-(1-(2-fluoro(1,1'-biphenyl)-4-yl)ethyl)-N-methyl-),
piceatannol, resveratrol, triamcinolone acetonide (pregna-1,4-diene-3,20-
dione,
9-fluoro-11,21-dihydroxy-16,17-((1-methylethylidene)bis(oxy))-, (1113,16
alpha)-
), ciclosporin (cyclosporin A), tacrolimus (15,19-epoxy-3H-pyrido(2,1-
c)(1,4)oxaazacyclotricosine-1,7,20,21 (4H,23H)-tetrone,
5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahyd ro-5,19-d ihydroxy-
3-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl)-14,16-dimethoxy-
4,10,12,18-tetramethyl-8-(2-propenyl)-, (3S-
(3R*(E(1S*,3S*,4S*)),4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*))
-), gusperimus (heptanamide, 7-((aminoiminomethyl)amino)-N-(2-((4-((3-
aminopropyl)amino)butyl)amino)-1-hydroxy-2-oxoethyl)-, (+/-)-), tixocortol
pivalate (pregn-4-ene-3,20-dione, 21-((2,2-dimethyl-1-oxopropyl)thio)-11,17-
dihydroxy-, (11 f3)-), alefacept (1-92 LFA-3 (antigen) (human) fusion protein
with
immunoglobulin G1 (human hinge-CH2-CH3 gamma1-chain), dimer),
halobetasol propionate (pregna-1,4-diene-3,20-dione, 21-chloro-6,9-difluoro-11-
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
hydroxy-16-methyl-17-(1-oxopropoxy)-, (6Alpha,11 f~,16f3)-), iloprost
trometamol
(pentanoic acid, 5-(hexahydro-5-hydroxy-4-(3-hydroxy-4-methyl-1-octen-6-
ynyl)-2(1 H)-pentalenylidene)-), beraprost (1 H-cyclopenta(b)benzofuran-5-
butanoic acid, 2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6-
ynyl)-), rimexolone (androsta-1,4-dien-3-one,11-hydroxy-16,17-dimethyl-17-(1-
oxopropyl)-, (11f5,16Alpha,17f3)-), dexamethasone (pregna-1,4-diene-3,20-
dione,9-fluoro-11,17,21-trihydroxy-16-methyl-, (11f3,16alpha)-), sulindac (cis-
5-
fluoro-2-methyl-1-((p-methylsulfinyl)benzylidene)indene-3-acetic acid),
proglumetacin (1 H-Indole-3-acetic acid, 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-, 2-(4-(3-((4-(benzoylamino)-5-(dipropylamino)-1,5-
dioxopentyl)oxy)propyl)-1-piperazinyl)ethylester, (+/-)-), alclometasone
dipropionate (pregna-1,4-diene-3,20-dione, 7-chloro-11-hydroxy-16-methyl-
17,21-bis(1-oxopropoxy)-, (7alpha,11f~,16alpha)-), pimecrolimus (15,19-epoxy-
3H-pyrido(2,1-c)(1,4)oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone, 3-(2-(4-
chloro-3-methoxycyclohexyl)-1-methyletheny)-8-ethyl-
5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-
14,16-d imethoxy-4,10,12,18-tetramethyl-, (3S-
(3R*(E(1 S*,3S*,4R*)),4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*))
-), hydrocortisone-17-butyrate (pregn-4-ene-3,20-dione, 11,21-dihydroxy-17-(1
oxobutoxy)-, (11f3)-), mitoxantrone (9,10-anthracenedione, 1,4-dihydroxy-5,8
bis((2-((2-hydroxyethyl)amino)ethyl)amino)-), mizoribine (1 H-imidazole-4-
carboxamide, 5-hydroxy-1-f3-D-ribofuranosyl-), prednicarbate (pregna-1,4-
' diene-3,20-dione, 17-((ethoxycarbonyl)oxy)-11-hydroxy-21-(1-oxopropoxy)-,
(11 f3)-), iobenzarit (benzoic acid, 2-((2-carboxyphenyl)amino)-4-chloro-),
glucametacin (D-glucose, 2-(((1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 H-
indol-3-yl)acetyl)amino)-2-deoxy-), fluocortolone monohydrate ((6 alpha)-
fluoro-
16alpha-methylpregna-1,4-dien-11 f5,21-diol-3,20-dione), fluocortin butyl
(pregna-1,4-dien-21-oic acid, 6-fluoro-11-hydroxy-16-methyl-3,20-dioxo-, butyl
ester, (6alpha,11f3,16alpha)-), difluprednate (pregna-1,4-diene-3,20-dione, 21-
(acetyloxy)-6,9-difluoro-11-hydroxy-17-(1-oxobutoxy)-, (6 alpha,11 f3)-),
81
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
diflorasone diacetate (pregna-1,4-diene-3,20-dione, 17,21-bis(acetyloxy)-6,9-
difluoro-11-hydroxy-16-methyl-, (6Alpha,11f3,16f3)-), dexamethasone valerate
(pregna-1,4-diene-3,20-dione, 9-fluoro-11,21-dihydroxy-16-methyl-17-((1-
oxopentyl)oxy)-, (11f3,16Alpha)-), methylprednisolone, deprodone propionate
(pregna-1,4-diene-3,20-dione, 11-hydroxy-17-(1-oxopropoxy)-, (11~)-),
bucillamine (L-cysteine, N-(2-mercapto-2-methyl-1-oxopropyl)-), amcinonide
(benzeneacetic acid, 2-amino-3-benzoyl-, monosodium salt, monohydrate),
acemetacin (1 H-indole-3-acetic acid, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-,
carboxymethyl ester), or an analogue or derivative thereof).
Further, analogues of rapamycin include tacrolimus and
derivatives thereof (e.g., EP0184162B1 and U.S. Patent No. 6,258,823)
everolimus and derivatives thereof (e.g., U.S. Patent No. 5,665,772). Further
representative examples of sirolimus analogues and derivatives can be found in
PCT Publication Nos. WO 97/10502, WO 96/41807, WO 96/35423, WO
96/03430, WO 96/00282, WO 95/16691, WO 95/15328, WO 95/07468, WO
95/04738, WO 95/04060, WO 94/25022, WO 94/21644, WO 94/18207, WO
94/10843, WO 94/09010, WO 94/04540, WO 94/02485, WO 94/02137, WO
94/02136, WO 93/25533, WO 93/18043, WO 93/13663, WO 93/11130, WO
93/10122, WO 93/04680, WO 92/14737, and WO 92/05179. Representative
U.S. patents include U.S. Patent Nos. 6,342,507; 5,985,890; 5,604,234;
5, 597, 715; 5, 583,139; 5, 563,172; 5, 561,228; 5, 561,137; 5, 541,193; 5,
541,189;
5,534,632; 5,527,907; 5,484,799; 5,457,194; 5,457,182; 5,362,735; 5,324,644;
5,318,895; 5,310,903; 5,310,901; 5,258,389; 5,252,732; 5,247,076; 5,225,403;
5,221,625; 5,210,030; 5,208,241; 5,200,411; 5,198,421; 5,147,877; 5,140,018;
5,116,756; 5,109,112; 5,093,338; and 5,091,389.
The structures of sirolimus, everolimus, and tacrolimus are
provided below:
82
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Name Code Name Company Structure
Everolimus SAR-943 Novartis See below
Sirolimus AY-22989 Wyeth See below
RAPAMUNE NSC-226080
Rapamycin
Tacrolimus FK506 Fujusawa See below
a,~'~,..-°
~4
Everolimus
0
1
..~5 0
Tacrolimus
83
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
'i'a a
--~., ~. ~- a .I
a
a
'a~' 41' ~~.,~..- I ai
a~
a
Sirolimus
Further sirolimus analogues and derivatives include tacrolimus
and derivatives thereof (e.g., EP0184162B1 and U.S. Patent No. 6,258,823)
everolimus and derivatives thereof (e.g., US Patent No. 5,665,772). Further
representative examples of sirolimus analogues and derivatives include ABT-
578 and others may be found in PCT Publication Nos. WO 97/10502, WO
96/41807, WO 96135423, WO 96/03430, WO 9600282, WO 95/16691, WO
9515328, WO 95/07468, WO 95/04738, WO 95/04060, WO 94/25022, WO
94/21644, WO 94/18207, WO 94/10843, WO 94/09010, WO 94/04540, WO
94/02485, WO 94/02137, WO 94/02136, WO 93/25533, WO 93/18043, WO
93/13663, WO 93/11130, WO 93/10122, WO 93/04680, WO 92114737, and WO
92/05179. Representative U.S. patents include U.S. Patent Nos. 6,342,507;
5,985,890; 5,604,234; 5,597,715; 5,583,139; 5,563,172; 5,561,228; 5,561,137;
5,541,193; 5,541,189; 5,534,632; 5,527,907; 5,484,799; 5,457,194; 5,457,182;
5,362,735; 5,324,644; 5,318,895; 5,310,903; 5,310,901; 5,258,389; 5,252,732;
5,247,076; 5,225,403; 5,221,625; 5,210,030; 5,208,241, 5,200,411; 5,198,421;
5,147,877; 5,140,018; 5,116,756; 5,109,112; 5,093,338; and 5,091,389.
In one aspect, the fibrosis-inhibiting agent may be, e.g.,
rapamycin (sirolimus), everolimus, biolimus, tresperimus, auranofin, 27-0-
demethylrapamycin, tacrolimus, gusperimus, pimecrolimus, or ABT-578.
84
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
19) Inosine monophosphate dehydroe~enase inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an inosine monophosphate dehydrogenase (IMPDH)
inhibitor (e.g., mycophenolic acid, mycophenolate mofetil (4-hexenoic acid, 6-
(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-
2-(4-morpholinyl)ethyl ester, (E)-), ribavirin (1H-1,2,4-triazole-3-
carboxamide,
1-f3-D-ribofuranosyl-), tiazofurin (4-thiazolecarboxamide, 2-f3-D-
ribofuranosyl-),
viramidine, aminothiadiazole, thiophenfurin, tiazofurin) or an analogue or
derivative thereof. Additional representative examples are included in U.S.
Patent Nos. 5,536,747, 5,807,876, 5,932,600, 6,054,472, 6,128,582, 6,344,465,
6,395,763, 6,399,773, 6,420,403, 6,479,628, 6,498,178, 6,514,979, 6,518,291,
6,541,496, 6,596,747, 6,617,323, 6,624,184, Patent Application Publication
Nos. 2002/0040022A1, 2002/0052513A1, 2002/0055483A1, 2002/0068346A1,
2002/0111378A1, 2002/0111495A1, 2002/0123520A1, 2002/0143176A1,
2002/0147160A1, 2002/0161038A1, 2002/0173491 A1, 2002/0183315A1,
2002/0193612A1, 2003/0027845A1, 2003/0068302A1, 2003/0105073A1,
2003/0130254A1, 2003/0143197A1, 2003/0144300A1, 2003/0166201 A1,
2003/0181497A1, 2003/0186974A1, 2003/0186989A1, 2003/0195202A1, and
PCT Publication Nos. WO 0024725A1, WO 00/25780A1, WO 00/26197A1, WO
00/51615A1, WO 00/56331 A1, WO 00/73288A1, WO 01/00622A1, WO
01/66706A1, WO 01/79246A2, WO 01/81340A2, WO 01/85952A2, WO
02/16382A1, WO 02/18369A2, WO 2051814A1, WO 2057287A2,
W02057425A2, WO 2060875A1, WO 2060896A1, WO 2060898A1, WO
2068058A2, WO 3020298A1, WO 3037349A1, WO 3039548A1, WO
3045901 A2, WO 3047512A2, WO 3053958A1, WO 3055447A2, WO
3059269A2, WO 3063573A2, WO 3087071 A1, WO 90/01545A1, WO
97/40028A1, WO 97/41211 A1, WO 98/40381 A1, and WO 99/55663A1 ).
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
20) Leukotriene Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a leukotreine inhibitor (e.g., ONO-
4057(benzenepropanoic acid, 2-(4-carboxybutoxy)-6-((6-(4-methoxyphenyl)-5-
hexenyl)oxy)-, (E)-), ONO-LB-448, pirodomast 1,8-naphthyridin-2(1 H)-one, 4-
hydroxy-1-phenyl-3-(1-pyrrolidinyl)-, Sch-40120 (benzo(b)(1,8)naphthyridin-
5(7H)-one, 10-(3-chlorophenyl)-6,8,9,10-tetrahydro-), L-656224 (4-
benzofuranol, 7-chloro-2-((4-methoxyphenyl)methyl)-3-methyl-5-propyl-), MAFP
(methyl arachidonyl fluorophosphonate), ontazolast (2-benzoxazolamine, N-(2-
cyclohexyl-1-(2-pyridinyl)ethyl)-5-methyl-, (S)-), amelubant (carbamic acid,
((4-
((3-((4-(1-(4-hydroxyphenyl)-1-
methylethyl)phenoxy)methyl)phenyl)methoxy)phenyl)iminomethyl)- ethyl ester),
SB-201993 (benzoic acid, 3-((((6-((1 E)-2-carboxyethenyl)-5-((8-(4-
methoxyphenyl)octyl)oxy)-2-pyridinyl)methyl)thio)methyl)-), LY-203647
(ethanone, 1-(2-hydroxy-3-propyl-4-(4-(2-(4-(1 H-tetrazol-5-yl)butyl)-2H-
tetrazol-
5-yl)butoxy)phenyl)-), LY-210073, LY-223982 (benzenepropanoic acid, 5-(3-
carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5-hexenyl)oxy)-, (E)-), LY-293111
(benzoic acid, 2-(3-(3-((5-ethyl-4'-fluoro-2-hydroxy(1,1'-biphenyl)-4-
yl)oxy)propoxy)-2-propylphenoxy)-), SM-9064 (pyrrolidine, 1-(4,11-dihydroxy-
13-(4-methoxyphenyl)-1-oxo-5,7,9-tridecatrienyl)-, (E,E,E)-), T-0757 (2,6-
octadienamide, N-(4-hydroxy-3,5-dimethylphenyl)-3,7-dimethyl-, (2E)-), or an
analogue or derivative thereof).
21 ) MCP-1 Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a MCP-1 antagonist (e.g., nitronaproxen (2-
napthaleneacetic acid, 6-methoxy-alpha-methyl 4-(nitrooxy)butyl ester (alpha
S)-), bindarit (2-(1-benzylindazol-3-ylmethoxy)-2-methylpropanoic acid), 1-
alpha-25 dihydroxy vitamin D3, or an analogue or derivative thereof).
86
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
22) MMP Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a matrix metalloproteinase (MMP) inhibitor (e.g., D-
9120, doxycycline (2-naphthacenecarboxamide, 4-(dimethylamino)-
1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-
dioxo- (4S-(4 alpha, 4a alpha, 5 Ipha, 5a alpha, 6 alpha, 12a alpha))-), BB-
2827, BB-1101 (2S-allyl-N1-hydroxy-3R-isobutyl-N4-(1 S-methylcarbamoyl-2-
phenylethyl)-succinamide), BB-2983, solimastat (N'-(2,2-dimethyl-1(S)-(N-(2-
pyridyl)carbamoyl)propyl)-N4-hydroxy-2(R)-isobutyl-3(S)-methoxysuccinamide),
batimastat (butanediamide, N4-hydroxy-N1-(2-(methylamino)-2-oxo-1-
(phenylmethyl)ethyl)-2-(2-methylpropyl)-3-((2-thienylthio)methyl)-, (2R-
(1(S*),2R*,3S*))-), CH-138, CH-5902, D-1927, D-5410, EF-13 (gamma-linolenic
acid lithium salt),CMT-3 (2-naphthacenecarboxamide, 1,4,4a,5,5a,6,11,12a-
octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxo-, (4aS,5aR,12aS)-), marimastat
(N-(2,2-dimethyl-1 (S)-(N-methylcarbamoyl)propyl)-N,3(S)-dihydroxy-2(R)-
isobutylsuccinamide), TIMP'S,ONO-4817, rebimastat (L-Valinamide, N-((2S)-2-
mercapto-1-oxo-4-(3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl)butyl)-L-leucyl-
N,3-
dimethyl-), PS-508, CH-715, nimesulide (methanesulfonamide, N-(4-nitro-2-
phenoxyphenyl)-), hexahydro-2-(2(R)-(1(RS)-(hydroxycarbamoyl)-4-
phenylbutyl)nonanoyl)-N-(2,2,6,6-etramethyl-4-piperidinyl)-3(S)-pyridazine
carboxamide, Rs-113-080, Ro-1130830, cipemastat (1-piperidinebutanamide,
f3-(cyclopentylmethyl)-N-hydroxy-gamma-oxo-alpha-((3,4,4-trimethyl-2,5-dioxo-
1-imidazolidinyl)methyl)-,(alpha R,f~R)-), 5-(4'-biphenyl)-5-(N-(4-
nitrophenyl)piperazinyl)barbituric acid, 6-methoxy-1,2,3,4-tetrahydro-
norharman-1-carboxylic acid, Ro-31-4724 (L-alanine, N-(2-(2-(hydroxyamino)-
2-oxoethyl)-4-methyl-1-oxopentyl)-L-leucyl-, ethyl ester), prinomastat (3-
thiomorpholinecarboxamide, N-hydroxy-2,2-dimethyl-4-((4-(4-pyridinyloxy)
phenyl)sulfonyl)-, (3R)-), AG-3433 (1H-pyrrole-3-propanic acid, 1-(4'-
cyano(1,1'-
bi phenyl)-4-yl )-b-((((3S )-tetra hyd ro-4,4-d imethyl-2-oxo-3-
furanyl)amino)carbonyl)-, phenylmethyl ester, (bS)-), PNU-142769 (2H-
87
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Isoindole-2-butanamide, 1,3-dihydro-N-hydroxy-alpha-((3S)-3-(2-methylpropyl)-
2-oxo-1-(2-phenylethyl)-3-pyrrolidinyl)-1,3-dioxo-, (alpha R)-), (S)-1-(2-
((((4,5-
dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)amino)-carbonyl)amino)-1-oxo-3-
(pentafluorophenyl)propyl)-4-(2-pyridinyl)piperazine, SU-5402 (1 H-pyrrole-3-
propanoic acid, 2-((1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl)-4-methyl-),
SC-77964, PNU-171829, CGS-27023A, N-hydroxy-2(R)-((4-methoxybenzene-
sulfonyl)(4-picolyl)amino)-2-(2-tetrahydrofuranyl)-acetamide, L-758354 ((1,1'-
biphenyl)-4-hexanoic acid, alpha-butyl-gamma-(((2,2-dimethyl-1-
((methylamino)carbonyl)propyl)amino)carbonyl)-4'-fluoro-, (alpha S-(alpha R*,
gammaS*(R*)))-, GI-155704A, CPA-926, TMI-005, XL-784, or an analogue or
derivative thereof). Additional representative examples are included in U.S.
Patent Nos. 5,665,777; 5,985,911; 6,288,261; 5,952,320; 6,441,189; 6,235,786;
6,294,573; 6,294,539; 6,563,002; 6,071,903; 6,358,980; 5,852,213; 6,124,502;
6,160,132; 6,197,791; 6,172,057; 6,288,086; 6,342,508; 6,228,869; 5,977,408;
5,929,097; 6,498,167; 6,534,491; 6,548,524; 5,962,481; 6,197,795; 6,162,814;
6,441,023; 6,444,704; 6,462,073; 6,162,821; 6,444,639; 6,262,080; 6,486,193;
6,329,550; 6,544,980; 6,352,976; 5,968,795; 5,789,434; 5,932,763; 6,500,847;
5,925,637; 6,225,314; 5,804,581; 5,863,915; 5,859,047; 5,861,428; 5,886,043;
6,288,063; 5,939,583; 6,166,082; 5,874,473; 5,886,022; 5,932,577; 5,854,277;
5,886,024; 6,495,565; 6,642,255; 6,495,548; 6,479,502; 5,696,082; 5,700,838;
6,444,639; 6,262,080; 6,486,193; 6,329,550; 6,544,980; 6,352,976; 5,968,795;
5,789,434; 5,932,763; 6,500,847; 5,925,637; 6,225,314; 5,804,581; 5,863,915;
5,859,047; 5,861,428; 5,886,043; 6,288,063; 5,939,583; 6,166,082; 5,874,473;
5,886,022; 5,932,577; 5,854,277; 5,886,024; 6,495,565; 6,642,255; 6,495,548;
6,479,502; 5,696,082; 5,700,838; 5,861,436; 5,691,382; 5,763,621; 5,866,717;
5,902,791; 5,962,529; 6,017,889; 6,022,873; 6,022,898; 6,103,739; 6,127,427;
6,258,851; 6,310,084; 6,358,987; 5,872,152; 5,917,090; 6,124,329; 6,329,373;
6,344,457; 5,698,706; 5,872,146; 5,853,623; 6,624,144; 6,462,042; 5,981,491;
5,955,435; 6,090,840; 6,114,372; 6,566,384; 5,994,293; 6,063,786; 6,469,020;
6,118,001; 6,187,924; 6,310,088; 5,994,312; 6,180,611; 6,110,896; 6,380,253;
88
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
5,455,262; 5,470,834; 6,147,114; 6,333,324; 6,489,324; 6,362,183; 6,372,758;
6,448,250; 6,492,367; 6,380,258; 6,583,299; 5,239,078; 5,892,112; 5,773,438;
5,696,147; 6,066,662; 6,600,057; 5,990,158; 5,731,293; 6,277,876; 6,521,606;
6,168,807; 6,506,414; 6,620,813; 5,684,152; 6,451,791; 6,476,027; 6,013,649;
6,503,892; 6,420,427; 6,300,514; 6,403,644; 6,177,466; 6,569,899; 5,594,006;
6,417,229; 5,861,510; 6,156,798; 6,387,931; 6,350,907; 6,090,852; 6,458,822;
6,509,337; 6,147,061; 6,114,568; 6,118,016; 5,804,593; 5,847,153; 5,859,061;
6,194,451; 6,482,827; 6,638,952; 5,677,282; 6,365,630; 6,130,254; 6,455,569;
6,057,369; 6,576,628; 6,110,924; 6,472,396; 6,548,667; 5,618,844; 6,495,578;
6,627,411; 5,514,716; 5,256,657; 5,773,428; 6,037,472; 6,579,890; 5,932,595;
6,013,792; 6,420,415; 5,532,265; 5,691,381; 5,639,746; 5,672,598; 5,830,915;
6,630,516; 5,324,634; 6,277,061; 6,140,099; 6,455,570; 5,595,885; 6,093,398;
6,379,667; 5,641,636; 5,698,404; 6,448,058; 6,008,220; 6,265,432; 6,169,103;
6,133,304; 6,541,521; 6,624,196; 6,307,089; 6,239,288; 5,756,545; 6,020,366;
6,117,869; 6,294,674; 6,037,361; 6,399,612; 6,495,568; 6,624,177; 5,948,780;
6,620,835; 6,284,513; 5,977,141; 6,153,612; 6,297,247; 6,559,142; 6,555,535;
6,350,885; 5,627,206; 5,665,764; 5,958,972; 6,420,408; 6,492,422; 6,340,709;
6,022,948; 6,274,703; 6,294,694; 6,531,499; 6,465,508; 6,437,177; 6,376,665;
5,268,384; 5,183,900; 5,189,178; 6,511,993; 6,617,354; 6,331,563; 5,962,466;
5,861,427; 5,830,869; and 6,087,359.
23) NF kappa B Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a NF kappa B (NFKB) inhibitor (e.g., AVE-0545, Oxi-104
(benzamide, 4-amino-3-chloro-N-(2-(diethylamino)ethyl)-), dexlipotam, R-
flurbiprofen ((1,1'-biphenyl)-4-acetic acid, 2-fluoro-alpha-methyl), SP100030
(2-
chloro-N-(3,5-di(trifluoromethyl)phenyl)-4-(trifluoromethyl)pyrimidine-5-
carboxamide), AVE-0545, Viatris, AVE-0547, Bay 11-7082, Bay 11-7085, 15
deoxy-prostaylandin J2, bortezomib (boronic acid, ((1 R)-3-methyl-1-(((2S)-1-
oxo-3-phenyl-2-((pyrazinylcarbonyl)amino)propyl)amino)butyl)-, benzamide an
89
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
d nicotinamide derivatives that inhibit NF-kappaB, such as those described in
U.S. Patent Nos. 5,561,161 and 5,340,565 (OxiGene), PG490-88Na, or an
analogue or derivative thereof).
24) NO Aaonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a NO antagonist (e.g., NCX-4016 (benzoic acid, 2-
(acetyloxy)-, 3-((nitrooxy)methyl)phenyl ester, NCX-2216, L-arginine or an
analogue or derivative thereof).
25) P38 MAP Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a p38 MAP kinase inhibitor (e.g., GW-2286, CGP-52411,
BIRB-798, SB220025, RO-320-1195, RWJ-67657, RWJ-68354, SCIO-469,
SCIO-323, AMG-548, CMC-146, SD-31145, CC-8866, Ro-320-1195, PD-98059
(4H-1-benzopyran-4-one, 2-(2-amino-3-methoxyphenyl)-), CGH-2466,
doramapimod, SB-203580 (pyridine, 4-(5-(4-fluorophenyl)-2-(4-
(methylsulfinyl)phenyl)-1 H-imidazol-4-yl)-), SB-220025 ((5-(2-amino-4-
pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole), SB-281832,
PD169316, SB202190, GSK-681323, EO-1606, GSK-681323, or an analogue
or derivative thereof). Additional representative examples are included in
U.S.
Patent Nos. 6,300,347; 6,316,464; 6,316,466; 6,376,527; 6,444,696; 6,479,507;
6,509,361; 6,579,874; 6,630,485, U.S. Patent Application Publication Nos.
2001 /0044538A1; 2002/0013354A1; 2002/0049220A1; 2002/0103245A1;
2002/0151491 A1; 2002/0156114A1; 2003/0018051 A1; 2003/0073832A1;
2003/0130257A1; 2003/0130273A1; 2003/0130319A1; 2003/0139388A1;
20030139462A1; 2003/0149031 A1; 2003/0166647A1; 2003/0181411 A1; and
PCT Publication Nos. WO 00/63204A2; WO 01/21591 A1; WO 01/35959A1;
WO 01/74811A2; WO 02/18379A2; WO 2064594A2; WO 2083622A2; WO
2094842A2; WO 2096426A1; WO 2101015A2; WO 2103000A2; WO
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
3008413A1; WO 3016248A2; WO 3020715A1; WO 3024899A2; WO
3031431 A1; W03040103A1; WO 3053940A1; WO 3053941 A2; WO
3063799A2; WO 3079986A2; WO 3080024A2; WO 3082287A1; WO
97/44467A1; WO 99/01449A1; and WO 99/58523A1.
26) Phosphodiesterase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a phosphodiesterase inhibitor (e.g., CDP-840 (pyridine,
4-((2R)-2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)-), CH-3697, CT-
2820, D-22888 (imidazo(1,5-a)pyrido(3,2-e)pyrazin-6(5H)-one, 9-ethyl-2-
methoxy-7-methyl-5-propyl-), D-4418 (8-methoxyquinoline-5-(N-(2,5-
dichloropyridin-3-yl))carboxamide), 1-(3-cyclopentyloxy-4-methoxyphenyl)-2-
(2,6-dichloro-4-pyridyl) ethanone oxime, D-4396, ONO-6126, CDC-998, CDC-
801, V-11294A (3-(3-(cyclopentyloxy)-4-methoxybenzyl)-6-(ethylamino)-8-
isopropyl-3H-purine hydrochloride), S,S'-methylene-bis(2-(8-cyclopropyl-3-
propyl-6-(4-pyridylmethylamino)-2-thio-3H-purine)) tetrahyrochloride, rolipram
(2-pyrrolidinone, 4-(3-(cyclopentyloxy)-4-methoxyphenyl)-), CP-293121, CP-
353164 (5-(3-cyclopentyloxy-4-methoxyphenyl)pyridine-2-carboxamide),
oxagrelate (6-phthalazinecarboxylic acid, 3,4-dihydro-1-(hydroxymethyl)-5,7-
dimethyl-4-oxo-, ethyl ester), PD-168787, ibudilast (1-propanone, 2-methyl-1-
(2-(1-methylethyl)pyrazolo(1,5-a)pyridin-3-yl)-), oxagrelate (6-
phthalazinecarboxylic acid, 3,4-dihydro-1-(hydroxymethyl)-5,7-dimethyl-4-oxo-,
ethyl ester), griseolic acid (alpha-L-talo-oct-4-enofuranuronic acid, 1-(6-
amino-
9H-purin-9-yl)-3,6-anhydro-6-C-carboxy-1,5-dideoxy-), KW-4490, KS-506, T-
440, roflumilast (benzamide, 3-(cyclopropylmethoxy)-N-(3,5-dichloro-4-
pyridinyl)-4-(difluoromethoxy)-), rolipram, milrinone, triflusinal (benzoic
acid, 2-
(acetyloxy)-4-(trifluoromethyl)-), anagrelide hydrochloride (imidazo(2,1-
b)quinazolin-2(3H)-one, 6,7-dichloro-1,5-dihydro-, monohydrochloride),
cilostazol (2(1 H)-quinolinone, 6-(4-(1-cyclohexyl-1 H-tetrazol-5-yl)butoxy)-
3,4-
dihydro-), propentofylline (1 H-purine-2,6-dione, 3,7-dihydro-3-methyl-1-(5-
91
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
oxohexyl)-7-propyl-), sildenafil citrate (piperazine, 1-((3-(4,7-dihydro-1-
methyl-7-
oxo-3-propyl-1 H-pyrazolo(4,3-d)pyrimidin-5-yl)-4-ethoxyphenyl)sulfonyl)-4-
methyl, 2-hydroxy-1,2,3-propanetricarboxylate- (1:1 )), tadalafil
(pyrazino(1',2':1,6)pyrido(3,4-b)indole1,4-dione, 6-(1,3-benzodioxol-5-yl)-
2,3,6,7,12,12a-hexahydro-2-methyl-, (6R-trans)), vardenafil (piperazine, 1-(3-
(1,4-dihydro-5-methyl(-4-oxo-7-propylimidazo(5,1-f)(1,2,4)-triazin-2-yl)-4-
ethoxyphenyl)sulfonyl)-4-ethyl-), milrinone ((3,4'-bipyridine)-5-carbonitrile,
1,6-
dihydro-2-methyl-6-oxo-), enoximone (2H-imidazol-2-one, 1,3-dihydro-4-methyl-
5-(4-(methylthio)benzoyl)-), theophylline (1 H-purine-2,6-dione, 3,7-dihydro-
1,3-
dimethyl-), ibudilast (1-propanone, 2-methyl-1-(2-(1-methylethyl)pyrazolo(1,5-
a)pyridin-3-yl)-), aminophylline (1H-purine-2,6-dione, 3,7-dihydro-1,3-
dimethyl-,
compound with 1,2-ethanediamine (2:1 )-), acebrophylline (7H-purine-7-acetic
acid, 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-,compd. with trans-4-(((2-
amino-
3,5-dibromophenyl)methyl)amino)cyclohexanol (1:1 )), plafibride (propanamide,
2-(4-chlorophenoxy)-2-methyl-N-(((4-morpholinylmethyl)amino)carbonyl)-),
ioprinone hydrochloride (3-pyridinecarbonitrile, 1,2-dihydro-5-imidazo(1,2-
a)pyridin-6-yl-6-methyl-2-oxo-, monohydrochloride-), fosfosal (benzoic acid, 2-
(phosphonooxy)-), amrinone ((3,4'-bipyridin)-6(1 H)-one, 5-amino-, or an
analogue or derivative thereof).
Other examples of phosphodiesterase inhibitors include
denbufylline (1H-purine-2,6-dione, 1,3-dibutyl-3,7-dihydro-7-(2-oxopropyl)-),
propentofylline (1 H-purine-2,6-dione, 3,7-dihydro-3-methyl-1-(5-oxohexyl)-7-
propyl-) and pelrinone (5-pyrimidinecarbonitrile, 1,4-dihydro-2-methyl-4-oxo-6-
((3-pyridinylmethyl)amino)-).
Other examples of phosphodiesterase III inhibitors include
enoximone (2H-imidazol-2-one, 1,3-dihydro-4-methyl-5-(4-(methylthio)benzoyl)-
), and saterinone (3-pyridinecarbonitrile, 1,2-dihydro-5-(4-(2-hydroxy-3-(4-(2-
methoxyphenyl)-1-piperazinyl)propoxy)phenyl)-6-methyl-2-oxo-).
Other examples of phosphodiesterase IV inhibitors include AWD-
12-281, 3-auinolinecarboxylic acid, 1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-
92
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
piperazinyl)-4-oxo-), tadalafil (pyrazino(1',2':1,6)pyrido(3,4-b)indole1,4-
dione, 6-
(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R-trans)), and
filaminast (ethanone, 1-(3-(cyclopentyloxy)-4-methoxyphenyl)-, O-
(aminocarbonyl)oxime, (1 E)-)
Another example of a phosphodiesterase V inhibitor is vardenafil
(piperazine, 1-(3-(1,4-dihydro-5-methyl(-4-oxo-7-propylimidazo(5,1-f)(1,2,4)-
triazin-2-yl)-4-ethoxyphenyl)sulfonyl)-4-ethyl-).
27) TGF beta Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a TGF beta Inhibitor (e.g., mannose-6-phosphate, LF-
984, tamoxifen (ethanamine, 2-(4-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-
dimethyl-, (Z)-), tranilast, or an analogue or derivative thereof).
28) Thromboxane A2 Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a thromboxane A2 antagonist (e.g., CGS-22652 (3-
pyridineheptanoic acid, y-(4-(((4-chlorophenyl)sulfonyl)amino)butyl)-, (±)-
),
ozagrel (2-propenoic acid, 3-(4-(1 H-imidazol-1-ylmethyl)phenyl)-, (E)-),
argatroban (2-piperidinecarboxylic acid, 1-(5-((aminoiminomethyl)amino)-1-oxo-
2-(((1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl)amino)pentyl)-4-methyl-
),
ramatroban (9H-carbazole-9-propanoic acid, 3-(((4-
fluorophenyl)sulfonyl)amino)-1,2,3,4-tetrahydro-, (R)-), torasemide (3-
pyridinesulfonamide, N-(((1-methylethyl)amino)carbonyl)-4-((3-
methylphenyl)amino)-), gamma linoleic acid ((Z,Z,Z)-6,9,12-octadecatrienoic
acid), seratrodast (benzeneheptanoic acid, zeta-(2,4,5-trimethyl-3,6-dioxo-1,4-
cyclohexadien-1-yl)-, (+/-)-, or an analogue or derivative thereof).
93
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
29) TNF alpha Antagonists and TACE Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a TNF alpha antagonist or TACE inhibitor (e.g., E-5531
(2-deoxy-6-0-(2-deoxy-3-0-(3(R)-(5(Z)-dodecenoyloxy)-decyl)-6-0-methyl-2-(3-
oxotetradecanamido)-4-O-phosphono-f3-D-glucopyranosyl)-3-0-(3(R)-
hydroxydecyl)-2-(3-oxotetradecanamido)-alpha-D-glucopyranose-1-O-
phosphate), AZD-4717, glycophosphopeptical, UR-12715 (B=benzoic acid, 2-
hydroxy-5-((4-(3-(4-(2-methyl-1 H-imidazol(4,5-c)pyridin-1-yl)methyl)-1-
piperidinyl)-3-oxo-1-phenyl-1-propenyl)phenyl)azo) (Z)), PMS-601, AM-87,
xyloadenosine (9H-purin-6-amine, 9-f3-D-xylofuranosyl-), RDP-58, RDP-59,
BB2275, benzydamine, E-3330 (undecanoic acid, 2-((4,5-dimethoxy-2-methyl-
3,6-dioxo-1,4-cyclohexadien-1-yl)methylene)-, (E)-), N-(D,L-2-
(hydroxyaminocarbonyl)methyl-4-methylpentanoyl)-L-3-(2'-naphthyl)alanyl-L-
alanine, 2-aminoethyl amide, CP-564959, MLN-608, SPC-839, ENMD-0997,
Sch-23863 ((2-(10,11-dihydro-5-ethoxy-5H-dibenzo (a,d) cyclohepten-S-yl)-N,
N-dimethyl-ethanamine), SH-636, PKF-241-466, PKF-242-484, TNF-484A,
cilomilast (cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-
carboxylic acid), GW-3333, GW-4459, BMS-561392, AM-87, cloricromene
(acetic acid, ((8-chloro-3-(2-(diethylamino)ethyl)-4-methyl-2-oxo-2H-1-
benzopyran-7-yl)oxy)-, ethyl ester), thalidomide (1H-Isoindole-1,3(2H)-dione,
2-
(2,6-dioxo-3-piperidinyl)-), vesnarinone (piperazine, 1-(3,4-dimethoxybenzoyl)-
4-(1,2,3,4-tetrahydro-2-oxo-6-puinolinyl)-), infliximab, lentinan, etanercept
(1-
235-tumor necrosis factor receptor (human) fusion protein with 236-467-
immunoglobulin G1 (human gamma1-chain Fc fragment)), diacerein (2-
anthracenecarboxylic acid, 4,5-bis(acetyloxy)-9,10-dihydro-9,10-dioxo-, or an
analogue or derivative thereof).
30) Tyrosine Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a tyrosine kinase inhibitor (e.g., SKI-606, ER-068224,
94
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
SD-208, N-(6-benzothiazolyl)-4-(2-(1-piperazinyl)pyrid-5-yl)-2-
pyrimidineamine,
celastrol (24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid, 3-hydroxy-
9,13-
dimethyl-2-oxo-, (9 beta.,13alpha,14f3,20 alpha)-), CP-127374 (geldanamycin,
17-demethoxy-17-(2-propenylamino)-), CP-564959, PD-171026, CGP-52411
(1 H-Isoindole-1,3(2H)-dione, 4,5-bis(phenylamino)-), CGP-53716 (benzamide,
N-(4-methyl-3-((4-(3-pyridinyl)-2-pyrimidinyl)amino)phenyl)-), imatinib (4-
((methyl-1-piperazinyl)methyl)-N-(4-methyl-3-((4-(3-pyridinyl)-2-
pyrimidinyl)amino)-phenyl)benzamide methanesulfonate), NVP-AAK980-NX,
KF-250706 (13-chloro,5(R),6(S)-epoxy-14,16-dihydroxy-11-(hydroyimino)-3(R)-
methyl-3,4,5,6,11,12-hexahydro-1H-2-benzoxacyclotetradecin-1-one), 5-(3-(3-
methoxy-4-(2-((E)-2-phenylethenyl)-4-oxazolylmethoxy)phenyl)propyl)-3-(2-
((E)-2-phenylethenyl)-4-oxazolylmethyl)-2,4-oxazolidinedione, genistein, Nv-
06,
or an analogue or derivative thereof).
31 ) Vitronectin Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a vitronectin inhibitor (e.g., O-(9,10-dimethoxy-
1,2,3,4,5,6-hexahydro-4-((1,4,5,6-tetrahydro-2-pyrimidinyl)hydrazono)-8-
benz(e)azulenyl)-N-((phenylmethoxy)carbonyl)-DL-homoserine 2,3-
dihydroxypropyl ester, (2S)-benzoylcarbonylamino-3-(2-((4S)-(3-(4,5-dihydro-
1 H-imidazol-2-ylamino)-propyl)-2,5-dioxo-imidazolidin-1-yl)-acetylamino)-
propionate, Sch-221153, S-836, SC-68448 ((3-((2-2-(((3-
((aminoiminomethyl)amino)-phenyl)carbonyl)amino)acetyl)amino)-3,5-
dichlorobenzenepropanoic acid), SD-7784, S-247, or an analogue or derivative
thereof).
' 32) Fibroblast Growth Factor Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a fibroblast growth factor inhibitor (e.g., CT-052923
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(((2H-benzo(d)1,3-dioxalan-5-methyl)amino)(4-(6,7-dimethoxyquinazolin-4-
yl)piperazinyl)methane-1-thione), or an analogue or derivative thereof).
33) Protein Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a protein kinase inhibitor (e.g., KP-0201448, NPC15437
(hexanamide, 2,6-diamino-N-((1-(1-oxotridecyl)-2-piperidinyl)methyl)-),
fasudil
(1 H-1,4-diazepine, hexahydro-1-(5-isoquinolinylsulfonyl)-), midostaurin
(benzamide, N-(2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-
epoxy-1 H,9H-diindolo(1,2,3-gh:3',2',1'-Im)pyrrolo(3,4-j)(1,7)benzodiazonin-11-
yl)-N-methyl-, (9Alpha,10f3,11 f3,13Alpha)-),fasudil (1 H-1,4-diazepine,
hexahydro-1-(5-isoquinolinylsulfonyl)-, dexniguldipine (3,5-
pyridinedicarboxylic
acid, 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-, 3-(4,4-diphenyl-1-
piperidinyl)propyl methyl ester, monohydrochloride, (R)-), LY-317615 (1 H-
pyrole-2,5=dione, 3-(1-methyl-1 H-indol-3-yl)-4-(1-(1-(2-pyridinylmethyl)-4-
piperidinyl)-1 H-indol-3-yl)-, monohydrochloride), perifosine (piperidinium, 4-
((hydroxyloctadecyloxy)phosphinyl)oxy)-1,1-dimethyl-, inner salt), LY-333531
(9H,18H-5,21:12,17-dimethenodibenzo(e,k)pyrrolo(3,4-
h)(1,4,13)oxadiazacyclohexadecine-18,20(19H)-dione,9-
((dimethylamino)methyl)-6,7,10,11-tetrahydro-, (S)-), Kynac; SPC-100270 (1,3-
octadecanediol, 2-amino-, (S-(R*,R*))-), Kynacyte, or an analogue or
derivative
thereof).
34) PDGF Receptor Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a PDGF receptor kinase inhibitor (e.g., RPR-127963E,
or an analogue or derivative thereof).
96
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
35) Endothelial Growth Factor Receptor Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an endothelial growth factor receptor kinase inhibitor
(e.g., CEP-7055, SU-0879 ((E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-
(aminothiocarbonyl)acrylonitrile), BIBF-1000, AG-013736 (CP-868596), AMG-
706, AVE-0005, NM-3 (3-(2-methylcarboxymethyl)-6-methoxy-8-hydroxy-
isocoumarin), Bay-43-9006, SU-011248, or an analogue or derivative thereof).
36) Retinoic Acid Receptor Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a retinoic acid receptor antagonist (e.g., etarotene
(Ro-
15-1570) (naphthalene, 6-(2-(4-(ethylsulfonyl)phenyl)-1-methylethenyl)-1,2,3,4-
tetrahydro-1,1,4,4-tetramethyl-, (E)-), (2E,4E)-3-methyl-5-(2-((E)-2-(2,6,6-
trimethyl-1-cyclohexen-1-yl)ethenyl)-1-cyclohexen-1-yl)-2,4-pentadienoic acid,
tocoretinate (retinoic acid, 3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-
trimethyltridecyl)-2H-1-benzopyran-6-yl ester, (2R*(4R*,8R*))-(~)-),
aliretinoin
(retinoic acid, cis-9, trans-13-), bexarotene (benzoic acid, 4-(1-(5,6,7,8-
tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl)-), tocoretinate
(retinoic acid, 3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-
2H-1-
benzopyran-6-yl ester, (2R*(4R*,8R*))-(~)-, or an analogue or derivative
thereof).
37) Platelet Derived Growth Factor Receptor Kinase Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a platelet derived growth factor receptor kinase
inhibitor
(e.g., leflunomide (4-isoxazolecarboxamide, 5-methyl-N-(4-
(trifluoromethyl)phenyl)-, or an analogue or derivative thereof).
97
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
38) Fibrinogen Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a fibrinogin antagonist (e.g., picotamide (1,3-
benzenedicarboxamide, 4-methoxy-N,N'-bis(3-pyridinylmethyl)-, or an analogue
or derivative thereof).
39) Antimycotic Agents
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an antimycotic agent (e.g., miconazole, sulconizole,
parthenolide, rosconitine, nystatin, isoconazole, fluconazole, ketoconasole,
imidazole, itraconazole, terpinafine, elonazole, bifonazole, clotrimazole,
conazole, terconazole (piperazine, 1-(4-((2-(2,4-dichlorophenyl)-2-(1H-1,2,4-
triazol-1-ylmethyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(1-methylethyl)-, cis-
),
isoconazole (1-(2-(2-6-dichlorobenzyloxy)-2-(2-,4-dichlorophenyl)ethyl)),
griseofulvin (spiro(benzofuran-2(3H),1'-(2)cyclohexane)-3,4'-dione, 7-chloro-
2',4,6-trimeth-oxy-6'methyl-, (1'S-trans)-), bifonazole (1H-imidazole, 1-
((1,1'-
biphenyl)-4-ylphenylmethyl)-), econazole nitrate (1-(2-((4-
chlorophenyl)methoxy)-2-(2,4-dichlorophenyl)ethyl)-1 H-imidazole nitrate),
croconazole (1 H-imidazole, 1-(1-(2-((3-chlorophenyl)methoxy)phenyl)ethenyl)-
),
sertaconazole (1H-Imidazole, 1-(2-((7-chlorobenzo(b)thien-3-yl)methoxy)-2-
(2,4-dichlorophenyl)ethyl)-), omoconazole (1 H-imidazole, 1-(2-(2-(4-
chlorophenoxy)ethoxy)-2-(2,4-dichlorophenyl)-1-methylethenyl)-, (Z)-),
flutrimazole (1 H-imidazole, 1-((2-fluorophenyl)(4-fluorophenyl)phenylmethyl)-
),
fluconazole (1 H-1,2,4-triazole-1-ethanol, alpha-(2,4-difluorophenyl)-alpha-(1
H-
1,2,4-triazol-1-ylmethyl)-), neticonazole (1H-Imidazole, 1-(2-(methylthio)-1-
(2-
(pentyloxy)phenyl)ethenyl)-, monohydrochloride, (E)-), butoconazole (1 H-
imidazole, 1-(4-(4-chlorophenyl)-2-((2,6-dichlorophenyl)thio)butyl)-, (+/-)-),
clotrimazole (1-((2-chlorophenyl)diphenylmethyl)-1 H-imidazole, or an analogue
or derivative thereof).
98
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
40) Bisphosphonates
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a bisphosphonate (e.g., clodronate, alendronate,
pamidronate, zoledronate, or an analogue or derivative thereof).
41 ) Phospholipase A1 Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a phospholipase A1 inhibitor (e.g., ioteprednol
etabonate
(androsta-1,4-diene-17-carboxylic acid, 17-((ethoxycarbonyl)oxy)-11-hydroxy-3-
oxo-, chloromethyl ester, (11 f3,17 alpha)-, or an analogue or derivative
thereof).
42) Histamine HllH2/H3 Receptor Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a histamine H1, H2, or H3 receptor antagonist (e.g.,
ranitidine (1,1-ethenediamine, N-(2-(((5-((dimethylamino)methyl)-2-
furanyl)methyl)thio)ethyl)-N'-methyl-2-nitro-), niperotidine (N-(2-((5-
((dimethylamino)methyl)furfuryl)thio)ethyl)-2-nitro-N'-piperonyl-1,1-
ethenediamine), famotidine (propanimidamide, 3-(((2-
((aminoiminomethyl)amino)-4-thiazolyl)methyl)thio)-N-(aminosulfonyl)-),
roxitadine acetate HCI (acetamide, 2-(acetyloxy)-N-(3-(3-(1-
piperidinylmethyl)phenoxy)propyl)-, monohydrochloride), lafutidine (acetamide,
2-((2-furanylmethyl)sulfinyl)-N-(4-((4-(1-piperidinylmethyl)-2-pyridinyl)oxy)-
2-
butenyl)-, (Z)-), nizatadine (1,1-ethenediamine, N-(2-(((2-
((dimethylamino)methyl)-4-thiazolyl)methyl)thio)ethyl)-N'-methyl-2-nitro-),
ebrotidine (benzenesulfonamide, N-(((2-(((2-((aminoiminomethyl)amino)-4-
thiazoly)methyl)thio)ethyl)amino)methylene)-4-bromo-), rupatadine (5H-
benzo(5,6)cyclohepta(1,2-b)pyridine, 8-chloro-6,11-dihydro-11-(1-((5-methyl-3-
pyridinyl)methyl)-4-piperidinylidene)-, trihydrochloride-), fexofenadine HCI
(benzeneacetic acid, 4-(1-hydroxy-4-(4(hydroxydiphenylmethyl)-1-
99
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
piperidinyl)butyl)-alpha, alpha-dimethyl-, hydrochloride, or an analogue or
derivative thereof).
43) Macrolide Antibiotics
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a macrolide antibiotic (e.g., dirithromycin
(erythromycin,
9-deoxo-11-deoxy-9,11-(imino(2-(2-methoxyethoxy)ethylidene)oxy)-, (9S(R))-),
flurithromycin ethylsuccinate (erythromycin, 8-fluoro-mono(ethyl butanedioate)
(ester)-), erythromycin stinoprate (erythromycin, 2'-propanoate, compound with
N-acetyl-L-cysteine (1:1 )), clarithromycin (erythromycin, 6-O-methyl-),
azithromycin (9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin-A),
telithromycin (3-de((2,6-dideoxy-3-C-methyl-3-O-methyl-alpha-L-ribo-
hexopyranosyl)oxy)-11,12-dideoxy-6-O-methyl-3-oxo-12,11-(oxycarbonyl((4-(4-
(3-pyridinyl)-1 H-imidazol-1-yl)butyl)imino))-), roxithromycin (erythromycin,
9-(O-
((2-methoxyethoxy)methyl)oxime)), rokitamycin (leucomycin V, 4B-butanoate
3B-propanoate), RV-11 (erythromycin monopropionate mercaptosuccinate),
midecamycin acetate (leucomycin V, 3B,9-diacetate 3,4B-dipropanoate),
midecamycin (leucomycin V, 3,4B-dipropanoate), josamycin (leucomycin V, 3-
acetate 4B-(3-methylbutanoate), or an analogue or derivative thereof).
44) GPllb Illa Receptor Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a GPllb Illa receptor antagonist (e.g., tirofiban
hydrochloride (L-tyrosine, N-(butylsulfonyl)-O-(4-(4-piperidinyl)butyl)-,
monohydrochloride-), eptifibatide (L-cysteinamide, N6-(aminoiminomethyl)-N2-
(3-mercapto-1-oxopropyl)-L-lysylglycyl-L-alpha-aspartyl-L-tryptophyl-L-prolyl-
,
cyclic(1->6)-disulfide), xemilofiban hydrochloride, or an analogue or
derivative thereof).
100
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
45) Endothelin Receptor Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an endothelin receptor antagonist (e.g., bosentan
(benzenesulfonamide, 4-(1,1-dimethylethyl)-N-(6-(2-hydroxyethoxy)-5-(2-
methoxyphenoxy)(2,2'-bipyrimidin)-4-yl)-, or an analogue or derivative
thereof).
46) Peroxisome Proliferator-Activated Receptor Agonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a peroxisome proliferator-activated receptor agonist
(e.g., gemfibrozil (pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl-),
fenofibrate (propanoic acid, 2-(4-(4-chlorobenzoyl)phenoxy)-2-methyl-, 1-
methylethyl ester), ciprofibrate (propanoic acid, 2-(4-(2,2-
dichlorocyclopropyl)phenoxy)-2-methyl-), rosiglitazone maleate (2,4-
thiazolidinedione, 5-((4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-,
(Z)-
2-butenedioate (1:1 )), pioglitazone hydrochloride (2,4-thiazolidinedione, 5-
((4-
(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)methyl)-, monohydrochloride (+/-)-),
etofylline clofibrate (propanoic acid, 2-(4-chlorophenoxy)-2-methyl-, 2-
(1,2,3,6-
tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purin-7-yl)ethyl ester), etofibrate (3-
pyridinecarboxylic acid, 2-(2-(4-chlorophenoxy)-2-methyl-1-oxopropoxy)ethyl
ester), clinofibrate (butanoic acid, 2,2'-(cyclohexylidenebis(4,1-
phenyleneoxy))bis(2-methyl-)), bezafibrate (propanoic acid, 2-(4-(2-((4-
chlorobenzoyl)amino)ethyl)phenoxy)-2-methyl-), binifibrate (3-
pyridinecarboxylic
acid, 2-(2-(4-chlorophenoxy)-2-methyl-1-oxopropoxy)-1,3-propanediyl ester), or
an analogue or derivative thereof).
In one aspect, the pharmacologically active compound is a
peroxisome proliferator-activated receptor alpha agonist, such as GW-590735,
GSK-677954, GSK501516, pioglitazone hydrochloride (2,4-thiazolidinedione, 5-
((4-(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)methyl)-, monohydrochloride (+/-)-,
or
an analogue or derivative thereof).
101
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
47) Estrogen Receptor Agents
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an estrogen receptor agent (e.g., estradiol, 17-~3-
estradiol, or an analogue or derivative thereof).
48) Somatostatin Analogues
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a somatostatin analogue (e.g., angiopeptin, or an
analogue or derivative thereof).
49) Neurokinin 1 Antagonists
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a neurokinin 1 antagonist (e.g., GW-597599, lanepitant
((1,4'-bipiperidine)-1'-acetamide, N-(2-(acetyl((2-methoxyphenyl)methyl)amino)-
1-(1 H-indol-3-ylmethyl)ethyl)- (R)-), nolpitantium chloride (1-
azoniabicyclo(2.2.2)octane, 1-(2-(3-(3,4-dichlorophenyl)-1-((3-(1-
methylethoxy)phenyl)acetyl)-3-piperidinyl)ethyl)-4-phenyl-, chloride, (S)-),
or
saredutant (benzamide, N-(4-(4-(acetylamino)-4-phenyl-1-piperidinyl)-2-(3,4-
dichlorophenyl)butyl)-N-methyl-, (S)-), or vofopitant (3-piperidinamine, N-((2-
methoxy-5-(5-(trifluoromethyl)-1 H-tetrazol-1-yl)phenyl)methyl)-2-phenyl-,
(2S,3S)-, or an analogue or derivative thereof).
50) Neurokinin 3 Antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a neurokinin 3 antagonist (e.g., talnetant (4-
quinolinecarboxamide, 3-hydroxy-2-phenyl-N-((1S)-1-phenylpropyl)-, or an
analogue or derivative thereof).
102
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
51 ) Neurokinin Antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a neurokinin antagonist (e.g., GSK-679769, GSK-
823296, SR-489686 (benzamide, N-(4-(4-(acetylamino)-4-phenyl-1-piperidinyl)-
2-(3,4-dichlorophenyl)butyl)-N-methyl-, (S)-), SB-223412; SB-235375 (4-
quinolinecarboxamide, 3-hydroxy-2-phenyl-N-((1S)-1-phenylpropyl)-), UK-
226471, or an analogue or derivative thereof).
52) VLA-4 Antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a VLA-4 antagonist (e.g., GSK683699, or an analogue
or derivative thereof).
53) Osteoclast Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a osteoclast inhibitor (e.g., ibandronic acid
(phosphonic
acid, (1-hydroxy-3-(methylpentylamino)propylidene) bis-), alendronate sodium,
or an analogue or derivative thereof).
54) DNA topoisomerase ATP Hydrolyzing Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a DNA topoisomerase ATP hydrolyzing inhibitor (e.g.,
enoxacin (1,8-naphthyridine-3-carboxylic acid, 1-ethyl-6-fluoro-1,4-dihydro-4-
oxo-7-(1-piperazinyl)-), levofloxacin (7H-Pyrido(1,2,3-de)-1,4-benzoxazine-6-
carboxylic acid, 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-
oxo-, (S)-), ofloxacin (7H-pyrido(1,2,3-de)-1,4-benzoxazine-6-carboxylic acid,
9-
fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-, (+/-)-),
pefloxacin (3-quinolinecarboxylic acid, 1-ethyl-6-fluoro-1,4-dihydro-7-(4-
methyl-
1-piperazinyl)-4-oxo-), pipemidic acid (pyrido(2,3-d)pyrimidine-6-carboxylic
acid,
8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)-), pirarubicin (5,12-
103
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
naphthacenedione, 10-((3-amino-2,3,6-trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-
alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-
(hydroxyacetyl)-1-methoxy-, (8S-(8 alpha,10 alpha(S*)))-), sparfloxacin (3-
quinolinecarboxylic acid, 5-amino-1-cyclopropyl-7-(3,5-dimethyl-1-piperazinyl)-
6,8-difluoro-1,4-dihydro-4-oxo-, cis-), AVE-6971, cinoxacin ((1,3)dioxolo(4,5-
g)cinnoline-3-carboxylic acid, 1-ethyl-1,4-dihydro-4-oxo-), or an analogue or
derivative thereof).
55) Angiotensin I Converting Enzyme Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an angiotensin I converting enzyme inhibitor (e.g.,
ramipril (cyclopenta(b)pyrrole-2-carboxylic acid, 1-(2-((1-(ethoxycarbonyl)-3-
phenylpropyl)amino)-1-oxopropyl)octahydro-, (2S-(1(R*(R*)),2 alpha, 3af3,
6a(3))-), trandolapril (1 H-indole-2-carboxylic acid, 1-(2-((1-carboxy-3-
phenylpropyl)amino)-1-oxopropyl)octahydro-, (2S-(1(R*(R*)),2 alpha,3a
alpha,7af3))-), fasidotril (L-alanine, N-((2S)-3-(acetylthio)-2-(1,3-
benzodioxol-5-
ylmethyl)-1-oxopropyl)-, phenylmethyl ester), cilazapril (6H-pyridazino(1,2-
a)(1,2)diazepine-1-carboxylic acid, 9-((1-(ethoxycarbonyl)-3-
phenylpropyl)amino)octahydro-10-oxo-, (1S-(1 alpha, 9 alpha(R*)))-), ramipril
(cyclopenta(b)pyrrole-2-carboxylic acid, 1-(2-((1-(ethoxycarbonyl)-3-
phenylpropyl)amino)-1-oxopropyl)octahydro-, (2S-(1(R*(R*)), 2
alpha,3af3,6af3))-, or an analogue or derivative thereof).
56) Angiotensin II Antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an angiotensin II antagonist (e.g., HR-720 (1 H-
imidazole-5-carboxylic acid, 2-butyl-4-(methylthio)-1-((2'-
((((propylamino)carbonyl)amino)sulfonyl)(1,1'-biphenyl)-4-yl)methyl)-,
dipotassium salt, or an analogue or derivative thereof).
104
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
57) Enkephalinase Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an enkephalinase inhibitor (e.g., Aventis 100240
(pyrido(2,1-a)(2)benzazepine-4-carboxylic acid, 7-((2-(acetylthio)-1-oxo-3-
phenylpropyl)amino)-1,2,3,4,6,7,8,12b-octahydro-6-oxo-, (4S-(4 alpha, 7
alpha(R*),12bf3))-), AVE-7688, or an analogue or derivative thereof).
58) Peroxisome Proliferator-Activated Receptor Gamma Agonist
Insulin Sensitizer
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is peroxisome proliferator-activated receptor gamma
agonist insulin sensitizer (e.g., rosiglitazone maleate (2,4-
thiazolidinedione, 5-
((4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-, (Z)-2-butenedioate
(1:1 ), farglitazar (GI-262570, GW-2570, GW-3995, GW-5393, GW-9765), LY-
929, LY-519818, LY-674, or LSN-862), or an analogue or derivative thereof).
59) Protein Kinase C Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a protein kinase C inhibitor, such as ruboxistaurin
mesylate (9H,18H-5,21:12,17-dimethenodibenzo(e,k)pyrrolo(3,4-
h)(1,4,13)oxadiazacyclohexadecine-18,20(19H)-dione,9-
((dimethylamino)methyl)-6,7,10,11-tetrahydro-, (S)-), safingol (1,3-
octadecanediol, 2-amino-, (S-(R*,R*))-), or enzastaurin hydrochloride (1 H-
pyrole-2,5-dione, 3-(1-methyl-1 H-indol-3-yl)-4-(1-(1-(2-pyridinylmethyl)-4-
piperidinyl)-1 H-indol-3-yl)-, monohydrochloride), or an analogue or
derivative
thereof.
60) ROCK (rho-associated kinase) Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a ROCK (rho-associated kinase) inhibitor, such as Y-
105
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
27632, HA-1077, H-1152 and 4-1-(aminoalkyl)-N-(4-pyridyl)
cyclohexanecarboxamide or an analogue or derivative thereof.
61 ) CXCR3 Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a CXCR3 inhibitor such as T-487, T0906487 or
analogue or derivative thereof.
62) Itk Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an Itk inhibitor such as BMS-509744 or an analogue or
derivative thereof.
63) Cytosolic phospholipase A2-alpha Inhibitors
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a cytosolic phospholipase A2-alpha inhibitor such as
efipladib (PLA-902) or analogue or derivative thereof.
64) PPAR Aaonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a PPAR Agonist (e.g., Metabolex ((-)-benzeneacetic
acid, 4-chloro-alpha-(3-(trifluoromethyl)-phenoxy)-, 2-(acetylamino)ethyl
ester),
balaglitazone (5-(4-(3-methyl-4-oxo-3,4-dihydro-quinazolin-2-yl-methoxy)-
benzyl)-thiazolidine-2,4-dione), ciglitazone (2,4-thiazolidinedione, 5-((4-((1-
methylcyclohexyl)methoxy)phenyl)methyl)-), DRF-10945, farglitazar, GSK-
677954, GW-409544, GW-501516, GW-590735, GW-590735, K-111, KRP-
101, LSN-862, LY-519818, LY-674, LY-929, muraglitazar; BMS-298585
(Glycine, N-((4-methoxyphenoxy)carbonyl)-N-((4-(2-(5-methyl-2-phenyl-4-
oxazolyl)ethoxy)phenyl)methyl)-), netoglitazone; isaglitazone (2,4-
thiazolidinedione, 5-((6-((2-fluorophenyl)methoxy)-2-naphthalenyl)methyl)-),
106
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Actos AD-4833; U-72107A (2,4-thiazolidinedione, 5-((4-(2-(5-ethyl-2-
pyridinyl)ethoxy)phenyl)methyl)-, monohydrochloride (+/-)-), JTT-501; PNU-
182716 (3,5-Isoxazolidinedione, 4-((4-(2-(5-methyl-2-phenyl-4-
oxazolyl)ethoxy)phenyl)methyl)-), AVANDIA (from SB Pharmco Puerto Rico,
Inc. (Puerto Rico); BRL-48482;BRL-49653;BRL-49653c; NYRACTA and Venvia
(both from (SmithKline Beecham (United Kingdom)); tesaglitazar ((2S)-2-
ethoxy-3-(4-(2-(4-((methylsulfonyl)oxy)phenyl)ethoxy)phenyl) propanoic acid),
troglitazone (2,4-Thiazolidinedione, 5-((4-((3,4-dihydro-6-hydroxy-2,5,7,8-
tetramethyl-2H-1-benzopyran-2-yl)methoxy)phenyl)methyl)-), and analogues
and derivatives thereof).
65) Immunosuppressants
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an immunosuppressant (e.g., batebulast
(cyclohexanecarboxylic acid, 4-(((aminoiminomethyl)amino)methyl)-, 4-(1,1-
dimethylethyl)phenyl ester, trans-), cyclomunine, exalamide (benzamide, 2-
(hexyloxy)-), LYN-001, CCI-779 (rapamycin 42-(3-hydroxy-2-(hydroxymethyl)-2-
methylpropanoate)), 1726; 1726-D; AVE-1726, or an analogue or derivative
thereof).
66) Erb Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an Erb inhibitor (e.g., canertinib dihydrochloride (N-
(4-(3-
(chloro-4-fluoro-phenylamino)-7-(3-morpholin-4-yl-propoxy)-quinazolin-6-yl)-
acrylamide dihydrochloride), CP-724714, or an analogue or derivative thereof).
67) Apoptosis Aaonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an apoptosis agonist (e.g., CEFLATONIN (CGX-635)
(from Chemgenex Therapeutics, Inc., Menlo Park, CA), CHML, LBH-589,
107
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
metoclopramide (benzamide, 4-amino-5-chloro-N-(2-(diethylamino)ethyl)-2-
methoxy-), patupilone (4,17-dioxabicyclo(14.1.0)heptadecane-5,9-dione, 7,11-
dihyd roxy-8,8,10,12,16-pentamethyl-3-( 1-methyl-2-(2-methyl-4-
thiazolyl)ethenyl, (1R,3S,7S,10R,11S,12S,16R)), AN-9; pivanex (butanoic acid,
(2,2-dimethyl-1-oxopropoxy)methyl ester), SL-100; SL-102; SL-11093; SL-
11098; SL-11099; SL-93; SL-98; SL-99, or an analogue or derivative thereof).
68) Lipocortin Agonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an lipocortin agonist (e.g., CGP-13774 (9Alpha-chloro-
6Alpha-fluoro-11 (3,17alpha-dihydroxy-16AIpha-methyl-3-oxo-1,4-androstadiene-
17f3-carboxylic acid-methylester-17-propionate), or analogue or derivative
thereof).
69) VCAM-1 antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a VCAM-1 antagonist (e.g., DW-908e, or an analogue or
derivative thereof).
70) Collagen Antae~onist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a collagen antagonist (e.g., E-5050
(Benzenepropanamide, 4-(2,6-dimethylheptyl)-N-(2-hydroxyethyl)-f3-methyl-),
lufironil (2,4-Pyridinedicarboxamide, N,N'-bis(2-methoxyethyl)-), or an
analogue
or derivative thereof).
71 ) Alpha 2 Integrin Antagonist
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is an alpha 2 integrin antagonist (e.g., E-7820, or an
analogue or derivative thereof).
108
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
72) TNF Alpha Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a TNF alpha inhibitor (e.g., ethyl pyruvate, Genz-
29155,
lentinan (Ajinomoto Co., Inc. (Japan)), linomide (3-quinolinecarboxamide, 1,2-
dihydro-4-hydroxy-N,1-dimethyl-2-oxo-N-phenyl-), UR-1505, or an analogue or
derivative thereof).
73) Nitric Oxide Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a nitric oxide inhibitor (e.g., guanidioethyldisulfide,
or an
analogue or derivative thereof).
74) Cathepsin Inhibitor
In another embodiment, the pharmacologically active fibrosis-
inhibiting compound is a cathepsin inhibitor (e.g., SB-462795 or an analogue
or
derivative thereof).
Anti-Infective Agents
The present invention also provides for the combination of a
polymeric composition and an agent which reduces the likelihood of infection
upon implantation of the composition or a medical implant.
Infection is a common complication of the implantation of foreign
bodies such as, for example, medical devices and implants. Foreign materials
provide an ideal site for micro- organisms to attach and colonize. It is also
hypothesized that there is an impairment of host defenses to infection in the
microenvironment surrounding a foreign material. These factors make medical
implants particularly susceptible to infection and make eradication of such an
infection difficult, if not impossible, in most cases. In many cases, an
infected
implant or device must be surgically removed from the body in order to
irradicate the infection.
109
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
The present invention provides agents (e.g., chemotherapeutic
agents) that can be released from a composition, and which have potent
antimicrobial activity at extremely low doses. A wide variety of anti-
infective
agents can be utilized in combination with the present compositions. Suitable
anti-infective agents may be readily determined based upon the assays
provided in Example 34). Discussed in more detail below are several
representative examples of agents that can be used as anti-infective agents,
such as: (A) anthracyclines (e.g., doxorubicin and mitoxantrone), (B)
fluoropyrimidines (e.g., 5-FU), (C) folic acid antagonists (e.g.,
methotrexate),
(D) podophylotoxins (e.g., etoposide), (E) camptothecins, (F) hydroxyureas,
and (G) platinum complexes (e.g., cisplatin).
A. Anthracyclines
In one aspect, the therapeutic anti-infective agent is an
anthracycline. Anthracyclines have the following general structure, where the
R
groups may be a variety of organic groups:
R
R
According to U.S. Patent 5,594,158, suitable R groups are as
follows: R~ is CH3 or CH20H; R2 is daunosamine or H; R3 and R4 are
independently one of OH, N02, NH2, F, CI, Br, 1, CN, H or groups derived from
these; R5 is hydrogen, ydroxyl, or methoxy; and R6_$ are all hydrogen.
Alternatively, R5 and R6 are hydrogen and R~ and R$ are alkyl or halogen, or
vice versa.
According to U.S. Patent 5,843,903, R~ may be a conjugated
peptide. According to U.S. Patent 4,296,105, R5 may be an ether linked alkyl
group. According to U.S. Patent 4,215,062, R5 may be OH or an ether linked
110
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
alkyl group. R~ may also be linked to the anthracycline ring by a group other
than C(O), such as an alkyl or branched alkyl group having the C(O) linking
moiety at its end, such as -CH2CH(CH2-X)C(O)-R~, wherein X is H or an alkyl
group (see, e.g., U.S_ Patent 4,215,062). R~ may alternately be a group linked
by the functional group =N-NHC(O)-Y, where Y is a group such as a phenyl or
substituted phenyl ring. Alternately R3 may have the following structure:
HsC O
~NH
Rio
in which R9 is OH either in or out of the plane of the ring, or is a second
sugar
moiety such as R3. Rio may be H or form a secondary amine with a group such
as an aromatic group, saturated or partially saturated 5 or 6 membered
heterocyclic having at least one ring nitrogen (see U.S. Patent 5,843,903).
Alternately, Rio may be derived from an amino acid, having the structure -
C(O)CH(NHR~~)(R~2), in which R~~ is H, or forms a C3_4 membered alkylene with
R~2. R~~ may be H, alkyl, aminoalkyl, amino, hydroxyl, mercapto, phenyl,
benzyl or methylthio (see U.S. Patent 4,296,105).
Exemplary anthracyclines are doxorubicin, daunorubicin,
idarubicin, epirubicin, pirarubicin, zorubicin, and carubicin. Suitable
compounds
have the structures:
R2
H
111
H 3C O
'NH2
R3
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
R~ R2 R3
Doxorubicin:OCH3 C(O)CH20H OH out of ring
plane
Epirubicin:
(4' epimer OCH3 C(O)CH2OH OH in ring plane
of
doxorubicin)
Daunorubicin:OCH3 C(O)CH3 OH out of ring
plane
Idarubicin: H C(O)CH3 OH out of ring
plane
Pirarubicin:OCH3 C(O)CH20H
0
~- i
Zorubicin: OCH3 C(CH3)(=N)NHC(O)C6H5 OH
Carubicin: OH C(O)CH3 OH out of ring
plane
Other suitable anthracyclines are anthramycin, mitoxantrone,
menogaril, nogalamycin, aclacinomycin A, olivomycin A, chromomycin A3, and
plicamycin having the structures:
R, RZ R
Menogaril H OCH3 H
OH o HN~NH~OH tJogalarrycin O-sugar H COOCH3
~ sugar H O OH'
3
~/ ~~J~I/0
OH O HN OH H3C0 ~~ OCH3
~NH~
Mitoxantrone
HO " HO- '_'~~ O
O ~1/
~CH~3~
R,O
~ ~'
Ho R~ Rz Ra Ra
OlivorcrycinCOCH(CH3)ZCH3COCN3H
A
ChromorrrycinCOCH3 CH3COCH3CH3
A3
PlicamycinH H H CH3
Other representative anthracyclines include, FCE 23762
doxorubicin derivative (Quaglia et al., J. Liq. Chromatogr. 77(18):3911-3923,
112
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
1994), annamycin (Zou et al., J. Pharm. Sci. 82(11 ):1151-1154, 1993), ruboxyl
(Rapoport et al., J. Controlled Release 58(2):153-162, 1999), anthracycline
disaccharide doxorubicin analogue (Pratesi et al., Clin. Cancer Res.
4(11 ):2833-2839, 1998), N-(trifluoroacetyl)doxorubicin and 4'-O-acetyl-N-
(trifluoroacetyl)doxorubicin (Berube & Lepage, Synth. Commun. 28(6):,1109-
1116, 1998), 2-pyrrolinodoxorubicin (Nagy et al., Proc. Nat'I Acad. Sci.
U.S.A.
95(4):1794-1799, 1998), disaccharide doxorubicin analogues (Arcamone et al.,
J. Nat'I Cancer Inst. 89(16):1217-1223, 1997), 4-demethoxy-7-O-(2,6-dideoxy-
4-O-(2,3,6-trideoxy-3-amino-a-L-lyxo-hexopyranosyl)-a-L-lyxo-hexopyranosyl)-
adriamicinone doxorubicin disaccharide analogue (Monteagudo et al.,
Carbohydr. Res. 300(1):11-16, 1997), 2-pyrrolinodoxorubicin (Nagy etal., Proc.
Nat'I Acad. Sci. U. S. A. 94(2):652-656, 1997), morpholinyl doxorubicin
analogues (Duran et al., Cancer Chemother. Pharmacol. 38(3}:210-216, 1996),
enaminomalonyl-~-alanine doxorubicin derivatives (Seitz et al., Tetrahedron
Lett. 36(9):1413-16, 1995), cephalosporin doxorubicin derivatives (Vrudhula et
al., J. Med. Chem. 38(8):1380-5, 1995), hydroxyrubicin (Solary et al., Int. J.
Cancer 58(1 ):85-94, 1994), methoxymorpholino doxorubicin derivative (Kuhl et
al., Cancer Chemother. Pharmacol. 33(1 ):10-16, 1993), (6-
maleimidocaproyl)hydrazone doxorubicin derivative (Willner et al.,
Bioconjugate
Chem. 4(6):521-7, 1993), N-(5,5-diacetoxypent-1-yl) doxorubicin (Cherif &
Farquhar, J. Med. Chem. 35(17):3208-14, 1992), FCE 23762
methoxymorpholinyl doxorubicin derivative (Ripamonti et al., Br. J. Cancer
65(5):703-7, 1992), N-hydroxysuccinimide ester doxorubicin derivatives
(Demant et al., Biochim. Biophys. Acta 1118(1 ):83-90, 1991 ),
polydeoxynucleotide doxorubicin derivatives (Ruggiero et al., Biochim.
Biophys.
Acta 1129(3):294-302, 1991 ), morpholinyl doxorubicin derivatives (EPA
434960), mitoxantrone doxorubicin analogue (Krapcho et al., J. Med. Chem.
34(8):2373-80. 1991 ), AD198 doxorubicin analogue (Traganos et al., Cancer
Res. 51(14):3682-9, 1991 ), 4-demethoxy-3'-N-trifluoroacetyldoxorubicin
(Horton
et al., Drug Des. Delivery 6(2):123-9, 1990), 4'-epidoxorubicin (Drzewoski et
al.,
113
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Pol. J. Pharmacol. Pharm. 40(2):159-65, 1988; Weenen et al., Eur. J. Cancer
Clin. Oncol. 20(7):919-26, 1984), alkylating cyanomorpholino doxorubicin
derivative (Scudder et al., J. Nat'I Cancer Inst. 80(16):1294-8, 1988),
deoxydihydroiodooxorubicin (EPA 275966), adriblastin (Kalishevskaya et al.,
Vestn. Mosk. Univ., 96(Biol. 1):21-7, 1988}, 4'-deoxydoxorubicin (Schoelzel et
al., Leuk. Res. 70(12):1455-9, 1986), 4-demethyoxy-4'-o-methyldoxorubicin
(Giuliani et al., Proc. Int. Congr. Chemother. 76:285-70-285-77, 1983), 3'-
deamino-3'-hydroxydoxorubicin (Norton et al., J. Antibiot. 37(8):853-8, 1984),
4-
demethyoxy doxorubicin analogues (Barbieri et al., Drugs Exp. Clin. Res.
10(2):85-90, 1984), N-L-leucyl doxorubicin derivatives (Trouet et al.,
Anthracyclines (Proc. Int. Symp. Tumor Pharmacother.), 179-81, 1983), 3'-
deamino-3'-(4-methoxy-1-piperidinyl) doxorubicin derivatives (U.S. 4,314,054),
3'-deamino-3'-(4-mortholinyl) doxorubicin derivatives (U.S. 4,301,277), 4'-
deoxydoxorubicin and 4'-o-methyldoxorubicin (Giuliani et al., Int. J. Cancer
27(1 ):5-13, 1981 ), aglycone doxorubicin derivatives (Chan & Watson, J.
Pharm.
Sci. 67(12):1748-52, 1978), SM 5887 (Pharma Japan 7468:20, 1995), MX-2
(Pharma Japan 7420:19, 1994), 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin (EP
275966), morpholinyl doxorubicin derivatives (EPA 434960), 3'-deamino-3'-(4-
methoxy-1-piperidinyl) doxorubicin derivatives (U.S. 4,314,054), doxorubicin-
14-valerate, morpholinodoxorubicin (U.S. 5,004,606), 3'-deamino-3'-(3"-cyano-
4"-morpholinyl doxorubicin; 3'-deamino-3'-(3"-cyano-4"-morpholinyl)-13-
dihydoxorubicin; (3'-deamino-3'-(3"-cyano-4"-morpholinyl) daunorubicin; 3'-
deamino-3'-(3"-cyano-4"-morpholinyl)-3-dihydrodaunorubicin; and 3'-deamino-
3'-(4"-morpholinyl-5-iminodoxorubicin and derivatives (U.S. 4,585,859), 3'-
deamino-3'-(4-methoxy-1-piperidinyl) doxorubicin derivatives (U.S. 4,314,054)
and 3-deamino-3-(4-morpholinyl) doxorubicin derivatives (U.S. 4,301,277).
B. Fluoropyrimidine analogues
In another aspect, the ant-infective therapeutic agent is a
fluoropyrimidine analog, such as 5-fluorouracil, or an analogue or derivative
114
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
thereof, including carmofur, doxifluridine, emitefur, tegafur, and
floxuridine.
Exemplary compounds have the structures:
0
R~~ F
N
O N
R~
R~ R2
5-FluorouracilH H
Carmofur C(O)NH(CH2)5CH3 H
DoxifluridineA~ H
Floxuridine A2 H
Emitefur CH20CH2CH3 B
Tegafur C H
CN
O ~ ~ 0 O
O N O
Cr
Other suitable fluoropyrimidine analogues include 5-FudR (5-
fluoro-deoxyuridine), or an analogue or derivative thereof, including 5-
iododeoxyuridine (5-IudR), 5-bromodeoxyuridine (5-BudR), fluorouridine
triphosphate (5-FUTP), and fluorodeoxyuridine monophosphate (5-dFUMP)
Exemplary compounds have the structures:
115
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
H
5-Fluoro-2'-deoxyuridine: R = F
5-Bromo-2'-deoxyuridine: R = Br
5-lodo-2'-deoxyuridine: R = I
Other representative examples of fluoropyrimidine analogues
include N3-alkylated analogues of 5-fluorouracil (Kozai et al., J. Chem. Soc.,
Per6cin Trans. 7(19):3145-3146, 1998), 5-fluorouracil derivatives with 1,4-
oxaheteroepane moieties (Gomez et al., Tetrahedron 54(43):13295-13312,
1998), 5-fluorouracil and nucleoside analogues (Li, Anticancer Res. 17(1A):21-
27, 1997), cis- and trans-5-fluoro-5,6-dihydro-6-alkoxyuracil (Van der Wilt et
al.,
Br. J. Cancer 68(4):702-7, 1993), cyclopentane 5-fluorouracil analogues
(Hronowski & Szarek, Can. J. Chem. 70(4):1162-9, 1992), A-OT-fluorouracil
(Zhang et al., Zongguo Yiyao Gongye Zazhi 20(11 ):513-15, 1989), N4-
trimethoxybenzoyl-5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine (Miwa
et al., Chem. Pharm. Bull. 38(4):998-1003, 1990), 1-hexylcarbamoyl-5-
fluorouracil (Hoshi et al., J. Pharmacobio-Dun. 3(9):478-81, 1980; Maehara et
al., Chemotherapy (Basel) 34(6):484-9, 1988), B-3839 (Prajda et al., In Vivo
2(2):151-4, 1988), uracil-1-(2-tetrahydrofuryl)-5-fluorouracil (Anai et al.,
Oncology 45(3):144-7, 1988), 1-(2'-deoxy-2'-fluoro-~i-D-arabinofuranosyl)-5-
fluorouracil (Suzuko et al., Mol. Pharmacol. 37(3):301-6, 1987), doxifluridine
(Matuura et al., Oyo Yakuri 29(5):803-31, 1985), 5'-deoxy-5-fluorouridine
(Bollag & Hartmann, Eur. J. Cancer 76(4):427-32, 1980), 1-acetyl-3-O-toluyl-5-
fluorouracil (Okada, Hiroshima J. Med. Sci. 28(1 ):49-66, 1979), 5-
fluorouracil-
116
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
m-formylbenzene-sulfonate (JP 55059173), N'-(2-furanidyl)-5-fluorouracil (JP
53149985) and 1-(2-tetrahydrofuryl)-5-fluorouracil (JP 52089680).
These compounds are believed to function as therapeutic agents
by serving as antimetabolites of pyrimidine.
C. Folic acid antagonists
In another aspect, the anti-infective therapeutic agent is a folic
acid antagonist, such as methotrexate or derivatives or analogues thereof,
including edatrexate, trimetrexate, raltitrexed, piritrexim, denopterin,
tomudex,
and pteropterin. Methotrexate analogues have the following general structure:
The identity of the R group may be selected from organic groups, particularly
those groups set forth in U.S. Patent Nos. 5,166,149 and 5,382,582. For
example, R~ may be N, R2 may be N or C(CH3), R3 and R3' may H or alkyl, e.g.,
CH3, R4 may be a single bond or NR, where R is H or alkyl group. R5,6,$ may be
H, OCH3, or alternately they can be halogens or hydro groups. R~ is a side
chain of the general structure:
H
HO
O
i
n
wherein n = 1 for methotrexate, n = 3 for pteropterin. The carboxyl groups in
the side chain may be esterified or form a salt such as a ~n2+ salt. R9 and
Rio
can be NH2 or may be alkyl substituted.
Exemplary folic acid antagonist compounds have the structures:
117
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
H2
Ro R~ Ra R3 R4 R5 Rs R~ Ra
MethotrexateNH2 N N H N(CH3) H H A (n=1 H
)
EdatrexateNH2 N N H CH(CH2CH3)H H A (n=1 H
)
TrimetrexateNHS CH C(CH3)H NH H OCH3 OCH3 OCH3
PteropterinOH N N H NH H H A (n=3)H
DenopterinOH N N CH3 N(CH3) H H A (n=1 H
)
PeritreximNH2 N C(CH3)H single OCH3 H H OCH3
bond
A: o
NH
HO
O
O OH
N CH3
HOOC~ O ~ H3
S N ~ ~ NH
HOOC NH
O
Tomudex
Other representative examples include 6-S-aminoacyloxymethyl
mercaptopurine derivatives (Harada et al., Chem. Pharm. Bull. 43(10):793-6,
1995), 6-mercaptopurine (6-MP) (Kashida et al., Biol. Pharm. Bull. 78(11
):1492-
7, 1995), 7,8-polymethyleneimidazo-1,3,2-diazaphosphorines (Nilov et al.,
Mendeleev Commun. 2:67, 1995), azathioprine (Chifotides et al., J. Inorg.
Biochem. 56(4):249-64, 1994), methyl-D-glucopyranoside mercaptopurine
derivatives (Da Silva et al., Eur. J. Med. Chem. 29(2):149-52, 1994) and s-
alkynyl mercaptopurine derivatives (Ratsino et al., Khim.-Farm. Zh. 75(8):65-
7,
1981 ); indoline ring and a modified ornithine or glutamic acid-bearing
methotrexate derivatives (Matsuoka et al., Chem. Pharm. Bull. 45(7):1146-
118
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
1150, 1997), alkyl-substituted benzene ring C bearing methotrexate derivatives
(Matsuoka et al., Chem. Pharm. Bull. 44(12):2287-2293, 1996), benzoxazine or
benzothiazine moiety-bearing methotrexate derivatives (Matsuoka et al., J.
Med. Chem. 40(1 ):105-111, 1997), 10-deazaaminopterin analogues (DeGraw
et al., J. Med. Chem. 40(3):370-376, 1997), 5-deazaaminopterin and 5,10-
dideazaaminopterin methotrexate analogues (Piper et al., J. Med. Chem.
40(3):377-384, 1997), indoline moiety-bearing methotrexate derivatives
(Matsuoka et al., Chem. Pharm. Bull. 44(7):1332-1337, 1996), lipophilic amide
methotrexate derivatives (Pignatello et al., World Meet. Pharm., Biopharm.
Pharm. Technol., 563-4, 1995), L-threo-(2S,4S)-4-fluoroglutamic acid and DL-
3,3-difluoroglutamic acid-containing methotrexate analogues (Hart et al., J.
Med. Chem. 39(1):56-65, 1996), methotrexate tetrahydroquinazoline analogue
(Gangjee, et al., J. Heterocycl. Chem. 32(1 ):243-8, 1995), N-(a-aminoacyl)
methotrexate derivatives (Cheung et al., Pteridines 3(1-2):101-2, 1992),
biotin
methotrexate derivatives (Fan et al., Pteridines 3(1-2):131-2, 1992), D-
glutamic
acid or D-erythrou, threo-4-fluoroglutamic acid methotrexate analogues
(McGuire et al., Biochem. Pharmacol. 42(12):2400-3, 1991 ), ~,y-methano
methotrexate analogues (Rosowsky et al., Pteridines 2(3):133-9, 1991 ), 10-
deazaaminopterin (10-EDAM) analogue (Braakhuis et al., Chem. Biol.
Pteridines, Proc. Int. Symp. Pteridines Folic Acid Deriv., 1027-30, 1989), y-
tetrazole methotrexate analogue (Kalman et al., Chem. Biol. Pteridines, Proc.
Int. Symp. Pteridines Folic Acid Deriv., 1154-7, 1989), N-(L-a-aminoacyl)
methotrexate derivatives (Cheung et al., Heterocycles 28(2):751-8, 1989), meta
and ortho isomers of aminopterin (Rosowsky et al., J. Med. Chem. 32(12):2582,
1989), hydroxymethylmethotrexate (DE 267495), y-fluoromethotrexate
(McGuire et al., Cancer Res. 49(16):4517-25, 1989), polyglutamyl methotrexate
derivatives (Kumar et al., Cancer Res. 46(10):5020-3, 1986), gem-
diphosphonate methotrexate analogues (WO 88/06158), a- and y-substituted
methotrexate analogues (Tsushima et al., Tetrahedron 44(17):5375-87, 1988),
5-methyl-5-deaza methotrexate analogues (4,725,687), N8-acyl-Na-(4-amino-4-
119
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
deoxypteroyl)-L-ornithine derivatives (Rosowsky et al., J. Med. Chem.
31(7):1332-7, 1988), 8-deaza methotrexate analogues (Kuehl et al., Cancer
Res. 48(6):1481-8, 1988), acivicin methotrexate analogue (Rosowsky et al., J.
Med. Chem. 30(8):1463-9, 1987), polymeric platinol methotrexate derivative
(Carraher et al., Polym. Sci. Technol. (Plenum), 35(Adv. Biomed. Polym.):311-
24, 1987), methotrexate-y-dimyristoylphophatidylethanolamine (Kinsky et al.,
Biochim. Biophys. Acta 917(2):211-18, 1987), methotrexate polyglutamate
analogues (Rosowsky et al., Chem. Biol. Pteridines, Pteridines Folic Acid
Deriv., Proc. Int. Symp. Pteridines Folic Acid Deriv.: Chem., Biol. Clin.
Aspects:
985-8, 1986), poly-y-glutamyl methotrexate derivatives (Kisliuk et al., Chem.
Biol. Pteridines, Pteridines Folic Acid Deriv., Proc. Int. Symp. Pteridines
Folic
Acid Deriv.: Chem., Biol. Clin. Aspects: 989-92, 1986), deoxyuridylate
methotrexate derivatives (Webber et al., Chem. Biol. Pteridines, Pteridines
Folic
Acid Deriv., Proc. Int. Symp. Pteridines Folic Acid Deriv.: Chem., Biol. Clin.
Aspects: 659-62, 1986), iodoacetyl lysine methotrexate analogue (Delcamp et
al., Chem. Biol. Pteridines, Pteridines Folic Acid Deriv., Proc. Int. Symp.
Pteridines Folic Acid Deriv.: Chem., Biol. Clin. Aspects: 807-9, 1986),
2,.omega.-diaminoalkanoid acid-containing methotrexate analogues (McGuire
et al., Biochem. Pharmacol. 35(15):2607-13, 1986), polyglutamate
methotrexate derivatives (Kamen & Winick, Methods Enzymol. 122(Vitam.
Coenzymes, Pt. G):339-46, 1986), 5-methyl-5-deaza analogues (Piper et al., J.
Med. Chem. 29(6):1080-7, 1986), quinazoline methotrexate analogue
(Mastropaolo et al., J. Med. Chem. 29(1 ):155-8, 1986), pyrazine methotrexate
analogue (Lever & Vestal, J. Heterocycl. Chem. 22(1 ):5-6, 1985), cysteic acid
and homocysteic acid methotrexate analogues (4,490,529), y-tert-butyl
methotrexate esters (Rosowsky et al., J. Med. Chem. 28(5):660-7, 1985),
fluorinated methotrexate analogues (Tsushima et al., Heterocycles 23(1 ):45-9,
1985), folate methotrexate analogue (Trombe, J. Bacteriol. 160(3):849-53,
1984), phosphonoglutamic acid analogues (Sturtz & Guillamot, Eur. J. Med.
Chem.--Chim. Ther. 19(3):267-73, 1984), poly (L-lysine) methotrexate
120
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
conjugates (Rosowsky et al., J. Med. Chem. 27(7):888-93, 1984), dilysine and
trilysine methotrexate derivates (Forsch ~ Rosowsky, J. Org. Chem.
49(7):1305-9, 1984), 7-hydroxymethotrexate (Fabre et al., Cancer Res.
43(10):4648-52, 1983), poly-y-glutamyl methotrexate analogues (Piper &
Montgomery, Adv. Exp. Med. Biol., 163(F~lyl Antifolyl Polyglutamates):95-100,
1983), 3',5'-dichloromethotrexate (Rosowsky & Yu, J. Med. Chem. 26(10):1448-
52, 1983), diazoketone and chloromethylketone methotrexate analogues
(Gangjee et al., J. Pharm. Sci. 71(6):717-19, 1982), 10-propargylaminopterin
and alkyl methotrexate homologs (Piper et al., J. Med. Chem. 25(7):877-80,
1982), lectin derivatives of methotrexate (Lin et al., JNCI 66(3):523-8, 1981
),
polyglutamate methotrexate derivatives (Galivan, Mol. Pharmacol. 17(1 ):105-
10, 1980), halogentated methotrexate derivatives (Fox, JNCI 58(4):J955-8,
1977), 8-alkyl-7,8-dihydro analogues (Chaykovsky et al., J. Med. Chem.
20(10):J1323-7, 1977), 7-methyl methotrexate derivatives and
dichloromethotrexate (Rosowsky & Chen, J. Med. Chem. 17(12):J1308-11,
1974), lipophilic methotrexate derivatives and 3',5'-dichloromethotrexate
(Rosowsky, J. Med. Chem. 16(10):J1190-3, 1973), deaza amethopterin
analogues (Montgomery et al., Ann. N. Y. Acad. Sci. 186:J227-34, 1971 ),
MX068 (Pharma Japan, 1658:18, 1999) and cysteic acid and homocysteic acid
methotrexate analogues (EPA 0142220).
These compounds are believed to act as antimetabolites of folic
acid.
D. Podophyllotoxins
In another aspect, the anti-infective therapeutic agent is a
Podophyllotoxin, or a derivative or an analogue thereof. Exemplary compounds
of this type are etoposide or teniposide, which have the following structures:
121
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Etoposide CH3
Teniposide s
OCH3
OH
Other representative examples of podophyllotoxins include Cu(II)-
VP-16 (etoposide) complex (Tawa et al., Bioorg. Med. Chem. 6(7):1003-1008,
1998), pyrrolecarboxamidino-bearing etoposide analogues (Ji et al., Bioorg.
Med. Chem. Lett. 7(5):607-612, 1997), 4a-amino etoposide analogues (Hu,
University of North Carolina Dissertation, 1992), y-lactone ring-modified
arylamino etoposide analogues (Zhou et al., J. Med. Chem. 37(2):287-92,
1994), N-glucosyl etoposide analogue (Allevi et al., Tetrahedron Lett.
34(45):7313-16, 1993), etoposide A-ring analogues (Kadow et al., Bioorg. Med.
Chem. Lett. 2(1 ):17-22, 1992), 4'-deshydroxy-4'-methyl etoposide (Saulnier et
al., Bioorg. Med. Chem. Lett. 2(10):1213-18, 1992), pendulum ring etoposide
analogues (Sinha et al., Eur. J. Cancer 26(5):590-3, 1990) and E-ring desoxy
etoposide analogues (Saulnier et al., J. Med. Chem. 32(7):1418-20, 1989).
These compounds are believed to act as topoisomerase II
inhibitors and/or DNA cleaving agents.
E. Camptothecins
In another aspect, the anti-infective therapeutic agent is
camptothecin, or an analogue or derivative thereof. Camptothecins have the
following general structure.
122
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In this structure, X is typically O, but can be other groups, e.g., NH
in the case of 21-lactam derivatives. R1 is typically H or OH, but may be
other
groups, e.g., a terminally hydroxylated C1_3 alkane. R2 is typically H or an
amino containing group such as (CH3)2NHCH2, but may be other groups e.g.,
NO2, NH2, halogen (as disclosed in, e.g., U.S. Patent 5,552,156) or a short
alkane containing these groups. R3 is typically H or a short alkyl such as
C2H5.
R4 is typically H but may be other groups, e.g., a methylenedioxy group with
R1,
Exemplary camptothecin compounds include topotecan,
irinotecan (CPT-11 ), 9-aminocamptothecin, 21-lactam-20(S)-camptothecin,
10,11-methylenedioxycamptothecin, SN-3~, 9-nitrocamptothecin, 10-
hydroxycamptothecin. Exemplary compounds have the structures:
R~ Rz R3
Camptothecin: H H H
Topotecan: OH (CH3)zNHCHz H
SN-38: OH H CZHS
X: O for most analogs, NH for 21-lactam analogs
Camptothecins have the five rings shown here. The ring labeled
E must be intact (the lactone rather than carboxylate form) for maximum
activity
and minimum toxicity.
Camptothecins are believed to function as topoisomerase I
inhibitors and/or DNA cleavage agents.
F. Hydroxyureas
The anti-infective therapeutic agent of the present invention may
be a hydroxyurea. Hydroxyureas have the following general structure:
123
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
O
R3 O-X
~N N~
R2 R~
Suitable hydroxyureas are disclosed in, for example, U.S. Patent
No. 6,080,874, wherein R~ is:
and R2 is an alkyl group having 1-4 carbons and R3 is one of H, acyl, methyl,
ethyl, and mixtures thereof, such as a methylether.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 5,665,768, wherein R~ is a cycloalkenyl group, for example N-(3-(5-(4-
fluorophenylthio)-furyl)-2-cyclopenten-1-yl)N-hydroxyurea; R2 is H or an alkyl
group having 1 to 4 carbons and R3 is H; X is H or a cation.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 4,299,778, wherein R~ is a phenyl group substituted with one or more
fluorine atoms; R2 is a cyclopropyl group; and R3 and X is H.
Other suitable hydroxyureas are disclosed in, e.g., U.S. Patent
No. 5,066,658, wherein R2 and R3 together with the adjacent nitrogen form:
(c~z)~
Y . N-
(CH2)m
where in m is 1 or 2, n is 0-2 and Y is an alkyl group.
In one aspect, the hydroxyurea has the structure:
0
OOH
H2N NH
Hydroxyurea
124
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
These compounds are thought to function by inhibiting DNA
synthesis.
G. Platinum complexes
In another aspect, the anti-infective therapeutic agent is a
platinum compound. In general, suitable platinum complexes may be of Pt(II)
or Pt(IV) and have this basic structure:
z2
wherein X and Y are anionic leaving groups such as sulfate, phosphate,
carboxylate, and halogen; R~ and R2 are alkyl, amine, amino alkyl any may be
further substituted, and are basically inert or bridging groups. For Pt(II)
complexes Z~ and Z2 are non-existent. For Pt(IV) Z~ and ~2 may be anionic
groups such as halogen, hydroxy, carboxylate, ester, sulfate or phosphate.
See, e.g., U.S. Patent Nos. 4,588,831 and 4,250,189.
Suitable platinum complexes may contain multiple Pt atoms. See,
e.g., U.S. Patent Nos. 5,409,915 and 5,380,897. For example bisplatinum and
triplatinum complexes of the type:
125
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Zt Zt
R X
X~ / ~ ~ , Rz
Y~ It\A~ ~t~Y
Zz Zz
Zt Zt Zt
X\ I /Rt X\ I A\ I /X
Pt Pt/ \ Pt
Y/ I ~ A~ I \ Y Rz~ I \ Y
Zz Zz Z2
Zt Zt
X\ ~ / Rz Rz~ ~ / X
Y~ It\A/ It~Y
Z2 ~ Z2
Z2~ I / R3
Pt
Y~~\ZI
X
Exemplary platinum compounds are cisplatin, carboplatin,
oxaliplatin, and miboplatin having the structures:
NH3
NH3 O O~
It
CIIt-NH3 I ~NH3
O
CI
O
Cisplatin Carboplatin
O O H H
\/
O NHz O N
Pt
/ \ ~ / ' H
O NH ~~~" O N
z O l
O H
Oxaliplatin Miboplatin
Other representative platinum compounds include
(CPA)~Pt(DOLYM) and (DACH)Pt(DOLYM) cisplatin (Choi et al., Arch.
Pharmacal Res. 22(2):151-156, 1999), Cis-(PtCl2(4,7-H-5-methyl-7-
oxo)1,2,4(triazolo(1,5-a)pyrimidine)2) (Navarro etal., J. Med. Chem. 47(3):332-
126
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
338, 1998), (Pt(cis-1,4-DACH)(trans-CI2)(CBDCA))~'/ZMeOH cisplatin
(Shamsuddin et al., Inorg. Chem. 36(25):5969-5971, 1997), 4-pyridoxate
diammine hydroxy platinum (Tokunaga et al., Pharm. Sci. 3(7):353-356, 1997),
Pt(II) ... Pt(II) (Pt2(NHCHN(C(CH2)(CH3)))4) (Navarro et al., Inorg. Chem.
35(26):7829-7835, 1996), 254-S cisplatin analogue (Koga et al., Neurol. Res.
18(3):244-247, 1996), o-phenylenediamine ligand bearing cisplatin analogues
(Koeckerbauer ~ Bednarski, J. Inorg. Biochem. 62(4):281-298, 1996), trans,
cis-(Pt(OAc)21~(en)) (Kratochwil et al., J. Med. Chem. 39(13):2499-2507,
1996),
estrogenic 1,2-diarylethylenediamine ligand (with sulfur-containing amino
acids
and glutathione) bearing cisplatin analogues (Bednarski, J. Inorg. Biochem.
62(1 ):75, 1996), cis-1,4-diaminocyclohexane cisplatin analogues (Shamsuddin
et al., J. Inorg. Biochem. 61(4):291-301, 1996), 5' orientational isomer of
cis-
(Pt(NH3)(4-aminoTEMP-O){d(GpG))) (Dunham & Lippard, J. Am. Chem. Soc.
117(43):10702-12, 1995), chelating diamine-bearing cisplatin analogues
(Koeckerbauer & Bednarski, J. Pharm. Sci. 84(7):819-23, 1995),1,2-
diarylethyleneamine ligand-bearing cisplatin analogues (Otto et al., J. Cancer
Res. Clin. Oncol. 121(1 ):31-8, 1995), (ethylenediamine)platinum(II) complexes
(Pasini et al., J. Chem. Soc., Dalton Trans. 4:579-85, 1995), CI-973 cisplatin
analogue (Yang et al., Int. J. Oncol. 5(3):597-602, 1994), cis-
diaminedichloroplatinum(II) and its analogues cis-1,1-
cyclobutanedicarbosylato(2R)-2-methyl-1,4-butanediamineplatinum(II) and cis-
diammine(glycolato)platinum (Claycamp & Zimbrick, J. Inorg. Biochem.
26(4):257-67, 1986; Fan et al., Cancer Res. 48(11 ):3135-9, 1988; Heiger-
Bernays et al., Biochemistry 29(36):8461-6, 1990; Kikkawa et al., J. Exp.
Clin.
Cancer Res. 12(4):233-40, 1993; Murray et al., Biochemistry 31(47):11812-17,
1992; Takahashi et al., Cancer Chemother. Pharmacol. 33(1 ):31-5, 1993), cis-
amine-cyclohexylamine-dichloroplatinum(II) (Yoshida et al., Biochem.
Pharmacol. 48(4):793-9, 1994), gem-diphosphonate cisplatin analogues (FR
2683529), (meso-1,2-bis(2,6-dichloro-4-hydroxyplenyl)ethylenediamine)
dichloroplatinum(II) (Bednarski et al., J. Med. Chem. 35(23):4479-85, 1992),
127
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
cisplatin analogues containing a tethered dansyl group (Hartwig et al., J. Am.
Chem. Soc. 114(21 ):8292-3, 1992), platinum(II) polyamines (Siegmann et al.,
Inorg. Met.-Containing Polym, Mater., (Proc. Am. Chem. Soc. Int. Symp.), 335-
61, 1990), cis-(3H)dichloro(ethylenediamine)platinum(II) (Eastman, Anal.
Biochem. 197(2):311-15, 1991 ), trans-diamminedichloroplatinum(II) and cis-
(Pt(NH3)~(N3-cytosine)CI) (Bellon & Lippard, Biophys. Chem. 35(2-3):179-88,
1990), 3H-cis-1,2-diaminocyclohexanedichloroplatinum(II) and 3H-cis-1,2-
diaminocyclohexanemalonatoplatinum (II) (Oswald et al., Res. Commun. Chem.
Pathol. Pharmacol. 64(1 ):41-58, 1989), diaminocarboxylatoplatinum (EPA
296321 ), trans-(D,1 )-1,2-diaminocyclohexane carrier ligand-bearing platinum
analogues (Wyrick & Chaney, J. Labelled Compd. Radiopharm. 25(4):349-57,
1988), aminoalkylaminoanthraquinone-derived cisplatin analogues (Kitov et al.,
Eur. J. Med. Chem. 23(4):381-3, 1988), spiroplatin, carboplatin, iproplatin
and
JM40 platinum analogues (Schroyen et al., Eur, J. Cancer Clin. Oncol.
24(8):1309-12, 1988), bidentate tertiary diamine-containing cisplatinum
derivatives (Orbell et al., Inorg. Chim. Acta 152(2):125-34, 1988),
platinum(11),
platinum(IV) (Liu & Wang, Shandong Yike Daxue Xuebao 24(1 ):35-41, 1986),
cis-diammine(1,1-cyclobutanedicarboxylato-)platinum(II) (carboplatin, JM8) and
ethylenediammine-malonatoplatinum(II) (JM40) (Begg et al., Radiother. Oncol.
9(2):157-65, 1987), JM8 and JM9 cisplatin analogues (Harstrick et al., Int. J.
Androl. 10(1 ); 139-45, 1987), (NPr4)2((PtCL4).cis-(PtCl2-(NH2Me)2))
(Brammer et al., J. Chem. Soc., Chem. Commun. 6:443-5, 1987), aliphatic
tricarboxylic acid platinum complexes (EPA 185225), and cis-dichloro(amino
acid)(tert-butylamine)platinum(II) complexes (Pasini & Bersanetti, Inorg,
Chim.
Acta 107(4):259-67, 1985). These compounds are thought to function by
binding to DNA, i.e., acting as alkylating agents of DNA.
Dosages of Anti-Infective Agents
The drug dose administered from the present compositions for
prevention or inhibition of infection in accordance with the present invention
will
128
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
depend on a variety of factors, including the type of formulation, the
location of
the treatment site, and the type of condition being treated. However, certain
principles can be applied in the application of this art. Drug dose can be
calculated as a function of dose per unit area (of the treatment site), total
drug
dose administered can be measured and appropriate surface concentrations of
active drug can be determined. Drugs are to be used at concentrations that
range from several times more than to 50%, 20%, 10%, 5%, or even less than
1 % of the concentration typically used in a single anti-infective systemic
dose
application. In certain aspects, the anti- infective agent is released from
the
polymer composition in effective concentrations in a time period that may be
measured from the time of infiltration into tissue adjacent to the device,
which
ranges from about less than 1 day to about 180 days. Generally, the release
time may also be from about less than 1 day to about 180 days; from about 7
days to about 14 days; from about 14 days to about 28 days; from about 28
days to about 56 days; from about 56 days to about 90 days; from about 90
days to about 180 days.
The exemplary anti-infective agents, used alone or in
combination, should be administered under the following dosing guidelines.
The total amount (dose) of anti-infective agent in the composition can be in
the
range of about 0.01 ~g-1 fig, or about 1 p,g-10 fig, or about 10 pg-1 mg, or
about 1 mg to 10 mg, or about 10 mg-100 mg, or about 100 mg to 250 mg, or
about 250 mg-1000 mg. The dose (amount) of anti-infective agent per unit area
of device or tissue surface to which the agent is applied may be in the range
of
about 0.01 ~g/mm2 - 1 p,g/mm2, or about 1 wg/mm2 - 10 pg/mm2, or about 10
~,g/mm2 -100 pg/mm2, or about 100 ~,g/mm2 to 250 ~g/mm~, or about 250
~g/mm2 - 1000 ~,g/mm2. As different polymer compositions will release the anti-
infective agent at differing rates, the above dosing parameters should be
utilized in combination with the release rate of the drug from the composition
such that a minimum concentration of about 10-$ M to 10-' M, or about 10-' M
to
129
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
10'6 M about 10-6 M to 10'5 M or about 10'5 M to 10'4 M of the agent is
maintained on the tissue surface.
(a) Anthracyclines. Utilizing the anthracycline doxorubicin as an
example, whether applied as a polymer coating, incorporated into the polymers
which make up the implant components, or applied without a carrier polymer,
the total dose of doxorubicin applied to the device or implant should not
exceed
25 mg (range of 0.1 ~g to 25 mg). In a particularly preferred embodiment, the
total amount of drug applied should be in the range of 1 pg to 5 mg. The dose
per unit area (i.e., the amount of drug as a function of the surface area of
the
portion of the implant to which drug is applied and/or incorporated) should
fall
within the range of 0.01 ~g - 100 p,g per mm2 of surface area. In a
particularly
preferred embodiment, doxorubicin should be applied to the implant surface at
a dose of 0.1 ~,g/mm2 - 10 p.g/mm2. As different polymer and non-polymer
coatings will release doxorubicin at differing rates, the above dosing
parameters
should be utilized in combination with the release rate of the drug from the
implant surface such that a minimum concentration of 10''- 104 M of
doxorubicin is maintained on the surface. It is necessary to insure that
surface
drug concentrations exceed concentrations of doxorubicin known to be lethal to
multiple species of bacteria and fungi (i.e., are in excess of 10-4 M;
although for
some embodiments lower concentrations are sufficient). In a preferred
embodiment, doxorubicin is released from the surface of the implant such that
anti-infective activity is maintained for a period ranging from several hours
to
several months. In a particularly preferred embodiment the drug is released in
effective concentrations for a period ranging from 1 week - 6 months. It
should
be readily evident based upon the discussions provided herein that analogues
and derivatives of doxorubicin (as described previously) with similar
functional
activity can be utilized for the purposes of this invention; the above dosing
parameters are then adjusted according to the relative potency of the analogue
or derivative as compared to the parent compound (e.g., a compound twice as
potent as doxorubicin is administered at half the above parameters, a
130
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
compound half as potent as doxorubicin is administered at twice the above
parameters, etc.).
Utilizing mitoxantrone as another example of an anthracycline,
whether applied as a polymer coating, incorporated into the polymers which
make up the device or implant, or applied without a carrier polymer, the total
dose of mitoxantrone applied should not exceed 5 mg (range of 0.01 ~.g to 5
mg). In a particularly preferred embodiment, the total amount of drug applied
should be in the range of 0.1 ~g to 1 mg. The dose per unit area (i.e., the
amount of drug as a function of the surface area of the portion of the implant
to
which drug is applied and/or incorporated) should fall within the range of
0.01
pg - 20 p.g per mm2 of surface area. In a particularly preferred embodiment,
mitoxantrone should be applied to the implant surface at a dose of 0.05
~,g/mm2
- 3 pg/mm2. As different polymer and non-polymer coatings will release
mitoxantrone at differing rates, the above dosing parameters should be
utilized
in combination with the release rate of the drug from the implant surface such
that a minimum concentration of 10-5- 10-6 M of mitoxantrone is maintained. It
is necessary to insure that drug concentrations on the implant surface exceed
concentrations of mitoxantrone known to be lethal to multiple species of
bacteria and fungi (i.e., are in excess of 10-5 M; although for some
embodiments
lower drug levels will be sufficient). In a preferred embodiment, mitoxantrone
is
released from the surface of the implant such that anti-infective activity is
maintained for a period ranging from several hours to several months. In a
particularly preferred embodiment the drug is released in effective
concentrations for a period ranging from 1 week - 6 months. It should be
readily evident based upon the discussions provided herein that analogues and
derivatives of mitoxantrone (as described previously) with similar functional
activity can be utilized for the purposes of this invention; the above dosing
parameters are then adjusted according to the relative potency of the analogue
or derivative as compared to the parent compound (e.g., a compound twice as
potent as mitoxantrone is administered at half the above parameters, a
131
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
compound half as potent as mitoxantrone is administered at twice the above
parameters, etc.).
(b) Fluoropyrimidines Utilizing the fluoropyrimidine 5-fluorouracil
as an example, whether applied as a polymer coating, incorporated into the
polymers which make up the device or implant, or applied without a carrier
polymer, the total dose of 5-fluorouracil applied should not exceed 250 mg
(range of 1.0 p.g to 250 mg). In a particularly preferred embodiment, the
total
amount of drug applied should be in the range of 10 pg to 25 mg. The dose per
unit area (i.e., the amount of drug as a function of the surface area of the
portion of the implant to which drug is applied and/or incorporated) should
fall
within the range of 0.1 ~,g -1 mg per mm2 of surface area. In a particularly
preferred embodiment, 5-fluorouracil should be applied to the implant surface
at
a dose of 1.0 p.g/mm2 - 50 p.glmm2. As different polymer and non-polymer
coatings will release 5-fluorouracil at differing rates, the above dosing
parameters should be utilized in combination with the release rate of the drug
from the implant surface such that a minimum concentration of 10-4- 10-' M of
5-fluorouracil is maintained. It is necessary to insure that surface drug
concentrations exceed concentrations of 5-fluorouracil known to be lethal to
numerous species of bacteria and fungi (i.e., are in excess of 10-4 M;
although
for some embodiments lower drug levels will be sufficient). In a preferred
embodiment, 5-fluorouracil is released from the implant surface such that anti-
infective activity is maintained for a period ranging from several hours to
several
months. In a particularly preferred embodiment the drug is released in
effective
concentrations for a period ranging from 1 week - 6 months. It should be
readily evident based upon the discussions provided herein that analogues and
derivatives of 5-fluorouracil (as described previously) with similar
functional
activity can be utilized for the purposes of this invention; the above dosing
parameters are then adjusted according to the relative potency of the analogue
or derivative as compared to the parent compound (e.g., a compound twice as
potent as 5-fluorouracil is administered at half the above parameters, a
132
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
compound half as potent as 5-fluorouracil is administered at twice the above
parameters, etc.).
(c) Podophylotoxins Utilizing the podophylotoxin etoposide as an
example, whether applied as a polymer coating, incorporated into the polymers
which make up the device or implant, or applied without a carrier polymer, the
total dose of etoposide applied should not exceed 25 mg (range of 0.1 ~.g to
25
mg). In a particularly preferred embodiment, the total amount of drug applied
should be in the range of 1 pg to 5 mg. The dose per unit area (i.e., the
amount
of drug as a function of the surface area of the portion of the implant to
which
drug is applied and/or incorporated) should fall within the range of 0.01 pg -
100
p,g per mm2 of surface area. In a particularly preferred embodiment, etoposide
should be applied to the implant surface at a dose of 0.1 p,g/mmz -10 ~glmm2.
As different polymer and non-polymer coatings will release etoposide at
differing rates, the above dosing parameters should be utilized in combination
with the release rate of the drug from the implant surface such that a
concentration of 10-5 - 10-6 M of etoposide is maintained. It is necessary to
insure that surface drug concentrations exceed concentrations of etoposide
known to be lethal to a variety of bacteria and fungi (i.e., are in excess of
10-5 M;
although for some embodiments lower drug levels will be sufFicient). In a
preferred embodiment, etoposide is released from the surface of the implant
such that anti-infective activity is maintained for a period ranging from
several
hours to several months. In a particularly preferred embodiment the drug is
released in effective concentrations for a period ranging from 1 week - 6
months. It should be readily evident based upon the discussions provided
herein that analogues and derivatives of etoposide (as described previously)
with similar functional activity can be utilized for the purposes of this
invention;
the above dosing parameters are then adjusted according to the relative
potency of the analogue or derivative as compared to the parent compound
(e.g., a compound twice as potent as etoposide is administered at half the
133
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
above parameters, a compound half as potent as etoposide is administered at
twice the above parameters, etc.).
It should be readily evident based upon the discussions provided
herein that combinations of anthracyclines (e.g., doxorubicin or
mitoxantrone),
fluoropyrimidines (e.g., 5-fluorouracil), folic acid antagonists (e.g.,
methotrexate
and/or podophylotoxins (e.g., etoposide) can be utilized to enhance the
antibacterial activity of the composition.
It should be readily evident based upon the discussions provided
herein that combinations of anthracyclines (e.g., doxorubicin or
mitoxantrone),
fluoropyrimidines (e.g., 5-fluorouracil), folic acid antagonists (e.g.,
methotrexate
and/or podophylotoxins (e.g., etoposide) may be utilized to enhance the
antibacterial activity of the composition.
Combination Therapies
In addition to incorporation of the above-mentioned therapeutic
agents (i.e., anti-infective agents or fibrosis-inhibiting agents), one or
more
other pharmaceutically active agents can be incorporated into the present
compositions to improve or enhance efficacy. In one aspect, the composition
may further include a compound which acts to have an inhibitory effect on
pathological processes in or around the treatment site. Representative
examples of additional therapeutically active agents include, by way of
example
and not limitation, anti-thrombotic agents, anti-proliferative agents, anti-
inflammatory agents, neoplastic agents, enzymes, receptor antagonists or
agonists, hormones, antibiotics, antimicrobial agents, antibodies, cytokine
inhibitors, IMPDH (inosine monophosplate dehydrogenase) inhibitors tyrosine
kinase inhibitors, MMP inhibitors, p38 MAP kinase inhibitors,
immunosuppressants, apoptosis antagonists, caspase inhibitors, and JNK
inhibitor.
The polymeric composition may further include an anti-thrombotic
agent and/or antiplatelet agent and/or a thrombolytic agent, which reduces the
134
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
likelihood of thrombotic events upon implantation of a medical implant.
Representative examples of anti-thrombotic and/or antiplatelet and/or
thrombolytic agents include heparin, heparin fragments, organic salts of
heparin, heparin complexes (e.g., benzalkonium heparinate,
tridodecylammonium heparinate), dextran, sulfonated carbohydrates such as
dextran sulfate, coumadin, coumarin, heparinoid, danaparoid, argatroban
chitosan sulfate, chondroitin sulfate, danaparoid, lepirudin, hirudin, AMP,
adenosine, 2-chloroadenosine, acetylsalicylic acid, phenylbutazone,
indomethacin, meclofenamate, hydrochloroquine, dipyridamole, iloprost,
streptokinase, factor Xa inhibitors, such as DX9065a, magnesium, and tissue
plasminogen activator. Further examples include plasminogen, lys-
plasminogen, alpha-2-antiplasmin, urokinase, aminocaproic acid, ticlopidine,
clopidogrel, trapidil (triazolopyrimidine), naftidrofuryl, auriritricarboxylic
acid and
glycoprotein Ilb/Illa inhibitors such as abcixamab, eptifibatide, and
tirogiban.
Other agents capable of affecting the rate of clotting include
glycosaminoglycans, danaparoid, 4-hydroxycourmarin, warfarin sodium,
dicumarol, phenprocoumon, indan-1,3-dione, acenocoumarol, anisindione, and
rodenticides including bromadiolone, brodifacoum, diphenadione,
chlorophacinone, and pidnone.
The polymeric formulation may be or include a hydrophilic
polymer gel that itself has anti-thrombogenic properties. For example, the
composition can be in the form of a coating that can comprise a hydrophilic,
biodegradable polymer that is physically removed from the surface of the
device over time, thus reducing adhesion of platelets to the device surface.
The gel composition can include a polymer or a blend of polymers.
Representative examples include alginates, chitosan and chitosan sulfate,
hyaluronic acid, dextran sulfate, PLURONIC polymers (e.g., F-127 or F87),
chain extended PLURONIC polymers, various polyester-polyether block
copolymers of various configurations (e.g., AB, ABA, or BAB, where A is a
polyester such as PLA, PGA, PLGA, PCL or the like), examples of which
135
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
include MePEG-PLA, PLA-PEG-PLA, and the like). In one embodiment, the
anti-thrombotic composition can include a crosslinked gel formed from a
combination of molecules (e.g., PEG) having two or more terminal electrophilic
groups and two or more nucleophilic groups.
The polymeric formulation may further include an agent from one
of the following classes of compounds: anti-inflammatory agents (e.g.,
dexamethasone, cortisone, fludrocortisone, prednisone, prednisolone, 6a-
methylprednisolone, triamcinolone, betamethasone, and aspirin); MMP
inhibitors (e.g., batimistat, marimistat, TIMP's representative examples of
which
are included in U.S. Patent Nos. 5,665,777; 5,985,911; 6,288,261; 5,952,320;
6,441,189; 6,235,786; 6,294,573; 6,294,539; 6,563,002; 6,071,903; 6,358,980;
5,852,213; 6,124,502; 6,160,132; 6,197,791; 6,172,057; 6,288,086; 6,342,508;
6,228,869; 5,977,408; 5,929,097; 6,498,167; 6,534,491; 6,548,524; 5,962,481;
6,197,795; 6,162,814; 6,441,023; 6,444,704; 6,462,073; 6,162,821; 6,444,639;
6,262,080; 6,486,193; 6,329,550; 6,544,980; 6,352,976; 5,968,795; 5,789,434;
5,932,763; 6,500,847; 5,925,637; 6,225,314; 5,804,581; 5,863,915; 5,859,047;
5,861,428; 5,886,043; 6,288,063; 5,939,583; 6,166,082; 5,874,473; 5,886,022;
5,932,577; 5,854,277; 5,886,024; 6,495,565; 6,642,255; 6,495,548; 6,479,502;
5,696,082; 5,700,838; 6,444,639; 6,262,080; 6,486,193; 6,329,550; 6,544,980;
6,352,976; 5,968,795; 5,789,434; 5,932,763; 6,500,847; 5,925,637; 6,225,314;
5,804,581; 5,863,915; 5,859,047; 5,861,428; 5,886,043; 6,288,063; 5,939,583;
6,166,082; 5,874,473; 5,886,022; 5,932,577; 5,854,277; 5,886,024; 6,495,565;
6,642,255; 6,495,548; 6,479,502; 5,696,082; 5,700,838; 5,861,436; 5,691,382;
5,763,621; 5,866,717; 5,902,791; 5,962,529; 6,017,889; 6,022,873; 6,022,898;
6,103,739; 6,127,427; 6,258,851; 6,310,084; 6,358,987; 5,872,152; 5,917,090;
6,124,329; 6,329,373; 6,344,457; 5,698,706; 5,872,146; 5,853,623; 6,624,144;
6,462,042; 5,981,491; 5,955,435; 6,090,840; 6,114,372; 6,566,384; 5,994,293;
6,063,786; 6,469,020; 6,118,001; 6,187,924; 6,310,088; 5,994,312; 6,180,611;
6,110,896; 6,380,253; 5,455,262; 5,470,834; 6,147,114; 6,333,324; 6,489,324;
6,362,183; 6,372,758; 6,448,250; 6,492,367; 6,380,258; 6,583,299; 5,239,078;
136
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
5,892,112; 5,773,438; 5,696,147; 6,066,662; 6,600,057; 5,990,158; 5,731,293;
6,277,876; 6,521,606; 6,168,807; 6,506,414; 6,620,813; 5,684,152; 6,451,791;
6,476,027; 6,013,649; 6,503,892; 6,420,427; 6,300,514; 6,403,644; 6,177,466;
6,569,899; 5,594,006; 6,417,229; 5,861,510; 6,156,798; 6,387,931; 6,350,907;
6,090,852; 6,458,822; 6,509,337; 6,147,061; 6,114,568; 6,118,016; 5,804,593;
5,847,153; 5,859,061; 6,194,451; 6,482,827; 6,638,952; 5,677,282; 6,365,630;
6,130,254; 6,455,569; 6,057,369; 6,576,628; 6,110,924; 6,472,396; 6,548,667;
5,618,844; 6,495,578; 6,627,411; 5,514,716; 5,256,657; 5,773,428; 6,037,472;
6,579,890; 5,932,595; 6,013,792; 6,420,415; 5,532,265; 5,639,746; 5,672,598;
5,830,915; 6,630,516; 5,324,634; 6,277,061; 6,140,099; 6,455,570; 5,595,885;
6,093,398; 6,379,667; 5,641,636; 5,698,404; 6,448,058; 6,008,220; 6,265,432;
6,169,103; 6,133,304; 6,541,521; 6,624,196; 6,307,089; 6,239,288; 5,756,545;
6,020,366; 6,117,869; 6,294,674; 6,037,361; 6,399,612; 6,495,568; 6,624,177;
5,948,780; 6,620,835; 6,284,513; 5,977,141; 6,153,612; 6,297,247; 6,559,142;
6,555,535; 6,350,885; 5,627,206; 5,665,764; 5,958,972; 6,420,408; 6,492,422;
6,340,709; 6,022,948; 6,274,703; 6,294,694; 6,531,499; 6,465,508; 6,437,177;
6,376,665; 5,268,384; 5,183,900; 5,189,178; 6,511,993; 6,617,354; 6,331,563;
5,962,466; 5,861,427; 5,830,869; and 6,087,359), cytolcine inhibitors
(chlorpromazine, mycophenolic acid, rapamycin, 1a-hydroxy vitamin D3),
IMPDH (inosine monophosplate dehydrogenase) inhibitors (e.g., mycophenolic
acid, ribaviran, aminothiadiazole, thiophenfurin, tiazofurin, viramidine)
(Representative examples are included in U.S. Patent, Nos. 5,536,747;
5,807,876; 5,932,600; 6,054,472; 6,128,582; 6,344,465; 6,395,763; 6,399,773;
6,420,403; 6,479,628; 6,498,178; 6,514,979; 6,518,291; 6,541,496; 6,596,747;
6,617,323; and 6,624,184, U.S. Patent Application Nos. 2002/0040022A1,
2002/0052513A1, 2002/0055483A1, 2002/0068346A1, 2002/0111378A1,
2002/0111495A1, 2002/0123520A1, 2002/0143176A1, 2002/0147160A1,
2002/0161038A1, 2002/0173491 A1, 2002/0183315A1, 2002/0193612A1,
2003/0027845A1, 2003/0068302A1, 2003/0105073A1, 2003/0130254A1,
2003/0143197A1, 2003/0144300A1, 2003/0166201 A1, 2003/0181497A1,
137
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
2003/0186974A1, 2003/0186989A1, and 2003/0195202A1, and PCT
Publication Nos. WO 00/24725A1, WO 00/25780A1, WO 00/26197A1, WO
00/51615A1, WO 00/56331 A1, WO 00/73288A1, WO 01 /00622A1, WO
01/66706A1, WO 01/79246A2, WO 01/81340A2, WO 01/85952A2, WO
02/16382A1, WO 02/18369A2, WO 02/051814A1, WO 02/057287A2, WO
02/057425A2, WO 02/060875A1, WO 02/060896A1, WO 02/060898A1, WO
02/068058A2, WO 03/020298A1, WO 03/037349A1, WO 03/039548A1, WO
03/045901A2, WO 03/047512A2, WO 03/053958A1, WO 03/055447A2, WO
03/059269A2, WO 03/063573A2, WO 03/087071 A1, WO 99/001545A1, WO
97/40028A1, WO 97/41211 A1, WO 98/40381 A1, and WO 99/55663A1 ), p38
MAP kinase inhibitors (MAPK) (e.g., GW-2286, CGP-52411, BIRB-798,
SB220025, RO-320-1195, RWJ-67657, RWJ-68354, SCIO-469)
(Representative examples are included in U.S. Patent Nos. 6,300,347;
6,316,464; 6,316,466; 6,376,527; 6,444,696; 6,479,507; 6,509,361; 6,579,874,
and 6,630,485, and U.S. Patent Application Publication Nos. 2001/0044538A1,
2002/0013354A1, 2002/0049220A1, 2002/0103245A1, 2002/0151491 A1,
2002/0156114A1, 2003/0018051 A1, 2003/0073832A1, 2003/0130257A1,
2003/0130273A1, 2003/0130319A1, 2003/0139388A1, 2003/0139462A1,
2003/0149031 A1, 2003/0166647A1, and 2003/0181411 A1, and PCT
Publication Nos. WO 00/63204A2, WO 01/21591A1, WO 01/35959A1, WO
01/74811A2, WO 02/18379A2, WO 02/064594A2, WO 02/083622A2, WO
02/094842A2,W0 02/096426A1, WO 02/101015A2, WO 02/103000A2, WO
03/008413A1, WO 03/016248A2, WO 03/020715A1, WO 03/024899A2, WO
03/031431A1, WO 03/040103A1, WO 03/053940A1, WO 03/053941 A2, WO
03/063799A2, WO 03/079986A2, WO 03/080024A2, WO 03/082287A1, WO
97/44467A1, WO 99/01449A1, and WO 99158523A1 ), and immunomodulatory
agents (rapamycin, everolimus, ABT-578, azathioprine azithromycin, analogues
of rapamycin, including tacrolimus and derivatives thereof (e.g., EP 018416281
and those described in U.S. Patent No. 6,258,823) and everolimus and
derivatives thereof (e.g., U.S. Patent No. 5,665,772). Further representative
138
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
examples of sirolimus analogues and derivatives include ABT-578 and those
found in PCT Publication Nos. WO 97/10502, WO 96/41807, WO 96/35423,
WO 96/03430, WO 96/00282, WO 95/16691, WO 95/15328, WO 95/07468,
WO 95/04738, WO 95/04060, WO 94/25022, WO 94/21644, WO 94/18207,
WO 94/10843, WO 94109010, WO 94/04540, WO 94/02485, WO 94/02137,
WO 94/02136, WO 93/25533, WO 93/18043, WO 93/13663, WO 93/11130, WO
93/10122, WO 93/04680, WO 92114737, and WO 92/05179 and in U.S. Patent
Nos. 6,342,507; 5,985,890; 5,604,234; 5,597,715; 5,583,139; 5,563,172;
5,561,228; 5,561,137; 5,541,193; 5,541,189; 5,534,632; 5,527,907; 5,484,799;
5,457,194; 5,457,182; 5,362,735; 5,324,644; 5,318,895; 5,310,903; 5,310,901;
5,258,389; 5,252,732; 5,247,076; 5,225,403; 5,221,625; 5,210,030; 5,208,241;
5,200,411; 5,198,421; 5,147,877; 5,140,018; 5,116,756; 5,109,112; 5,093,338;
and 5,091,389.
Other examples of biologically active agents which may be
included in the compositions of the invention include tyrosine kinase
inhibitors,
such as imantinib, ZK-222584, CGP-52411, CGP-53716, NVP-AAK980-NX,
CP-127374, CP-564959, PD-171026, PD-173956, PD-180970, SU-0879, and
SKI-606; MMP inhibitors such as nimesulide, PKF-241-466, PKF-242-484,
CGS-27023A, SAR-943, primomastat, SC-77964, PNU-171829, AG-3433,
PNU-142769, SU-5402, and Dexlipotam; p38 MAP kinase inhibitors such as
include CGH-2466 and PD-98-59; immunosuppressants such as argyrin B,
macrocyclic lactone, ADZ-62-826, CCI-779, tilomisole, amcinonide, FK-778,
AVE-1726, and MDL-28842; cytokine inhibitors such as TNF-484A, PD-
172084, CP-293121, CP-353164, and PD-168787; NFKB inhibitors, such as,
AVE-0547, AVE-0545, and IPL-576092; HMGCoA reductase inhibitors, such
as, pravestatin, atorvastatin, fluvastatin, dalvastatin, glenvastatin,
pitavastatin,
CP-83101, U-20685; apoptosis antagonist (e.g., troloxamine, TCH-346 (N-
methyl-N-propargyl-10-aminomethyl-dibenzo(b,f)oxepin); and caspase
inhibitors (e.g., PF-5901 (benzenemethanol, alpha-pentyl-3-(2-
quinolinylmethoxy)-), and JNK inhibitor (e.g., AS-602801 ).
139
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another aspect, the composition may further include an
antibiotic (e.g., amoxicillin, trimethoprim-sulfamethoxazole, azithromycin,
clarithromycin, amoxicillin-clavulanate, cefprozil, cefuroxime, cefpodoxime,
or
cefd i n i r).
In certain aspects, a polymeric composition comprising a fibrosis-
inhibiting agent is combined with an agent that can modify metabolism of the
agent in vivo to enhance efficacy of the fibrosis-inhibiting agent. One class
of
therapeutic agents that can be used to alter drug metabolism includes agents
capable of inhibiting oxidation of the anti-scarring agent by cytochrome P450
(CYP). In one embodiment, compositions are provided that include a fibrosis-
inhibiting agent (e.g., paclitaxel, rapamycin, everolimus) and a CYP
inhibitor,
which may be combined (e.g., coated) with any of the devices described herein,
including, without limitation, stents, grafts, patches, valves, wraps, and
films.
Representative examples of CYP inhibitors include flavones, azole antifungals,
macrolide antibiotics, HIV protease inhibitors, and anti-sense oligomers.
Devices comprising a combination of a fibrosis-inhibiting agent and a CYP
inhibitor may be used to treat a variety of proliferative conditions that can
lead
to undesired scarring of tissue, including intimal hyperplasia, surgical
adhesions, and tumor growth.
In another aspect, a polymeric composition comprising an anti-
infective agent (e.g., anthracyclines (e.g., doxorubicin or mitoxantrone),
fluoropyrimidines (e.g., 5-fluorouracil), folic acid antagonists (e.g.,
methotrexate
and/or podophylotoxins (e.g., etoposide)) can be combined with traditional
antibiotic and/or antifungal agents to enhance efficacy. The anti-infective
agent
may be further combined with anti-thrombotic and/or antiplatelet agents (for
example, heparin, dextran sulfate, danaparoid, lepirudin, hirudin, AMP,
adenosine, 2-chloroadenosine, aspirin, phenylbutazone, indomethacin,
meclofenamate, hydrochloroquine, dipyridamole, iloprost, ticlopidine,
clopidogrel, abcixamab, eptifibatide, tirofiban, streptokinase, and/or tissue
plasminogen activator) to enhance efficacy.
140
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Although the above therapeutic agents have been provided for the
purposes of illustration, it should be understood that the present invention
is not
so limited. For example, although agents are specifically referred to above,
the
present invention should be understood to include analogues, derivatives and
conjugates of such agents. As an illustration, paclitaxel should be understood
to refer to not only the common chemically available form of paclitaxel, but
analogues (e.g., TAXOTERE, as noted above) and paclitaxel conjugates (e.g.,
paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylos). In addition, as will
be
evident to one of skill in the art, although the agents set forth above may be
noted within the context of one class, many of the agents listed in fact have
multiple biological activities. Further, more than one therapeutic agent may
be
utilized at a time (i.e., in combination), or delivered sequentially.
H. Compositions and Methods for Generating Compositions Which
Comprise a Therapeutic Agent
The present invention provides various compositions which can
be used to inhibit fibrosis and/or infection of tissue in the vicinity of a
treatment
site (e.g., a surgical site). Within various embodiments, fibrosis and/or
infection
is inhibited by local or systemic release of specific pharmacological agents
that
become localized at the site or intervention. Within other embodiments,
fibrosis
and/or infection can be inhibited by local or systemic release of specific
pharmacological agents that become localized adjacent to a device or implant
that has been introduced into a host. In certain embodiments, compositions are
provided which inhibit fibrosis in and around an implanted device, or prevent
"stenosis" of a device/implant in situ, thus enhancing the efficacy. In other
embodiments, anti-infective compositions are provided which inhibit or prevent
infection in and around an implanted device.
There are numerous methods available for optimizing delivery of
the therapeutic agent to the site of the intervention. Several of these are
described below.
141
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
1 ) Systemic, Regional and Local Delivery of Therapeutic Agents
A variety of drug-delivery technologies are available for systemic,
regional and local delivery of anti-infective and/or anti-fibrosis therapeutic
agents.
For systemic delivery of therapeutic agents, several routes of
administration would be suitable to provide systemic exposure of the
therapeutic agent, including: (a) intravenous, (b) oral, (c) subcutaneous, (d)
intraperitoneal, (e) intrathecal, (f) inhaled and intranasal, (g) sublingual
or
transbuccal, (h) rectal, (i) intravaginal, (j) intra-arterial, (k)
intracardiac, (I)
transdermal, (m) intra-ocular and (n) intramuscular. The therapeutic agent may
be administered as a sustained low dose therapy to prevent disease
progression, prolong disease remission, or decrease symptoms in active
disease. Alternatively, the therapeutic agent may be administered in higher
doses as a "pulse" therapy to induce remission in acutely active disease. The
minimum dose capable of achieving these endpoints can be used and can vary
according to patient, severity of disease, formulation of the administered
agent,
potency and tolerability of. the therapeutic agent, and route of
administration.
For regional and local delivery of therapeutic agents, several
techniques would be suitable to achieve preferentially elevated levels of
therapeutic agents in the vicinity of the area to be treated. These include:
(a)
using drug-delivery catheters and/or a syringe and needle for local, regional
or
systemic delivery of fibrosis-inhibiting agents to the tissue surrounding the
device or implant (typically, drug delivery catheters are advanced through the
circulation or inserted directly into tissues under radiological guidance
until they
reach the desired anatomical location; the fibrosis-inhibiting agent can then
be
released from the catheter lumen in high local concentrations in order to
deliver
therapeutic doses of the drug to the tissue surrounding the device or
implant);
(b) drug localization techniques such as magnetic, ultrasonic or MRI-guided
drug delivery; (c) chemical modification of the therapeutic drug or
formulation
designed to increase uptake of the agent into damaged tissues (e.g.,
antibodies
142
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
directed against damaged or healing tissue components such as macrophages,
neutrophils, smooth muscle cells, fibroblasts, extracellular matrix
components,
neovascular tissue); (d) chemical modification of the therapeutic drug or
formulation designed to localize the drug to areas of bleeding or disrupted
vasculature; and/or (e) direct injection, for example subcutaneous,
intramuscular, intra-articular, etc, of the therapeutic agent, for example,
under
normal or endoscopic vision.
2) Infiltration of Therapeutic Agents into the Tissue Surrounding a
Device or Implant
Alternatively, the tissue cavity or surgical pocket into which a
device or implant is placed can be treated with an anti-infective and/or
fibrosis-
inhibiting therapeutic agent prior to, during, or after the procedure. This
can be
accomplished in several ways including: (a) topical application of the agent
into
the anatomical space or surface where the device will be placed (particularly
useful for this embodiment is the use of polymeric carriers which release the
agent over a period ranging from several hours to several weeks.
Compositions that can be used for this application include, e.g., fluids,
microspheres, pastes, gels, microparticulates, sprays, aerosols, solid
implants
and other formulations which release a therapeutic agent into the region where
the device or implant will be implanted); (b) microparticulate forms of the
therapeutic agent are also useful for directed delivery into the implantation
site;
(c) sprayable collagen-containing formulations such as COSTASIS and
crosslinked derivatized polyethylene glycol) -collagen compositions
(described, e.g., in U.S. Patent Nos. 5,874,500 and 5,565,519 and referred to
herein as "CT3" (both from Angiotech Pharmaceuticals, Inc., Canada), either
alone, or loaded with a therapeutic agent, applied to the implantation site
(or the
implantldevice surface); (d) sprayable PEG-containing formulations such as
COSEAL or ADHIBIT (Angiotech Pharmaceuticals, Inc.), SPRAYGEL or
DURASEAL (both from Confluent Surgical, Inc., Boston, MA), either alone, or
143
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
loaded with a therapeutic agent, applied to the implantation site (or the
implant/device surface); (e) fibrin-containing formulations such as FLOSEAL or
TISSEEL (both from Baxter Healthcare Corporation, Fremont, CA), applied to
the implantation site (or the implantldevice surface); (f) hyaluronic acid-
s containing formulations such as RESTYLANE or PERLANE (both from Q-Med
AB, Sweden), HYLAFORM (Inamed Corporation (Santa Barbara, CA)),
SYNVISC (Biomatrix, Inc., Ridgefield, NJ), SEPRAFILM or SEPRACOAT (both
from Genzyme Corporation, Cambridge, MA) loaded with a therapeutic agent
applied to the implantation site (or the implant/device surface); (g)
polymeric
gels for surgical implantation such as REPEL (Life Medical Sciences, Inc.,
Princeton, NJ) or FLOGEL (Baxter Healthcare Corporation) loaded with a
therapeutic agent applied to the implantation site (or the implant/device
surface); (h) orthopedic "cements" used to hold prostheses and tissues in
place
with a therapeutic agent applied to the implantation site (or the
implant/device
surface); (i) surgical adhesives containing cyanoacrylates such as
DERMABOND (Johnson & Johnson, Inc., New Brunswick, NJ), INDERMIL
(U.S. Surgical Company, Norwalk, CT), GLUSTITCH (Blacklock Medical
Products Inc., Canada), TISSUMEND If (Veterniary Products Laboratories"
Phoenix, AZ), VETBOND (3M Company, St. Paul, MN), HISTOACRYL BLUE
(Davis & Geck, St. Louis, MO) and ORABASE SMOOTHE-N-SEAL Liquid
Protectant (Colgate-Palmolive Company, New York, NY) loaded with a
therapeutic agent, applied to the implantation site (or the implant/device
surface); andlor (j) protein-based sealants or adhesives such as BIOGLUE
(Cryolife, Inc.) and TISSUEBOND (TissueMed, Ltd.) loaded with a therapeutic
agent, applied to the implantation site (or the implant/device surface).
A preferred polymeric matrix which can be used to help prevent
the formation of fibrous tissue, either alone or in combination with a
fibrosis
inhibiting agent/composition, is formed from reactants comprising either one
or
both of pentaerythritol polyethylene glycol)ether tetra-sulfhydryl] (4-armed
thiol
PEG, which includes structures having a linking groups) between a sulfhydryl
144
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
groups) and the terminus of the polyethylene glycol backbone) and
pentaerythritol polyethylene glycol)ether tetra-succinimidyl glutarate] (4-
armed
NHS PEG, which again includes structures having a linking groups) between a
NHS groups) and the terminus of the polyethylene glycol backbone) as
reactive reagents. Another preferred composition comprises either one or both
of pentaerythritol polyethylene glycol)ether tetra-amino] (4-armed amino PEG,
which includes structures having a linking groups) between an amino groups)
and the terminus of the polyethylene glycol backbone) and pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate] (4-armed NHS PEG,
which again includes structures having a linking groups) between a NHS
groups) and the terminus of the polyethylene glycol backbone) as reactive
reagents. Chemical structures for these reactants are shown in, e.g., U.S.
Patent 5,874,500. Optionally, collagen or a collagen derivative (e.g.,
methylated collagen) is added to the polyethylene glycol)-containing
reactants) to form a preferred crosslinked matrix that can serve as a
polymeric
carrier for a therapeutic agent or a stand-alone composition to help prevent
the
formation of fibrous tissue.
3) Sustained-Release Preparations of Therapeutic Agents
As described previously, desired therapeutic agents may be
admixed with, blended with, conjugated to, or, otherwise modified to contain a
polymer composition (which may be either biodegradable or non-
biodegradable) or a non-polymeric composition in order to release the
therapeutic agent over a prolonged period of time. For many of the
aforementioned embodiments, localized delivery as well as localized sustained
delivery of the fibrosis-inhibiting and/or anti-infective agent may be
required.
For example, a desired therapeutic agent may be admixed with, blended with,
conjugated to, or, otherwise modified to contain a polymeric composition
(which
may be either biodegradable or non-biodegradable) or non-polymeric
composition in order to release the therapeutic agent over a period of time.
145
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Representative examples of biodegradable polymers suitable for
the delivery of the aforementioned therapeutic agents include albumin,
collagen, gelatin, hyaluronic acid, starch, cellulose and cellulose
derivatives
(e.g., regenerated cellulose, methylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate
phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose
phthalate),
casein, dextrans, polysaccharides, fibrinogen, poly(ether ester) multiblock
copolymers, based on polyethylene glycol) and poly(butylene terephthalate),
tyrosine-derived polycarbonates (e.g., U.S. Patent No. 6,120,491),
poly(hydroxyl acids), poly(D,L-lactide), poly(D,L-lactide-co-glycolide),
poly(glycolide), poly(hydroxybutyrate), polydioxanone, poly(alkylcarbonate)
and
poly(orthoesters), polyesters, poly(hydroxyvaleric acid), polydioxanone,
polyesters, poly(malic acid), poly(tartronic acid), poly(acrylamides),
polyanhydrides, polyphosphazenes, poly(amino acids), poly(alkylene oxide)-
polyester) block copolymers (e.g., X-Y, X-Y-X, Y-X-Y, R-(Y-X)", or R-(X-Y)",
where X is a polyalkylene oxide (e.g., poly(ethylene glycol, polypropylene
glycol) and block copolymers of polyethylene oxide) and polypropylene oxide)
(e.g., PLURONIC and PLURONIC R series of polymers from BASF
Corporation, Mount Olive, NJ) and Y is a polyester, where the polyester may
comprise the residues of one or more of the monomers selected from lactide,
lactic acid, glycolide, glycolic acid, e-caprolactone, gamma-caprolactone,
hydroxyvaleric acid, hydroxybutyric acid, beta-butyrolactone, gamma-
butyrolactone, gamma-valerolactone, y-decanolactone, ~-decanolactone,
trimethylene carbonate, 1,4-dioxane-2-one or 1,5-dioxepan-Zone (e.g., PLGA,
PLA, PCL, polydioxanone and copolymers thereof) and R is a multifunctional
initiator), and the copolymers as well as blends thereof (see generally,
Illum, L.,
Davids, S.S. (eds.) "Polymers in Controlled Drug Delivery" Wright, Bristol,
1987;
Arshady, J. Controlled Release 7 7:1-22, 1991; Pitt, Int. J. Phar. 59:173-196,
1990; Holland et al., J. Controlled Release 4:155-0180, 1986).
146
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Representative examples of non-degradable polymers suitable for
the delivery of the aforementioned therapeutic agents include polyethylene-co-
vinyl acetate) ("EVA") copolymers, aromatic polyesters, such as polyethylene
terephthalate), silicone rubber, acrylic polymers (polyacrylate, polyacrylic
acid,
polymethylacrylic acid, polymethylmethacrylate, poly(butyl methacrylate)),
poly(alkylcynoacrylate) (e.g., poly(ethylcyanoacrylate),
poly(butyicyanoacrylate)
poly(hexylcyanoacrylate) poly(octylcyanoacrylate)), acrylic resin,
polyethylene,
polypropylene, polyamides (nylon 6,6), polyurethanes (e.g., CHRONOFLEX AL
and CHRONOFLEX AR (both from CardioTech International, Inc., Woburn,
MA), TECOFLEX, and BIONATE (Polymer Technology Group, Inc., Emeryville,
CA))" polyester urethanes), poly(ether urethanes), polyester-urea), polyethers
(poly(ethylene oxide), polypropylene oxide), polyoxyalkylene ether block
copolymers based on ethylene oxide and propylene oxide such as the
PLURONIC polymers (e.g., F-127 or F87) from BASF Corporation (Mount
Olive, NJ), and poly(tetramethylene glycol), styrene-based polymers
(polystyrene, polystyrene sulfonic acid), poly(styrene)-block-
poly(isobutylene)-
block-poly(styrene), poly(styrene)-poly(isoprene) block copolymers), and vinyl
polymers (polyvinylpyrrolidone, polyvinyl alcohol), polyvinyl acetate
phthalate)
as well as copolymers and blends thereof. Polymers may also be developed
which are either anionic (e.g., alginate, carrageenan, carboxymethyl
cellulose,
poly(acrylamido-2-methyl propane sulfonic acid) and copolymers thereof,
poly(methacrylic acid and copolymers thereof and poly(acrylic acid) and
copolymers thereof, as well as blends thereof, or cationic (e.g., chitosan,
poly-
L-lysine, polyethylenimine, and poly(allyl amine)) and blends thereof (see
generally, Dunn et al., J. Applied Polymer Sci. 50:353-365, 1993; Cascone et
al., J. Materials Sci.: Materials in Medicine 5:770-774, 1994; Shiraishi et
al.,
Biol. Pharm. Bull. 16(11 ):1164-1168, 1993; Thacharodi and Rao, Int'I J.
Pharm.
120:115-118, 1995; Miyazaki et al., Int'I J. Pharm. 115:257-263, 1995).
Some examples of preferred polymeric carriers for the practice of
this invention include polyethylene-co-vinyl acetate), polyurethanes, poly
(D,L-
147
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
lactic acid) oligomers and polymers, poly (L-lactic acid) oligomers and
polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid,
copolymers of lactide and glycolide, poly (caprolactone), poly
(valerolactone),
polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid) with a
polyethylene glycol (e.g., MePEG), block copolymers of the form X-Y, X-Y-X, Y-
X-Y, R-(Y-X)", or R-(X-Y)", where X is a polyalkylene oxide (e.g.,
poly(ethylene
glycol, polypropylene glycol) and block copolymers of polyethylene oxide) and
polypropylene oxide) (e.g., PLURONIC and PLURONIC R series of polymers
from BASF Corporation, Mount Olive, NJ) and Y is a polyester, where the
polyester may comprise the residues of one or more of the monomers selected
from lactide, lactic acid, glycolide, glycolic acid, e-caprolactone, gamma-
caprolactone, hydroxyvaleric acid, hydroxybutyric acid, beta-butyrolactone,
gamma-butyrolactone, gamma-valerolactone, y-decanolactone, ~-
decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or 1,5-dioxepan-
Zone and R is a multifunctional initiator), silicone rubbers,
poly(styrene)block-
poly(isobutylene)-block-poly(styrene), poly(acrylate) polymers and blends,
admixtures, or co-polymers of any of the above. Other preferred polymers
include collagen, poly(alkylene oxide)-based polymers, polysaccharides such
as hyaluronic acid, chitosan and fucans, and copolymers of polysaccharides
with degradable polymers.
Other representative polymers capable of sustained localized
delivery of anti-infective andlor fibrosis-inhibiting therapeutic agents
include
carboxylic polymers, polyacetates, polycarbonates, polyethers, polyethylenes,
polyvinylbutyrals, polysilanes, polyureas, polyoxides, polystyrenes,
polysulfides,
polysulfones, polysulfonides, polyvinylhalides, pyrrolidones, rubbers, thermal-
setting polymers, cross-linkable acrylic and methacrylic polymers, ethylene
acrylic acid copolymers, styrene acrylic copolymers, vinyl acetate polymers
and
copolymers, vinyl acetal polymers and copolymers, epoxies, melamines, other
amino resins, phenolic polymers, and copolymers thereof, water-insoluble
cellulose ester polymers (including cellulose acetate propionate, cellulose
14~
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
acetate, cellulose acetate butyrate, cellulose nitrate, cellulose acetate
phthalate, and mixtures thereof), polyvinylpyrrolidone, polyethylene glycols,
polyethylene oxide, polyvinyl alcohol, polyethers, polysaccharides,
hydrophilic
polyurethane, polyhydroxyacrylate, dextran, xanthan, hydroxypropyl cellulose,
and homopolymers and copolymers of N-vinylpyrrolidone, N-vinyllactam, N-
vinyl butyrolactam, N-vinyl caprolactam, other vinyl compounds having polar
pendant groups, acrylate and methacrylate having hydrophilic esterifying
groups, hydroxyacrylate, and acrylic acid, and combinations thereof; cellulose
esters and ethers, ethyl cellulose, hydroxyethyl cellulose, cellulose nitrate,
cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate,
natural and synthetic elastomers, rubber, acetal, styrene polybutadiene,
acrylic
resin, polyvinylidene chloride, polycarbonate, homopolymers and copolymers of
vinyl compounds, polyvinylchloride, and polyvinylchloride acetate.
Representative examples of patents relating to drug-delivery
polymers and their preparation include PCT Publication Nos. WO 98/19713,
WO 01/17575, WO 01/41821, WO 01/41822, and WO 01/15526 (as well as the
corresponding U.S. applications), U.S. Patent Nos. 4,500,676, 4,582,865,
4,629,623, 4,636,524, 4,713,448, 4,795,741, 4,913,743, 5,069,899, 5,099,013,
5,128,326, 5,143,724, 5,153,174, 5,246,698, 5,266,563, 5,399,351, 5,525,348,
5,800,412, 5,837,226, 5,942,555, 5,997,517, 6,007,833, 6,071,447, 6,090,995,
6,106,473, 6,110,483, 6,121,027, 6,156,345, 6,214,901, 6,368,611 6,630,155,
6,528,080, RE37,950, 6,46,1631, 6,143,314, 5,990,194, 5,792,469, 5,780,044,
5,759,563, 5,744,153, 5,739,176, 5,733,950, 5,681,873, 5,599,552, 5,340,849,
5,278,202, 5,278,201, 6,589,549, 6,287,588, 6,201,072, 6,117,949, 6,004,573,
5,702,717, 6,413,539, 5,714,159, 5,612,052, and U.S. Patent Application
Publication Nos. 2003/0068377, 2002/0192286, 2002/0076441, and
2002/0090398.
It should be obvious to one of skill in the art that the polymers as
described herein can also be blended or copolymerized in various compositions
as required to deliver therapeutic doses of biologically active agents.
149
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Polymeric carriers for anti-infective and/or fibrosis-inhibiting
therapeutic agents can be fashioned in a variety of forms, with desired
release
characteristics and/or with specific properties depending upon the composition
being utilized. For example, polymeric carriers may be fashioned to release a
therapeutic agent upon exposure to a specific triggering event such as pH
(see,
e.g., Heller et al., "Chemically Self-Regulated Drug Delivery Systems," in
Polymers in Medicine 111, Elsevier Science Publishers B.V., Amsterdam, 1988,
pp. 175-188; Kang et al., J. Applied Polymer Sci. 48:343-354, 1993; Dong et
al.,
J. Controlled Release 19:171-178, 1992; Dong and Hoffman, J. Controlled
Release 15:141-152, 1991; Kim et al., J. Controlled Release 28:143-152, 1994;
Cornejo-Bravo et al., J. Controlled Release 33:223-229, 1995; Wu and Lee,
Pharm. Res. 10(10):1544-1547, 1993; Serres et al., Pharm. Res. 13(2):196-
201, 1996; Peppas, "Fundamentals of pH- and Temperature-Sensitive Delivery
Systems," in Gurny et al. (eds.), Pulsatile Drug Delivery, Wissenschaftliche
Verlagsgesellschaft mbH, Stuttgart, 1993, pp. 41-55; Doelker, "Cellulose
Derivatives," 1993, in Peppas and Langer (eds.), Biopolymers I, Springer-
Verlag, Berlin). Representative examples of pH-sensitive polymers include poly
(acrylic acid) and its derivatives (including for example, homopolymers such
as
poly(aminocarboxylic acid); poly(acrylic acid); poly(methyl acrylic acid),
copolymers of such homopolymers, and copolymers of poly(acrylic acid) and/or
acrylate or acrylamide (monomers such as those discussed above. Other pH
sensitive polymers include polysaccharides such as cellulose acetate
phthalate;
hydroxypropylmethylcellulose phthalate; hydroxypropylmethylcellulose acetate
succinate; cellulose acetate trimellilate; and chitosan. Yet other pH
sensitive
polymers include any mixture of a pH sensitive polymer and a water-soluble
polymer.
Likewise, ant-infective and/or fibrosis-inhibiting therapeutic agents
can be delivered via polymeric carriers which are temperature sensitive (see,
e.g., Chen et al., "Novel Hydrogels of a Temperature-Sensitive PLURONIC
Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery,"
150
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
in Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22:167-168, Controlled
Release Society, Inc., 1995; Okano, "Molecular Design of Stimuli-Responsive
Hydrogels for Temporal Controlled Drug Delivery," in Proceed. Intern. Symp.
Control. Rel. Bioact. Mater. 22:111-112, Controlled Release Society, Inc.,
1995;
Johnston et al., Pharm. Res. 9(3):425-433, 1992; Tung, Int'I J. Pharm. 107:85-
90, 1994; Harsh and Gehrke, J. Controlled Release 17:175-186, 1991; Bae et
al., Pharm. Res. 8(4):531-537, 1991; Dinarvand and D'Emanuele, J. Controlled
Release 36:221-227, 1995; Yu and Grainger, "Novel Thermo-sensitive
Amphiphilic Gels: Poly N-isopropylacrylamide-co-sodium acrylate-co-n-N-
alkylacrylamide Network Synthesis and Physicochemical Characterization,"
Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science &
Technology, Beaverton, OR, pp. 820-821; ~hou and Smid, "Physical Hydrogels
of Associative Star Polymers," Polymer Research Institute, Dept. of Chemistry,
College of Environmental Science and Forestry, State Univ. of New York,
Syracuse, NY, pp. 822-823; Hoffman et al., "Characterizing Pore Sizes and
Water 'Structure' in Stimuli-Responsive Hydrogels," Center for Bioengineering,
Univ. of Washington, Seattle, WA, p. 828; Yu and Grainger, "Thermo-sensitive
Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic,
Anionic and Ampholytic Hydrogels," Dept. of Chemical & Biological Sci.,
Oregon Graduate Institute of Science & Technology, Beaverton, OR, pp. 829-
830; Kim et al., Pharm. Res. 9(3):283-290, 1992; Bae et al., Pharm. Res.
8(5):624-628,' 1991; Kono et al., J. Controlled Release 30:69-75, 1994;
Yoshida
et al., J. Controlled Release 32:97-102, 1994; Okano et al., J. Controlled
Release 36:125-133, 1995; Chun and Kim, J. Controlled Release 38:39-47,
1996; D'Emanuele and Dinarvand, Int'I J. Pharm. 118:237-242, 1995; Katono
et al., J. Controlled Release 16:215-228, 1991; Hoffman, "Thermally Reversible
Hydrogels Containing Biologically Active Species," in Migliaresi et al.
(eds.),
Polymers in Medicine III, Elsevier Science Publishers B.V., Amsterdam, 1988,
pp. 161-167; Hoffman, "Applications of Thermally Reversible Polymers and
Hydrogels in Therapeutics and Diagnostics," in Third International Symposium
151
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
on Recent Advances in Drug Delivery Systems, Salt Lake City, UT, Feb. 24-27,
1987, pp. 297-305; Gutowska et al., J. Controlled Release 22:95-104, 1992;
Palasis and Gehrke, J. Controlled Release 18:1-12, 1992; Paavola et al.,
Pharm. Res. 12(12):1997-2002, 1995).
Representative examples of thermogelling polymers, and the
gelatin temperature (LCST (°C)) include homopolymers such as
poly(N-methyl-N-n-propylacrylamide), 19.8; poly(N-n-propylacrylamide), 21.5;
poly(N-methyl-N-isopropylacrylamide), 22.3; poly(N-n-propylmethacrylamide),
28.0; poly(N-isopropylacrylamide), 30.9; poly(N, n-diethylacrylamide), 32.0;
poly(N-isopropylmethacrylamide), 44.0; poly(N-cyclopropylacrylamide), 45.5;
poly(N-ethylmethyacrylamide), 50.0; poly(N-methyl-N-ethylacrylamide), 56.0;
poly(N-cyclopropylmethacrylamide), 59.0; poly(N-ethylacrylamide), 72Ø
Moreover thermogelling polymers may be made by preparing copolymers
between (among) monomers of the above, or by combining such
homopolymers with other water-soluble polymers such as acrylmonomers (e.g.,
acrylic acid and derivatives thereof, such as methylacrylic acid, acrylate
monomers and derivatives thereof, such as butyl methacrylate, butyl acrylate,
lauryl acrylate, and acrylamide monomers and derivatives thereof, such as
N-butyl acrylamide and acrylamide).
Other representative examples of thermogelling polymers include
cellulose ether derivatives such as hydroxypropyl cellulose, 41 °C;
methyl
cellulose, 55°C; hydroxypropylmethyl cellulose, 66°C; and
ethylhydroxyethyl
cellulose, polyalkylene oxide-polyester block copolymers of the structure X-Y,
Y-X-Y and X-Y-X where X in a polyalkylene oxide and Y is a biodegradable
polyester (e.g., PLG-PEG-PLG) and PLURONICs such as F-127, 10 - 15°C;
L-122, 19°C; L-92, 26°C; L-81, 20°C; and L-61,
24°C.
Representative examples of patents relating to thermally gelling
polymers and the preparation include U.S. Patent Nos. 6,451,346; 6,201,072;
6,117,949; 6,004,573; 5,702,717; and 5,484,610; and PCT Publication Nos.
152
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
WO 99/07343; WO 99/18142; WO 03/17972; WO 01/82970; WO 00/18821;
WO 97/15287; WO 01/41735; WO 00/00222 and WO 00/38651.
Anti-infective and/or fibrosis-inhibiting therapeutic agents may be
linked by occlusion in the polymer, dissolution in the polymer, bound by
covalent linkages, bound by ionic interactions, or encapsulated in
microcapsules. Within certain embodiments of the invention, therapeutic
compositions are provided in non-capsular formulations such as microspheres
(ranging from nanometers to micrometers in size), pastes, threads of various
size, films, or sprays. In one aspect, the anti-scarring agent may be
incorporated into biodegradable magnetic nanospheres. The nanospheres may
be used, for example, to replenish an anti-scarring agent into an implanted
intravascular device, such as a stent containing a weak magnetic alloy (see,
e.g., Z. Forties, B. B. Yellen, G. Friedman, K. Barbee. "An approach to
targeted
drug delivery based on uniform magnetic fields," IEEE Trans. Magn. 39(5):
3372-3377 (2003)).
Within certain aspects of the present invention, therapeutic
compositions of anti-infective and/or fibrosis-inhibiting agents may be
fashioned
in the form of microspheres, microparticles and/or nanoparticles having any
size ranging from 50 nm to 500 Vim, depending upon the particular use. These
compositions can be. These compositions can be formed by spray-drying
methods, milling methods, coacervation methods, W/O emulsion methods,
W/O/W emulsion methods, and solvent evaporation methods. In other aspects,
these compositions can include microemulsions, emulsions, liposomes and
micelles. Alternatively, such compositions may also be readily applied as a
"spray", which solidifies into a film or coating for use as a device/implant
surface
coating or to line the tissues of the implantation site. Such sprays may be
prepared from microspheres of a wide array of sizes, including for example,
from 0.1 pm to 3 Vim, from 10 ~m to 30 p,m, and from 30 pm to 100 p.m.
Therapeutic compositions that include anti-infective and/or anti-
fibrosis agents may also be prepared in a variety of "paste" or gel forms. For
153
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
example, within one embodiment of the invention, therapeutic compositions are
provided which are liquid at one temperature (e.g., temperature greater than
37°C, such as 40°C, 45°C, 50°C, 55°C or
60°C), and solid or semi-solid at
another temperature (e.g., ambient body temperature, or any temperature lower
than 37°C). Such "thermopastes" may be readily made utilizing a variety
of
techniques (see, e.g., PCT Publication WO 98/24427). Other pastes may be
applied as a liquid, which solidify in vivo due to dissolution of a water-
soluble
component of the paste and precipitation of encapsulated drug into the
aqueous body environment. These "pastes" and "gels" containing therapeutic
agents are particularly useful for application to the surface of tissues that
will be
in contact with the implant or device.
Within further aspects of the present invention, polymeric carriers
are provided which are adapted to contain and release a hydrophobic ant-
infective and/or fibrosis-inhibiting compound, and/or the carrier containing
the
hydrophobic compound in combination with a carbohydrate, protein or
polypeptide. Within certain embodiments, the polymeric carrier contains or
comprises regions, pockets, or granules of one or more hydrophobic
compounds. For example, within one embodiment of the invention,
hydrophobic compounds may be incorporated within a matrix which contains
the hydrophobic therapeutic compound, followed by incorporation of the matrix
within the polymeric carrier. A variety of matrices can be utilized in this
regard,
including for example, carbohydrates and polysaccharides such as starch,
cellulose, dextran, methylcellulose, sodium alginate, heparin, chitosan and
hyaluronic acid, proteins or polypeptides such as albumin, collagen and
gelatin.
Within alternative embodiments, hydrophobic compounds may be contained
within' a hydrophobic core, and this core contained within a hydrophilic
shell.
The anti-infective and/or fibrosis-inhibiting therapeutic agent may
be delivered as a solution. The therapeutic agent can be incorporated directly
into the solution to provide a homogeneous solution or dispersion. In certain
embodiments, the solution is an aqueous solution. The aqueous solution may
154
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
further include buffer salts, as well as viscosity modifying agents (e.g.,
hyaluronic acid, alginates, carboxymethylcellulose (CMG), and the like). In
another aspect of the invention, the solution can include a biocompatible
solvent or liquid oligomers and/or polymers, such as ethanol, DMSO, glycerol,
PEG-200, PEG-300 or NMP. These compositions may further comprise a
polymer such a degradable polyester, where the polyester may comprise the
residues of one or more of the monomers selected from lactide, lactic acid,
glycolide, glycolic acid, e-caprolactone, gamma-caprolactone, hydroxyvaleric
acid, hydroxybutyric acid, beta-butyrolactone, gamma-butyrolactone, gamma-
valerolactone, y-decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-
dioxane-2-one or 1,5-dioxepan-Zone, or block copolymers of the form X-Y, Y-X-
Y, R-(Y-X)", R-(X-Y)" and X-Y-X (where X in a polyalkylene oxide (e.g.,
polyethylene glycol, polypropylene glycol) and block copolymers of
polyethylene oxide) and polypropylene oxide) (e.g., PLURONIC and
PLURONIC R series of polymers from BASF Corporation, Mount Olive, NJ) and
Y is a biodegradable polyester, where the polyester may comprise the residues
of one or more of the monomers selected from lactide, lactic acid, glycolide,
glycolic acid, e-caprolactone, gamma-caprolactone, hydroxyvaleric acid,
hydroxybutyric acid, beta-butyrolactone, gamma-butyrolactone, gamma-
valerolactone, y-decanolactone, b-decanolactone, trimethylene carbonate, 1,4-
dioxane-2-one or 1,5-dioxepan-Zone (e.g., PLG-PEG-PLG) and R is a
multifunctional initiator).
Within another aspect of the invention, the therapeutic anti-
infective and/or fibrosis-inhibiting agent can further comprise a secondary
carrier. The secondary carrier can be in the form of microspheres (e.g., PLGA,
PLLA, PDLLA, PCL, gelatin, polydioxanone, poly(alkylcyanoacrylate)),
nanospheres (PLGA, PLLA, PDLLA, PCL, gelatin, polydioxanone,
poly(alkylcyanoacrylate)), liposomes, emulsions, microemulsions, micelles
(SDS, block copolymers of the form X-Y, Y-X-Y, R-(Y-X)", R-(X-Y)~ and X-Y-X
(where X in a polyalkylene oxide (e.g., poly(ethylene glycol, polypropylene
155
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
glycol) and block copolymers of polyethylene oxide) and polypropylene oxide)
(e.g., PLURONIC and PLURONIC R series of polymers from BASF
Corporation, Mount Olive, NJ) and Y is a biodegradable polyester, where the
polyester may comprise the residues of one or more of the monomers selected
from lactide, lactic acid, glycolide, glycolic acid, e-caprolactone, gamma-
caprolactone, hydroxyvaleric acid, hydroxybutyric acid, beta-butyrolactone,
gamma-butyrolactone, gamma-valerolactone, y-decanolactone, b-
decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or 1,5-dioxepan-
2one (e.g., PLG-PEG-PLG) and R is a multifunctional initiator), zeolites or
cyclodextrins.
Other carriers that may likewise be utilized to contain and deliver
anti-infective and/or fibrosis-inhibiting therapeutic agents described herein
include: hydroxypropyl cyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108:69-
75, 1994), liposomes (see, e.g., Sharma et al., Cancer Res. 53:5877-5881,
1993; Sharma and Straubinger, Pharm. Res. 11(60):889-896, 1994; WO
93/18751; U.S. Patent No. 5,242,073), liposomelgel (WO 94/26254),
nanocapsules (Bartoli et al., J. Microencapsulation 7(2):191-197, 1990),
micelles (Alkan-Onyuksel et al., Pharm. Res. 11(2):206-212, 1994), implants
(Jampel et al., Invest. Ophthalm. Vis. Science 34(11 ):3076-3083, 1993; Walter
et al., Cancer Res. 54:22017-2212, 1994), nanoparticles (Violante and
Lanzafame PAACR), nanoparticles - modified (U.S. Patent No. 5,145,684),
nanoparticles (surface modified) (U.S. Patent No. 5,399,363), micelle
(surfactant) (U.S. Patent No. 5,403,858), synthetic phospholipid compounds
(U.S. Patent No. 4,534,899), gas borne dispersion (U.S. Patent No. 5,301,664),
liquid emulsions, foam, spray, gel, lotion, cream, ointment, dispersed
vesicles,
particles or droplets solid- or liquid- aerosols, microemulsions (U.S. Patent
No.
5,330,756), polymeric shell (nano- and micro- capsule) (U.S. Patent No.
5,439,686), emulsion (Tarr et al., Pharm Res. 4: 62-165, 1987), nanospheres
(Hagan et al., Proc. Intern. Symp. Control Rel. Bioact. Mater. 22, 1995; Kwon
et al., Pharm Res. 12(2):192-195; Kwon et al., Pharm Res. 10(7):970-974;
156
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Yokoyama et al., J. Contr. Rel. 32:269-277, 1994; Gref et al., Science
263:1600-1603, 1994; Bazile et al., J. Pharm. Sci. 84:493-498, 1994) and
implants (U.S. Patent No. 4,882,168).
Within another aspect of the present invention, polymeric carriers
can be materials that are formed in situ. In one embodiment, the precursors
can
be monomers or macromers that contain unsaturated groups that can be
polymerized and/or cross-linked. The monomers or macromers can then, for
example, be injected into the treatment area or onto the surface of the
treatment area and polymerized in situ using a radiation source (e.g., visible
or
UV light) or a free radical system (e.g., potassium persulfate and ascorbic
acid
or iron and hydrogen peroxide). The polymerization step can be performed
immediately prior to, simultaneously to or post injection of the reagents into
the
treatment site. Representative examples of compositions that undergo free
radical polymerization reactions are described in WO 01/44307, WO 01/68720,
WO 02/072166, WO 03/043552, WO 93/17669, WO 00/64977; U.S. Patent
Nos. 5,900,245, 6,051,248, 6,083,524, 6,177,095, 6,201,065, 6,217,894,
6,639,014, 6,352,710, 6,410,645, 6,531,147, 5,567,435, 5,986,043, 6,602,975;
U.S. Patent Application Publication Nos. 2002/012796A1, 2002/0127266A1,
2002/0151650A1, 2003/0104032A1, 2002/0091229A1, and 2003/0059906A1.
In certain aspects, it is desirable to use compositions that can be
administered as liquids, but subsequently form hydrogels at the site of
administration. Such in situ hydrogel forming compositions can be
administered as liquids from a variety of different devices, and are more
adaptable for administration to any site, since they are not preformed.
Examples of in situ forming hydrogels include photoactivatable mixtures of
water-soluble co-polyester prepolymers and polyethylene glycol to create
hydrogel barriers. Block copolymers of polyalkylene oxide polymers (e.g.,
PLURONIC compounds from BASF Corporation, Mount Olive, NJ) and
poloxamers have been designed that are soluble in cold water, but form
157
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
insoluble hydrogels that adhere to tissues at body temperature (Leach, et al.,
Am. J. Obstet. Gynecol. 162:1317-1319 (1990)).
As mentioned elsewhere herein, the present invention provides
for polymeric crosslinked matrices, and polymeric carriers, that may be used
to
assist in the prevention of the formation or growth of fibrous connective
tissue.
The composition may contain and deliver fibrosis-inhibiting agents in the
vicinity
of the implanted device. The following compositions are particularly useful
when it is desired to infiltrate around the device, with or without a fibrosis-
inhibiting agent. Such polymeric materials may be prepared from, e.g., (a)
synthetic materials, (b) naturally-occurring materials, or (c) mixtures of
synthetic
and naturally occurring materials. The matrix may be prepared from, e.g., (a)
a
one-component; i.e., self-reactive, compound, or (b) two or more compounds
that are reactive with one another. Typically, these materials are fluid prior
to
delivery, and thus can be sprayed or otherwise extruded from a delivery device
(e.g., a syringe) in order to deliver the composition. After delivery, the
component materials react with each other, andlor with the body, to provide
the
desired affect. In some instances, materials that are reactive with one
another
must be kept separated prior to delivery to the patient, and are mixed
together
just prior to being delivered to the patient, in order that they maintain a
fluid
form prior to delivery. In a preferred aspect of the invention, the components
of
the matrix are delivered in a liquid state to the desired site in the body,
whereupon in situ polymerization occurs.
First and Second Synthetic Polymers
In one embodiment, crosslinked polymer compositions (in other
words, crosslinked matrices) are prepared by reacting a first synthetic
polymer
containing two or more nucleophilic groups with a second synthetic polymer
containing two or more electrophilic groups, where the electrophilic groups
are
capable of covalently binding with the nucleophilic groups. In one embodiment,
the first and second polymers are each non-immunogenic. In another
158
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
embodiment, the matrices are not susceptible to enzymatic cleavage by, e.g., a
matrix metalloproteinase (e.g., collagenase) and are therefore expected to
have
greater long-term persistence in vivo than collagen-based compositions.
As used herein, the term "polymer" refers inter alia to polyalkyls,
polyamino acids, polyalkyleneoxides and polysaccharides. Additionally, for
external or oral use, the polymer may be polyacrylic acid or carbopol. As used
herein, the term "synthetic polymer" refers to polymers that are not naturally
occurring and that are produced via chemical synthesis. As such, naturally
occurring proteins such as collagen and naturally occurring polysaccharides
such as hyaluronic acid are specifically excluded. Synthetic collagen, and
synthetic hyaluronic acid, and their derivatives, are included. Synthetic
polymers containing either nucleophilic or electrophilic groups are also
referred
to herein as "multifunctionally activated synthetic polymers." The term
"multifunctionally activated" (or, simply, "activated") refers to synthetic
polymers
which have, or have been chemically modified to have, two or more nucleophilic
or electrophilic groups which are capable of reacting with one another (i.e.,
the
nucleophilic groups react with the electrophilic groups) to form covalent
bonds.
Types of multifunctionally activated synthetic polymers include difunctionally
activated, tetrafunctionally activated, and star-branched polymers.
Multifunctionally activated synthetic polymers for use in the
present invention must contain at least two, more preferably, at least three,
functional groups in order to form a three-dimensional crosslinked network
with
synthetic polymers containing multiple nucleophilic groups (i.e., "multi-
nucleophilic polymers"). In other words, they must be at least difunctionally
activated, and are more preferably trifunctionally or tetrafunctionally
activated.
If the first synthetic polymer is a difunctionally activated synthetic
polymer, the
second synthetic polymer must contain three or more functional groups in order
to obtain a three-dimensional crosslinked network. Most preferably, both the
first and the second synthetic polymer contain at least three functional
groups.
159
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Synthetic polymers containing multiple nucleophilic groups are
also referred to generically herein as "multi-nucleophilic polymers." For use
in
the present invention, multi-nucleophilic polymers must contain at least two,
more preferably, at least three, nucleophilic groups. If a synthetic polymer
containing only two nucleophilic groups is used, a synthetic polymer
containing
three or more electrophilic groups must be used in order to obtain a three-
dimensional crosslinked network.
Preferred multi-nucleophilic polymers for use in the compositions
and methods of the present invention include synthetic polymers that contain,
or have been modified to contain, multiple nucleophilic groups such as primary
amino groups and thiol groups. Preferred multi-nucleophilic polymers include:
(i) synthetic polypeptides that have been synthesized to contain two or more
primary amino groups or thiol groups; and (ii) polyethylene glycols that have
been modified to contain two or more primary amino groups or thiol groups. In
general, reaction of a thiol group with an electrophilic group tends to
proceed
more slowly than reaction of a primary amino group with an electrophilic
group.
In one embodiment, the multi-nucleophilic polypeptide is a
synthetic polypeptide that has been synthesized to incorporate amino acid
residues containing primary amino groups (such as lysine) and/or amino acids
containing thiol groups (such as cysteine). Poly(lysine), a synthetically
produced polymer of the amino acid lysine (145 MW), is particularly preferred.
Poly(lysine)s have been prepared having anywhere from 6 to about 4,000
primary amino groups, corresponding to molecular weights of about 870 to
about 580,000.
Poly(lysine)s for use in the present invention preferably have a
molecular weight within the range of about 1,000 to about 300,000; more
preferably, within the range of about 5,000 to about 100,000; most preferably,
within the range of about 8,000 to about 15,000. Poly(lysine)s of varying
molecular weights are commercially available from Peninsula Laboratories, Inc.
(Belmont, Calif.) and Aldrich Chemical (Milwaukee, WI).
160
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Polyethylene glycol can be chemically modified to contain multiple
primary amino or thiol groups according to methods set forth, for example, in
Chapter 22 of Polyethylene Glycol) Chemistry: Biotechnical and Biomedical
Applications, J. Milton Harris, ed., Plenum Press, N.Y. (1992). Polyethylene
glycols which have been modified to contain two or more primary amino groups
are referred to herein as "multi-amino PEGs." Polyethylene glycols which have
been modified to contain two or more thiol groups are referred to herein as
"multi-thiol PEGs." As used herein, the term "polyethylene glycol(s)" includes
modified and or derivatized polyethylene glycol(s).
Various forms of multi-amino PEG are commercially available
from Shearwater Polymers (Huntsville, Ala.) and from Huntsman Chemical
Company (Utah) under the name "Jeffamine." Multi-amino PEGs useful in the
present invention include Huntsman's Jeffamine diamines ("D" series) and
triamines ("T" series), which contain two and three primary amino groups per
molecule, respectively.
Polyamines such as ethylenediamine (H2N-CH2-CH2-NH2),
tetramethylenediamine (H2N-(CH2)4-NH2), pentamethylenediamine (cadaverine)
(H2N-(CH2)5-NH2), hexamethylenediamine (H2N-(CH~)6-NH2), di(2-
aminoethyl)amine (HN-(CH2-CH2-NH2)2), and tris(2-aminoethyl)amine (N-(CH2-
CH2-NH2)3) may also be used as the synthetic polymer containing multiple
nucleophilic groups.
Synthetic polymers containing multiple electrophilic groups are
also referred to herein as "multi-electrophilic polymers." For use in the
present
invention, the multifunctionally activated synthetic polymers must contain at
least two, more preferably, at least three, electrophilic groups in order to
form a
three-dimensional crosslinked network with multi-nucleophilic polymers.
Preferred multi-electrophilic polymers for use in the compositions of the
invention are polymers which contain two or more succinimidyl groups capable
of forming covalent bonds with nucleophilic groups on other molecules.
Succinimidyl groups are highly reactive with materials containing primary
amino
161
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(NH2) groups, such as multi-amino PEG, poly(lysine), or collagen. Succinimidyl
groups are slightly less reactive with materials containing thiol (SH) groups,
such as multi-thiol PEG or synthetic polypeptides containing multiple cysteine
residues.
As used herein, the term "containing two or more succinimidyl
groups" is meant to encompass polymers which are preferably commercially
available containing two or more succinimidyl groups, as well as those that
must be chemically derivatized to contain two or more succinimidyl groups. As
used herein, the term "succinimidyl group" is intended to encompass
sulfosuccinimidyl groups and other such variations of the "generic"
succinimidyl
group. The presence of the sodium sulfite moiety on the sulfosuccinimidyl
group serves to increase the solubility of the polymer.
Hydrophilic polymers and, in particular, various derivatized
polyethylene glycols, are preferred for use in the compositions of the present
invention. As used herein, the term "PEG" refers to polymers having the
repeating structure (OCH2-CH2)~. Structures for some specific,
tetrafunctionally
activated forms of PEG are shown in FIGS. 4 to 13 of U.S. Patent 5,874,500,
incorporated herein by reference. Examples of suitable PEGS include PEG
succinimidyl propionate (SE-PEG), PEG succinimidyl succinamide (SSA-PEG),
and PEG succinimidyl carbonate (SC-PEG). In one aspect of the invention, the
crosslinked matrix is formed in situ by reacting pentaerythritol polyethylene
glycol)ether tetra-sulfhydryl] (4-armed thiol PEG) and pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate] (4-armed NHS PEG) as
reactive reagents. Structures for these reactants are shown in U.S. Patent
5,874,500. Each of these materials has a core with a structure that may be
seen by adding ethylene oxide-derived residues to each of the hydroxyl groups
in pentaerythritol, and then derivatizing the terminal hydroxyl groups
(derived
from the ethylene oxide) to contain either thiol groups (so as to form 4-armed
thiol PEG) or N-hydroxysuccinimydyl groups (so as to form 4-armed NHS
PEG), optionally with a linker group present between the ethylene oxide
derived
162
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
backbone and the reactive functional group, where this product is commercially
available as COSEAL from Angiotech Pharmaceuticals Inc. Optionally, a group
"D" may be present in one or both of these molecules, as discussed in more
detail below.
As discussed above, preferred activated polyethylene glycol
derivatives for use in the invention contain succinimidyl groups as the
reactive
group. However, different activating groups can be attached at sites along the
length of the PEG molecule. For example, PEG can be derivatized to form
functionally activated PEG propionaldehyde (A-PEG), or functionally activated
PEG glycidyl ether (E-PEG), or functionally activated PEG-isocyanate (I-PEG),
or functionally activated PEG-vinylsulfone (V-PEG).
Hydrophobic polymers can also be used to prepare the
compositions of the present invention. Hydrophobic polymers for use in the
present invention preferably contain, or can be derivatized to contain, two or
more electrophilic groups, such as succinimidyl groups, most preferably, two,
three, or four electrophilic groups. As used herein, the term "hydrophobic
polymer" refers to polymers which contain a relatively small proportion of
oxygen or nitrogen atoms.
Hydrophobic polymers which already contain two or more
succinimidyl groups include, without limitation, disuccinimidyl suberate
(DSS),
bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidylpropionate)
(DSP),
bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and 3,3'--
dithiobis(sulfosuccinimidylpropionate (DTSPP), and their analogs and
derivatives. The above-referenced polymers are commercially available from
Pierce (Rockford, IIL), under catalog Nos. 21555, 21579, 22585, 21554, and
21577, respectively.
Preferred hydrophobic polymers for use in the invention generally
have a carbon chain that is no longer than about 14 carbons. Polymers having
carbon chains substantially longer than 14 carbons generally have very poor
solubility in aqueous solutions and, as such, have very long reaction times
163
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
when mixed with aqueous solutions of synthetic polymers containing multiple
nucleophilic groups.
Certain polymers, such as polyacids, can be derivatized to contain
two or more functional groups, such as succinimidyl groups. Polyacids for use
in the present invention include, without limitation, trimethylolpropane-based
tricarboxylic acid, di(trimethylol propane)-based tetracarboxylic acid,
heptanedioic acid, octanedioic acid (suberic acid), and hexadecanedioic acid
(thapsic acid). Many of these polyacids are commercially available from
DuPont Chemical Company (Wilmington, DE). According to a general method,
polyacids can be chemically derivatized to contain two or more succinimidyl
groups by reaction with an appropriate molar amount of N-hydroxysuccinimide
(NHS) in the presence of N,N'-dicyclohexylcarbodiimide (DCC).
Polyalcohols such as trimethylolpropane and di(trimethylol
propane) can be converted to carboxylic acid form using various methods, then
further derivatized by reaction with NHS in the presence of DCC to produce
trifunctionally and tetrafunctionally activated polymers, respectively, as
described in U.S. application Ser. No. 08/403,358. Polyacids such as
heptanedioic acid (HOOC-(CH2)5-COOH), octanedioic acid (HOOC-(CH2)s-
COOH), and hexadecanedioic acid (HOOC-(CH2)~4-COOH) are derivatized by
the addition of succinimidyl groups to produce difunctionally activated
polymers.
Polyamines such as ethylenediamine, tetramethylenediamine,
pentamethylenediamine (cadaverine), hexamethylenediamine, bis (2-
aminoethyl)amine, and tris(2-aminoethyl)amine can be chemically derivatized to
polyacids, which can then be derivatized to contain two or more succinimidyl
groups by reacting with the appropriate molar amounts of N-
hydroxysuccinimide in the presence of DCC, as described in U.S. application
Ser. No. 08/403,358. Many of these polyamines are commercially available
from DuPont Chemical Company.
In a preferred embodiment, the first synthetic polymer will contain
multiple nucleophilic groups (represented below as "X") and it will react with
the
164
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
second synthetic polymer containing multiple electrophilic groups (represented
below as "Y"), resulting in a covalently bound polymer network, as follows:
Polymer-Xm + Polymer-Y" -~ Polymer-Z-Polymer
wherein m <_2, n <_2, and m + n _<5;
where exemplary X groups include -NH2, -SH, -OH, -PH2, CO-NH-
NHS, etc., where the X groups may be the same or different in polymer-Xm;
where exemplary Y groups include -C02-N(COCH2)2, -CO2H, -
CHO, -CHOCH~ (epoxide), -N=C=O, -S02-CH=CH2, -N(COCH)2 (i.e., a five-
membered heterocyclic ring with a double bond present between the two CH
groups), -S-S-(C5H4N), etc., where the Y groups may be the same or different
in
polymer-Y~; and
where Z is the functional group resulting from the union of a
nucleophilic group (X) and an electrophilic group (Y).
As noted above, it is also contemplated by the present invention
that X and Y may be the same or different, i.e., a synthetic polymer may have
two different electrophilic groups, or two different nucleophilic groups, such
as
with glutathione.
In one embodiment, the backbone of at least one of the synthetic
polymers comprises alkylene oxide residues, e.g., residues from ethylene
oxide, propylene oxide, and mixtures thereof. The term 'backbone' refers to a
significant portion of the polymer.
For example, the synthetic polymer containing alkylene oxide
residues may be described by the formula X-polymer-X or Y-polymer-Y,
wherein X and Y are as defined above, and the term "polymer" represents -
(CH2CH2 O)"- or -(CH(CH3)CH2 O)n or -(CH2-CH2-O)"-(CH(CH3)CH2-O)n . In
these cases the synthetic polymer would be difunctional.
The required functional group X or Y is commonly coupled to the
polymer backbone by a linking group (represented below as "Q"), many of
which are known or possible. There are many ways to prepare the various
functionalized polymers, some of which are listed below:
165
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Polymer-Q~-X + Polymer-Q2-Y --> Polymer-Q~-Z-Q2-Polymer
Exemplary Q groups include -O-(GH~)~ ; -S-(CH2)~ ; -NH-(CH2)n ;
-02C-NH-(CH2)n ; -02C-(CH2)n ; -02C-(CR~H)n ; and -O-R2-CO-NH-, which
provide synthetic polymers of the partial structures: polymer-O-(CH2)n (X or
Y);
polymer-S-(CH2)n (X or Y); polymer-NH-(CH2)"(X or Y); polymer-02C-NH-
(CH2)~ (X or Y); polymer-02C-(CH~)~ (X or Y); polymer-02C-(CR~H)"-(X or Y);
and polymer-O-R2-CO-NH-(X or Y), respectively. In these structures, n = 1-10,
R~ = H or alkyl (i.e., CH3, C2H5, etc.); R~ = CH2, or CO-NH-CH~CH2; and Q~ and
Q2 may be the same or different.
For example, when Q2 = OCH2CH~ (there is no Q~ in this case); Y
- -C02-N(COCH2)2; and X = -NH2, -SH, or -OH, the resulting reactions and Z
groups would be as follows:
Polymer-NHS + Polymer-O-CH2-CH2-C02-N(COCH~)~
Polymer-NH-CO-CHI-CHI-O-Polymer;
Polymer-SH + Polymer-O-CHI-CH2-C02-N(COCH2)2 -
Polymer-S-COCH2CH2-O-Polymer; and
Polymer-OH + Polymer-O-CH2-CH2-C02-N(COCH2)2
Polymer-O-COCH2CH2-O-Polymer.
An additional group, represented below as "D", can be inserted
between the polymer and the linking group, if present. One purpose of such a
D group is to affect the degradation rate of the crosslinked polymer
composition
in vivo, for example, to increase the degradation rate, or to decrease the
degradation rate. This may be useful in many instances, for example, when
drug has been incorporated into the matrix, and it is desired to increase or
decrease polymer degradation rate so as to influence a drug delivery profile
in
the desired direction. An illustration of a crosslinking reaction involving
first and
second synthetic polymers each having D and Q groups is shown below.
Polymer-D-Q-X + Polymer-D-Q-Y -~ Polymer-D-Q-Z-Q-D-Polymer
Some useful biodegradable groups "D" include polymers formed
from one or more a-hydroxy acids, e.g., lactic acid, glycolic acid, and the
166
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
cyclization products thereof (e.g., lactide, glycolide), E-caprolactone, and
amino
acids. The polymers may be referred to as polylactide, polyglycolide, poly(co-
lactide-glycolide); poly-~-caprolactone, polypeptide (also known as poly amino
acid, for example, various di- or tri-peptides) and poly(anhydride)s.
In a general method for preparing the crosslinked polymer
compositions used in the context of the present invention, a first synthetic
polymer containing multiple nucleophilic groups is mixed with a second
synthetic polymer containing multiple electrophilic groups. Formation of a
three-dimensional crosslinked network occurs as a result of the reaction
between the nucleophilic groups on the first synthetic polymer and the
electrophilic groups on the second synthetic polymer.
The concentrations of the first synthetic polymer and the second
synthetic polymer used to prepare the compositions of the present invention
will
vary depending upon a number of factors, including the types and molecular
weights of the particular synthetic polymers used and the desired end use
application. In general, when using multi-amino PEG as the first synthetic
polymer, it is preferably used at a concentration in the range of about 0.5 to
about 20 percent by weight of the final composition, while the second
synthetic
polymer is used at a concentration in the range of about 0.5 to about 20
percent
by weight of the final composition. For example, a final composition having a
total weight of 1 gram (1000 milligrams) would contain between about 5 to
about 200 milligrams of multi-amino PEG, and between about 5 to about 200
milligrams of the second synthetic polymer.
Use of higher concentrations of both first and second synthetic
polymers will result in the formation of a more tightly crosslinked network,
producing a stiffer, more robust gel. Compositions intended for use in tissue
augmentation will generally employ concentrations of first and second
synthetic
polymer that fall toward the higher end of the preferred concentration range.
Compositions intended for use as bioadhesives or in adhesion prevention do
not need to be as firm and may therefore contain lower polymer concentrations.
167
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Because polymers containing multiple electrophilic groups will
also react with water, the second synthetic polymer is generally stored and
used in sterile, dry form to prevent the loss of crosslinking ability due to
hydrolysis which typically occurs upon exposure of such electrophilic groups
to
aqueous media. Processes for preparing synthetic hydrophilic polymers
containing multiple electrophylic groups in sterile, dry form are set forth in
U.S.
Patent 5,643,464. For example, the dry synthetic polymer may be compression
molded into a thin sheet or membrane, which can then be sterilized using
gamma or, preferably, e-beam irradiation. The resulting dry membrane or
sheet can be cut to the desired size or chopped into smaller size
particulates.
In contrast, polymers containing multiple nucleophilic groups are generally
not
water-reactive and can therefore be stored in aqueous solution.
In certain embodiments, one or both of the electrophilic- or
nucleophilic-terminated polymers described above can be combined with a
synthetic or naturally occurring polymer. The presence of the synthetic or
naturally occurring polymer may enhance the mechanical and/or adhesive
properties of the in situ forming compositions. Naturally occurring polymers,
and polymers derived from naturally occurring polymer that may be included in
in situ forming materials include naturally occurring proteins, such as
collagen,
collagen derivatives (such as methylated collagen), fibrinogen, thrombin,
albumin, fibrin, and derivatives of and naturally occurring polysaccharides,
such
as glycosaminoglycans, including deacetylated and desulfated
glycosaminoglycan derivatives.
In one aspect, a composition comprising naturally-occurring
protein and both of the first and second synthetic polymer as described above
is used to form the crosslinked matrix according to the present invention. In
one aspect, a composition comprising collagen and both of the first and second
synthetic polymer as described above is used to form the crosslinked matrix
according to the present invention. In one aspect, a composition comprising
methylated collagen and both of the first and second synthetic polymer as
168
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
described above is used to form the crosslinked matrix according to the
present
invention. In one aspect, a composition comprising fibrinogen and both of the
first and second synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
~ composition comprising thrombin and both of the first and second synthetic
polymer as described above is used to form the crosslinked matrix according to
the present invention. In one aspect, a composition comprising albumin and
both of the first and second synthetic polymer as described above is used to
form the crosslinked matrix according to the present invention. In one aspect,
a
composition comprising fibrin and both of the first and second synthetic
polymer
as described above is used to form the crosslinked matrix according to the
present invention. In one aspect, a composition comprising naturally occurring
polysaccharide and both of the first and second synthetic polymer as described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising glycosaminoglycan and both of the
first and second synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
composition comprising deacetylated glycosaminoglycan and both of the first
and second synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
composition comprising desulfated glycosaminoglycan and both of the first and
second synthetic polymer as described above is used to form the crosslinked
matrix according to the present invention.
In one aspect, a composition comprising naturally-occurring
protein and the first synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
composition comprising collagen and the first synthetic polymer as described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising methylated collagen and the first
synthetic polymer as described above is used to form the crosslinked matrix
169
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
according to the present invention. In one aspect, a composition comprising
fibrinogen and the first synthetic polymer as described above is used to form
the crosslinked matrix according to the present invention. In one aspect, a
composition comprising thrombin and the first synthetic polymer as described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising albumin and the first synthetic
polymer
as described above is used fo form the crosslinked matrix according to the
present invention. In one aspect, a composition comprising fibrin and the
first
synthetic polymer as described above is used to form the crosslinked matrix
according to the present invention. In one aspect, a composition comprising
naturally occurring polysaccharide and the first synthetic polymer as
described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising glycosaminoglycan and the first
synthetic polymer as described above is used to form the crosslinked matrix
according to the present invention. In one aspect, a composition comprising
deacetylated glycosaminoglycan and the first synthetic polymer as described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising desulfated glycosaminoglycan and the
first synthetic polymer as described above is used to form the crosslinked
matrix according to the present invention.
In one aspect, a composition comprising naturally-occurring
protein and the second synthetic polymer as described above is used to form
the crosslinked matrix according to the present invention. In one aspect, a
composition comprising collagen and the second synthetic polymer as
described above is used to form the crosslinked matrix according to the
present
invention. In one aspect, a composition comprising methylated collagen and
the second synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
composition comprising fibrinogen and the second synthetic polymer as
described above is used to form the crosslinked matrix according to the
present
170
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
invention. In one aspect, a composition comprising thrombin and the second
synthetic polymer as described above is used to form the crosslinked matrix
according to the present invention. In one aspect, a composition comprising
albumin and the second synthetic polymer as described above is used to form
the crosslinked matrix according to the present invention. In one aspect, a
composition comprising fibrin and the second synthetic polymer as described
above is used to form the crosslinked matrix according to the present
invention.
In one aspect, a composition comprising naturally occurring polysaccharide and
the second synthetic polymer as described above is used to form the
crosslinked matrix according to the present invention. In one aspect, a
composition comprising glycosaminoglycan and the second synthetic polymer
as described above is used to form the crosslinked matrix according to the
present invention. In one aspect, a composition comprising deacetylated
glycosaminoglycan and the second synthetic polymer as described above is
used to form the crosslinked matrix according to the present invention. In one
aspect, a composition comprising desulfated glycosaminoglycan and the
second synthetic polymer as described above is used to form the crosslinked
matrix according to the present invention.
The presence of protein or polysaccharide components which
contain functional groups that can react with the functional groups on
multiple
activated synthetic polymers can result in formation of a crosslinked
synthetic
polymer-naturally occurring polymer matrix upon mixing and/or crosslinking of
the synthetic polymer(s). In particular, when the naturally occurring polymer
(protein or polysaccharide) also contains nucleophilic groups such as primary
amino groups, the electrophilic groups on the second synthetic polymer will
react with the primary amino groups on these components, as well as the
nucleophilic groups on the first synthetic polymer, to cause these other
components to become part of the polymer matrix. For example, lysine-rich
proteins such as collagen may be especially reactive with electrophilic groups
on synthetic polymers.
171
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In one aspect, the naturally occurring protein is polymer may be
collagen. As used herein, the term "collagen" or "collagen material" refers to
all
forms of collagen, including those which have been processed or otherwise
modified and is intended to encompass collagen of any type, from any source,
including, but not limited to, collagen extracted from tissue or produced
recombinantly, collagen analogues, collagen derivatives, modified collagens,
and denatured collagens, such as gelatin.
In general, collagen from any source may be included in the
compositions of the invention; for example, collagen may be extracted and
purified from human or other mammalian source, such as bovine or porcine
corium and human placenta, or may be recombinantly or otherwise produced.
The preparation of purified, substantially non-antigenic collagen in solution
from
bovine skin is well known in the art. U.S. Patent No. 5,428,022 discloses
methods of extracting and purifying collagen from the human placenta. U.S.
Patent No. 5,667,839, discloses methods of producing recombinant human
collagen in the milk of transgenic animals, including transgenic cows.
Collagen
of any type, including, but not limited to, types I, II, III, IV, or any
combination
thereof, may be used in the compositions of the invention, although type I is
generally preferred. Either atelopeptide or telopeptide-containing collagen
may
be used; however, when collagen from a xenogeneic source, such as bovine
collagen, is used, atelopeptide collagen is generally preferred, because of
its
reduced immunogenicity compared to telopeptide-containing collagen.
Collagen that has not been previously crosslinked by methods
such as heat, irradiation, or chemical crosslinking agents is preferred for
use in
the compositions of the invention, although previously crosslinked collagen
may
be used. Non-crosslinked atelopeptide fibrillar collagen is commercially
available from Inamed Aesthetics (Santa Barbara, CA) at collagen
concentrations of 35 mg/ml and 65 mg/ml under the trademarks ZYDERM I
Collagen and ZYDERM II Collagen, respectively. Glutaraldehyde crosslinked
atelopeptide fibrillar collagen is commercially available from Inamed
172
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Corporation (Santa Barbara, CA) at a collagen concentration of 35 mg/ml under
the trademark ZYPLAST Collagen.
Collagens for use in the present invention are generally in
aqueous suspension at a concentration between about 20 mg/ml to about 120
mg/ml; preferably, between about 30 mg/ml to about 90 mg/ml.
Because of its tacky consistency, nonfibrillar collagen may be
preferred for use in compositions that are intended for use as bioadhesives.
The term "nonfibrillar collagen" refers to any modified or unmodified collagen
material that is in substantially nonfibrillar form at pH 7, as indicated by
optical
clarity of an aqueous suspension of the collagen.
Collagen that is already in nonfibrillar form may be used in the
compositions of the invention. As used herein, the term "nonfibrillar
collagen" is
intended to encompass collagen types that are nonfibrillar in native form, as
well as collagens that have been chemically modified such that they are in
nonfibrillar form at or around neutral pH. Collagen types that are
nonfibrillar (or
microfibrillar) in native form include types IV, VI, and VII.
Chemically modified collagens that are in nonfibrillar form at
neutral pH include succinylated collagen and methylated collagen, both of
which can be prepared according to the methods described in U.S. Pat. No.
4,164,559, issued Aug. 14, 1979, to Miyata et al., which is hereby
incorporated
by reference in its entirety. Due to its inherent tackiness, methylated
collagen is
particularly preferred for use in bioadhesive compositions, as disclosed in
U.S.
application Ser. No. 08/476,825.
Collagens for use in the crosslinked polymer compositions of the
present invention may start out in fibrillar form, then be rendered
nonfibrillar by
the addition of one or more fiber disassembly agent. The fiber disassembly
agent must be present in an amount sufficient to render the collagen
substantially nonfibrillar at pH 7, as described above. Fiber disassembly
agents
for use in the present invention include, without limitation, various
biocompatible
alcohols, amino acids (e.g., arginine), inorganic salts (e.g., sodium chloride
and
173
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
potassium chloride), and carbohydrates (e.g., various sugars including
sucrose).
In one aspect, the polymer may be collagen or a collagen
derivative, for example methylated collagen. An example of an in situ forming
composition uses pentaerythritol polyethylene glycol)ether tetra-sulfhydryl]
(4-
armed thiol PEG), pentaerythritol polyethylene glycol)ether tetra-succinimidyl
glutarate] (4-armed NHS PEG) and methylated collagen as the reactive
reagents. This composition, when mixed with the appropriate buffers can
produce a crosslinked hydrogel. (See, e.g., U.S. Patent Nos. 5,874,500;
6,051,648; 6,166,130; 5,565,519 and 6,312,725).
In another aspect, the naturally occurring polymer may be a
glycosaminoglycan. Glycosaminoglycans, e.g., hyaluronic acid, contain both
anionic and cationic functional groups along each polymeric chain, which can
form intramolecular and/or intermolecular ionic crosslinks, and are
responsible
for the thixotropic (or shear thinning) nature of hyaluronic acid.
In certain aspects, the glycosaminoglycan may be derivatized.
For example, glycosaminoglycans can be chemically derivatized by, e.g.,
deacetylation, desulfation, or both in order to contain primary amino groups
available for reaction with electrophilic groups on synthetic polymer
molecules.
Glycosaminoglycans that can be derivatized according to either or both of the
aforementioned methods include the following: hyaluronic acid, chondroitin
sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C,
chitin
(can be derivatized to chitosan), keratan sulfate, keratosulfate, and heparin.
Derivatization of glycosaminoglycans by deacetylation and/or desulfation and
covalent binding of the resulting glycosaminoglycan derivatives with synthetic
hydrophilic polymers is described in further detail in commonly assigned,
allowed U.S. patent application Ser. No. 08/146,843, filed Nov. 3, 1993.
In general, the collagen is added to the first synthetic polymer,
then the collagen and first synthetic polymer are mixed thoroughly to achieve
a
homogeneous composition. The second synthetic polymer is then added and
174
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
mixed into the collagen/first synthetic polymer mixture, where it will
covalently
bind to primary amino groups or thiol groups on the first synthetic polymer
and
primary amino groups on the collagen, resulting in the formation of a
homogeneous crosslinked network. Various deacetylated and/or desulfated
glycosaminoglycan derivatives can be incorporated into the composition in a
similar manner as that described above for collagen. In addition, the
iritroduction of hydrocolloids such as carboxymethylcellulose may promote
tissue adhesion and/or swellability.
Administration of the Crosslinked Synthetic Polymer Compositions
The compositions of the present invention having two synthetic
polymers may be administered before, during or after crosslinking of the first
and second synthetic polymer. Certain uses, which are discussed in greater
detail below, such as tissue augmentation, may require the compositions to be
crosslinked before administration, whereas other applications, such as tissue
adhesion, require the compositions to be administered before crosslinking has
reached "equilibrium." The point at which crosslinking has reached equilibrium
is defined herein as the point at which the composition no longer feels tacky
or
sticky to the touch.
In order to administer the composition prior to crosslinking, the
first synthetic polymer and second synthetic polymer may be contained within
separate barrels of a dual-compartment syringe. In this case, the two
synthetic
polymers do not actually mix until the point at which the two polymers are
extruded from the tip of the syringe needle into the patient's tissue. This
allows
the vast majority of the crosslinking reaction to occur in situ, avoiding the
problem of needle blockage which commonly occurs if the two synthetic
polymers are mixed too early and crosslinking between the two components is
already too advanced prior to delivery from the syringe needle. The use of a
dual-compartment syringe, as described above, allows for the use of smaller
175
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
diameter needles, which is advantageous when performing soft tissue
augmentation in delicate facial tissue, such as that surrounding the eyes.
Alternatively, the first synthetic polymer and second synthetic
polymer may be mixed according to the methods described above prior to
delivery to the tissue site, then injected to the desired tissue site
immediately
(preferably, within about 60 seconds) following mixing.
In another embodiment of the invention, the first synthetic polymer
and second synthetic polymer are mixed, then extruded and allowed to
crosslink into a sheet or other solid form. The crosslinked solid is then
dehydrated to remove substantially all unbound water. The resulting dried
solid
may be ground or comminuted into particulates, then suspended in a
nonaqueous fluid carrier, including, without limitation, hyaluronic acid,
dextran
sulfate, dextran, succinylated noncrosslinked collagen, methylated
noncrosslinked collagen, glycogen, glycerol, dextrose, maltose, triglycerides
of
fatty acids (such as corn oil, soybean oil, and sesame oil), and egg yolk
phospholipid. The suspension of particulates can be injected through a small-
gauge needle to a tissue site. Once inside the tissue, the crosslinked polymer
particulates will rehydrate and swell in size at least five-fold.
Hydrophilic Polymer + Plurality of Crosslinkable Components
As mentioned above, the first and/or second synthetic polymers
may be combined with a hydrophilic polymer, e.g., collagen or methylated
collagen, to form a composition useful in the present invention. In one
general
embodiment, the compositions useful in the present invention include a
hydrophilic polymer in combination with two or more crosslinkable components.
This embodiment is described in further detail in this section.
The Hydrophilic Polymer Component:
The hydrophilic polymer component may be a synthetic or
naturally occurring hydrophilic polymer. Naturally occurring hydrophilic
176
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polymers include, but are not limited to: proteins such as collagen and
derivatives thereof, fibronectin, albumins, globulins, fibrinogen, and fibrin,
with
collagen particularly preferred; carboxylated polysaccharides such as
polymannuronic acid and polygalacturonic acid; aminated polysaccharides,
particularly the glycosaminoglycans, e.g., hyaluronic acid, chitin,
chondroitin
sulfate A, B, or C, keratin sulfate, keratosulfate and heparin; and activated
polysaccharides such as dextran and starch derivatives. Collagen (e.g.,
methylated collagen) and glycosaminoglycans are preferred naturally occurring
hydrophilic polymers for use herein.
In general, collagen from any source may be used in the
composition of the method; for example, collagen may be extracted and purified
from human or other mammalian source, such as bovine or porcine corium and
human placenta, or may be recombinantly or otherwise produced. The
preparation of purified, substantially non-antigenic collagen in solution from
bovine skin is well known in the art. See, e.g., U.S. Pat. No. 5,428,022, to
Palefsky et al., which discloses methods of extracting and purifying collagen
from the human placenta. See also U.S. Patent No. 5,667,839, to Berg, which
discloses methods of producing recombinant human collagen in the milk of
transgenic animals, including transgenic cows. Unless otherwise specified, the
term "collagen" or "collagen material" as used herein refers to all forms of
collagen, including those that have been processed or otherwise modified.
Collagen of any type, including, but not limited to, types I, II, III, IV,
or any combination thereof, may be used in the compositions of the invention,
although type I is generally preferred. Either atelopeptide or telopeptide-
containing collagen may be used; however, when collagen from a source, such
as bovine collagen, is used, atelopeptide collagen is generally preferred,
because of its reduced immunogenicity compared to telopeptide-containing
collagen.
Collagen that has not been previously crosslinked by methods
such as heat, irradiation, or chemical crosslinking agents is preferred for
use in
177
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
the compositions of the invention, although previously crosslinked collagen
may
be used. Non-crosslinked atelopeptide fibrillar collagen is commercially
available from McGhan Medical Corporation (Santa Barbara, Calif.) at collagen
concentrations of 35 mg/ml and 65 mg/ml under the trademarks ZYDERM~ I
Collagen and ZYDERM~ II Collagen, respectively. Glutaraldehyde-crosslinked
atelopeptide fibrillar collagen is commercially available from McGhan Medical
Corporation at a collagen concentration of 35 mg/ml under the trademark
ZYPLAST~
Collagens for use in the present invention are generally, although
not necessarily, in aqueous suspension at a concentration between about 20
mg/ml to about 120 mg/ml, preferably between about 30 mg/ml to about 90
mg/ml.
Although intact collagen is preferred, denatured collagen,
commonly known as gelatin, can also be used in the compositions of the
invention. Gelatin may have the added benefit of being degradable faster than
collagen.
Because of its greater surface area and greater concentration of
reactive groups, nonfibrillar collagen is generally preferred. The term
"nonfibrillar collagen" refers to any modified or unmodified collagen material
that
is in substantially nonfibrillar form at pH 7, as indicated by optical clarity
of an
aqueous suspension of the collagen.
Collagen that is already in nonfibrillar form may be used in the
compositions of the invention. As used herein, the term "nonfibrillar
collagen" is
intended to encompass collagen types that are nonfibrillar in native form, as
well as collagens that have been chemically modified such that they are in
nonfibrillar form at or around neutral pH. Collagen types that are
nonfibrillar (or
microfibrillar) in native form include types IV, VI, and VII.
Chemically modified collagens that are in nonfibrillar form at
neutral pH include succinylated collagen, propylated collagen, ethylated
collagen, methylated collagen, and the like, both of which can be prepared
178
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
according to the methods described in U.S. Pat. No. 4,164,559, to Miyata et
al.,
which is hereby incorporated by reference in its entirety. Due to its inherent
tackiness, methylated collagen is particularly preferred, as disclosed in U.S.
Patent No. 5,614,587 to Rhee et al.
Collagens for use in the crosslinkable compositions of the present
invention may start out in fibrillar form, then be rendered nonfibrillar by
the
addition of one or more fiber disassembly agents. The fiber disassembly agent
must be present in an amount sufficient to render the collagen substantially
nonfibrillar at pH 7, as described above. Fiber disassembly agents for use in
the present invention include, without limitation, various biocompatible
alcohols,
amino acids, inorganic salts, and carbohydrates, with biocompatible alcohols
being particularly preferred. Preferred biocompatible alcohols include
glycerol
and propylene glycol. Non-biocompatible alcohols, such as ethanol, methanol,
and isopropanol, are not preferred for use in the present invention, due to
their
potentially deleterious effects on the body of the patient receiving them.
Preferred amino acids include arginine. Preferred inorganic salts include
sodium chloride and potassium chloride. Although carbohydrates, such as
various sugars including sucrose, may be used in the practice of the present
invention, they are not as preferred as other types of fiber disassembly
agents
because they can have cytotoxic effects in vivo.
As fibrillar collagen has less surface area and a lower
concentration of reactive groups than nonfibrillar, fibrillar collagen is less
preferred. However, as disclosed in U.S. Patent 5,614,587, fibrillar collagen,
or
mixtures of nonfibrillar and fibrillar collagen, may be preferred for use in
compositions intended for long-term persistence in vivo, if optical clarity is
not a
requirement.
Synthetic hydrophilic polymers may also be used in the present
invention. Useful synthetic hydrophilic polymers include, but are not limited
to:
polyalkylene oxides, particularly polyethylene glycol and polyethylene oxide)-
polypropylene oxide) copolymers, including block and random copolymers;
179
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polyols such as glycerol, polyglycerol (particularly highly branched
polyglycerol), propylene glycol and trimethylene glycol substituted with one
or
more polyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated glycerol,
mono- and di-polyoxyethylated propylene glycol, and mono- and di-
polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,
polyoxyethylated glucose; acrylic acid polymers and analogs and copolymers
thereof, such as polyacrylic acid per se, polymethacrylic acid,
poly(hydroxyethyl-methacrylate), poly(hydroxyethylacrylate),
poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxide acrylate)
and
copolymers of any of the foregoing, and/or with additional acrylate species
such
as aminoethyl acrylate and mono-2-(acryloxy)-ethyl succinate; polymaleic acid;
poly(acrylamides) such as polyacrylamide per se, poly(methacrylamide),
poly(dimethylacrylamide), and poly(N-isopropyl-acrylamide); poly(olefinic
alcohol)s such as polyvinyl alcohol); poly(N-vinyl lactams) such as polyvinyl
pyrrolidone), poly(N-vinyl caprolactam), and copolymers thereof;
polyoxazolines, including poly(methyloxazoline) and poly(ethyloxazoline); and
polyvinylamines. It must be emphasized that the aforementioned list of
polymers is not exhaustive, and a variety of other synthetic hydrophilic
polymers may be used, as will be appreciated by those skilled in the art.
The Crosslinkable Components:
The compositions of the invention also comprise a plurality of
crosslinkable components. Each of the crosslinkable components participates
in a reaction that results in a crosslinked matrix. Prior to completion of the
crosslinking reaction, the crosslinkable components provide the necessary
adhesive qualities that enable the methods of the invention.
The crosslinkable components are selected so that crosslinking
gives rise to a biocompatible, nonimmunogenic matrix useful in a variety of
contexts including adhesion prevention, biologically active agent delivery,
tissue
augmentation, and other applications. The crosslinkable components of the
180
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
invention comprise: a component A, which has m nucleophilic groups, wherein
m > 2 and a component B, which has n electrophilic groups capable of reaction
with the m nucleophilic groups, wherein n > 2 and m + n > 4. An optional third
component, optional component C, which has at least one functional group that
is either electrophilic and capable of reaction with the nucleophilic groups
of
component A, or nucleophilic and capable of reaction with the electrophilic
groups of component B may also be present. Thus, the total number of
functional groups present on components A, B and C, when present, in
combination is > 5; that is, the total functional groups given by m + n + p
must
be > 5, where p is the number of functional groups on component C and, as
indicated, is > 1. Each of the components is biocompatible and
nonimmunogenic, and at least one component is comprised of a hydrophilic
polymer. Also, as will be appreciated, the composition may contain additional
crosslinkable components D, E, F, etc., having one or more reactive
nucleophilic or electrophilic groups and thereby participate in formation of
the
crosslinked biomaterial via covalent bonding to other components.
The m nucleophilic groups on component A may all be the same,
or, alternatively, A may contain two or more different nucleophilic groups.
Similarly, the n electrophilic groups on component B may all be the same, or
two or more different electrophilic groups may be present. The functional
groups) on optional component C, if nucleophilic, may or may not be the same
as the nucleophilic groups on component A, and, conversely, if electrophilic,
the
functional groups) on optional component C may or may not be the same as
the electrophilic groups on component B.
Accordingly, the components may be represented by the
structural formulae
(I) R~(-[Q~]q-X)m (component A),
(II) R2(-[Q2]rY)" (component B), and
(III) R3(-[Q3]S Fn)P (optional component C),
wherein:
181
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
R~, R~ and R3 are independently selected from the group
consisting of C2 to C~4 hydrocarbyl, heteroatom-containing C~ to C~4
hydrocarbyl, hydrophilic polymers, and hydrophobic polymers, providing that at
least one of R~, R2 and R3 is a hydrophilic polymer, preferably a synthetic
hydrophilic polymer;
X represents one of the m nucleophilic groups of component A,
and the various X moieties on A may be the same or different;
Y represents one of the n electrophilic groups of component B,
and the various Y moieties on A may be the same or different;
Fn represents a functional group on optional component C;
Q~, Q2 and Q3 are linking groups;
m >2, n >2, m + n is >_4, q, and r are independently zero or 1,
and when optional component C is present, p >_1, and s is independently zero
or 1.
Reactive Groups:
X may be virtually any nucleophilic group, so long as reaction can
occur with the electrophilic group Y. Analogously, Y may be virtually any
electrophilic group, so long as reaction can take place with X. The only
limitation is a practical one, in that reaction between X and Y should be
fairly
rapid and take place automatically upon admixture with an aqueous medium,
without need for heat or potentially toxic or non-biodegradable reaction
catalysts or other chemical reagents. It is also preferred although not
essential
that reaction occur without need for ultraviolet or other radiation. Ideally,
the
reactions between X and Y should be complete in under 60 minutes, preferably
under 30 minutes. Most preferably, the reaction occurs in about 5 to 15
minutes or less.
Examples of nucleophilic groups suitable as X include, but are not
limited to, -NH2, -NHR~, -N(R4)2, -SH, -OH, -COOH, -C6H4-OH, -PH2, -PHRS, -
P(R5)2, -NH-NH2, -CO-NH-NH2, -C5H4N, etc. wherein R4 and R5 are
182
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
hydrocarbyl, typically alkyl or monocyclic aryl, preferably alkyl, and most
preferably lower alkyl. Organometallic moieties are also useful nucleophilic
groups for the purposes of the invention, particularly those that act as
carbanion
donors. Organometallic nucleophiles are not, however, preferred. Examples of
organometallic moieties include: Grignard functionalities -R6MgHal wherein R6
is a carbon atom (substituted or unsubstituted), and Hal is halo, typically
bromo,
iodo or chloro, preferably bromo; and lithium-containing functionalities,
typically
alkyllithium groups; sodium-containing functionalities.
It will be appreciated by those of ordinary skill in the art that
certain nucleophilic groups must be activated with a base so as to be capable
of reaction with an electrophile. For example, when there are nucleophilic
sulfhydryl and hydroxyl groups in the crosslinkable composition, the
composition must be admixed with an aqueous base in order to remove a
proton and provide an -S- or -O' species to enable reaction with an
electrophile.
Unless it is desirable for the base to participate in the crosslinking
reaction, a
nonnucleophilic base is preferred. In some embodiments, the base may be
present as a component of a buffer solution. Suitable bases and corresponding
crosslinking reactions are described infra in Section E.
The selection of electrophilic groups provided within the
crosslinkable composition, i.e., on component B, must be made so that reaction
is possible with the specific nucleophilic groups. Thus, when the X moieties
are
amino groups, the Y groups are selected so as to react with amino groups.
Analogously, when the X moieties are sulfhydryl moieties, the corresponding
electrophilic groups are sulfhydryl-reactive groups, and the like.
By way of example, when X is amino (generally although not
necessarily primary amino), the electrophilic groups present on Y are amino
reactive groups such as, but not limited to: (1 ) carboxylic acid esters,
including
cyclic esters and "activated" esters; (2) acid chloride groups (-CO-CI); (3)
anhydrides (-(CO)-O-(CO)-R); (4) ketones and aldehydes, including a,(3-
unsaturated aldehydes and ketones such as -CH=CH-CH=O and -CH=CH-
183
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
C(CH3)=O; (5) halides; (6) isocyanate (-N=C=O); (7) isothiocyanate (-N=C=S);
(8) epoxides; (9} activated hydroxyl groups (e.g., activated with conventional
activating agents such as carbonyldiimidazole or sulfonyl chloride); and (10)
olefins, including conjugated olefins, such as ethenesulfonyl (-S02CH=CH2)
and analogous functional groups, including acrylate (-C02-C=CH2),
methacrylate (-C02-C(CH3)=CH2)), ethyl acrylate (-C02-C(CH2CH3)=CH2), and
ethyleneimino (-CH=CH-C=NH). Since a carboxylic acid group per se is not
susceptible to reaction with a nucleophilic amine, components containing
carboxylic acid groups must be activated so as to be amine-reactive.
Activation
may be accomplished in a variety of ways, but often involves reaction with a
suitable hydroxyl-containing compound in the presence of a dehydrating agent
such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For
example, a carboxylic acid can be reacted with an alkoxy-substituted N-
hydroxy-succinimide or N-hydroxysulfosuccinimide in the presence of DCC to
form reactive electrophilic groups, the N-hydroxysuccinimide ester and the N-
hydroxysulfosuccinimide ester, respectively. Carboxylic acids may also be
activated by reaction with an acyl halide such as an acyl chloride (e.g.,
acetyl
chloride), to provide a reactive anhydride group. In a further example, a
carboxylic acid may be converted to an acid chloride group using, e.g.,
thionyl
chloride or an acyl chloride capable of an exchange reaction. Specific
reagents
and procedures used to carry out such activation reactions will be known to
those of ordinary skill in the art and are described in the pertinent texts
and
literature.
Analogously, when X is sulfhydryl, the electrophilic groups present
on Y are groups that react with a sulfhydryl moiety. Such reactive groups
include those that form thioester linkages upon reaction with a sulfhydryl
group,
such as those described in PCT Publication No. WO 00/62827 to Wallace et al.
As explained in detail therein, such "sulfhydryl reactive" groups include, but
are
not limited to: mixed anhydrides; ester derivatives of phosphorus; ester
derivatives of p-nitrophenol, p-nitrothiophenol and pentafluorophenol; esters
of
184
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
substituted hydroxylamines, including N-hydroxyphthalimide esters, N-
hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, and N-
hydroxygfutarimide esters; esters of 1-hydroxybenzotriazole; 3-hydroxy-3,4-
dihydro-benzotriazin-4-one; 3-hydroxy-3,4-dihydro-quinazoline-4-one;
carbonylimidazole derivatives; acid chlorides; ketenes; and isocyanates. With
these sulfhydryl reactive groups, auxiliary reagents can also be used to
facilitate bond formation, e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
can be used to facilitate coupling of sulfhydryl groups to carboxyl-containing
groups.
In addition to the sulfhydryl reactive groups that form thioester
linkages, various other sulfhydryl reactive functionalities can be utilized
that
form other types of linkages. For example, compounds that contain methyl
imidate derivatives form imido-thioester linkages with sulfhydryl groups.
Alternatively, sulfhydryl reactive groups can be employed that form disulfide
bonds with sulfhydryl groups; such groups generally have the structure -S-S-Ar
where Ar is a substituted or unsubstituted nitrogen-containing heteroaromatic
moiety or a non-heterocyclic aromatic group substituted with an electron-
withdrawing moiety, such that Ar may be, for example, 4-pyridinyl, o-
nitrophenyl, m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-
benzoic
acid, 2-nitro-4-pyridinyl, etc. In such instances, auxiliary reagents, i.e.,
mild
oxidizing agents such as hydrogen peroxide, can be used to facilitate
disulfide
bond formation.
Yet another class of sulfhydryl reactive groups forms thioether
bonds with sulfhydryl groups. Such groups include, inter alia, maleimido,
substituted maleimido, haloalkyl, epoxy, imino, and aziridino, as well as
olefins
(including conjugated olefins) such as ethenesulfonyl, etheneimino, acrylate,
methacrylate, and a,(3-unsaturated aldehydes and ketones. This class of
sulfhydryl reactive groups are particularly preferred as the thioether bonds
may
provide faster crosslinking and longer in vivo stability.
185
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
When X is -OH, the electrophilic functional groups on the
remaining components) must react with hydroxyl groups. The hydroxyl group
may be activated as described above with respect to carboxylic acid groups, or
it may react directly in the presence of base with a sufficiently reactive
electrophile such as an epoxide group, an aziridine group, an acyl halide, or
an
anhydride.
When X is an organometallic nucleophile such as a Grignard
functionality or an alkyllithium group, suitable electrophilic functional
groups for
reaction therewith are those containing carbonyl groups, including, by way of
example, ketones and aldehydes.
It will also be appreciated that certain functional groups can react
as nucleophiles or as electrophiles, depending on the selected reaction
partner
and/or the reaction conditions. For example, a carboxylic acid group can act
as
a nucleophile in the presence of a fairly strong base, but generally acts as
an
electrophile allowing nucleophilic attack at the carbonyl carbon and
concomitant
replacement of the hydroxyl group with the incoming nucleophile.
The covalent linkages in the crosslinked structure that result upon
covalent binding of specific nucleophilic components to specific electrophilic
components in the crosslinkable composition include, solely by way of example,
the following (the optional linking groups Q~ and Q~ are omitted for clarity):
TABLE
REPRESENTATIVE
NUCLEOPHILIC REPRESENTATIVE
COMPONENT ELECTROPHILIC
COMPONENT RESULTING LINI~CAGE
(A, optional
component C (B, FNE~)
element FNNU)
R~-NH2 R2-O-(CO)-O-N(COCH2) R~-NH-(CO)-O-R2
(succinimidyl carbonate
terminus)
1~6
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
REPRESENTATIVE
NUCLEOPHILIC REPRESENTATIVE
COMPONENT ELECTROPHILIC
(A, optional COMPONENT RESULTING LINKAGE
component C (B, FNE~)
element FNNU)
R~-SH R2-O-(CO)-O-N(COCH2) R~-S-(CO)-O-R~
R~-OH R~-O-(CO)-O-N(COCH~) R~-O-(CO)-R2
R~-NH2 R2-O(CO)-CH=CH2 R~-NH-CH~CH~-(CO)-O-R2
(acrylate terminus)
R~-SH R2-O-(CO)-CH=CH2 R~-S-CH2CH2-(CO)-O-R2
R~-OH R2-O-(CO)-CH=CH2 R~-O-CH2CH2-(CO)-O-R~
R~-NH2 R2-O(CO)-(CH2)3-CO~- R~-NH-(CO)-(CH2)3-(CO)-
N(COCH2) ORS
(succinimidyl glutarate
terminus)
R~-SH R2-O(CO)-(CH2)3-C02- R~-S-(CO)-(CH~)3-(CO)-
N(COCH2) OR2
R~-OH R2-O(CO)-(CH2)3-C02- R~-O-(CO)-(CH2)3-(CO)-
N(COCH2) OR2
R~-NH2 R2-O-CH2-C02-N(COCH~) R'-NH-(CO)-CH2-OR2
(succinimidyl acetate
terminus)
R~-SH R2-O-CH2-CO2-N(COCH2) R~-S-(CO)-CH2-OR2
R~-OH R2-O-CH2-CO2-N(COCH2) R~-O-(CO)-CH2-OR2
R~-NH2 R2-O-NH(CO)-(CH2)2-C02-R~-NH-~CO)-(CH2)2-(CO)-
N(COCH2) NH-OR
(succinimidyl succinamide
terminus)
R~-SH R2-O-NH(CO)-(CH2)2-C02-R~-S-(CO)-(CH2)2-(CO)-
N(COCH2) NH-OR2
R~-OH R2-O-NH(CO)-(CH2)2-C02-R~-O-(CO)-(CH2)2-(CO)-
N(COCH2) NH-OR2
R~-NH2 R2-O- (CH2)2-CHO R~-NH-(CO)-(CH2)2-OR2
(propionaldehyde terminus)
187
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
REPRESENTATIVE
NUCLEOPHILIC REPRESENTATIVE
COMPONENT ELECTROPHILIC
RESULTING LINKAGE
(A, optional COMPONENT
component C (B, FNE~)
element FNNU)
R~-NH2 j ~ R~-NH-CH2-CH(OH)-CH~-
2 OR and
R -O-CH2=CH-CH2
R~-N[CH2-CH(OH)-CH2-
(glycidyl ether terminus)~R~12
R~-NH2 R2-O-(CH2)~-N=C=O R~-NH-(CO)-NH-CH2-OR2
(isocyanate terminus)
R~-NH2 R~-NH-CH2CH2-S02-R~
R2-S02-CH=CH2
(vinyl sulfone terminus)
R~-SH R~-S02-CH=CH2 R~-S-CH~CH~-S02-R2
Linking Groups:
The functional groups X and Y and FN on optional component C
may be directly attached to the compound core (R~, R2 or R3 on optional
component C, respectively), or they may be indirectly attached through a
linking
group, with longer linking groups also termed "chain extenders." In structural
formulae (I), (II) and (III), the optional linking groups are represented by
Q~, Q2
and Q3, wherein the linking groups are present when q, r and s are equal to 1
(with R, X, Y, Fn, m n and p as defined previously).
Suitable linking groups are well known in the art. See, for
example, International Patent Publication No. WO 97/22371. Linking groups
are useful to avoid steric hindrance problems that are sometimes associated
with the formation of direct linkages between molecules. Linking groups may
additionally be used to link several multifunctionally activated compounds
together to make larger molecules. In a preferred embodiment, a linking group
can be used to alter the degradative properties of the compositions after
188
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
administration and resultant gel formation. For example, linking groups can be
incorporated into components A, B, or optional component C to promote
hydrolysis, to discourage hydrolysis, or to provide a site for enzymatic
degradation.
Examples of linking groups that provide hydrolyzable sites,
include, inter alias ester linkages; anhydride linkages, such as obtained by
incorporation of glutarate and succinate; ortho ester linkages; ortho
carbonate
linkages such as trimethylene carbonate; amide linkages; phosphoester
linkages; a-hydroxy acid linkages, such as may be obtained by incorporation of
lactic acid and glycolic acid; lactone-based linkages, such as may be obtained
by incorporation of caprolactone, valerolactone, y-butyrolactone and p-
dioxanone; and amide linkages such as in a dimeric, oligomeric, or poly(amino
acid) segment. Examples of non-degradable linking groups include
succinimide, propionic acid and carboxymethylate linkages. See, for example,
PCT WO 99/07417. Examples of enzymatically degradable linkages include
Leu-Gly-Pro-Ala, which is degraded by collagenase; and Gly-Pro-Lys, which is
degraded by plasmin.
Linking groups can also enhance or suppress the reactivity of the
various nucleophilic and electrophilic groups. For example, electron-
withdrawing groups within one or two carbons of a sulfhydryl group would be
expected to diminish its effectiveness in coupling, due to a lowering of
nucleophilicity. Carbon-carbon double bonds and carbonyl groups will also
have such an effect. Conversely, electron-withdrawing groups adjacent to a
carbonyl group (e.g., the reactive carbonyl of glutaryl-N-hydroxysuccinimidyl)
would increase the reactivity of the carbonyl carbon with respect to an
incoming
nucleophile. By contrast, sterically bulky groups in the vicinity of a
functional
group can be used to diminish reactivity and thus coupling rate as a result of
steric hindrance.
By way of example, particular linking groups and corresponding
component structure are indicated in the following Table:
189
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
TABLE
LINKING GROUP COMPONENT STRUCTURE
-O-(CH2)n- Component A: R~-O-(CH2)n-X
Component B: R~-O-(CH2)n Y
Optional Component C: R3-O-(CH2)n-Z
-S-(CH2)n Component A: R~-S-(CH2)n X
Component B: R2-S-(CH2)~ Y
Optional Component C: R3-S-(CH~)n Z
-NH-(CH2)~ Component A: R~-NH-(CH2)n-X
Component B: R2-NH-(CH2)n-Y
Optional Component C: R3-NH-(CH~)n-Z
-O-(CO)-NH-(CH2)n- Component A: 'R~-O-(CO)-NH-(CH2)n X
Component B: R2-O-(CO)-NH-(CH2)~ Y
Optional Component C: R3-O-(CO)-NH-(CH2)n-Z
-NH-(CO)-O-(CH2)n- Component A: R~-NH-(CO)-O-(CH2)n X
Component B: R~-NH-(CO)-O-(CH2)n-Y
Optional Component C: R3-NH-(CO)-O-(CH2)n-Z
-O-(CO)-(CH2)n- Component A: R~-O-(CO)-(CH2)n-X
Component B: R2-O-(CO)-(CH2)n Y
Optional Component C: R3-O-(CO)-(CH2)n-Z
-(CO)-O-(CH2)n Component A: R~-(CO)-O-(CH2)n-X
Component B: R2-(CO)-O-(CH2)"Y
Optional Component C: R3-(CO)-O-(CH2)n
Z
-O-(CO)-O-(CHZ)~ Component A: R~-O-(CO)-O-(CH2)n X
Component B: R2-O-(CO)-O-(CH2)n Y
Optional Component C: R3-O-(CO)-O-(CH2)n-Z
-O-(CO)-CHR~- Component A: R~-O-(CO)-CHR~-X
Component B: R2-O-(CO)-CHR~-Y
Optional Component C: R3-O-(CO)-CHR7-Z
-O-R$-(CO)-NH- Component A: R~-O-R$-(CO)-NH-X
Component B: R2- O-R$-(CO)-NH-Y
Optional Component C: R3- O-R$-(CO)-NH-Z
190
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In the above Table, n is generally in the range of 1 to about 10, R'
is generally hydrocarbyl, typically alkyl or aryl, preferably alkyl, and most
preferably lower alkyl, and R$ is hydrocarbylene, heteroatom-containing
hydrocarbylene, substituted hydrocarbylene, or substituted heteroatom-
containing hydrocarbylene) typically alkylene or arylene (again, optionally
substituted and/or containing a heteroatom), preferably lower alkylene (e.g.,
methylene, ethylene, n-propylene, n-butylene, etc.), phenylene, or
amidoalkylene (e.g., -(CO)-NH-CH2).
Other general principles that should be considered with respect to
linking groups are as follows: If higher molecular weight components are to be
used, they preferably have biodegradable linkages as described above, so that
fragments larger than 20,000 mol. wt. are not generated during resorption in
the
body. In addition, to promote water miscibility and/or solubility, it may be
desired to add sufficient electric charge or hydrophilicity. Hydrophilic
groups
can be easily introduced using known chemical synthesis, so long as they do
not give rise to unwanted swelling or an undesirable decrease in compressive
strength. In particular, polyalkoxy segments may weaken gel strength.
The Component Core:
The "core" of each crosslinkable component is comprised of the
molecular structure to which the nucleophilic or electrophilic groups are
bound.
Using the formulae (I) R~-[Q~]q-X)m, for component A, (II) R2(-[Q2]r-Y)" for
component B, and (III)
R3(-[Q3]S Fn)p for optional component C, the "core" groups are R~,
R2 and R3. Each molecular core of the reactive components of the
crosslinkable composition is generally selected from synthetic and naturally
occurring hydrophilic polymers, hydrophobic polymers, and C2-C~4 hydrocarbyl
groups zero to 2 heteroatoms selected from N, O and S, with the proviso that
at
least one of the crosslinkable components A, B, and optionally C, comprises a
191
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
molecular core of a synthetic hydrophilic polymer. In a preferred embodiment,
at least one of A and B comprises a molecular core of a synthetic hydrophilic
polymer.
Hydrophilic Crosslinkable Components
In one aspect, the crosslinkable components) is (are) hydrophilic
polymers. The term "hydrophilic polymer" as used herein refers to a synthetic
polymer having an average molecular weight and composition effective to
render the polymer "hydrophilic" as defined above. As discussed above,
synthetic crosslinkable hydrophilic polymers useful herein include, but are
not
limited to: polyalkylene oxides, particularly polyethylene glycol and
polyethylene oxide)-polypropylene oxide) copolymers, including block and
random copolymers; polyols such as glycerol, polyglycerol (particularly highly
branched polyglycerol), propylene glycol and trimethylene glycol substituted
with one or more polyalkylene oxides, e.g., mono-, di- and tri-
polyoxyethylated
glycerol, mono- and di-polyoxyethylated propylene glycol, and mono- and di-
polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,
polyoxyethylated glucose; acrylic acid polymers and analogs and copolymers
thereof, such as polyacrylic acid per se, polymethacrylic acid,
poly(hydroxyethyl-methacrylate), poly(hydroxyethylacrylate),
poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxide acrylate)
and
copolymers of any of the foregoing, and/or with additional acrylate species
such
as aminoethyl acrylate and mono-2-(acryloxy)-ethyl succinate; polymaleic acid;
poly(acrylamides) such as polyacrylamide per se, poly(methacrylamide),
poly(dimethylacrylamide), and poly(N-isopropyl-acrylamide); poly(olefinic
alcohol)s such as polyvinyl alcohol); poly(N-vinyl lactams) such as polyvinyl
pyrrolidone), poly(N-vinyl caprolactam), and copolymers thereof;
polyoxazolines, including poly(methyloxazoline) and poly(ethyloxazoline); and
polyvinylamines. It must be emphasized that the aforementioned list of
192
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polymers is not exhaustive, and a variety of other synthetic hydrophilic
polymers may be used, as will be appreciated by those skilled in the art.
The synthetic crosslinkable hydrophilic polymer may be a
homopolymer, a block copolymer, a random copolymer, or a graft copolymer.
In addition, the polymer may be linear or branched, and if branched, may be
minimally to highly branched, dendrimeric, hyperbranched, or a star polymer.
The polymer may include biodegradable segments and blocks, either
distributed throughout the polymer's molecular structure or present as a
single
block, as in a block copolymer. Biodegradable segments are those that
degrade so as to break covalent bonds. Typically, biodegradable segments are
segments that are hydrolyzed in the presence of water and/or enzymatically
cleaved in situ. Biodegradable segments may be composed of small molecular
segments such as ester linkages, anhydride linkages, ortho ester linkages,
ortho carbonate linkages, amide linkages, phosphonate linkages, etc. Larger
biodegradable "blocks" will generally be composed of oligomeric or polymeric
segments incorporated within the hydrophilic polymer. Illustrative oligomeric
and polymeric segments that are biodegradable include, by way of example,
poly(amino acid) segments, poly(orthoester) segments, poly(orthocarbonate)
segments, and the like.
Other suitable synthetic crosslinkable hydrophilic polymers
include chemically synthesized polypeptides, particularly polynucleophilic
polypeptides that have been synthesized to incorporate amino acids containing
primary amino groups (such as lysine) and/or amino acids containing thiol
groups (such as cysteine). Poly(lysine), a synthetically produced polymer of
the
amino acid lysine (145 MW), is particularly preferred. Poly(lysine)s have been
prepared having anywhere from 6 to about 4,000 primary amino groups,
corresponding to molecular weights of about 870 to about 580,000.
Poly(lysine)s for use in the present invention preferably have a molecular
weight within the range of about 1,000 to about 300,000, more preferably
within
the range of about 5,000 to about 100,000, and most preferably, within the
193
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
range of about 8,000 to about 15,000. Poly(lysine)s of varying molecular
weights are commercially available from Peninsula Laboratories, Inc. (Belmont,
Calif.).
The synthetic crosslinkable hydrophilic polymer may be a
homopolymer, a block copolymer, a random copolymer, or a graft copolymer.
In addition, the polymer may be linear or branched, and if branched, may be
minimally to highly branched, dendrimeric, hyperbranched, or a star polymer.
The polymer may include biodegradable segments and blocks, either
distributed throughout the polymer's molecular structure or present as a
single
block, as in a block copolymer. Biodegradable segments are those that
degrade so as to break covalent bonds. Typically, biodegradable segments are
segments that are hydrolyzed in the presence of water and/or enzymatically
cleaved in situ. Biodegradable segments may be composed of small molecular
segments such as ester linkages, anhydride linkages, ortho ester linkages,
ortho carbonate linkages, amide linkages, phosphonate linkages, etc. Larger
biodegradable "blocks" will generally be composed of oligomeric or polymeric
segments incorporated within the hydrophilic polymer. Illustrative oligomeric
and polymeric segments that are biodegradable include, by way of example,
poly(amino acid) segments, poly(orthoester) segments, poly(orthocarbonate)
segments, and the like.
Although a variety of different synthetic crosslinkable hydrophilic
polymers can be used in the present compositions, as indicated above,
preferred synthetic crosslinkable hydrophilic polymers are polyethylene glycol
(PEG) and polyglycerol (PG), particularly highly branched polyglycerol.
Various
forms of PEG are extensively used in the modification of biologically active
molecules because PEG lacks toxicity, antigenicity, and immunogenicity (i.e.,
is
biocompatible), can be formulated so as to have a wide range of solubilities,
and do not typically interfere with the enzymatic activities and/or
conformations
of peptides. A particularly preferred synthetic crosslinkable hydrophilic
polymer
for certain applications is a polyethylene glycol (PEG) having a molecular
194
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
weight within the range of about 100 to about 100,000 mol. wt., although for
highly branched PEG, far higher molecular weight polymers can be employed --
up to 1,000,000 or more -- providing that biodegradable sites are incorporated
ensuring that all degradation products will have a molecular weight of less
than
about 30,000. For most PEGs, however, the preferred molecular weight is
about 1,000 to about 20,000 mol. wt., more preferably within the range of
about
7,500 to about 20,000 mol. wt. Most preferably, the polyethylene glycol has a
molecular weight of approximately 10,000 mol. wt.
Naturally occurring crosslinkable hydrophilic polymers include, but
are not limited to: proteins such as collagen, fibronectin, albumins,
globulins,
fibrinogen, and fibrin, with collagen particularly preferred; carboxylated
polysaccharides such as polymannuronic acid and polygalacturonic acid;
aminated polysaccharides, particularly the glycosaminoglycans, e.g.,
hyaluronic
acid, chitin, chondroitin sulfate A, B, or C, keratin sulfate, keratosulfate
and
heparin; and activated polysaccharides such as dextran and starch derivatives.
Collagen and glycosaminoglycans are examples of naturally occurring
hydrophilic polymers for use herein, with methylated collagen being a
preferred
hydrophilic polymer.
Any of the hydrophilic polymers herein must contain, or be
activated to contain, functional groups, i.e., nucleophilic or electrophilic
groups,
which enable crosslinking. Activation of PEG is discussed below; it is to be
understood, however, that the following discussion is for purposes of
illustration
and analogous techniques may be employed with other polymers.
With respect to PEG, first of all, various functionalized
polyethylene glycols have been used effectively in fields such as protein
modification (see Abuchowski et al., Enzymes as Drugs, John Wiley & Sons:
New York, N.Y. (1981 ) pp. 367-383; and Dreborg et al., Crit. Rev. Therap.
Drug
Carrier Syst. (1990) 6:315), peptide chemistry (see Mutter et al., The
Peptides,
Academic: New York, N.Y. 2:285-332; and Zalipsky et al., Int. J. Peptide
Protein
Res. (1987) 30:740), and the synthesis of polymeric drugs (see Zalipsky et
al.,
195
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Eur. Polym. J. (1983) 19:1177; and Ouchi et al., J. Macromol. Sci. Chem.
(1987) A24:1011 ).
Activated forms of PEG, including multifunctionally activated PEG,
are commercially available, and are also easily prepared using known methods.
For example, see Chapter 22 of Polyethylene Glycol) Chemistry: Biotechnical
and Biomedical Applications, J. Milton Harris, ed., Plenum Press, NY (1992);
and Shearwater Polymers, Inc. Catalog, Polyethylene Glycol Derivatives,
Huntsville, Alabama (1997-1998).
Structures for some specific, tetrafunctionally activated forms of
PEG are shown in FIGS. 1 to 10 of U.S. Patent 5,874,500, as are generalized
reaction products obtained by reacting the activated PEGs with multi-amino
PEGs, i.e., a PEG with two or more primary amino groups. The activated PEGs
illustrated have a pentaerythritol (2,2-bis(hydroxymethyl)-1,3-propanediol)
core.
Such activated PEGs, as will be appreciated by those in the art, are readily
prepared by conversion of the exposed hydroxyl groups in the PEGylated polyol
(i.e., the terminal hydroxyl groups on the PEG chains) to carboxylic acid
groups
(typically by reaction with an anhydride in the presence of a nitrogenous
base),
followed by esterification with N-hydroxysuccinimide, N-
hydroxysulfosuccinimide, or the like, to give the polyfunctionally activated
PEG.
Hydrophobic Polymers:
The crosslinkable compositions of the invention can also include
hydrophobic polymers, although for most uses hydrophilic polymers are
preferred. Polylactic acid and polyglycolic acid are examples of two
hydrophobic polymers that can be used. With other hydrophobic polymers, only
short-chain oligomers should be used, containing at most about 14 carbon
atoms, to avoid solubility-related problems during reaction.
196
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Low Molecular Weight Components:
As indicated above, the molecular core of one or more of the
crosslinkable components can also be a low molecular weight compound, i.e., a
C2-C~4 hydrocarbyl group containing zero to 2 heteroatoms selected from N, O,
S and combinations thereof. Such a molecular core can be substituted with
nucleophilic groups or with electrophilic groups.
When the low molecular weight molecular core is substituted with
primary amino groups, the component may be, for example, ethylenediamine
(H2N-CH2CH~-NH2), tetramethylenediamine (H2N-(CH4)-NHS),
pentamethylenediamine (cadaverine) (H2N-(CH5)-NH2), hexamethylenediamine
(H2N-(CH6)-NH2), bis(2-aminoethyl)amine (HN-[CH2CH2-NH2]2), or tris(2-
aminoethyl)amine (N-[CH2CH2-NH~~3).
Low molecular weight diols and polyols include
trimethylolpropane, di(trimethylol propane), pentaerythritol, and diglycerol,
all of
which require activation with a base in order to facilitate their reaction as
nucleophiles. Such diols and polyols may also be functionalized to provide di-
and poly-carboxylic acids, functional groups that are, as noted earlier
herein,
also useful as nucleophiles under certain conditions. Polyacids for use in the
present compositions include, without limitation, trimethylolpropane-based
tricarboxylic acid, di(trimethylol propane)-based tetracarboxylic acid,
heptanedioic acid, octanedioic acid (suberic acid), and hexadecanedioic acid
(thapsic acid), all of which are commercially available and/or readily
synthesized using known techniques.
Low molecular weight di- and poly-electrophiles include, for
example, disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3),
dithiobis(succinimidylpropionate) (DSP), bis(2-succinimidooxycarbonyloxy)
ethyl sulfone (BSOCOES), and 3,3'-dithiobis(sulfosuccinimidylpropionate
(DTSPP), and their analogs and derivatives. The aforementioned compounds
are commercially available from Pierce (Rockford, IIL). Such di- and poly-
electrophiles can also be synthesized from di- and polyacids, for example by
197
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
reaction with an appropriate molar amount of N-hydroxysuccinimide in the
presence of DCC. Polyols such as trimethylolpropane and di(trimethylol
propane) can be converted to carboxylic acid form using various known
techniques, then further derivatized by reaction with NHS in the presence of
DCC to produce trifunctionally and tetrafunctionally activated polymers.
Delivery Systems:
Suitable delivery systems for the homogeneous dry powder
composition (containing at least two crosslinkable polymers) and the two
buffer
solutions may involve a multi-compartment spray device, where one or more
compartments contains the powder and one or more compartments contain the
buffer solutions needed to provide for the aqueous environment, so that the
composition is exposed to the aqueous environment as it leaves the
compartment. Many devices that are adapted for delivery of multi-component
tissue sealants/hemostatic agents are well known in the art and can also be
used in the practice of the present invention. Alternatively, the composition
can
be delivered using any type of controllable extrusion system, or it can be
delivered manually in the form of a dry powder, and exposed to the aqueous
environment at the site of administration.
The homogeneous dry powder composition and the two buffer
solutions may be conveniently formed under aseptic conditions by placing each
of the three ingredients (dry powder, acidic buffer solution and basic buffer
solution) into separate syringe barrels. For example, the composition, first
buffer solution and second buffer solution can be housed separately in a
multiple-compartment syringe system having a multiple barrels, a mixing head,
and an exit orifice. The first buffer solution can be added to the barrel
housing
the composition to dissolve the composition and form a homogeneous solution,
which is then extruded into the mixing head. The second buffer solution can be
simultaneously extruded into the mixing head. Finally, the resulting
composition can then be extruded through the orifice onto a surface.
198
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
For example, the syringe barrels holding the dry powder and.the
basic buffer may be part of a dual-syringe system, e.g., a double barrel
syringe
as described in U.S. Patent 4,359,049 to Redl et al. In this embodiment, the
acid buffer can be added to the syringe barrel that also holds the dry powder,
so as to produce the homogeneous solution. In other words, the acid buffer
may be added (e.g., injected) into the syringe barrel holding the dry powder
to
thereby produce a homogeneous solution of the first and second components.
This homogeneous solution can then be extruded into a mixing head, while the
basic buffer is simultaneously extruded into the mixing head. Within the
mixing
head, the homogeneous solution and the basic buffer are mixed together to
thereby form a reactive mixture. Thereafter, the reactive mixture is extruded
through an orifice and onto a surface (e.g., tissue), where a film is formed,
which can function as a sealant or a barrier, or the like. The reactive
mixture
begins forming a three-dimensional matrix immediately upon being formed by
the mixing of the homogeneous solution and the basic buffer in the mixing
head. Accordingly, the reactive mixture is preferably extruded from the mixing
head onto the tissue very quickly after it is formed so that the three-
dimensional
matrix forms on, and is able to adhere to, the tissue.
Other systems for combining two reactive liquids are well known
in the art, and include the systems described in U.S. Patent Nos. 6,454,786 to
Holm et al.; 6,461,325 to Delmotte et al.; 5,585,007 to Antanavich et al.;
5,116,315 to Capozzi et al.; and 4,631,055 to Redl et al.
Storage and Handling:
Because crosslinkable components containing electrophilic
groups react with water, the electrophilic component or components are
generally stored and used in sterile, dry form to prevent hydrolysis.
Processes
for preparing synthetic hydrophilic polymers containing multiple electrophilic
groups in sterile, dry form are set forth in commonly assigned U.S. Patent No.
5,643,464 to Rhee et al. For example, the dry synthetic polymer may be
199
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
compression molded into a thin sheet or membrane, which can then be
sterilized using gamma or, preferably, e-beam irradiation. The resulting dry
membrane or sheet can be cut to the desired size or chopped into smaller size
particulates.
Components containing multiple nucleophilic groups are generally
not water-reactive and can therefore be stored either dry or in aqueous
solution.
If stored as a dry, particulate, solid, the various components of the
crosslinkable
composition may be blended and stored in a single container. Admixture of all
components with water, saline, or other aqueous media should not occur until
immediately prior to use.
In an alternative embodiment, the crosslinking components can
be mixed together in a single aqueous medium in which they are both
unreactive, i.e., such as in a low pH buffer. Thereafter, they can be sprayed
onto the targeted tissue site along with a high pH buffer, after which they
will
rapidly react and form a gel.
Suitable liquid media for storage of crosslinleable compositions
include aqueous buffer solutions such as monobasic sodium phosphateldibasic
sodium phosphate, sodium carbonatelsodium bicarbonate, glutamate or
acetate, at a concentration of 0.5 to 300 mM. In general, a sulfhydryl-
reactive
component such as PEG substituted with maleimido groups or succinimidyl
esters is prepared in water or a dilute buffer, with a pH of between around 5
to
6. Buffers with pKs between about 8 and 10.5 for preparing a polysulfhydryl
component such as sulfhydryl-PEG are useful to achieve fast gelation time of
compositions containing mixtures of sulfhydryl-PEG and SG-PEG. These
include carbonate, borate and AMPSO (3-[(1,1-dimethyl-2-
hydroxyethyl)amino]2-hydroxy-propane-sulfonic acid). In contrast, using a
combination of maleimidyl PEG and sulfhydryl-PEG, a pH of around 5 to 9 is
preferred for the liquid medium used to prepare the sulfhydryl PEG.
200
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Collagen + Fibrinogen and/or Thrombin (e.e~., Costasis)
In yet another aspect, the polymer composition may include
collagen in combination with fibrinogen and/or thrombin. (See, e.g., U.S.
Patent
Nos. 5,290,552; 6,096,309; and 5,997,811 ). For example, an aqueous
composition may include a fibrinogen and FXIII, particularly plasma, collagen
in
an amount sufficient to thicken the composition, thrombin in an amount
sufficient to catalyze polymerization of fibrinogen present in the
composition,
and Ca2+ and, optionally, an antifibrinolytic agent in amount sufficient to
retard
degradation of the resulting adhesive clot. The composition may be formulated
as a two-part composition that may be mixed together just prior to use, in
which
fibrinogen/FXIII and collagen constitute the first component, and thrombin
together with an antifibrinolytic agent, and Ca2+ constitute the second
component.
Plasma, which provides a source of fibrinogen, may be obtained
from the patient for which the composition is to be delivered. The plasma can
be used "as is" after standard preparation which includes centrifuging out
cellular components of blood. Alternatively, the plasma can be further
processed to concentrate the fibrinogen to prepare a plasma cryoprecipitate.
The plasma cryoprecipitate can be prepared by freezing the plasma for at least
about an hour at about -20 °C., and then storing the frozen plasma
overnight at
about 4 °C. to slowly thaw. The thawed plasma is centrifuged and the
plasma
cryoprecipitate is harvested by removing approximately four-fifths of the
plasma
to provide a cryoprecipitate comprising the remaining one-fifth of the plasma.
Other fibrinogen/FXIII preparations may be used, such as cryoprecipitate,
patient autologous fibrin sealant, fibrinogen analogs or other single donor or
commercial fibrin sealant materials. Approximately 0.5 ml to about 1.0 ml of
either the plasma or the plasma-cryoprecipitate provides about 1 to 2 ml of
adhesive composition which is sufficient for use in middle ear surgery. Other
plasma proteins (e.g., albumin, plasminogen, von Willebrands factor, Factor
201
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
VIII, etc.) may or may not be present in the fibrinogen/FXII separation due to
wide variations in the formulations and methods to derive them.
Collagen, preferably hypoallergenic collagen, is present in the
composition in an amount sufficient to thicken the composition and augment the
cohesive properties of the preparation. The collagen may be atelopeptide
collagen or telopeptide collagen, e.g., native collagen. In addition to
thickening
the composition, the collagen augments the fibrin by acting as a
macromolecular lattice work or scaffold to which the fibrin network adsorbs.
This gives more strength and durability to the resulting glue clot with a
relatively
low concentration of fibrinogen in comparison to the various concentrated
autogenous fibrinogen glue formulations (i.e., AFGs).
The form of collagen which is employed may be described as at
least "near native" in its structural characteristics. It may be further
characterized as resulting in insoluble fibers at a pH above 5; unless
crosslinked or as part of a complex composition, e.g., bone, it will generally
consist of a minor amount by weight of fibers with diameters greater than 50
nm, usually from about 1 to 25 volume % and there will be substantially
little, if
any, change in the helical structure of the fibrils. In addition, the collagen
composition must be able to enhance gelation in the surgical adhesion
composition.
A number of commercially available collagen preparations may be
used. ZYDERM Collagen Implant (ZCI) has a fibrillar diameter distribution
consisting of 5 to 10 nm diameter fibers at 90% volume content and the
remaining 10% with greater than about 50 nm diameter fibers. ZCI is available
as a fibrillar slurry and solution in phosphate buffered isotonic saline, pH
7.2,
and is injectable with fine gauge needles. As distinct from ZCI, cross-linked
collagen available as ZYPLAST may be employed. ZYPLAST is essentially an
exogenously crosslinked (glutaraldehyde) version of ZCI. The material has a
somewhat higher content of greater than about 50 nm diameter fibrils and
202
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
remains insoluble over a wide pH range. Crosslinking has the effect of
mimicking in vivo endogenous crosslinking found in many tissues.
Thrombin acts as a catalyst for fibrinogen to provide fibrin, an
insoluble polymer and is present in the composition in an amount sufficient to
catalyze polymerization of fibrinogen present in the patient plasma. Thrombin
also activates FXIII, a plasma protein that catalyzes covalent crosslinks in
fibrin,
rendering the resultant clot insoluble. Usually the thrombin is present in the
adhesive composition in concentration of from about 0.01 to about 1000 or
greater NIH units (NIHu) of activity, usually about i to about 500 NIHu, most
usually about 200 to about 500 NIHu. The thrombin can be from a variety of
host animal sources, conveniently bovine. Thrombin is commercially available
from a variety of sources including Parke-Davis, usually lyophilized with
buffer
salts and stabilizers in vials which provide thrombin activity ranging from
about
1000 NIHu to 10,000 NIHu. The thrombin is usually prepared by reconstituting
the powder by the addition of either sterile distilled water or isotonic
saline.
Alternately, thrombin analogs or reptile-sourced coagulants may be used.
The composition may additionally comprise an effective amount of
an antifibrinolytic agent to enhance the integrity of the glue clot as the
healing
processes occur. A number of antifibrinolytic agents are well known and
include aprotinin, C1-esterase inhibitor and s-amino-n-caproic acid (EACA). s-
amino-n-caproic acid, the only antifibrinolytic agent approved by the FDA, is
effective at a concentration of from about ~ mg/ml to about 40 mg/ml of the
final
adhesive composition, more usually from about 20 to about 30 mg/ml. EACA is
commercially available as a solution having a concentration of about 250
mg/ml. Conveniently, the commercial solution is diluted with distilled water
to
provide a solution of the desired concentration. That solution is desirably
used
to reconstitute lyophilized thrombin to the desired thrombin concentration.
Other examples of in situ forming materials based on the
crosslinking of proteins are described, e.g., in U.S. Patent Nos. RE38158;
4,839,345; 5,514,379, 5,583,114; 6,458,147; 6,371,975; 5,290,552; 6,096,309;
203
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
U.S. Patent Application Publication Nos. 2002/0161399; 2001 /0018598 and
PCT Publication Nos. WO 03/090683; WO 01/45761; WO 99/66964 and WO
96/03159). '
Self-Reactive Compounds
In one aspect, the therapeutic agent is released from a
crosslinked matrix formed, at least in part, from a self-reactive compound. As
used herein, a self-reactive compound comprises a core substituted with a
minimum of three reactive groups. The reactive groups may be directed
attached to the core of the compound, or the reactive groups may be indirectly
attached to the compound's core, e.g., the reactive groups are joined to the
core through one or more linking groups.
Each of the three reactive groups that are necessarily present in a
self-reactive compound can undergo a bond-forming reaction with at least one
of the remaining two reactive groups. For clarity it is mentioned that when
these compounds react to form a crosslinked matrix, it will most often happen
that reactive groups on one compound will reactive with reactive groups on
another compound. That is, the term "self-reactive" is not intended to mean
that each self-reactive compound necessarily reacts with itself, but rather
that
when a plurality of identical self-reactive compounds are in combination and
undergo a crosslinking reaction, then these compounds will react with one
another to form the matrix. The compounds are "self-reactive" in the sense
that
they can react with other compounds having the identical chemical structure as
themselves.
The self-reactive compound comprises at least four components:
a core and three reactive groups. In one embodiment, the self-reactive
compound can be characterized by the formula (I), where R is the core, the
reactive groups are represented by X~, X2 and X3, and a linker (L) is
optionally
present between the core and a functional group.
204
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
X2
(L2)q
(1 )
X1-(L1)p R-(Ls)r Xs
The core R is a polyvalent moiety having attachment to at least
three groups (i.e., it is at least trivalent) and may be, or may contain, for
example, a hydrophilic polymer, a hydrophobic polymer, an amphiphilic
polymer, a C2_~4 hydrocarbyl, or a C~_~4 hydrocarbyl which is heteroatom-
containing. The linking groups L~, L2, and L3 may be the same or different.
The
designators p, q and r are either 0 (when no linker is present) or 1 (when a
linker is present). The reactive groups X~, X2 and X3 may be the same or
different. Each of these reactive groups reacts with at least one other
reactive
group to form a three-dimensional matrix. Therefore X~ can react with X2
and/or X3, X2 can react with X~ and/or X3, X3 can react with X~ and/or X2 and
so
forth. A trivalent core will be directly or indirectly bonded to three
functional
groups, a tetravalent core will be directly or indirectly bonded to four
functional
groups, etc.
Each side chain typically has one reactive group. However, the
invention also encompasses self-reactive compounds where the side chains
contain more than one reactive group. Thus, in another embodiment of the
invention, the self-reactive compound has the formula (II):
L X~ - (~4)a - '~~ ' (L5)b l c
where: a and b are integers from 0-1; c is an integer from 3-12; R' is
selected
from hydrophilic polymers, hydrophobic polymers, amphiphilic polymers, C2_~4
hydrocarbyls, and heteroatom-containing C~_~4 hydrocarbyls; X' and Y' are
reactive groups and can be the same or different; and L4 and L5 are linking
groups. Each reactive group inter-reacts with the other reactive group to form
a
three-dimensional matrix. The compound is essentially non-reactive in an
initial
environment but is rendered reactive upon exposure to a modification in the
initial environment that provides a modified environment such that a plurality
of
205
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
the self-reactive compounds inter-react in the modified environment to form a
three-dimensional matrix. In one preferred embodiment, R is a hydrophilic
polymer. In another preferred embodiment, X' is a nucleophilic group and Y' is
an electrophilic group.
The following self-reactive compound is one example of a
compound of formula (II):
where R4 has the formula:
0
0
0 0
0
H2N p-N
x
O
Thus, in formula (II), a and b are 1; c is 4; the core R' is the
hydrophilic polymer, tefirafunctionally activated polyethylene glycol, (C(CH2-
O-
)4; X' is the electrophilic reactive group, succinimidyl; Y' is the
nucleophilic
reactive group -CH-NH2; L4 is -C(O)-O-; and L5 is -(CH2- CH2-O-CH2)x CH2-O-
C(O)-(CH2)2-.
The self-reactive compounds of the invention are readily
synthesized by techniques that are well known in the art. An exemplary
synthesis is set forth below:
206
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
O
HO
O
HN O
O
O
Mitsunobo
or
DCC
o
o
o
R40 O
O HN O
x
O
RaO O
OR4 /
H2, Pd/C
207
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
R4
O
Mitsunobo
or
HO DCC
o
The reactive groups are selected so that the compound is
essentially non-reactive in an initial environment. Upon exposure to a
specific
modification in the initial environment, providing a modified environment, the
compound is rendered reactive and a plurality of self-reactive compounds are
then able to inter-react in the modified environment to form a three-
dimensional
matrix. Examples of modification in the initial environment are detailed
below,
but include the addition of an aqueous medium, a change in pH, exposure to
ultraviolet radiation, a change in temperature, or contact with a redox
initiator.
The core and reactive groups can also be selected so as to
provide a compound that has one of more of the following features: are
biocompatible, are non-immunogenic, and do not leave any toxic, inflammatory
or immunogenic reaction products at the site of administration. Similarly, the
208
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
core and reactive groups can also be selected so as to provide a resulting
matrix that has one or more of these features.
In one embodiment of the invention, substantially immediately or
immediately upon exposure to the modified environment, the self-reactive
compounds inter-react form a three-dimensional matrix. The term "substantially
immediately" is intended to mean within less than five minutes, preferably
within
less than two minutes, and the term "immediately" is intended to mean within
less than one minute, preferably within less than 30 seconds.
In one embodiment, the self-reactive compound and resulting
matrix are not subject to enzymatic cleavage by matrix metalloproteinases such
as collagenase, and are therefore not readily degradable in vivo. Further, the
self-reactive compound may be readily tailored, in terms of the selection and
quantity of each component, to enhance certain properties, e.g., compression
strength, swellability, tack, hydrophilicity, optical clarity, and the like.
In one preferred embodiment, R is a hydrophilic polymer. In
another preferred embodiment, X is a nucleophilic group, Y is an electrophilic
group and Z is either an electrophilic or a nucleophilic group. Additional
embodiments are detailed below.
A higher degree of inter-reaction, e.g., crosslinking, may be useful
when a less swellable matrix is desired or increased compressive strength is
desired. In those embodiments, it may be desirable to have n be an integer
from 2-12. In addition, when a plurality of self-reactive compounds are
utilized,
the compounds may be the same or different.
A. Reactive Groups
Prior to use, the self-reactive compound is stored in an initial
environment that insures that the compound remain essentially non-reactive
until use. Upon modification of this environment, the compound is rendered
reactive and a plurality of compounds will then inter-react to form the
desired
209
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
matrix. The initial environment, as well as the modified environment, is thus
determined by the nature of the reactive groups involved.
The number of reactive groups can be the same or different.
However, in one embodiment of the invention, the number of reactive groups
are approximately equal. As used in this context, the term "approximately"
refers to a 2:1 to 1:2 ratio of moles of one reactive group to moles of a
different
reactive groups. A 1:1:1 molar ratio of reactive groups is generally
preferred.
In general, the concentration of the self-reactive compounds in the
modified environment, when liquid in nature, will be in the range of about 1
to
50 wt%, generally about 2 to 40 wt%. The preferred concentration of the
compound in the liquid will depend on a number of factors, including the type
of
compound (i.e., type of molecular core and reactive groups), its molecular
weight, and the end use of the resulting three-dimensional matrix. For
example, use of higher concentrations of the compounds, or using highly
functionalized compounds, will result in the formation of a more tightly
crosslinked network, producing a stiffer, more robust gel. As such,
compositions intended for use in tissue augmentation will generally employ
concentrations of self-reactive compounds that fall toward the higher end of
the
preferred concentration range. Compositions intended for use as bioadhesives
or in adhesion prevention do not need to be as firm and may therefore contain
lower concentrations of the self-reactive compounds.
1 ) Electrophilic and Nucleophilic Reactive Groups
In one embodiment of the invention, the reactive groups are
electrophilic and nucleophilic groups, which undergo a nucleophilic
substitution
reaction, a nucleophilic addition reaction, or both. The term "electrophilic"
refers to a reactive group that is susceptible to nucleophilic attack, i.e.,
susceptible to reaction with an incoming nucleophilic group. Electrophilic
groups herein are positively charged or electron-deficient, typically electron-
deficient. The term "nucleophilic" refers to a reactive group that is electron
rich,
210
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
has an unshared pair of electrons acting as a reactive site, and reacts with a
positively charged or electron-deficient site. For such reactive groups, the
modification in the initial environment comprises the addition of an aqueous
medium and/or a change in pH.
In one embodiment of the invention, X1 (also referred to herein as
X) can be a nucleophilic group and X2 (also referred to herein as Y) can be an
electrophilic group or vice versa, and X3 (also referred to herein as Z) can
be
either an electrophilic or a nucleophilic group.
X may be virtually any nucleophilic group, so long as reaction can
occur with the electrophilic group Y and also with Z, when Z is electrophilic
(~EL)~ Analogously, Y may be virtually any electrophilic group, so long as
reaction can take place with X and also with Z when Z is nucleophilic (ZNU).
The only limitation is a practical one, in that reaction between X and Y, and
X
and ZED, or Y and ZNU should be fairly rapid and take place automatically upon
admixture with an aqueous medium, without need for heat or potentially toxic
or
non-biodegradable reaction catalysts or other chemical reagents. It is also
preferred although not essential that reaction occur without need for
ultraviolet
or other radiation. In one embodiment, the reactions between X and Y, and
between either X and ZED or Y and ZNU, are complete in under 60 minutes,
preferably under 30 minutes. Most preferably, the reaction occurs in about 5
to
15 minutes or less.
Examples of nucleophilic groups suitable as X or FnNU include, but
are not limited to: -NH2, -NHR~, -N(R~)2, -SH, -OH, -COOH, -C6H4-OH, -H,
-PH2,
-PHR~, -P(R~)2, -NH-NH2, -CO-NH-NH2, -C5H4N, etc. wherein R~ is a
hydrocarbyl group and each R1 may be the same or different. R~ is typically
alkyl or monocyclic aryl, preferably alkyl, and most preferably lower alkyl.
Organometallic moieties are also useful nucleophilic groups for the purposes
of
the invention, particularly those that act as carbanion donors. Examples of
organometallic moieties include: Grignard functionalities -R2MgHal wherein R2
211
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
is a carbon atom (substituted or unsubstituted), and Hal is halo, typically
bromo,
iodo or chloro, preferably bromo; and lithium-containing functionalities,
typically
alkyllithium groups; sodium-containing functionalities.
It will be appreciated by those of ordinary skill in the art that
certain nucleophilic groups must be activated with a base so as to be capable
of reaction with an electrophilic group. For example, when there are
nucleophilic sulfhydryl and hydroxyl groups in the self-reactive compound, the
compound must be admixed with an aqueous base in order to remove a proton
and provide an -S- or -O- species to enable reaction with the electrophilic
group.
Unless it is desirable for the base to participate in the reaction, a non-
nucleophilic base is preferred. In some embodiments, the base may be present
as a component of a buffer solution. Suitable bases and corresponding
crosslinking reactions are described herein.
The selection of electrophilic groups provided on the self-reactive
compound, must be made so that reaction is possible with the specific
nucleophilic groups. Thus, when the X reactive groups are amino groups, the Y
and any ZED groups are selected so as to react with amino groups.
Analogously, when the X reactive groups are sulfhydryl moieties, the
corresponding electrophilic groups are sulfhydryl-reactive groups, and the
like.
In general, examples of electrophilic groups suitable as Y or ZED include, but
are
not limited to, -CO-CI, -(CO)-O-(CO)-R (where R is an alkyl group),
-CH=CH-CH=O and -CH=CH-C(CH3)=O, halo, -N=C=O, -N=C=S,
-S02CH=CH2, -O(CO)-C=CH2, -O(CO)-C(CH3)=CH2, -S-S-(C5H4N),
-O(CO)-C(CH2CH3)=CH2, -CH=CH-C=NH, -COOH, -(CO)O-N(COCH2)2, -CHO,
-(CO)O-N(COCH2)2-S(O)20H, and -N(COCH)2.
When X is amino (generally although not necessarily primary
amino), the electrophilic groups present on Y and ZED are amine-reactive
groups. Exemplary amine-reactive groups include, by way of example and not
limitation, the following groups, or radicals thereof: (1 ) carboxylic acid
esters,
including cyclic esters and "activated" esters; (2) acid chloride groups (-CO-
CI);
212
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(3) anhydrides (-(CO)-O-(CO)-R, where R is an alkyl group); (4) ketones and
aldehydes, including a,~i-unsaturated aldehydes and ketones such as
-CH=CH-CH=O and -CH=CH-C(CH3)=O; (5) halo groups; (6) isocyanate group
(-N=C=O); (7) thioisocyanato group (-N=C=S); (8) epoxides; (9) activated
hydroxyl groups (e.g., activated with conventional activating agents such as
carbonyldiimidazole or sulfonyl chloride); and (10) olefins, including
conjugated
olefins, such as ethenesulfonyl (-S02CH=CH2) and analogous functional
groups, including acrylate (-O(CO)-C=CH2), methacrylate
(-O(CO)-C(CH3)=CH2), ethyl acrylate (-O(CO)-C(CH2CH3)=CHI), and
ethyleneimino (-CH=CH-C=NH).
In one embodiment the amine-reactive groups contain an
electrophilically reactive carbonyl group susceptible to nucleophilic attack
by a
primary or secondary amine, for example the carboxylic acid esters and
aldehydes noted above, as well as carboxyl groups (-COOH).
Since a carboxylic acid group per se is not susceptible to reaction
with a nucleophilic amine, components containing,carboxylic acid groups must
be activated so as to be amine-reactive. Activation may be accomplished in a
variety of ways, but often involves reaction with a suitable hydroxyl-
containing
compound in the presence of a dehydrating agent such as
dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For example, a
carboxylic acid can be reacted with an alkoxy-substituted N-hydroxy-
succinimide'or N-hydroxysulfosuccinimide in the presence of DCC to form
reactive electrophilic groups, the N-hydroxysuccinimide ester and the N-
hydroxysulfosuccinimide ester, respectively. Carboxylic acids may also be
activated by reaction with an acyl halide such as an acyl chloride (e.g.,
acetyl
chloride), to provide a reactive anhydride group. In a further example, a
carboxylic acid may be converted to an acid chloride group using, e.g.,
thionyl
chloride or an acyl chloride capable of an exchange reaction. Specific
reagents
and procedures used to carry out such activation reactions will be known to
213
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
those of ordinary skill in the art and are described in the pertinent texts
and
literature.
Accordingly, in one embodiment, the amine-reactive groups are
selected from succinimidyl ester (-O(CO)-N(COCH2)2), sulfosuccinimidyl ester
(-O(CO)-N(COCH2)2-S(O)20H), maleimido (-N(COCH)2), epoxy, isocyanato,
thioisocyanato, and ethenesulfonyl.
Analogously, when X is sulfhydryl, the electrophilic groups present
on Y and ZED are groups that react with a sulfhydryl moiety. Such reactive
groups include those that form thioester linkages upon reaction with a
sulfhydryl
group, such as those described in WO 00/62827 to Wallace et al. As explained
in detail therein, sulfhydryl reactive groups include, but are not limited to:
mixed
anhydrides; ester derivatives of phosphorus; ester derivatives of p-
nitrophenol,
p-nitrothiophenol and pentafluorophenol; esters of substituted hydroxylamines,
including N-hydroxyphthalimide esters, N-hydroxysuccinimide esters, N-
hydroxysulfosuccinimide esters, and N-hydroxyglutarimide esters; esters of 1-
hydroxybenzotriazole; 3-hydroxy-3,4-dihydro-benzotriazin-4-one; 3-hydroxy-
3,4-dihydro-quinazoline-4-one; carbonylimidazole derivatives; acid chlorides;
ketenes; and isocyanates. With these sulfhydryl reactive groups, auxiliary
reagents can also be used to facilitate bond formation, e.g., 1-ethyl-3-[3-
dimethylaminopropyl]carbodiimide can be used to facilitate coupling of
sulfhydryl groups to carboxyl-containing groups.
In addition to the sulfhydryl reactive groups that form thioester
linkages, various other sulfhydryl reactive functionalities can be utilized
that
form other types of linkages. For example, compounds that contain methyl
imidate derivatives form imido-thioester linkages with sulfhydryl groups.
Alternatively, sulfhydryl reactive groups can be employed that form disulfide
bonds with sulfhydryl groups; such groups generally have the structure -S-S-Ar
where Ar is a substituted or unsubstituted nitrogen-containing heteroaromatic
moiety or a non-heterocyclic aromatic group substituted with an electron-
withdrawing moiety, such that Ar may be, for example, 4-pyridinyl, o-
214
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
nitrophenyl, m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-
benzoic
acid, 2-nitro-4-pyridinyl, etc. In such instances, auxiliary reagents, i.e.,
mild
oxidizing agents such as hydrogen peroxide, can be used to facilitate
disulfide
bond formation.
Yet another class of sulfhydryl reactive groups forms thioether
bonds with sulfhydryl groups. Such groups include, inter alia, maleimido,
substituted maleimido, haloalkyl, epoxy, imino, and aziridino, as well as
olefins
(including conjugated olefins) such as ethenesulfonyl, etheneimino, acrylate,
methacrylate, and a,(3-unsaturated aldehydes and ketones.
When X is -OH, the electrophilic functional groups on the
remaining components) must react with hydroxyl groups. The hydroxyl group
may be activated as described above with respect to carboxylic acid groups, or
it may react directly in the presence of base with a sufficiently reactive
electrophilic group such as an epoxide group, an aziridine group, an acyl
halide,
an anhydride, and so forth.
When X is an organometallic nucleophilic group such as a
Grignard functionality or an alkyllithium group, suitable electrophilic
functional
groups for reaction therewith are those containing carbonyl groups, including,
by way of example, ketones and aldehydes.
It will also be appreciated that certain functional groups can react
as nucleophilic or as electrophilic groups, depending on the selected reaction
partner andlor the reaction conditions. For example, a carboxylic acid group
can act as a nucleophilic group in the presence of a fairly strong base, but
generally acts as an electrophilic group allowing nucleophilic attack at the
carbonyl carbon and concomitant replacement of the hydroxyl group with the
incoming nucleophilic group.
These, as well as other embodiments are illustrated below, where
the covalent linkages in the matrix that result upon covalent binding of
specific
nucleophilic reactive groups to specific electrophilic reactive groups on the
self-
reactive compound include, solely by way of example, the following Table:
215
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Table
Representative
Nucleophilic Representative Electrophilic
Group (?C, Group (Y, ZED) Resulting Linkage
GNU)
-NH2 -O-(CO)-O-N(COCH~)a -NH-(CO)-O-
succinimidyl carbonate
terminus
-SH -O-(CO)-O-N(COCH~)2 -S-(CO)-O-
-OH -O-(CO)-O-N(COCH~)2 -O-(CO)-
-NH2 -O(CO)-CH=CH2 -NH-CH2CH2-(CO)-O-
acrylate terminus
-S H -O-(CO)-CH=CH2 -S-CH2CH2-(CO)-O-
-OH -O-(CO)-CH=CH2 -O-CH2CH2-(CO)-O-
-NH~ -O(CO)-(CH2)3-C02-N(COCH2)2-NH-(CO)-(CH2)3-(CO)-O-
succinimidyl glutarate
terminus
-SH -O(CO)-(CH2)3-C02-N(COCH2)2-S-(CO)-(CH2)3-(CO)-O-
-OH -O(CO)-(CH2)3-C02-N(COCH2)2-O-(CO)-(CH2)3-(CO)-O-
-N H2 -O-CH2-C02-N(COCH2)2 -N H-( CO)-CH2-O-
succinimidyl acetate terminus
-S H -O-CH2-C02-N(COC H2)2 -S-(CO)-C H2-O-
-OH -O-CH2-CO~-N(COCH2)2 -O-(CO)-CH2-O-
-NH2 -O-NH(CO)-(CH2)2-C02- -NH-(CO)-(CH2)2-(CO)-
N(COCH2)2 NH-O-
succinimidyl succinamide
terminus
-SH -O-NH(CO)-(CH2)2-C02- -S-(CO)-(CH2)2-(CO)-NH-
N(COCH2)2 O-
OH -O-NH(CO)-(CH2)2-C02- -O-(CO)-(CH2)2-(CO)-NH-
N(COCH2)2 O-
-NH2 -O- (CH2)2-CHO -NH-(CO)-(CH2)2-O-
propionaldehyde terminus
-NH2 % ~ -NH-CH2-CH(OH)-CH2-O-
-O-CH2-CH CH2 and
glycidyl ether terminus -N[CH2-CH(OH)-CH2-O-J2
216
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Representative
Nucleophilic Representative Electrophilic
Group (X, Group (Y, ZED) Resulting Linkage
~N~)
-NH2 -O-(CH2)2-N=C=O -NH-(CO)-NH-CH2-O-
(isocyanate terminus)
-NH2 -S02-CH=CH2 -NH-CH~CH2-S02-
vinyl sulfone terminus
-SH -S02-CH=CH2 -S-CH2CH2-S02-
For self-reactive compounds containing electrophilic and
nucleophilic reactive groups, the initial environment typically can be dry and
sterile. Since electrophilic groups react with water, storage in sterile, dry
form
will prevent hydrolysis. The dry synthetic polymer may be compression molded
into a thin sheet or membrane, which can then be sterilized using gamma or e-
beam irradiation. The resulting dry membrane or sheet can be cut to the
desired size or chopped into smaller size particulates. The modification of a
dry
initial environment will typically comprise the addition of an aqueous medium.
In one embodiment, the initial environment can be an aqueous
medium such as in a low pH bufFer, i.e., having a pH less than about 6.0, in
which both electrophilic and nucleophilic groups are non-reactive. Suitable
liquid media for storage of such compounds include aqueous buffer solutions
such as monobasic sodium phosphate/dibasic sodium phosphate, sodium
carbonate/sodium bicarbonate, glutamate or acetate, at a concentration of 0.5
to 300 mM. Modification of an initial low pH aqueous environment will
typically
comprise increasing the pH to at least pH 7.0, more preferably increasing the
pH to at least pH 9.5.
In another embodiment the modification of a dry initial
environment comprises dissolving the self-reactive compound in a first buffer
solution having a pH within the range of about 1.0 to 5.5 to form a
homogeneous solution, and (ii) adding a second buffer solution having a pH
within the range of about 6.0 to 11.0 to the homogeneous solution. The buffer
solutions are aqueous and can be any pharmaceutically acceptable basic or
217
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
acid composition. The term "buffer" is used in a general sense to refer to an
acidic or basic aqueous solution, where the solution may or may not be
functioning to provide a buffering effect (i.e., resistance to change in pH
upon
addition of acid or base) in the compositions of the present invention. For
example, the self-reactive compound-can be in the form of a homogeneous dry
powder. This powder is then combined with a buffer solution having a pH within
the range of about 1.0 to 5.5 to form a homogeneous acidic aqueous solution,
and this solution is then combined with a buffer solution having a pH within
the
range of about 6.0 to 11.0 to form a reactive solution. For example, 0.375
grams of the dry powder can be combined with 0.75 grams of the acid buffer to
provide, after mixing, a homogeneous solution, where this solution is combined
with 1.1 grams of the basic buffer to provide a reactive mixture that
substantially
immediately forms a three-dimensional matrix.
Acidic buffer solutions having a pH within the range of about 1.0
to 5.5, include by way of illustration and not limitation, solutions of:
citric acid,
hydrochloric acid, phosphoric acid, sulfuric acid, AMPSO (3-[(1,1-dimethyl-2-
hydroxyethyl)aminoJ2-hydroxy-propane-sulfonic acid), acetic acid, lactic acid,
and combinations thereof. In a preferred embodiment, the acidic buffer
solution, is a solution of citric acid, hydrochloric acid, phosphoric acid,
sulfuric
acid, and combinations thereof. Regardless of the precise acidifying agent,
the
acidic buffer preferably has a pH such that it retards the reactivity of the
nucleophilic groups on the core. For example, a pH of 2.1 is generally
sufficient
to retard the nucleophilicity of thiol groups. A lower pH is typically
preferred
when the core contains amine groups as the nucleophilic groups. In general,
the acidic buffer is an acidic solution that, when contacted with nucleophilic
groups, renders those nucleophilic groups relatively non-nucleophilic.
An exemplary acidic buffer is a solution of hydrochloric acid,
having a concentration of about 6.3 mM, and a pH in the range of 2.1 to 2.3.
This buffer may be prepared by combining concentrated hydrochloric acid with
water, i.e., by diluting concentrated hydrochloric acid with water. Similarly,
this
218
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
buffer A may also be conveniently prepared by diluting 1.23 grams of
concentrated hydrochloric acid to a volume of 2 liters, or diluting 1.84 grams
of
concentrated hydrochloric acid to a volume to 3 liters, or diluting 2.45 grams
of
concentrated hydrochloric acid to a volume of 4 liters, or diluting 3.07 grams
concentrated hydrochloric acid to a volume of 5 liters, or diluting 3.68 grams
of
concentrated hydrochloric acid to a volume to 6 liters. For safety reasons,
the
concentrated acid is preferably added to water.
Basic buffer solutions having a pH within the range of about 6.0 to
11.0, include by way of illustration and not limitation, solutions of:
glutamate,
acetate, carbonate and carbonate salts (e.g., sodium carbonate, sodium
carbonate monohydrate and sodium bicarbonate), borate, phosphate and
phosphate salts (e.g., monobasic sodium phosphate monohydrate and dibasic
sodium phosphate), and combinations thereof. In a preferred embodiment, the
basic buffer solution is a solution of carbonate salts, phosphate salts, and
combinations thereof.
In general, the basic buffer is an aqueous solution that neutralizes
the effect of the acidic buffer, when it is added to the homogeneous solution
of
the compound and first buffer, so that the nucleophilic groups on the core
regain their nucleophilic character (that has been masked by the action of the
acidic buffer), thus allowing the nucleophilic groups to inter-react with the
electrophilic groups on the core.
An exemplary basic buffer is an aqueous solution of carbonate
and phosphate salts. This buffer may be prepared by combining a base
solution with a salt solution. The salt solution may be prepared by combining
34.7 g of monobasic sodium phosphate monohydrate, 49.3 g of sodium
carbonate monohydrate, and sufficient water to provide a solution volume of 2
liter. Similarly, a 6 liter solution may be prepared by combining 104.0 g of
monobasic sodium phosphate monohydrate, 147.94 g of sodium carbonate
monohydrate, and sufficient water to provide 6 liter of the salt solution. The
basic buffer may be prepared by combining 7.2 g of sodium hydroxide with
219
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
180.0 g of water. The basic buffer is typically prepared by adding the base
solution as needed to the salt solution, ultimately to provide a mixture
having
the desired pH, e.g., a pH of 9.65 to 9.75.
In general, the basic species present in the basic buffer should be
sufficiently basic to neutralize the acidity provided by the acidic buffer,
but
should not be so nucleophilic itself that it will react substantially with the
electrophilic groups'on the core. For this reason, relatively "soft" bases
such as
carbonate and phosphate are preferred in this embodiment of the invention.
To illustrate the preparation of a three-dimensional matrix of the
present invention, one may combine an admixture of the self-reactive
compound with a first, acidic, buffer (e.g., an acid solution, e.g., a dilute
hydrochloric acid solution) to form a homogeneous solution. This
homogeneous solution is mixed with a second, basic, buffer (e.g., a basic
solution, e.g., an aqueous solution containing phosphate and carbonate salts)
whereupon the reactive groups on the core of the self-reactive compound
substantially immediately inter-react with one another to form a three-
dimensional matrix.
2) Redox Reactive Groups
In one embodiment of the invention, the reactive groups are vinyl
groups such as styrene derivatives, which undergo a radical polymerization
upon initiation with a redox initiator. The term "redox" refers to a reactive
group
that is susceptible to oxidation-reduction activation. The term "vinyl" refers
to a
reactive group that is activated by a redox initiator, and forms a radical
upon
reaction. X, Y and Z can be the same or different vinyl groups, for example,
methacrylic groups.
For self-reactive compounds containing vinyl reactive groups, the
initial environment typically will be an aqueous environment. The modification
of the initial environment involves the addition of a redox initiator.
220
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
3) Oxidative Coupling Reactive Groups
In one embodiment of the invention, the reactive groups undergo
an oxidative coupling reaction. For example, X, Y and Z can be a halo group
such as chloro, with an adjacent electron-withdrawing group on the halogen-
bearing carbon (e.g., on the "L" linking group). Exemplary electron-
withdrawing
groups include nitro, aryl, and so forth.
For such reactive groups, the modification in the initial
environment comprises a change in pH. For example, in the presence of a
base such as KOH, the self-reactive compounds then undergo a de-hydro,
chloro coupling reaction, forming a double bond between the carbon atoms, as
illustrated below:
c1
c-Ar c1
-Ar
Ar-C- ~ -C-Ar
CI I KOH Ar-C-R-C-Ar
CI ~ CI
C-Ar
CI
-Ar
Ar-C-R-CI+Ar
Ar-C-R-C-Ar CI CI
CI CI
For self reactive compounds containing oxidative coupling
reactive groups, the initial environment typically can be can be dry and
sterile,
or a non-basic medium. The modification of the initial environment will
typically
comprise the addition of a base.
4) Photoinitiated Reactive Groups
In one embodiment of the invention, the reactive groups are
photoinitiated groups. For such reactive groups, the modification in the
initial
environment comprises exposure to ultraviolet radiation.
In one embodiment of the invention, X can be an azide (-N3)
group and Y can be an alkyl group such as -CH(CH3)2 or vice versa. Exposure
to ultraviolet radiation will then form a bond between the groups to provide
for
the following linkage: -NH-C(CH3)2-CH2-. In another embodiment of the
221
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
invention, X can be a benzophenone (-(C6H4)-C(O)-(C6H5)) group and Y can be
an alkyl group such as -CH(CH3)2 or vice versa. Exposure to ultraviolet
radiation will then form a bond between the groups to provide for the
following
linkage:
OH
~ ~ ~ HsC~CHs~
For self-reactive compounds containing photoinitiated reactive
groups, the initial environment typically will be in an ultraviolet radiation-
shielded environment. This can be for example, storage within a container that
is impermeable to ultraviolet radiation.
The modification of the initial environment will typically comprise
exposure to ultraviolet radiation.
5) Temperature-sensitive Reactive Groins
In one embodiment of the invention, the reactive groups are
temperature-sensitive groups, which undergo a thermochemical reaction. For
such reactive groups, the modification in the initial environment thus
comprises
a change in temperature. The term "temperature-sensitive" refers to a reactive
group that is chemically inert at one temperature or temperature range and
reactive at a different temperature or temperature range.
In one embodiment of the invention, X, Y, and Z are the same or
different vinyl groups.
For self-reactive compounds containing reactive groups that are
temperature-sensitive, the initial environment typically will be within the
range of
about 10 to 30°C.
The modification of the initial environment will typically comprise
changing the temperature to within the range of about 20 to 40°C.
222
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
B. Linking Groups
The reactive groups may be directly attached to the core, or they
may be indirectly attached through a linking group, with longer linking groups
also termed "chain extenders." In the formula (I) shown above, the optional
linker groups are represented by L~, L2, and L3, wherein the linking groups
are
present when p, q and r are equal to 1.
Suitable linking groups are well known in the art. See, for
example, WO 97/22371 to Rhee et al. Linking groups are useful to avoid steric
hindrance problems that can sometimes associated with the formation of direct
linkages between molecules. Linking groups may additionally be used to link
several self-reactive compounds together to make larger molecules. In one
embodiment, a linking group can be used to alter the degradative properties of
the compositions after administration and resultant gel formation. For
example,
linking groups can be used to promote hydrolysis, to discourage hydrolysis, or
to provide a site for enzymatic degradation.
Examples of linking groups that provide hydrolyzable sites,
include, inter alias ester linkages; anhydride linkages, such as those
obtained by
incorporation of glutarate and succinate; ortho ester linkages; ortho
carbonate
linkages such as trimethylene carbonate; amide linkages; phosphoester
linkages; a-hydroxy acid linkages, such as those obtained by incorporation of
lactic acid and glycolic acid; lactone-based linkages, such as those obtained
by
incorporation of caprolactone, valerolactone, y-butyrolactone and p-dioxanone;
and amide linkages such as in a dimeric, oligomeric, or poly(amino acid)
segment. Examples of non-degradable linking groups include succinimide,
propionic acid and carboxymethylate linkages. See, for example, WO 99/07417
to Coury et al. Examples of enzymatically degradable linkages include Leu-
Gly-Pro-Ala, which is degraded by collagenase; and Gly-Pro-Lys, which is
degraded by plasmin.
Linking groups can also be included to enhance or suppress the
reactivity of the various reactive groups. For example, electron-withdrawing
223
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
groups within one or two carbons of a sulfhydryl group would be expected to
diminish its effectiveness in coupling, due to a lowering of nucleophilicity.
Carbon-carbon double bonds and carbonyl groups will also have such an effect.
Conversely, electron-withdrawing groups adjacent to a carbonyl group (e.g.,
the
reactive carbonyl of glutaryl-N-hydroxysuccinimidyl) would increase the
reactivity of the carbonyl carbon with respect to an incoming nucleophilic
group.
By contrast, sterically bulky groups in the vicinity of a reactive group can
be
used to diminish reactivity and thus reduce the coupling rate as a result of
steric
hindrance.
By way of example, particular linking groups and corresponding
formulas are indicated in the following Table:
Table
Linking group Component structure
-O-(CH2),~ -O-(CH2)X X
-O-(CH2)X Y
-O-(CH2)X Z
-S-(CH2)X -S-(CH2)X X
-S-(CH2),~ Y
-S-(CH2)x Z
-NH-(CH2)X -NH-(CH2)X X
-NH-(CH2),~ Y
-NH-(CH2)x Z
-O-(CO)-NH-(CH2)X -O-(CO)-NH-(CH2)x X
-O-(CO)-NH-(CH2)X Y
-O-(CO)-NH-(CH2)x Z
-NH-(CO)-O-(CH2)x -NH-(CO)-O-(CH2)X X
-NH-(CO)-O-(CH2)x Y
-NH-(CO)-O-(CH2)X Z
224
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Linking group Component structure
-O-(CO)-(CH2)X -O-(CO)-(CH2)x X
-O-(CO)-(CH~)X Y
-O-(CO)-(CH2),~ Z
-(CO)-O-(CH2)X -(CO)-O-(CH2)~-X
-(CO)-O-(CH2)n-Y
-(CO)-O-(CH2)" Z
-O-(CO)-O-(CH2),~ -O-(CO)-O-(CH~),~ X
-O-(CO)-O-(CH2)X Y
-O-(CO)-O-(CH2)x Z
-O-( CO )-C H R2- -O-( CO )-C H R2-X
-O-(CO)-CH R2-Y
-O-(CO)-CH R~-Z
-O-R3-( C O )-N H- -O-R3-( CO )-N H-X
- O-R3-(CO)-NH-Y
- O-R3-(CO)-NH-Z
In the above Table, x is generally in the range of 1 to about 10; R2
is generally hydrocarbyl, typically alkyl or aryl, preferably alkyl, and most
preferably lower alkyl; and R3 is hydrocarbylene, heteroatom-containing
hydrocarbylene, substituted hydrocarbylene, or substituted heteroatom-
containing hydrocarbylene) typically alkylene or arylene (again, optionally
substituted and/or containing a heteroatom), preferably lower alkylene (e.g.,
methylene, ethylene, n-propylene, n-butylene, etc.), phenylene, or
amidoalkylene (e.g., -(CO)-NH-CH2).
Other general principles that should be considered with respect to
linking groups are as follows. If a higher molecular weight self-reactive
compound is to be used, it will preferably have biodegradable linkages as
described above, so that fragments larger than 20,000 mol. wt. are not
generated during resorption in the body. In addition, to promote water
225
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
miscibility and/or solubility, it may be desired to add sufficient electric
charge or
hydrophilicity. Hydrophilic groups can be easily introduced using known
chemical synthesis, so long as they do not give rise to unwanted swelling or
an
undesirable decrease in compressive strength. In particular, polyalkoxy
segments may weaken gel strength.
C. The Core
The "core" of each self-reactive compound is comprised of the
molecular structure to which the reactive groups are bound. The molecular
core can a polymer, which includes synthetic polymers and naturally occurring
polymers. In one embodiment, the core is a polymer containing repeating
monomer units. The polymers can be hydrophilic, hydrophobic, or amphiphilic.
The molecular core can also be a low molecular weight components such as a
C2_~4 hydrocarbyl or a heteroatom-containing C2_~4 hydrocarbyl. The
heteroatom-containing C2_~4 hydrocarbyl can have 1 or 2 heteroatoms selected
from N, O and S. In a preferred embodiment, the self-reactive compound
comprises a molecular core of a synthetic hydrophilic polymer.
1 ) Hydrophilic Polymers
As mentioned above, the term "hydrophilic polymer" as used
herein refers to a polymer having an average molecular weight and composition
that naturally renders, or is selected to render the polymer as a whole
"hydrophilic." Preferred polymers are highly pure or are purified to a highly
pure
state such that the polymer is or is treated to become pharmaceutically pure.
Most hydrophilic polymers can be rendered water soluble by incorporating a
sufficient number of oxygen (or less frequently nitrogen) atoms available for
forming hydrogen bonds in aqueous solutions.
Synthetic hydrophilic polymers may be homopolymers, block
copolymers including di-block and tri-block copolymers, random copolymers, or
graft copolymers. In addition, the polymer may be linear or branched, and if
226
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
branched, may be minimally to highly branched, dendrimeric, hyperbranched,
or a star polymer. The polymer may include biodegradable segments and
blocks, either distributed throughout the polymer's molecular structure or
present as a single block, as in a block copolymer. Biodegradable segments
preferably degrade so as to break covalent bonds. Typically, biodegradable
segments are segments that are hydrolyzed in the presence of water and/or
enzymatically cleaved in situ. Biodegradable segments may be composed of
small molecular segments such as ester linkages, anhydride linkages, ortho
ester linkages, ortho carbonate linkages, amide linkages, phosphonate
linkages, etc. Larger biodegradable "blocks" will generally be composed of
,oligomeric or polymeric segments incorporated within the hydrophilic polymer.
Illustrative oligomeric and polymeric segments that are biodegradable include,
by way of example, poly(amino acid) segments, poly(orthoester) segments,
poly(orthocarbonate) segments, and the like. ~ther biodegradable segments
that may form part of the hydrophilic polymer core include polyesters such as
polylactide, polyethers such as polyalkylene oxide, polyamides such as a
protein, and polyurethanes. For example, the core of the self-reactive
compound can be a diblock copolymer of tetrafunctionally activated
polyethylene glycol and polylactide.
Synthetic hydrophilic polymers that are useful herein include, but
are not limited to: polyalkylene oxides, particularly polyethylene glycol
(PEG)
and polyethylene oxide)-polypropylene oxide) copolymers, including block and
random copolymers; polyols such as glycerol, polyglycerol (PG) and
particularly
highly branched polyglycerol, propylene glycol; poly(oxyalkylene)-substituted
diols, and poly(oxyalkylene)-substituted polyols such as mono-, dl- and tri-
polyoxyethylated glycerol, mono- and di-polyoxyethylated propylene glycol, and
mono- and di-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,
polyoxyethylated glucose; poly(acrylic acids) and analogs and copolymers
thereof, such as polyacrylic acid per se, polymethacrylic acid,
poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),
227
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
poly(methylalkylsulfoxide methacrylates), poly(methylalkylsulfoxide acrylates)
and copolymers of any of the foregoing, and/or with additional acrylate
species
such as aminoethyl acrylate and mono-2-(acryloxy)-ethyl succinate; polymaleic
acid; poly(acrylamides) such as polyacrylamide per se, poly(methacrylamide),
poly(dimethylacrylamide), poly(N-isopropyl-acrylamide), and copolymers
thereof; poly(olefinic alcohols) such as polyvinyl alcohols) and copolymers
thereof; poly(N-vinyl lactams) such as polyvinyl pyrrolidones), poly(N-vinyl
caprolactams), and copolymers thereof; polyoxazolines, including
poly(methyloxazoline) and poly(ethyloxazoline); and polyvinylamines; as well
as
copolymers of any of the foregoing. It must be emphasized that the
aforementioned list of polymers is not exhaustive, and a variety of other
synthetic hydrophilic polymers may be used, as will be appreciated by those
skilled in the art.
Those of ordinary skill in the art will appreciate that synthetic
polymers such as polyethylene glycol cannot be prepared practically to have
exact molecular weights, and that the term "molecular weight" as used herein
refers to the weight average molecular weight of a number of molecules in any
given sample, as commonly used in the art. Thus, a sample of PEG 2,000
might contain a statistical mixture of polymer molecules ranging in weight
from,
for example, 1,500 to 2,500 daltons with one molecule differing slightly from
the
next over a range. Specification of a range of molecular weights indicates
that
the average molecular weight may be any value between the limits specified,
and may include molecules outside those limits. Thus, a molecular weight
range of about 800 to about 20,000 indicates an average molecular weight of at
least about 800, ranging up to about 20 kDa.
Other suitable synthetic hydrophilic polymers include chemically
synthesized polypeptides, particularly polynucleophilic polypeptides that have
been synthesized to incorporate amino acids containing primary amino groups
(such as lysine) and/or amino acids containing thiol groups (such as
cysteine).
Poly(lysine), a synthetically produced polymer of the amino acid lysine (145
228
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
MW), is particularly preferred. Poly(lysine)s have been prepared having
anywhere from 6 to about 4,000 primary amino groups, corresponding to
molecular weights of about 870 to about 580,000. Poly(lysine)s for use in the
present invention preferably have a molecular weight within the range of about
1,000 to about 300,000, more preferably within the range of about 5,000 to
about 100,000, and most preferably, within the range of about 8,000 to about
15,000. Poly(lysine)s of varying molecular weights are commercially available
from Peninsula Laboratories, Inc. (Belmont, Calif.).
Although a variety of different synthetic hydrophilic polymers can
be used in the present compounds, preferred synthetic hydrophilic polymers are
PEG and PG, particularly highly branched PG. Various forms of PEG are
extensively used in the modification of biologically active molecules because
PEG lacks toxicity, antigenicity, and immunogenicity (i.e., is biocompatible),
can
be formulated so as to have a wide range of solubilities, and does not
typically
interfere with the enzymatic activities and/or conformations of peptides. A
particularly preferred synthetic hydrophilic polymer for certain applications
is a
PEG having a molecular weight within the range of about 100 to about 100,000,
although for highly branched PEG, far higher molecular weight polymers can be
employed, up to 1,000,000 or more, providing that biodegradable sites are
incorporated ensuring that all degradation products will have a molecular
weight
of less than about 30,000. For most PEGs, however, the preferred molecular
weight is about 1,000 to about 20,000, more preferably within the range of
about 7,500 to about 20,000. Most preferably, the polyethylene glycol has a
molecular weight of approximately 10,000.
Naturally occurring hydrophilic polymers include, but are not
limited to: proteins such as collagen, fibronectin, albumins, globulins,
fibrinogen,
fibrin and thrombin, with collagen particularly preferred; carboxylated
polysaccharides such as polymannuronic acid and polygalacturonic acid;
aminated polysaccharides, particularly the glycosaminoglycans, e.g.,
hyaluronic
acid, chitin, chondroitin sulfate A, B, or C, keratin sulfate, keratosulfate
and
229
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
heparin; and activated polysaccharides such as dextran and starch derivatives.
Collagen and glycosaminoglycans are preferred naturally occurring hydrophilic
polymers for use herein.
Unless otherwise specified, the term "collagen" as used herein
refers to all forms of collagen, including those, which have been processed or
otherwise modified. Thus, collagen from any source may be used in the
compounds of the invention; for example, collagen may be extracted and
purified from human or other mammalian source, such as bovine or porcine
corium and human placenta, or may be recombinantly or otherwise produced.
The preparation of purified, substantially non-antigenic collagen in solution
from
bovine skin is well known in the art. For example, U.S. Patent No. 5,428,022
to
Palefsky et al. discloses methods of extracting and purifying collagen from
the
human placenta, and U.S. Patent No. 5,667,839 to Berg discloses methods of
producing recombinant human collagen in the milk of transgenic animals,
including transgenic cows. Non-transgenic, recombinant collagen expression in
yeast and other cell lines) is described in U.S. Patent No. 6,413,742 to Olsen
et
al., 6,428,978 to Olsen et al., and 6,653,450 to Berg et al.
Collagen of any type, including, but not limited to, types I, II, III, IV,
or any combination thereof, may be used in the compounds of the invention,
although type I is generally preferred. Either atelopeptide or telopeptide-
containing collagen may be used; however, when collagen from a natural
source, such as bovine collagen, is used, atelopeptide collagen is generally
preferred, because of its reduced immunogenicity compared to telopeptide-
containing collagen.
Collagen that has not been previously crosslinked by methods
such as heat, irradiation, or chemical crosslinking agents is preferred for
use in
the invention, although previously crosslinked collagen may be used.
Collagens for use in the present invention are generally, although
not necessarily, in aqueous suspension at a concentration between about 20
mg/ml to about 120 mg/ml, preferably between about 30 mg/ml to about 90
230
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
mglml. Although intact collagen is preferred, denatured collagen, commonly
known as gelatin, can also be used. Gelatin may have the added benefit of
being degradable faster than collagen.
Nonfibrillar collagen is generally preferred for use in compounds
of the invention, although fibrillar collagens may also be used. The term
"nonfibrillar collagen" refers to any modified or unmodified collagen material
that
is in substantially nonfibrillar form, i.e., molecular collagen that is not
tightly
associated with other collagen molecules so as to form fibers. Typically, a
solution of nonfibrillar collagen is more transparent than is a solution of
fibrillar
collagen. Collagen types that are nonfibrillar (or microfibrillar) in native
form
include types IV, VI, and VII.
Chemically modified collagens that are in nonfibrillar form at
neutral pH include succinylated collagen and methylated collagen, both of
which can be prepared according to the methods described in U.S. Patent No.
4,164,559 to Miyata et al. Methylated collagen, which contains reactive amine
groups, is a preferred nucleophile-containing component in the compositions of
the present invention. In another aspect, methylated collagen is a component
that is present in addition to first and second components in the matrix-
forming
reaction of the present invention. Methylated collagen is described in, for
example, in U.S. Patent No. 5,614,587 to Rhee et al.
Collagens for use in the compositions of the present invention
may start out in fibrillar form, then can be rendered nonfibrillar by the
addition of
one or more fiber disassembly agent. The fiber disassembly agent must be
present in an amount sufficient to render the collagen substantially
nonfibrillar
at pH 7, as described above. Fiber disassembly agents for use in the present
invention include, without limitation, various biocompatible alcohols, amino
acids, inorganic salts, and carbohydrates, with biocompatible alcohols being
particularly preferred. Preferred biocompatible alcohols include glycerol and
propylene glycol. Non-biocompatible alcohols, such as ethanol, methanol, and
isopropanol, are not preferred for use in the present invention, due to their
231
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
potentially deleterious effects on the body of the patient receiving them.
Preferred amino acids include arginine. Preferred inorganic salts include
sodium chloride and potassium chloride. Although carbohydrates, such as
various sugars including sucrose, may be used in the practice of the present
invention, they are not as preferred as other types of fiber disassembly
agents
because they can have cytotoxic effects in vivo.
Fibrillar collagen is less preferred for use in the compounds of the
invention. However, as disclosed in U.S. Patent No. 5,614,587 to Rhee et al.,
fibrillar collagen, or mixtures of nonfibrillar and fibrillar collagen, may be
preferred for use in compounds intended for long-term persistence in vivo.
2) Hydrophobic Polymers
The core of the self-reactive compound may also comprise a
hydrophobic polymer, including low molecular weight polyfunctional species,
although for most uses hydrophilic polymers are preferred. Generally,
"hydrophobic polymers" herein contain a relatively small proportion of oxygen
andlor nitrogen atoms. Preferred hydrophobic polymers for use in the invention
generally have a carbon chain that is no longer than about 14 carbons.
Polymers having carbon chains substantially longer than 1'4 carbons generally
have very poor solubility in aqueous solutions and, as such, have very long
reaction times when mixed with aqueous solutions of synthetic polymers
containing, for example, multiple nucleophilic groups. Thus, use of short-
chain
oligomers can avoid solubility-related problems during reaction. Polylactic
acid
and polyglycolic acid are examples of two particularly suitable hydrophobic .
polymers.
3) Amphiphilic Polymers
Generally, amphiphilic polymers have a hydrophilic portion and a
hydrophobic (or lipophilic) portion. The hydrophilic portion can be at one end
of
the core and the hydrophobic portion at the opposite end, or the hydrophilic
and
232
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
hydrophobic portions may be distributed randomly (random copolymer) or in the
form of sequences or grafts (block copolymer) to form the amphiphilic polymer
core of the self-reactive compound. The hydrophilic and hydrophobic portions
may include any of the aforementioned hydrophilic and hydrophobic polymers.
Alternately, the amphiphilic polymer core can be a hydrophilic
polymer that has been modified with hydrophobic moieties (e.g., alkylated PEG
or a hydrophilic polymer modified with one or more fatty chains ), or a
hydrophobic polymer that has been modified with hydrophilic moieties (e.g.,
"PEGylated" phospholipids such as polyethylene glycolated phospholipids).
4) Low Molecular Weight Components
As indicated above, the molecular core of the self-reactive
compound can also be a low molecular weight compound, defined herein as
being a C2_~4 hydrocarbyl or a heteroatom-containing C~_~4 hydrocarbyl, which
contains 1 to 2 heteroatoms selected from N, O, S and combinations thereof.
Such a molecular core can be substituted with any of the reactive groups
described herein.
Alkanes are suitable Cg_q4 hydrocarbyl molecular cores.
Exemplary alkanes, for substituted with a nucleophilic primary amino group and
a Y electrophilic group, include, ethyleneamine (H2N-CH2CH2-Y),
tetramethyleneamine (H2N-(CH4)-Y), pentamethyleneamine (H2N-(CH5)-Y), and
hexamethyleneamine (H2N-(CH6)-Y).
Low molecular weight diols and polyols are also suitable C~_14
hydrocarbyls and include trimethylolpropane, di(trimethylol propane),
pentaerythritol, and diglycerol. Polyacids are also suitable C2_~4
hydrocarbyls,
and include trimethylolpropane-based tricarboxylic acid, di(trimethylol
propane)-
based tetracarboxylic acid, heptanedioic acid, octanedioic acid (suberic
acid),
and hexadecanedioic acid (thapsic acid).
Low molecular weight di- and poly-electrophiles are suitable
heteroatom-containing C2_~4 hydrocarbyl molecular cores. These include, for
233
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
example, disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3),
dithiobis(succinimidylpropionate) (DSP), bis(2-succinimidooxycarbonyloxy)
ethyl sulfone (BSOCOES), and 3,3'-dithiobis(sulfosuccinimidylpropionate
(DTSPP), and their analogs and derivatives.
In one embodiment of the invention, the self-reactive compound of
the invention comprises a low-molecular weight material core, with a plurality
of
acrylate moieties and a plurality of thiol groups.
D. Preparation
The self-reactive compounds are readily synthesized to contain a
hydrophilic, hydrophobic or amphiphilic polymer core or a low molecular weight
core, functionalized with the desired functional groups, i.e., nucleophilic
and
electrophilic groups, which enable crosslinking. For example, preparation of a
self-reactive compound having a polyethylene glycol (PEG) core is discussed
below. However, it is to be understood that the following discussion is for
purposes of illustration and analogous techniques may be employed with other
polymers.
With respect to PEG, first of all, various functionalized PEGs have
been used effectively in fields such as protein modification (see Abuchowski
et
al., Enzymes as Drugs, John Wiley & Sons: New York, N.Y. (1981 ) pp. 367-
383; and Dreborg et al. (1990) Crit. Rev. Therap. Drug Carrier Syst. 6:315),
peptide chemistry (see Mutter et al., The Peptides, Academic: New York, N.Y.
2:285-332; and Zalipsky et al. (1987) Int. J. Peptide Protein Res. 30:740),
and
the synthesis of polymeric drugs (see Zalipsky et al. (1983) Eur. Polym. J.
19:1177; and Ouchi et al. (1987) J. Maeromol. Sci. Cflem. A24:1011 ).
Functionalized forms of PEG, including multi-functionalized PEG,
are commercially available, and are also easily prepared using known methods.
For example, see Chapter 22 of Polyethylene Glycol) Chemistry: Biotechnical
and Biomedical Applications, J. Milton Harris, ed., Plenum Press, NY (1992).
234
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Multi-functionalized forms of PEG are of particular interest and
include, PEG succinimidyl glutarate, PEG succinimidyl propionate, succinimidyl
butylate, PEG succinimidyl acetate, PEG succinimidyl succinamide, PEG
succinimidyl carbonate, PEG propionaldehyde, PEG glycidyl ether, PEG-
isocyanate, and PEG-vinylsulfone. Many such forms of PEG are described in
U.S. Patent No. 5,328,955 and 6,534,591, both to Rhee et al. Similarly,
various
forms of multi-amino PEG are commercially available from sources such as
PEG Shop, a division of SunBio of South Korea (www.sunbio.com), Nippon Oil
and Fats (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku,
Tokyo), Nektar Therapeutics (San Carlos, California, formerly Shearwater
Polymers, Huntsville, Alabama) and from Huntsman's Performance Chemicals
Group (Houston, Texas) under the name Jeffamine~ polyoxyalkyleneamines.
Multi-amino PEGs useful in the present invention include the Jeffamine
diamines ("D" series) and triamines ("T" series), which contain two and three
primary amino groups per molecule. Analogous poly(sulfhydryl) PEGs are also
available from Nektar Therapeutics, e.g., in the form of pentaerythritol
polyethylene glycol) ether tetra-sulfhydryl (molecular weight 10,000). These
multi-functionalized forms of PEG can then be modified to include the other
desired reactive groups.
Reaction with succinimidyl groups to convert terminal hydroxyl
groups to reactive esters is one technique for preparing a core with
electrophilic
groups. This core can then be modified include nucleophilic groups such as
primary amines, thiols, and hydroxyl groups. Other agents to convert hydroxyl
groups include carbonyldiimidazole and sulfonyl chloride. However, as
discussed herein, a wide variety of electrophilic groups may be advantageously
employed for reaction with corresponding nucleophilic groups. Examples of
such electrophilic groups include acid chloride groups; anhydrides, ketones,
aldehydes, isocyanate, isothiocyanate, epoxides, and olefins, including
conjugated olefins such as ethenesulfonyl (-S02CH=CH2) and analogous
functional groups.
235
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Other in situ Crosslinkina Materials
Numerous other types of in situ forming materials have been
described which may be used in combination with an anti-scarring agent in
accordance with the invention. The in situ forming material may be a
biocompatible crosslinked polymer that is formed from water soluble precursors
having electrophilic and nucleophilic groups capable of reacting and
crosslinking in situ (see, e.g., U.S. Patent No. 6,566,406). The in situ
forming
material may be hydrogel that may be formed through a combination of physical
and chemical crosslinking processes, where physical crosslinking is mediated
by one or more natural or synthetic components that stabilize the hydrogel-
forming precursor solution at a deposition site for a period of time
sufficient for
more resilient chemical crosslinks to form (see, e.g., U.S. Patent No.
6,818,018). The in situ forming material may be formed upon exposure to an
aqueous fluid from a physiological environment from dry hydrogel precursors
(see, e.g., U.S. Patent No. 6,703,047). The in situ forming material may be a
hydrogel matrix that provides controlled release of relatively low molecular
weight therapeutic species by first dispersing or dissolving the therapeutic
species within relatively hydrophobic rate modifying agents to form a mixture;
the mixture is formed into microparticles that are dispersed within
bioabsorbable hydrogels, so as to release the water soluble therapeutic agents
in a controlled fashion (see, e.g., 6,632,457). The in situ forming material
may
be a multi-component hydrogel system (see, e.g., U.S. Patent No. 6,379, 373).
The in situ forming material may be a multi-arm block copolymer that includes
a
central core molecule, such as a residue of a polyol, and at least three
copolymer arms covalently attached to the central core molecule, each
copolymer arm comprising an inner hydrophobic polymer segment covalently
attached to the central core molecule and an outer hydrophilic polymer segment
covalently attached to the hydrophobic polymer segment, wherein the central
core molecule and the hydrophobic polymer segment define a hydrophobic core
region (see, e.g., 6,730,334). The in situ forming material may include a gel-
236
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
forming macromer that includes at least four polymeric blocks, at feast two of
which are hydrophobic and at least one of which is hydrophilic, and including
a
crosslinkable group (see, e.g., 6,639,014). The in situ forming material may
be
a water-soluble macromer that includes at least one hydrolysable linkage
formed from carbonate or dioxanone groups, at least one water-soluble
polymeric block, and at least one polymerizable group (see, e.g., U.S. Patent
No. 6,177,095). The in situ forming material may comprise polyoxyalkylene
block copolymers that form weak physical crosslinks to provide gels having a
paste-like consistency at physiological temperatures. (see, e.g., U.S. Patent
No.
4,911,926). The in situ forming material may be a thermo-irreversible gel made
from polyoxyalkylene polymers and ionic polysaccharides (see, e.g., U.S.
Patent No. 5,126,141 ). The in situ forming material may be a gel forming
composition that includes chitin derivatives (see, e.g., U.S. Patent No.
5,093,319), chitosan-coagulum (see, e.g., U.S. Patent No. 4,532,134), or
hyaluronic acid (see, e.g., U.S. Patent No. 4,141,973). The in situ forming
material may be an in situ modification of alginate (see, e.g., U.S. Patent
No.
5,266,326 ). The in situ forming material may be formed from ethylenically
unsaturated water soluble macromers that can be crosslinked in contact with
tissues, cells, and bioactive molecules to form gels (see, e.g., U.S. Patent
No.
5,573,934). The in situ forming material may include urethane prepolymers
used in combination with an unsaturated cyano compound containing a cyano
group attached to a carbon atom, such as cyano(meth)acrylic acids and esters
thereof (see, e.g., U.S. Patent No. 4,740,534). The in situ forming material
may
be a biodegradable hydrogel that polymerizes by a photoinitiated free radical
polymerization from water soluble macromers (see, e.g., U.S. Patent No.
5,410,016). The in situ forming material may be formed from a two component
mixture including a first part comprising a serum albumin protein in an
aqueous
buffer having a pH in a range of about 8.0-11.0, and a second part comprising
a
water-compatible or water-soluble bifunctional crosslinking agent. (see, e.g.,
U.S. Patent No. 5,583,114).
237
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another aspect, in situ forming materials that can be used
include those based on the crosslinking of proteins. For example, the in situ
forming material may be a biodegradable hydrogel composed of a recombinant
or natural human serum albumin and polyethylene) glycol polymer solution
whereby upon mixing the solution cross-links to form a mechanical non-liquid
covering structure which acts as a sealant. See e.g., U.S. Patent No.
6,458,147
and 6,371,975. The in situ forming material may be composed of two separate
mixtures based on fibrinogen and thrombin which are dispensed together to
form a biological adhesive when intermixed either prior to or on the
application
site to form a fibrin sealant. See e.g., U.S. Patent No. 6,764,467. The in
situ
forming material may be composed of ultrasonically treated collagen and
albumin which form a viscous material that develops adhesive properties when
crosslinked chemically with glutaraldehyde and amino acids or peptides. See
e.g., U.S. Patent No. 6,310,036. The in situ forming material may be a
hydrated adhesive gel composed of an aqueous solution consisting essentially
of a protein having amino groups at the side chains (e.g., gelatin, albumin)
which is crosslinked with an N-hydroxyimidoester compound. See e.g., U.S.
Patent No. 4,839,345. The in situ forming material may be a hydrogel prepared
from a protein or polysaccharide backbone (e.g., albumin or polymannuronic
acid) bonded to a cross-linking agent (e.g., polyvalent derivatives of
polyethylene or polyalkylene glycol). See e.g., U.S. Patent No. 5,514,379. The
in situ forming material may be composed of a polymerizable collagen
composition that is applied to the tissue and then exposed to an initiator to
polymerize the collagen to form a seal over a wound opening in the tissue. See
e.g., U.S. Patent No. 5,874,537. The in situ forming material may be a two
component mixture composed of a protein (e.g., serum albumin) in an aqueous
bufFer having a pH in the range of about 8.0-11.0 and a water-soluble
bifunctional polyethylene oxide type crosslinking agent, which transforms from
a
liquid to a strong, flexible bonding composition to seal tissue in situ. See
e.g.,
U.S. Patents 5,583,114 and RE38158 and PCT Publication No. WO 96/03159.
238
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
The in situ forming material may be composed of a protein, a surfactant, and a
lipid in a liquid carrier, which is crosslinked by adding a crosslinker and
used as
a sealant or bonding agent in situ. See e.g., U.S. Patent Application No.
2004/0063613A1 and PCT Publication Nos. WO 01/45761 and WO 03/090683.
The in situ forming material may be composed of two enzyme-free liquid
components that are mixed by dispensing the components into a catheter tube
deployed at the vascular puncture site, wherein, upon mixing, the two liquid
components chemically cross-link to form a mechanical non-liquid matrix that
seals a vascular puncture site. See e.g., U.S. Patent Application Nos.
2002/0161399A1 and 2001/0018598A1. The in situ forming material may be a
cross-linked albumin composition composed of an albumin preparation and a
carbodiimide preparation which are mixed under conditions that permit
crosslinking of the albumin for use as a bioadhesive or sealant. See e.g., PCT
Publication No. WO 99/66964. The in situ forming material may be composed
of collagen and a peroxidase and hydrogen peroxide, such that the collagen is
crosslinked to from a semi-solid gel that seals a wound. See e.g., PCT
Publication No. WO 01/35882.
In another aspect, in situ forming materials that can be used
include those based on isocyanate or isothiocyanate capped polymers. For
example, the in situ forming material may be composed of isocyanate-capped
polymers that are liquid compositions which form into a solid adhesive coating
by in situ polymerization and crosslinking upon contact with body fluid or
tissue.
See e.g., PCT Publication No. WO 04/021983. The in situ forming material
may be a moisture-curing sealant composition composed of an active
isocyanato-terminated isocyanate prepolymer containing a polyol component
with a molecular weight of 2,000 to 20,000 and an isocyanurating catalyst
agent. See e.g., U.S. Patent No. 5,206,331.
In another embodiment, the reagents can undergo an
electrophilic-nucleophilic reaction to produce a crosslinked matrix. Polymers
containing and/or terminated with nucleophilic groups such as amine,
239
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
sulfhydryl, hydroxyl, -PH2 or C~-NH-NH2 can be used as the nucleophilic
reagents and polymers containing and/or terminated with electrophilic groups
such as succinimidyl, carboxylic acid, aldehyde, epoxide, isocyanate, vinyl,
vinyl sulfone, maleimide, -S-S-(C5H4N) or activated esters, such as are used
in
peptide synthesis can be used as the electrophilic reagents. For example, a 4-
armed thiol derivatized polyethylene glycol) (e.g., pentaerythritol
polyethylene
glycol)ether tetra-sulfhydryl) can be reacted with a 4 armed NHS-derivatized
polyethylene glycol (e.g., pentaerythritol polyethylene glycol)ether tetra-
succinimidyl glutarate) under basic conditions (pH > about 8). Representative
examples of compositions that undergo such electrophilic-nucleophilic
crosslinking reactions are described, for example, in U.S. Patent. Nos.
5,752,974; 5,807,581; 5,874,500; 5,936,035; 6,051,648; 6,165,489; 6,312,725;
6,458,889; 6,495,127; 6,534,591; 6,624,245; 6,566,406; 6,610,033; 6,632,457;
and PCT Application Publication Nos. WO 04/060405 and WO 04/060346.
In another embodiment, the electrophilic- or nucleophilic-
terminated polymers can further comprise a polymer that can enhance the
mechanical and/or adhesive properties of the in situ forming compositions.
This
polymer can be a degradable or non-degradable polymer. For example, the
polymer may be collagen or a collagen derivative, for example methylated
collagen. An example of an in situ forming composition uses pentaerythritol
polyethylene glycol)ether tetra-sulfhydryl) (4-armed thiol PEG),
pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate) (4-armed NHS PEG) and
methylated collagen as the reactive reagents. This composition, when mixed
with the appropriate buffers can produce a crosslinked hydrogel. (See, e.g.,
U.S. Patent Nos. 5,874,500; 6,051,648; 6,166,130; 5,565,519 and 6,312,725).
In another embodiment, the reagents that can form a covalent
bond with the tissue to which it is applied may be used. Polymers containing
and/or terminated with electrophilic groups such as succinimidyl, aldehyde,
epoxide, isocyanate, vinyl, vinyl sulfone, maleimide, -S-S-(C5H4N) or
activated
esters, such as are used in peptide synthesis may be used as the reagents.
240
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
For example, a 4 armed NHS-derivatized polyethylene glycol (e.g.,
pentaerythritol polyethylene glycol)ether tetra-succinimidyl glutarate) may be
applied to the tissue in the solid form or in a solution form. In the
preferred
embodiment, the 4 armed NHS-derivatized polyethylene glycol is applied to the
tissue under basic conditions (pH > about 8). Other representative examples of
compositions of this nature that may be used are disclosed in PCT Application
Publication No. WO 04/060405 and WO 04/060346, and U.S. Patent
Application No. 10/749,123.
In another embodiment, the in situ forming material polymer can
be a polyester. Polyesters that can be used in in situ forming compositions
include poly(hydroxyesters). In another embodiment, the polyester can
comprise the residues of one or more of the monomers selected from lactide,
lactic acid, glycolide, glycolic acid, e-caprolactone, gamma-caprolactone,
hydroxyvaleric acid, hydroxybutyric acid, beta-bufyrolactone, gamma-
butyrolactone, gamma-valerolactone, y-decanolactone, b-decanolactone,
trimethylene carbonate, 1,4-dioxane-2-one or 1,5-dioxepan-Zone.
Representative examples of these types of compositions are described in U.S.
Patent. Nos. 5,874,500; 5,936,035; 6,312,725; 6,495,127 and PCT Publication
Nos. WO 2004/028547.
In another embodiment, the electrophilic-terminated polymer can
be partially or completely replaced by a small molecule or oligomer that
comprises an electrophilic group (e.g., disuccinimidyl glutarate).
In another embodiment, the nucleophilic-terminated polymer can
be partially or completely replaced by a small molecule or oligomer that
comprises a nucleophilic group (e.g., dicysteine, dilysine, trilysine, etc.).
Other examples of in situ forming materials that can be used
include those based on the crosslinking of proteins (described in, for
example,
U.S. Patent Nos. RE38158; 4,839,345; 5,514,379, 5,583,114; 6,310,036;
6,458,147; 6,371,975; US Patent Application Publication Nos.
2004/0063613A1, 2002/0161399A1, and 2001 /0018598A1, and PCT
241
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Publication Nos. WO 03/090683, WO 01/45761, WO 99/66964, and WO
96/03159) and those based on isocyanate or isothiocyanate capped polymers
(see, e.g., PCT Publication No. WO 04/021983).
Other examples of in situ forming materials can include reagents
that comprise one or more cyanoacrylate groups. These reagents can be used
to prepare a poly(alkylcyanoacrylate) or poly(carboxyalkylcyanoacrylate)
(e.g.,
poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate),
poly(hexylcyanoacrylate), poly(methoxypropylcyanoacrylate), and
poly(octylcyanoacrylate)).
Examples of commercially available cyanoacrylates that can be
used in the present invention include DERMABOND, INDERMIL, GLUSTITCH,
VETBOND, HISTOACRYL, TISSUMEND, HISTOACRYL BLUE and ORABASE
SOOTHE-N-SEAL LIQUID PROTECTANT.
In another embodiment, the cyanoacrylate compositions may
further comprise additives to stabilize the reagents and/or alter the rate of
reaction of the cyanoacrylate, and/or plasticize the poly(cyanoacrylate),
and/or
alter the rate of degradation of the poly(cyanoacrylate). For example, a
trimethylene carbonate based polymer or an oxalate polymer of polyethylene
glycol) or a s-caprolactone based copolymer may be mixed with a 2-
alkoxyalkylcyanoacrylate (e.g., 2-methoxypropylcyanoacrylate). Representative
examples of these compositions are described in U.S. Patent Nos. 5,350,798
and 6,299,631.
In another embodiment, the cyanoacrylate composition can be
prepared by capping heterochain polymers with a cyanoacrylate group. The
cyanoacrylate-capped heterochain polymer preferably has at least two
cyanoacrylate ester groups per chain. The heterochain polymer can comprise
an absorbable poly(ester), polyester-carbonate), poly(ether-carbonate) and
poly(ether-ester). The poly(ether-esters described in U.S. Patent Nos.
5,653,992 and 5,714,159 can also be used as the heterochain polymers. A
triaxial poly(s-caprolactone-co-trimethylene carbonate) is an example of a
242
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polyester-carbonate) that can be used. The heterochain polymer may be a
polyether. Examples of polyethers that can be used include polyethylene
glycol), polypropylene glycol) and block copolymers of polyethylene glycol)
and polypropylene glycol) (e.g., PLURONICS group of polymers including but
not limited to PLURONIC F127 or F63). Representative examples of these
compositions are described in U.S. Patent No. 6,699,940.
Within another aspect of the invention, the biologically active ant-
infective and/or fibrosis-inhibiting agent can be delivered with a non-
polymeric
compound (e.g., a carrier). These non-polymeric carriers can include sucrose
derivatives (e.g., sucrose acetate isobutyrate, sucrose oleate), sterols such
as
cholesterol, stigmasterol, (3-sitosterol, and estradiol; cholesteryl esters
such as
cholesteryl stearate; C~2 -C24 fatty acids such as lauric acid, myristic acid,
palmitic acid, stearic acid, arachidic acid, behenic acid, and lignoceric
acid; C~$
-C36 mono-, di- and triacylglycerides such as glyceryl monooleate, glyceryl
monolinoleate, glyceryl monolaurate, glyceryl monodocosanoate, glyceryl
monomyristate, glyceryl monodicenoate, glyceryl dipalmitate, glyceryl
didocosanoate, glyceryl dimyristate, glyceryl didecenoate, glyceryl
tridocosanoate, glyceryl trimyristate, glyceryl tridecenoate, glycerol
tristearate
and mixtures thereof; sucrose fatty acid esters such as sucrose distearate and
sucrose palmitate; sorbitan fatty acid esters such as sorbitan monostearate,
sorbitan monopalmitate and sorbitan tristearate; C~6 -C~$ fatty alcohols such
as
cetyl alcohol, myristyl alcohol, stearyl alcohol, and cetostearyl alcohol;
esters of
fatty alcohols and fatty acids such as cetyl palmitate and cetearyl palmitate;
anhydrides of fatty acids such as stearic anhydride; phospholipids including
phosphatidylcholine (lecithin), phosphatidylserine, phosphatidylethanolamine,
phosphatidylinositol, and lysoderivatives thereof; sphingosine and derivatives
thereof; spingomyelins such as stearyl, palmitoyl, and tricosanyl
spingomyelins;
ceramides such as stearyl and palmitoyl ceramides; glycosphingolipids; lanolin
and lanolin alcohols, calcium phosphate, sinfiered and unscintered
hydoxyapatite, zeolites; and combinations and mixtures thereof.
243
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Representative examples of patents relating to non-polymeric
delivery systems and the preparation include U.S. Patent Nos. 5,736,152;
5,888,533; 6,120,789; 5,968,542; and 5,747,058.
Within certain embodiments of the invention, the therapeutic
compositions are provided that include (i) a fibrosis-inhibiting agent and/or
(ii)
an anti-infective agent. The therapeutic compositions may include one or more
additional therapeutic agents (such as described above), for example, anti-
inflammatory agents, anti-thrombotic agents, and/ or anti-platelet agents.
Other
agents that may be combined with the therapeutic compositions include, e.g.,
additional ingredients such as surfactants (e.g., PLURONICS, such as F-127,
L-122, L-101, L-92, L-81, and L-61 ), preservatives, anti-oxidants.
In one aspect, the present invention provides compositions
comprising i) an anti-fibrotic agent and ii) a polymer or a compound that
forms a
polymer in situ. The following are some, but by no means all, of the preferred
anti-fibrotic agents and classes of anti-fibrotic agents that may be included
in
the inventive compositions:
1 a. An anti-fibrotic agent that inhibits cell regeneration.
2a. An anti-fibrotic agent that inhibits angiogenesis.
3a. An anti-fibrotic agent that inhibits fibroblast migration.
4a. An anti-fibrotic agent that inhibits fibroblast proliferation.
5a. An anti-fibrotic agent that inhibits deposition of extracellular
matrix.
6a. An anti-fibrotic agent inhibits tissue remodeling.
7a. An anti-fibrotic agent that is an angiogenesis inhibitor.
244
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
8a. An anti-fibrotic agent that is a 5-lipoxygenase inhibitor or
antagonist.
9a. An anti-fibrotic agent that is a chemokine receptor
antagonist.
1 Oa. An anti-fibrotic agent that is a cell cycle inhibitor.
11 a. An anti-fibrotic agent that is a taxane.
12a. An anti-fibrotic agent that is an anti-microtubule agent.
13a. An anti-fibrotic agent that is paclitaxel.
14a. An anti-fibrotic agent that is a cathepsin inhibitor.
15a. An anti-fibrotic agent that is an analogue or derivative of
paclitaxel.
16a. An anti-fibrotic agent that is a vinca alkaloid.
17a. An anti-fibrotic agent that is camptothecin or an analogue
or derivative thereof.
18a. An anti-fibrotic agent that is a podophyllotoxin.
19a. An anti-fibrotic agent that is etoposide or an analogue or
derivative thereof.
20a. An anti-fibrotic agent that is an anthracycline.
21 a. An anti-fibrotic agent that is doxorubicin or an analogue or
derivative thereof.
22a. An anti-fibrotic agent that mitoxantrone or an analogue or
derivative thereof.
245
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
23a. An anti-fibrotic agent that is a platinum compound.
24a. An anti-fibrotic agent that is a nitrosourea.
25a. An anti-fibrotic agent that is a nitroimidazole.
26a. An anti-fibrotic agent that is a folic acid antagonist.
27a. An anti-fibrotic agent that is a cytidine analogue.
28a. An anti-fibrotic agent that is a pyrimidine analogue.
29a. An anti-fibrotic agent that is a fluoropyrimidine analogue.
30a. An anti-fibrotic agent that is a purine analogue.
31 a. An anti-fibrotic agent that is a nitrogen mustard or an
analogue or derivative thereof.
32a. An anti-fibrotic agent that is a hydroxyurea.
33a. An anti-fibrotic agent that is a mytomicin or an analogue or
derivative thereof.
34a. An anti-fibrotic agent that is an alkyl sulfonate.
35a. An anti-fibrotic agent that is a benzamide or an analogue or
derivative thereof.
36a. An anti-fibrotic agent that is a nicotinamide or an analogue
or derivative thereof.
37a. An anti-fibrotic agent that is a halogenated sugar or an
analogue or derivative thereof.
38a. An anti-fibrotic agent that is a DNA alkylating agent.
246
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
39a. An anti-fibrotic agent that is an anti-microtubule agent.
40a. An anti-fibrotic agent that is a topoisomerase inhibitor.
41 a. An anti-fibrotic agent that is a DNA cleaving agent.
42a. An anti-fibrotic agent that is an antimetabolite.
43a. An anti-fibrotic agent inhibits adenosine deaminase.
44a. An anti-fibrotic agent inhibits purine ring synthesis.
45a. An anti-fibrotic agent that is a nucleotide interconversion
inhibitor.
46a. An anti-fibrotic agent inhibits dihydrofolate reduction.
47a. An anti-fibrotic agent blocks thymidine monophosphate.
48a. An anti-fibrotic agent causes DNA damage.
49a. An anti-fibrotic agent that is a DNA intercalation agent.
50a. An anti-fibrotic agent that is a RNA synthesis inhibitor.
51 a. An anti-fibrotic agent that is a pyrimidine synthesis inhibitor.
52a. An anti-fibrotic agent that inhibits ribonucleotide synthesis
or function.
53a. An anti-fibrotic agent that inhibits thymidine
monophosphate synthesis or function.
54a. An anti-fibrotic agent that inhibits DNA synthesis.
55a. An anti-fibrotic agent that causes DNA adduct formation.
56a. An anti-fibrotic agent that inhibits protein synthesis.
247
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
57a. An anti-fibrotic agent that inhibits microtubule function.
58a. - An anti-fibrotic agent that is a cyclin dependent protein
kinase inhibitor.
59a. An anti-fibrotic agent that is an epidermal growth factor
kinase inhibitor.
60a. An anti-fibrotic agent that is an elastase inhibitor.
61 a. An anti-fibrotic agent that is a factor Xa inhibitor.
62a. An anti-fibrotic agent that is a farnesyltransferase inhibitor.
63a. An anti-fibrotic agent that is a fibrinogen antagonist.
64a. An anti-fibrotic agent that is a guanylate cyclase stimulant.
65a. An anti-fibrotic agent that is a heat shock protein 90
antagonist.
66a. An anti-fibrotic agent that is geldanamycin or an analogue
or derivative thereof.
67a. An anti-fibrotic agent that is a guanylate cyclase stimulant.
68a. An anti-fibrotic agent that is a HMGCoA reductase inhibitor.
69a. An anti-fibrotic agent that is simvastatin or an analogue or
derivative thereof.
70a. An anti-fibrotic agent that is a hydroorotate dehydrogenase
inhibitor.
71 a. An anti-fibrotic agent that is an IKK2 inhibitor.
72a. An anti-fibrotic agent that is an IL-1 antagonist.
248
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
73a. An anti-fibrotic agent that is an ICE antagonist.
74a. An anti-fibrotic agent that is an IRAK antagonist.
75a. An anti-fibrotic agent that is an IL-4 agonist.
76a. An anti-fibrotic agent that is an immunomodulatory agent.
77a. An anti-fibrotic agent that is sirolimus or an analogue or
derivative thereof.
78a. An anti-fibrotic agent that is a nitric oxide inhibitor.
79a. An anti-fibrotic agent that is everolimus or an analogue or
derivative thereof.
80a. An anti-fibrotic agent that is tacrolimus or an analogue or
derivative thereof.
81a. An anti-fibrotic agent that is a TNF alpha inhibitor.
82a. An anti-fibrotic agent that is biolmus or an analogue or
derivative thereof.
83a. An anti-fibrotic agent that is tresperimus or an analogue or
derivative thereof.
84a. An anti-fibrotic agent that is auranofin or an analogue or
derivative thereof.
85a. An anti-fibrotic agent that is 27-0-demethylrapamycin or an
analogue or derivative thereof.
86a. An anti-fibrotic agent that is gusperimus or an analogue or
derivative thereof.
249
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
87a. An anti-fibrotic agent that is pimecrolimus or an analogue
or derivative thereof.
88a. An anti-fibrotic agent that is ABT-578 or an analogue or
derivative thereof.
89a. An anti-fibrotic agent that is an inosine monophosphate
dehydrogenase (IMPDH) inhibitor.
90a. An anti-fibrotic agent that is mycophenolic acid or an
analogue or derivative thereof.
91 a. An anti-fibrotic agent that is 1-alpha-25 dihydroxy vitamin
D3 or an analogue or derivative thereof.
92a. An anti-fibrotic agent that is a leukotriene inhibitor.
93a. An anti-fibrotic agent that is a MCP-1 antagonist.
94a. An anti-fibrotic agent that is a MMP inhibitor.
95a. An anti-fibrotic agent that is an NF kappa B inhibitor.
96a. An anti-fibrotic agent that is an NF kappa B inhibitor,
wherein the NF kappa B inhibitor is Bay 11-7082.
97a. An anti-fibrotic agent that is an NO antagonist.
98a. An anti-fibrotic agent that is a p38 MAP kinase inhibitor.
99a. An anti-fibrotic agent that is a p38 MAP kinase inhibitor,
wherein the p38 MAP kinase inhibitor is SB 202190.
100a. An anti-fibrotic agent that is a phosphodiesterase inhibitor.
101 a. An anti-fibrotic agent that is a TGF beta inhibitor.
250
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
102x. An anti-fibrotic agent that is a thromboxane A2 antagonist.
103a. An anti-fibrotic agent that is a TNF alpha antagonist.
104a. An anti-fibrotic agent that is a TACE inhibitor.
105a. An anti-fibrotic agent that is a tyrosine kinase inhibitor.
106a. An anti-fibrotic agent that is a vitronectin inhibitor.
107a. An anti-fibrotic agent that is a fibroblast growth factor
inhibitor.
108a. An anti-fibrotic agent that is a protein kinase inhibitor.
109a. An anti-fibrotic agent that is a PDGF receptor kinase
inhibitor.
110a. An anti-fibrotic agent that is an endothelial growth factor
receptor kinase inhibitor.
111 a. An anti-fibrotic agent that is a retinoic acid receptor
antagonist.
112a. An anti-fibrotic agent that is a platelet derived growth factor
receptor kinase inhibitor.
113a. An anti-fibrotic agent that is a fibrinogen antagonist.
114a. An anti-fibrotic agent that is an antimycotic agent.
115a. An anti-fibrotic agent that is an antimycotic agent, wherein
the antimycotic agent that is sulconizole.
116a. An anti-fibrotic agent that is a bisphosphonate.
117a. An anti-fibrotic agent that is a phospholipase A1 inhibitor.
251
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
118a. An anti-fibrotic agent that is a histamine H1/H2/H3 receptor
antagonist.
119a. An anti-fibrotic agent that is a macrolide antibiotic.
120a. An anti-fibrotic agent that is a GPllb/Illa receptor
antagonist.
121 a. An anti-fibrotic agent that is an endothelin receptor
antagonist.
122a. An anti-fibrotic agent that is a peroxisome proliferator-
activated receptor agonist.
123a. An anti-fibrotic agent that is an estrogen receptor agent.
124a. An anti-fibrotic agent that is a somastostatin analogue.
125a. An anti-fibrotic agent that is a neurokinin 1 antagonist.
126a. An anti-fibrotic agent that is a neurokinin 3 antagonist.
127a. An anti-fibrotic agent that is a VLA-4 antagonist.
128a. An anti-fibrotic agent that is an osteoclast inhibitor.
129a. An anti-fibrotic agent that is a DNA topoisomerase ATP
hydrolyzing inhibitor.
130a. An anti-fibrotic agent that is an angiotensin I converting
enzyme inhibitor.
131 a. An anti-fibrotic agent that is an angiotensin II antagonist.
132a. An anti-fibrotic agent that is an enkephalinase inhibitor.
252
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
133a. An anti-fibrotic agent that is a peroxisome proliferator-
activated receptor gamma agonist insulin sensitizer.
134a. An anti-fibrotic agent that is a protein kinase C inhibitor.
135a. An anti-fibrotic agent that is a ROCK (rho-associated
kinase) inhibitor.
136a. An anti-fibrotic agent that is a CXCR3 inhibitor.
137a. An anti-fibrotic agent that is an Itk inhibitor.
138a. An anti-fibrotic agent that is a cytosolic phospholipase A2-
alpha inhibitor.
139a. An anti-fibrotic agent that is a PPAR agonist.
140a. An anti-fibrotic agent that is an immunosuppressant.
141 a. An anti-fibrotic agent that is an Erb inhibitor.
142a. An anti-fibrotic agent that is an apoptosis agonist.
143a. An anti-fibrotic agent that is a lipocortin agonist.
144a. An anti-fibrotic agent that is a VCAM-1 antagonist.
145a. An anti-fibrotic agent that is a collagen antagonist.
As mentioned above, the present invention provides compositions
comprising each of the foregoing 146 (i.e., 1 a through 145a) listed anti-
fibrotic
agents or classes of anti-fibrotic agents, with each of the following 98
(i.e., 1 b
through 97b) polymers and compounds:
1 b. A crosslinked polymer.
2b. A polymer that reacts with mammalian tissue.
253
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
3b. A polymer that is a naturally occurring
polymer.
4b. A polymer that is a protein.
5b. A polymer that is a carbohydrate.
6b. A polymer that is biodegradable.
7b. A polymer that is crosslinked and
biodegradable.
8b. A polymer that nonbiodegradable.
9b. Collagen.
10b. Methylated collagen.
11 b. Fibrinogen.
12b. Thrombin.
13b. Albumin.
14b. Plasminogen.
15b. von Willebrands factor.
16b. Factor VIII.
17b. Hypoallergenic collagen.
18b. Atelopeptidic collagen.
19b. Telopeptide collagen.
20b. Crosslinked collagen.
21 b. Aprotinin.
22b. Gelatin.
23b. A protein conjugate.
254
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
24b. A gelatin conjugate.
25b. Hyaluronic acid.
26b. A hyaluronic acid derivative.
27b. A synthetic polymer.
28b. A polymer formed from reactants comprising a synthetic
isocyanate-containing compound.
29b. A synthetic isocyanate-containing compound.
30b. A polymer formed from reactants comprising a synthetic
thiol-containing compound.
31 b. A synthetic thiol-containing compound.
32b. A polymer formed from reactants comprising a synthetic
compound containing at least two thiol groups.
33b. A synthetic compound containing at least two thiol groups.
34b. A polymer formed from reactants comprising a synthetic
compound containing at least three thiol groups.
35b. A synthetic compound containing at least three thiol
groups.
36b. A polymer formed from reactants comprising a synthetic
compound containing at least four thiol groups.
37b. A synthetic compound containing at least four thiol groups.
38b. A polymer formed from reactants comprising a synthetic
amino-containing compound.
39b. A synthetic amino-containing compound.
255
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
40b. A polymer formed from reactants comprising a synthetic
compound containing at least two amino groups.
41 b. A synthetic compound containing at least two amino
groups.
42b. A polymer formed from reactants comprising a synthetic
compound containing at least three amino groups.
43b. A synthetic compound containing at least three amino
groups.
44b. A polymer formed from reactants comprising a synthetic
compound containing at least four amino groups.
45b. A synthetic compound containing at least four amino
groups.
46b. A polymer formed from reactants comprising a synthetic
compound comprising a carbonyl-oxygen-succinimidyl group.
47b. A synthetic compound comprising a carbonyl-oxygen-
succinimidyl group.
48b. A polymer formed from reactants comprising a synthetic
compound comprising at least two carbonyl-oxygen-succinimidyl groups.
49b. A synthetic compound comprising at least two carbonyl-
oxygen-succinimidyl groups.
50b. A polymer formed from reactants comprising a synthetic
compound comprising at least three carbonyl-oxygen-succinimidyl groups.
51 b. A synthetic compound comprising at least three carbonyl-
oxygen-succinimidyl groups.
52b. A polymer formed from reactants comprising a synthetic
compound comprising at least four carbonyl-oxygen-succinimidyl groups.
256
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
53b. A synthetic compound comprising at least four carbonyl-
oxygen-succinimidyl groups.
54b. A polymer formed from from reactants comprising a
synthetic polyalkylene oxide-containing compound.
55b. A synthetic polyalkylene oxide-containing compound.
56b. A polymer formed from reactants comprising a synthetic
compound comprising both polyalkylene oxide and biodegradable polyester
blocks.
57b. A synthetic compound comprising both polyalkylene oxide
and biodegradable polyester blocks.
58b. A polymer formed from reactants comprising a synthetic
polyalkylene oxide-containing compound having reactive amino groups.
59b. A synthetic polyalkylene oxide-containing compound
having reactive amino groups.
60b. A polymer formed from reactants comprising a synthetic
polyalkylene oxide-containing compound having reactive thiol groups.
61 b. A synthetic polyalkylene oxide-containing compound
having reactive thiol groups.
62b. A polymer formed from reactants comprising a synthetic
polyalkylene oxide-containing compound having reactive carbonyl-oxygen-
succinimidyl groups.
63b. A synthetic polyalkylene oxide-containing compound
having reactive carbonyl-oxygen-succinimidyl groups.
64b. A polymer formed from reactants comprising a synthetic
compound comprising a biodegradable polyester block.
257
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
65b. A synthetic compound comprising a biodegradable
polyester block.
66b. A polymer formed from reactants comprising a synthetic
polymer formed in whole or part from lactic acid or lactide.
67b. A synthetic polymer formed in whole or part from lactic acid
or lactide.
68b. A polymer formed from reactants comprising a synthetic
polymer formed in whole or part from glycolic acid or glycolide.
69b. A synthetic polymer formed in whole or part from glycolic
acid or glycolide.
70b. A polymer formed from reactants comprising polylysine.
71 b. Polylysine.
72b. A polymer formed from reactants comprising (a) protein
and (b) a compound comprising a polyalkylene oxide portion.
73b. A polymer formed from reactants comprising (a) protein
and (b) polylysine.
74b. A polymer formed from reactants comprising (a) protein
and (b) a compound having at least four thiol groups.
75b. A polymer formed from reactants comprising (a) protein
and (b) a compound having at least four amino groups.
76b. A polymer formed from reactants comprising (a) protein
and (b) a compound having at least four carbonyl-oxygen-succinimide groups.
77b. A polymer formed from reactants comprising (a) protein
and (b) a compound having a biodegradable region formed from reactants
selected from lactic acid, lactide, glycolic acid, glycolide, and epsilon-
caprolactone.
258
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
78b. A polymer formed from reactants comprising (a) collagen
and (b) a compound comprising a polyalkylene oxide portion.
79b. A polymer formed from reactants comprising (a) collagen
and (b) polylysine.
80b. A polymer formed from reactants comprising (a) collagen
and (b) a compound having at least four thiol groups.
81 b. A polymer formed from reactants comprising (a) collagen
and (b) a compound having at least four amino groups.
82b. A polymer formed from reactants comprising (a) collagen
and (b) a compound having at least four carbonyl-oxygen-succinimide groups.
83b. A polymer formed from reactants comprising (a) collagen
and (b) a compound having a biodegradable region formed from reactants
selected from lactic acid, lactide, glycolic acid, glycolide, and epsilon-
caprolactone.
84b. A polymer formed from reactants comprising (a)
methylated collagen and (b) a compound comprising a polyalkylene oxide
portion.
85b. A polymer formed from reactants comprising (a)
methylated collagen and (b) polylysine.
86b. A polymer formed from reactants comprising (a)
methylated collagen and (b) a compound having at least four thiol groups.
87b. A polymer formed from reactants comprising (a)
methylated collagen and (b) a compound having at least four amino groups.
88b. A polymer formed from reactants comprising (a)
methylated collagen and (b) a compound having at least four carbonyl-oxygen-
succinimide groups.
259
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
89b. A polymer formed from reactants comprising (a)
methylated collagen and (b) a compound having a biodegradable region formed
from reactants selected from lactic acid, lactide, glycolic acid, glycolide,
and
epsilon-caprolactone.
90b. A polymer formed from reactants comprising hyaluronic
acid.
91 b. A polymer formed from reactants comprising a hyaluronic
acid derivative.
92b. A polymer formed from reactants comprising pentaerythritol
polyethylene glycol)ether tetra-sulfhydryl of number average molecular weight
between 3,000 and 30,000.
93b. Pentaerythritol polyethylene glycol)ether tetra-sulfhydryl of
number average molecular weight between 3,000 and 30,000.
94b. A polymer formed from reactants comprising pentaerythritol
polyethylene glycol)ether tetra-amino of number average molecular weight
between 3,000 and 30,000.
95b. Pentaerythritol polyethylene glycol)ether tetra-amino of
number average molecular weight between 3,000 and 30,000.
96b. A polymer formed from reactants comprising (a) a synthetic
compound having a number average molecular weight between 3,000 and
30,000 and comprising a polyalkylene oxide region and multiple nucleophilic
groups, and (b) a synthetic compound having a number average molecular
weight between 3,000 and 30,000 and comprising a polyalkylene oxide region
and multiple electrophilic groups.
97b. A mixture of (a) a synthetic compound having a number
average molecular weight between 3,000 and 30,000 and comprising a
polyalkylene oxide region and multiple nucleophilic groups, and (b) a
synthetic
compound having a number average molecular weight between 3,000 and
260
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
30,000 and comprising a polyalleylene oxide region and multiple electrophilic
groups.
As mentioned above, the present invention provides compositions
comprising each of the foregoing 146 (1 a through 145a) listed anti-fibrotic
agents or classes of anti-fibrotic agents, with each of the foregoing 98 (1 b
through 97b) polymers and compounds: Thus, in separate aspects, the
invention provides 146 times 98 = 14,308 described compositions. In other
words, each of the following is a distinct aspect of the present invention:
1 a+1 b; 1 a + 2b; 1 a + 3b; 1 a+4b; 1 a+5b; 1 a+6b; 1 a+7b; 1 a+8b; 1 a+9b; 1
a+1 Ob;
1 a+11 b; 1 a+12b; 1 a+13b; 1 a+14b; 1 a+15b; 1 a+16b; 1 a+17b; 1 a+18b; 1
a+19b;
1 a+20b; 1 a+21 b; 1 a+22b; 1 a+23b; 1 a+24b; 1 a+25b; 1 a+26b; 1 a+27b; 1
a+28b;
1 a+29b; 1 a+30b; 1 a+31 b; 1 a+32b; 1 a+33b; 1 a+34b; 1 a+35b; 1 a+36b; 1
a+37b;
1 a+38b; 1 a+39b; 1 a+40b; 1 a+41 b; 1 a+42b; 1 a+43b; 1 a+44b; 1 a+45b; 1
a+46b;
1 a+47b; 1 a+48b; 1 a+49b; 1 a+50b; 1 a+51 b; 1 a+52b; 1 a+53b; 1 a+54b; 1
a+55b;
1 a+55b; 1 a+57b; 1 a+58b; 1 a+59b; 1 a+60b; 1 a+61 b; 1 a+62b; 1 a+63b; 1
a+64b;
1 a+65b; 1 a+66b; 1 a+67b; 1 a+68b; 1 a+69b; 1 a+70b; 1 a+71 b; 1 a+72b; 1
a+73b;
1 a+74b; 1 a+75b; 1 a+76b; 1 a+77b; 1 a+78b; 1 a+79b; 1 a+80b; 1 a+81 b; 1
a+82b;
1 a+83b; 1 a+84b; 1 a+85b; 1 a+86b; 1 a+87b; 1 a+88b; 1 a+89b; 1 a+90b; 1 a+91
b;
1 a+92b; 1 a+93b; 1 a+94b; 1 a+95b; 1 a+96b; 1 a+97b; 2a+1 b; 2a + 2b; 2a +
3b;
2a+4b; 2a+5b; 2a+6b; 2a+7b; 2a+8b; 2a+9b; 2a+1 Ob; 2a+11 b; 2a+12b;
2a+13b; 2a+14b; 2a+15b; 2a+16b; 2a+17b; 2a+18b; 2a+19b; 2a+20b; 2a+21 b;
2a+22b; 2a+23b; 2a+24b; 2a+25b; 2a+26b; 2a+27b; 2a+28b; 2a+29b; 2a+30b;
2a+31 b; 2a+32b; 2a+33b; 2a+34b; 2a+35b; 2a+36b; 2a+37b; 2a+38b; 2a+39b;
2a+40b; 2a+41 b; 2a+42b; 2a+43b; 2a+44b; 2a+45b; 2a+46b; 2a+47b; 2a+48b;
2a+49b; 2a+50b; 2a+51 b; 2a+52b; 2a+53b; 2a+54b; 2a+55b; 2a+55b; 2a+57b;
2a+58b; 2a+59b; 2a+60b; 2a+61 b; 2a+62b; 2a+63b; 2a+64b; 2a+65b; 2a+66b;
2a+67b; 2a+68b; 2a+69b; 2a+70b; 2a+71 b; 2a+72b; 2a+73b; 2a+74b; 2a+75b;
2a+76b; 2a+77b; 2a+78b; 2a+79b; 2a+80b; 2a+81 b; 2a+82b; 2a+83b; 2a+84b;
2a+85b; 2a+86b; 2a+87b; 2a+88b; 2a+89b; 2a+90b; 2a+91 b; 2a+92b; 2a+93b;
2a+94b; 2a+95b; 2a+96b; 2a+97b; 3a+22b; 3a+23b; 3a+24b; 3a+25b; 3a+26b;
261
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
3a+27b; 3a+28b; 3a+29b; 3a+30b; 3a+31 b; 3a+32b; 3a+33b; 3a+34b; 3a+35b;
3a+36b; 3a+37b; 3a+38b; 3a+39b; 3a+40b; 3a+41 b; 3a+42b; 3a+43b; 3a+44b;
3a+45b; 3a+46b; 3a+47b; 3a+48b; 3a+49b; 3a+50b; 3a+51 b; 3a+52b; 3a+53b;
3a+54b; 3a+55b; 3a+55b; 3a+57b; 3a+58b; 3a+59b; 3a+60b; 3a+61 b; 3a+62b;
3a+63b; 3a+64b; 3a+65b; 3a+66b; 3a+67b; 3a+68b; 3a+69b; 3a+70b; 3a+71 b;
3a+72b; 3a+73b; 3a+74b; 3a+75b; 3a+76b; 3a+77b; 3a+78b; 3a+79b; 3a+80b;
3a+81 b; 3a+82b; 3a+83b; 3a+84b; 3a+85b; 3a+86b; 3a+87b; 3a+88b; 3a+89b;
3a+90b; 3a+91 b; 3a+92b; 3a+93b; 3a+94b; 3a+95b; 3a+96b; 3a+97b; 4a+12b;
4a+13b; 4a+14b; 4a+15b; 4a+16b; 4a+17b; 4a+18b; 4a+19b; 4a+20b; 4a+21 b;
4a+22b; 4a+23b; 4a+24b; 4a+25b; 4a+26b; 4a+27b; 4a+28b; 4a+29b; 4a+30b;
4a+31 b; 4a+32b; 4a+33b; 4a+34b; 4a+35b; 4a+36b; 4a+37b; 4a+38b; 4a+39b;
4a+40b; 4a+41 b; 4a+42b; 4a+43b; 4a+44b; 4a+45b; 4a+46b; 4a+47b; 4a+48b;
4a+49b; 4a+50b; 4a+51 b; 4a+52b; 4a+53b; 4a+54b; 4a+55b; 4a+55b; 4a+57b;
4a+58b; 4a+59b; 4a+60b; 4a+61 b; 4a+62b; 4a+63b; 4a+64b; 4a+65b; 4a+66b;
4a+67b; 4a+68b; 4a+69b; 4a+70b; 4a+71 b; 4a+72b; 4a+73b; 4a+74b; 4a+75b;
4a+76b; 4a+77b; 4a+78b; 4a+79b; 4a+80b; 4a+81 b; 4a+82b; 4a+83b; 4a+84b;
4a+85b; 4a+86b; 4a+87b; 4a+88b; 4a+89b; 4a+90b; 4a+91 b; 4a+92b; 4a+93b;
4a+94b; 4a+95b; 4a+96b; 4a+97b; 5a+12b; 5a+13b; 5a+14b; 5a+15b; 5a+16b;
5a+17b; 5a+18b; 5a+19b; 5a+20b; 5a+21 b; 5a+22b; 5a+23b; 5a+24b; 5a+25b;
5a+26b; 5a+27b; 5a+28b; 5a+29b; 5a+30b; 5a+31 b; 5a+32b; 5a+33b; 5a+34b;
5a+35b; 5a+36b; 5a+37b; 5a+38b; 5a+39b; 5a+40b; 5a+41 b; 5a+42b; 5a+43b;
5a+44b; 5a+45b; 5a+46b; 5a+47b; 5a+48b; 5a+49b; 5a+50b; 5a+51 b; 5a+52b;
5a+53b; 5a+54b; 5a+55b; 5a+55b; 5a+57b; 5a+58b; 5a+59b; 5a+60b; 5a+61 b;
5a+62b; 5a+63b; 5a+64b; 5a+65b; 5a+66b; 5a+67b; 5a+68b; 5a+69b; 5a+70b;
5a+71 b; 5a+72b; 5a+73b; 5a+74b; 5a+75b; 5a+76b; 5a+77b; 5a+78b; 5a+79b;
5a+80b; 5a+81 b; 5a+82b; 5a+83b; 5a+84b; 5a+85b; 5a+86b; 5a+87b; 5a+88b;
5a+89b; 5a+90b; 5a+91 b; 5a+92b; 5a+93b; 5a+94b; 5a+95b; 5a+96b; 5a+97b;
6a+1 b; 6a + 2b; 6a + 3b; 6a+4b; 6a+5b; 6a+6b; 6a+7b; 6a+8b; 6a+9b; 6a+1 Ob;
6a+11 b; 6a+12b; 6a+13b; 6a+14b; 6a+15b; 6a+16b; 6a+17b; 6a+18b; 6a+19b;
6a+20b; 6a+21 b; 6a+22b; 6a+23b; 6a+24b; 6a+25b; 6a+26b; 6a+27b; 6a+28b;
262
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
6a+29b; 6a+30b; 6a+31 b; 6a+32b; 6a+33b; 6a+34b; 6a+35b; 6a+36b; 6a+37b;
6a+38b; 6a+39b; 6a+40b; 6a+41 b; 6a+42b; 6a+43b; 6a+44b; 6a+45b; 6a+46b;
6a+47b; 6a+48b; 6a+49b; 6a+50b; 6a+51 b; 6a+52b; 6a+53b; 6a+54b; 6a+55b;
6a+55b; 6a+57b; 6a+58b; 6a+59b; 6a+60b; 6a+61 b; 6a+62b; 6a+63b; 6a+64b;
6a+65b; 6a+66b; 6a+67b; 6a+68b; 6a+69b; 6a+70b; 6a+71 b; 6a+72b; 6a+73b;
6a+74b; 6a+75b; 6a+76b; 6a+77b; 6a+78b; 6a+79b; 6a+80b; 6a+81 b; 6a+82b;
6a+83b; 6a+84b; 6a+85b; 6a+86b; 6a+87b; 6a+88b; 6a+89b; 6a+90b; 6a+91 b;
6a+92b; 6a+93b; 6a+94b; 6a+95b; 6a+96b; 6a+97b; 7a+1 b; 7a + 2b; 7a + 3b;
7a+4b; 7a+5b; 7a+6b; 7a+7b; 7a+8b; 7a+9b; 7a+1 Ob; 7a+11 b; 7a+12b;
7a+13b; 7a+14b; 7a+15b; 7a+16b; 7a+17b; 7a+18b; 7a+19b; 7a+20b; 7a+21 b;
7a+22b; 7a+23b; 7a+24b; 7a+25b; 7a+26b; 7a+27b; 7a+28b; 7a+29b; 7a+30b;
7a+31 b; 7a+32b; 7a+33b; 7a+34b; 7a+35b; 7a+36b; 7a+37b; 7a+38b; 7a+39b;
7a+40b; 7a+41 b; 7a+42b; 7a+43b; 7a+44b; 7a+45b; 7a+46b; 7a+47b; 7a+48b;
7a+49b; 7a+50b; 7a+51 b; 7a+52b; 7a+53b; 7a+54b; 7a+55b; 7a+55b; 7a+57b;
7a+58b; 7a+59b; 7a+60b; 7a+61 b; 7a+62b; 7a+63b; 7a+64b; 7a+65b; 7a+66b;
7a+67b; 7a+68b; 7a+69b; 7a+70b; 7a+71 b; 7a+72b; 7a+73b; 7a+74b; 7a+75b;
7a+76b; 7a+77b; 7a+78b; 7a+79b; 7a+80b; 7a+81 b; 7a+82b; 7a+83b; 7a+84b;
7a+85b; 7a+86b; 7a+87b; 7a+88b; 7a+89b; 7a+90b; 7a+91 b; 7a+92b; 7a+93b;
7a+94b; 7a+95b; 7a+96b; 7a+97b; 8a+12b; 8a+13b; 8a+14b; 8a+15b; 8a+16b;
8a+17b; 8a+18b; 8a+19b; 8a+20b; 8a+21 b; 8a+22b; 8a+23b; 8a+24b; 8a+25b;
8a+26b; 8a+27b; 8a+28b; 8a+29b; 8a+30b; 8a+31 b; 8a+32b; 8a+33b; 8a+34b;
8a+35b; 8a+36b; 8a+37b; 8a+38b; 8a+39b; 8a+40b; 8a+41 b; 8a+42b; 8a+43b;
8a+44b; 8a+45b; 8a+46b; 8a+47b; 8a+48b; 8a+49b; 8a+50b; 8a+51 b; 8a+52b;
8a+53b; 8a+54b; 8a+55b; 8a+55b; 8a+57b; 8a+58b; 8a+59b; 8a+60b; 8a+61 b;
8a+62b; 8a+63b; 8a+64b; 8a+65b; 8a+66b; 8a+67b; 8a+68b; 8a+69b; 8a+70b;
8a+71 b; 8a+72b; 8a+73b; 8a+74b; 8a+75b; 8a+76b; 8a+77b; 8a+78b; 8a+79b;
8a+80b; 8a+81 b; 8a+82b; 8a+83b; 8a+84b; 8a+85b; 8a+86b; 8a+87b; 8a+88b;
8a+89b; 8a+90b; 8a+91 b; 8a+92b; 8a+93b; 8a+94b; 8a+95b; 8a+96b; 8a+97b;
9a+1 b; 9a + 2b; 9a + 3b; 9a+4b; 9a+5b; 9a+6b; 9a+7b; 9a+8b; 9a+9b; 9a+1 Ob;
9a+11 b; 9a+12b; 9a+13b; 9a+14b; 9a+15b; 9a+16b; 9a+17b; 9a+18b; 9a+19b;
263
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
9a+20b; 9a+21 b; 9a+22b; 9a+23b; 9a+24b; 9a+25b; 9a+26b; 9a+27b; 9a+28b;
9a+29b; 9a+30b; 9a+31 b; 9a+32b; 9a+33b; 9a+34b; 9a+35b; 9a+36b; 9a+37b;
9a+38b; 9a+39b; 9a+40b; 9a+41 b; 9a+42b; 9a+43b; 9a+44b; 9a+45b; 9a+46b;
9a+47b; 9a+48b; 9a+49b; 9a+50b; 9a+51 b; 9a+52b; 9a+53b; 9a+54b; 9a+55b;
9a+55b; 9a+57b; 9a+58b; 9a+59b; 9a+60b; 9a+61 b; 9a+62b; 9a+63b; 9a+64b;
9a+65b; 9a+66b; 9a+67b; 9a+68b; 9a+69b; 9a+70b; 9a+71 b; 9a+72b; 9a+73b;
9a+74b; 9a+75b; 9a+76b; 9a+77b; 9a+78b; 9a+79b; 9a+80b; 9a+81 b; 9a+82b;
9a+83b; 9a+84b; 9a+85b; 9a+86b; 9a+87b; 9a+88b; 9a+89b; 9a+90b; 9a+91 b;
9a+92b; 9a+93b; 9a+94b; 9a+95b; 9a+96b; 9a+97b; 1 Oa+1 b; 1 Oa + 2b; 1 Oa +
3b; 1 Oa+4b; 10a+5b; 10a+6b; 10a+7b; 1 Oa+8b; 10a+9b; 10a+1 Ob; 1 Oa+11 b;
10a+12b; 10a+13b; 10a+14b; 10a+15b; 10a+16b; 10a+17b; 10a+18b;
10a+19b; 1 Oa+20b; 10a+21 b; 1 Oa+22b; 10a+23b; 1 Oa+24b; 10a+25b;
1 Oa+26b; 10a+27b; 1 Oa+28b; 10a+29b; 10a+30b; 10a+31 b; 10a+32b;
10a+33b;10a+34b;10a+35b;10a+36b;10a+37b;10a+38b;10a+39b;
10a+40b; 1 Oa+41 b; 1 Oa+42b; 1 Oa+43b; 10a+44b; 10a+45b; 10a+46b;
1 Oa+47b; 1 Oa+48b; 1 Oa+49b; 1 Oa+50b; 1 Oa+51 b; 1 Oa+52b; 1 Oa+53b;
10a+54b; 10a+55b; 10a+55b; 10a+57b; 10a+58b; 10a+59b; 10a+60b;
1 Oa+61 b; 1 Oa+62b; 10a+63b; 1 Oa+64b; 10a+65b; 1 Oa+66b; 1 Oa+67b;
1 Oa+68b; 10a+69b; 1 Oa+70b; 10a+71 b; 10a+72b; 10a+73b; 10a+74b;
10a+75b; 1 Oa+76b; 10a+77b; 1 Oa+78b; 10a+79b; 10a+80b; 1 Oa+81 b;
10a+82b; 10a+83b; 10a+84b; 10a+85b; 10a+86b; 10a+87b; 10a+88b;
1 Oa+89b; 10a+90b; 1 Oa+91 b; 10a+92b; 1 Oa+93b; 1 Oa+94b; 10a+95b;
1 Oa+96b; 1 Oa+97b; 11 a+1 b; 11 a + 2b; 11 a + 3b; 11 a+4b; 11 a+5b; 11 a+6b;
11 a+7b; 11 a+8b; 11 a+9b; 11 a+1 Ob; 11 a+11 b; 11 a+12b; 11 a+13b; 11 a+14b;
11 a+15b; 11 a+16b; 11 a+17b; 11 a+18b; 11 a+19b; 11 a+20b; 11 a+21 b;
11 a+22b; 11 a+23b; 11 a+24b; 11 a+25b; 11 a+26b; 11 a+27b; 11 a+28b;
11 a+29b; 11 a+30b; 11 a+31 b; 11 a+32b; 11 a+33b; 11 a+34b; 11 a+35b;
11 a+36b; 11 a+37b; 11 a+38b; 11 a+39b; 11 a+40b; 11 a+41 b; 11 a+42b;
11 a+43b; 11 a+44b; 11 a+45b; 11 a+46b; 11 a+47b; 11 a+48b; 11 a+49b;
11 a+50b; 11 a+51 b; 11 a+52b; 11 a+53b; 11 a+54b; 11 a+55b; 11 a+55b;
264
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
11 a+57b; 11 a+58b; 11 a+59b; 11 a+60b; 11 a+61 b; 11 a+62b; 11 a+63b;
11a+64b;11a+65b;11a+66b;11a+67b;11a+68b;11a+69b;11a+70b;
11 a+71 b; 11 a+72b; 11 a+73b; 11 a+74b; 11 a+75b; 11 a+76b; 11 a+77b;
11 a+78b; 11 a+79b; 11 a+80b; 11 a+81 b; 11 a+82b; 11 a+83b; 11 a+84b;
11 a+85b; 11 a+86b; 11 a+87b; 11 a+88b; 11 a+89b; 11 a+90b; 11 a+91 b;
11 a+92b; 11 a+93b; 11 a+94b; 11 a+95b; 11 a+96b; 11 a+97b; 12a+1 b; 12a + 2b;
12a + 3b; 12a+4b; 12a+5b; 12a+6b; 12a+7b; 12a+8b; 12a+9b; 12a+10b;
12a+11 b; 12a+12b; 12a+13b; 12a+14b; 12a+15b; 12a+16b; 12a+17b;
12a+18b; 12a+19b; 12a+20b; 12a+21 b; 12a+22b; 12a+23b; 12a+24b;
12a+25b; 12a+26b; 12a+27b; 12a+28b; 12a+29b; 12a+30b; 12a+31 b;
12a+32b;12a+33b;12a+34b;12a+35b;12a+36b;12a+37b;12a+38b;
12a+39b; 12a+40b; 12a+41 b; 12a+42b; 12a+43b; 12a+44b; 12a+45b;
12a+46b; 12a+47b; 12a+48b; 12a+49b; 12a+50b; 12a+51 b; 12a+52b;
12a+53b;12a+54b;12a+55b;12a+55b;12a+57b;12a+58b;12a+59b;
12a+60b; 12a+61 b; 12a+62b; 12a+63b; 12a+64b; 12a+65b; 12a+66b;
12a+67b; 12a+68b; 12a+69b; 12a+70b; 12a+71 b; 12a+72b; 12a+73b;
12a+74b;12a+75b;12a+76b;12a+77b;12a+78b;12a+79b;12a+80b;
12a+81 b; 12a+82b; 12a+83b; 12a+84b; 12a+85b; 12a+86b; 12a+87b;
12a+88b; 12a+89b; 12a+90b; 12a+91 b; 12a+92b; 12a+93b; 12a+94b;
12a+95b; 12a+96b; 12a+97b; 13a+1 b; 13a + 2b; 13a + 3b; 13a+4b; 13a+5b;
13a+6b; 13a+7b; 13a+8b; 13a+9b; 13a+10b; 13a+11 b; 13a+12b; 13a+13b;
13a+14b; 13a+15b; 13a+16b; 13a+17b; 13a+18b; 13a+19b; 13a+20b;
13a+21 b; 13a+22b; 13a+23b; 13a+24b; 13a+25b; 13a+26b; 13a+27b;
13a+28b; 13a+29b; 13a+30b; 13a+31 b; 13a+32b; 13a+33b; 13a+34b;
13a+35b; 13a+36b; 13a+37b; 13a+38b; 13a+39b; 13a+40b; 13a+41 b;
13a+42b; 13a+43b; 13a+44b; 13a+45b; 13a+46b; 13a+47b; 13a+48b;
13a+49b; 13a+50b; 13a+51 b; 13a+52b; 13a+53b; 13a+54b; 13a+55b;
13a+55b; 13a+57b; 13a+58b; 13a+59b; 13a+60b; 13a+61 b; 13a+62b;
13a+63b; 13a+64b; 13a+65b; 13a+66b; 13a+67b; 13a+68b; 13a+6gb;
13a+70b; 13a+71 b; 13a+72b; 13a+73b; 13a+74b; 13a+75b; 13a+76b;
265
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
13a+77b; 13a+78b; 13a+79b; 13a+80b; 13a+81 b; 13a+82b; 13a+83b;
13a+84b;13a+85b;13a+86b;13a+87b;13a+88b;13a+89b;13a+90b;
13a+91 b; 13a+92b; 13a+93b; 13a+94b; 13a+95b; 13a+96b; 13a+97b; 14a+1 b;
14a + 2b; 14a + 3b; 14a+4b; 14a+5b; 14a+6b; 14a+7b; 14a+8b; 14a+9b;
14a+1 Ob; 14a+11 b; 14a+12b; 14a+13b; 14a+14b; 14a+15b; 14a+16b;
14a+17b; 14a+18b; 14a+19b; 14a+20b; 14a+21 b; 14a+22b; 14a+23b;
14a+24b;14a+25b;14a+26b;14a+27b;14a+28b;14a+29b;14a+30b;
14a+31 b; 14a+32b; 14a+33b; 14a+34b; 14a+35b; 14a+36b; 14a+37b;
14a+38b; 14a+39b; 14a+40b; 14a+41 b; 14a+42b; 14a+43b; 14a+44b;
14a+45b; 14a+46b; 14a+47b; 14a+48b; 14a+49b; 14a+50b; 14a+51 b;
14a+52b;14a+53b;14a+54b;14a+55b;14a+55b;14a+57b;14a+58b;
14a+59b; 14a+60b; 14a+61 b; 14a+62b; 14a+63b; 14a+64b; 14a+65b;
14a+66b; 14a+67b; 14a+68b; 14a+69b; 14a+70b; 14a+71 b; 14a+72b;
14a+73b; 14a+74b; 14a+75b; 14a+76b; 14a+77b; 14a+78b; 14a+79b;
14a+80b; 14a+81 b; 14a+82b; 14a+83b; 14a+84b; 14a+85b; 14a+86b;
14a+87b; 14a+88b; 14a+89b; 14a+90b; 14a+91 b; 14a+92b; 14a+93b;
14a+94b; 14a+95b; 14a+96b; 14a+97b; 15a+1 b; 15a + 2b; 15a + 3b; 15a+4b;
15a+5b; 15a+6b; 15a+7b; 15a+8b; 15a+9b; 15a+1 Ob; 15a+11 b; 15a+12b;
15a+13b; 15a+14b; 15a+15b; 15a+16b; 15a+17b; 15a+18b; 15a+19b;
15a+20b; 15a+21 b; 15a+22b; 15a+23b; 15a+24b; 15a+25b; 15a+26b;
15a+27b; 15a+28b; 15a+29b; 15a+30b; 15a+31 b; 15a+32b; 15a+33b;
15a+34b; 15a+35b; 15a+36b; 15a+37b; 15a+38b; 15a+39b; 15a+40b;
15a+41 b; 15a+42b; 15a+43b; 15a+44b; 15a+45b; 15a+46b; 15a+47b;
15a+48b; 15a+49b; 15a+50b; 15a+51 b; 15a+52b; 15a+53b; 15a+54b;
15a+55b; 15a+55b; 15a+57b; 15a+58b; 15a+59b; 15a+60b; 15a+61 b;
15a+62b; 15a+63b; 15a+64b; 15a+65b; 15a+66b; 15a+67b; 15a+68b;
15a+69b; 15a+70b; 15a+71 b; 15a+72b; 15a+73b; 15a+74b; 15a+75b;
15a+76b; 15a+77b; 15a+78b; 15a+79b; 15a+80b; 15a+81 b; 15a+82b;
15a+83b; 15a+84b; 15a+85b; 15a+86b; 15a+87b; 15a+88b; 15a+89b;
15a+90b; 15a+91 b; 15a+92b; 15a+93b; 15a+94b; 15a+95b; 15a+96b;
266
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
15a+97b; 16a+1 b; 16a + 2b; 16a + 3b; 16a+4b; 16a+5b; 16a+6b; 16a+7b;
16a+8b; 16a+9b; 16a+1 Ob; 16a+11 b; 16a+12b; 16a+13b; 16a+14b; 16a+15b;
16a+16b; 16a+17b; 16a+18b; 16a+19b; 16a+20b; 16a+21 b; 16a+22b;
16a+23b; 16a+24b; 16a+25b; 16a+26b; 16a+27b; 16a+28b; 16a+29b;
16a+30b; 16a+31 b; 16a+32b; 16a+33b; 16a+34b; 16a+35b; 16a+36b;
16a+37b; 16a+38b; 16a+39b; 16a+40b; 16a+41 b; 16a+42b; 16a+43b;
16a+44b; 16a+45b; 16a+46b; 16a+47b; 16a+48b; 16a+49b; 16a+50b;
16a+51 b; 16a+52b; 16a+53b; 16a+54b; 16a+55b; 16a+55b; 16a+57b;
16a+58b; 16a+59b; 16a+60b; 16a+61 b; 16a+62b; 16a+63b; 16a+64b;
16a+65b; 16a+66b; 16a+67b; 16a+68b; 16a+69b; 16a+70b; 16a+71 b;
16a+72b; 16a+73b; 16a+74b; 16a+75b; 16a+76b; 16a+77b; 16a+78b;
16a+79b; 16a+80b; 16a+81 b; 16a+82b; 16a+83b; 16a+84b; 16a+85b;
16a+86b; 16a+87b; 16a+88b; 16a+89b; 16a+90b; 16a+91 b; 16a+92b;
16a+93b; 16a+94b; 16a+95b; 16a+96b; 16a+97b; 17a+1 b; 17a + 2b; 17a + 3b;
17a+4b; 17a+5b; 17a+6b; 17a+7b; 17a+8b; 17a+9b; 17a+1 Ob; 17a+11 b;
17a+12b; 17a+13b; 17a+14b; 17a+15b; 17a+16b; 17a+17b; 17a+18b;
17a+19b; 17a+20b; 17a+21 b; 17a+22b; 17a+23b; 17a+24b; 17a+25b;
17a+26b; 17a+27b; 17a+28b; 17a+29b; 17a+30b; 17a+31 b; 17a+32b;
17a+33b; 17a+34b; 17a+35b; 17a+36b; 17a+37b; 17a+38b; 17a+39b;
17a+40b; 17a+41 b; 17a+42b; 17a+43b; 17a+44b; 17a+45b; 17a+46b;
17a+47b; 17a+48b; 17a+49b; 17a+50b; 17a+51 b; 17a+52b; 17a+53b;
17a+54b; 17a+55b; 17a+55b; 17a+57b; 17a+58b; 17a+59b; 17a+60b;
17a+61 b; 17a+62b; 17a+63b; 17a+64b; 17a+65b; 17a+66b; 17a+67b;
17a+68b; 17a+69b; 17a+70b; 17a+71 b; 17a+72b; 17a+73b; 17a+74b;
17a+75b; 17a+76b; 17a+77b; 17a+78b; 17a+79b; 17a+80b; 17a+81 b;
17a+82b; 17a+83b; 17a+84b; 17a+85b; 17a+86b; 17a+87b; 17a+88b;
17a+89b; 17a+90b; 17a+91 b; 17a+92b; 17a+93b; 17a+94b; 17a+95b;
17a+96b; 17a+97b; 18a+1 b; 18a + 2b; 18a + 3b; 18a+4b; 18a+5b; 18a+6b;
18a+7b; 18a+8b; 18a+9b; 18a+10b; 18a+11 b; 18a+12b; 18a+13b; 18a+14b;
18a+15b; 18a+16b; 18a+17b; 18a+18b; 18a+19b; 18a+20b; 18a+21 b;
267
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
18a+22b;18a+23b;18a+24b;18a+25b;18a+26b;18a+27b;18a+28b;
18a+29b; 18a+30b; 18a+31 b; 18a+32b; 18a+33b; 18a+34b; 18a+35b;
18a+36b; 18a+37b; 18a+38b; 18a+39b; 18a+40b; 18a+41 b; 18a+42b;
18a+43b; 18a+44b; 18a+45b; 18a+46b; 18a+47b; 18a+48b; 18a+49b;
18a+50b; 18a+51 b; 18a+52b; 18a+53b; 18a+54b; 18a+55b; 18a+55b;
18a+57b; 18a+58b; 18a+59b; 18a+60b; 18a+61 b; 18a+62b; 18a+63b;
18a+64b;18a+65b;18a+66b;18a+67b;18a+68b;18a+69b;18a+70b;
18a+71 b; 18a+72b; 18a+73b; 18a+74b; 18a+75b; 18a+76b; 18a+77b;
18a+78b; 18a+79b; 18a+80b; 18a+81 b; 18a+82b; 18a+83b; 18a+84b;
18a+85b; 18a+86b; 18a+87b; 18a+88b; 18a+89b; 18a+90b; 18a+91 b;
18a+92b; 18a+93b; 18a+94b; 18a+95b; 18a+96b; 18a+97b; 19a+1 b; 19a + 2b;
19a + 3b; 19a+4b; 19a+5b; 19a+6b; 19a+7b; 19a+8b; 19a+9b; 19a+10b;
19a+11 b; 19a+12b; 19a+13b; 19a+14b; 19a+15b; 19a+16b; 19a+17b;
19a+18b; 19a+19b; 19a+20b; 19a+21 b; 19a+22b; 19a+23b; 19a+24b;
19a+25b; 19a+26b; 19a~27b; 19a+28b; 19a+29b; 19a+30b; 19a+31 b;
19a+32b;19a+33b;19a+34b;19a+35b;19a+36b;19a+37b;19a+38b;
19a+39b; 19a+40b; 19a+41 b; 19a+42b; 19a+43b; 19a+44b; 19a+45b;
19a+46b; 19a+47b; 19a+48b; 19a+49b; 19a+50b; 19a+51 b; 19a+52b;
19a+53b;19a+54b;19a+55b;19a+55b;19a+57b;19a+58b;19a+59b;
19a+60b; 19a+61 b; 19a+62b; 19a+63b; 19a+64b; 19a+65b; 19a+66b;
19a+67b; 19a+68b; 19a+69b; 19a+70b; 19a+71 b; 19a+72b; 19a+73b;
19a+74b;19a+75b;19a+76b;19a+77b;19a+78b;19a+79b;19a+80b;
19a+81 b; 19a+82b; 19a+83b; 19a+84b; 19a+85b; 19a+86b; 19a+87b;
19a+88b; 19a+89b; 19a+90b; 19a+91 b; 19a+92b; 19a+93b; 19a+94b;
19a+95b; 19a+96b; 19a+97b; 20a+1 b; 20a + 2b; 20a + 3b; 20a+4b; 20a+5b;
20a+6b; 20a+7b; 20a+8b; 20a+9b; 20a+10b; 20a+11 b; 20a+12b; 20a+13b;
20a+14b; 20a+15b; 20a+16b; 20a+17b; 20a+18b; 20a+19b; 20a+20b;
20a+21 b; 20a+22b; 20a+23b; 20a+24b; 20a+25b; 20a+26b; 20a+27b;
20a+28b; 20a+29b; 20a+30b; 20a+31 b; 20a+32b; 20a+33b; 20a+34b;
20a+35b; 20a+36b; 20a+37b; 20a+38b; 20a+39b; 20a+40b; 20a+41 b;
268
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
20a+42b; 20a+43b; 20a+44b; 20a+45b; 20a+46b; 20a+47b; 20a+48b;
20a+49b; 20a+50b; 20a+51 b; 20a+52b; 20a+53b; 20a+54b; 20a+55b;
20a+55b; 20a+57b; 20a+58b; 20a+59b; 20a+60b; 20a+61 b; 20a+62b;
20a+63b; 20a+64b; 20a+65b; 20a+66b; 20a+67b; 20a+68b; 20a+69b;
20a+70b; 20a+71 b; 20a+72b; 20a+73b; 20a+74b; 20a+75b; 20a+76b;
20a+77b; 20a+78b; 20a+79b; 20a+80b; 20a+81 b; 20a+82b; 20a+83b;
20a+84b; 20a+85b; 20a+86b; 20a+87b; 20a+88b; 20a+89b; 20a+90b;
20a+91 b; 20a+92b; 20a+93b; 20a+94b; 20a+95b; 20a+96b; 20a+97b; 21 a+1 b;
21 a + 2b; 21 a + 3b; 21 a+4b; 21 a+5b; 21 a+6b; 21 a+7b; 21 a+8b; 21 a+9b;
21 a+1 Ob; 21 a+11 b; 21 a+12b; 21 a+13b; 21 a+14b; 21 a+15b; 21 a+16b;
21 a+17b; 21 a+18b; 21 a+19b; 21 a+20b; 21 a+21 b; 21 a+22b; 21 a+23b;
21 a+24b; 21 a+25b; 21 a+26b; 21 a+27b; 21 a+28b; 21 a+29b; 21 a+30b;
21 a+31 b; 21 a+32b; 21 a+33b; 21 a+34b; 21 a+35b; 21 a+36b; 21 a+37b;
21 a+38b; 21 a+39b; 21 a+40b; 21 a+41 b; 21 a+42b; 21 a+43b; 21 a+44b;
21 a+45b; 21 a+46b; 21 a+47b; 21 a+48b; 21 a+49b; 21 a+50b; 21 a+51 b;
21 a+52b; 21 a+53b; 21 a+54b; 21 a+55b; 21 a+55b; 21 a+57b; 21 a+58b;
21 a+59b; 21 a+60b; 21 a+61 b; 21 a+62b; 21 a+63b; 21 a+64b; 21 a+65b;
21 a+66b; 21 a+67b; 21 a+68b; 21 a+69b; 21 a+70b; 21 a+71 b; 21 a+72b;
21 a+73b; 21 a+74b; 21 a+75b; 21 a+76b; 21 a+77b; 21 a+78b; 21 a+79b;
21 a+80b; 21 a+81 b; 21 a+82b; 21 a+83b; 21 a+84b; 21 a+85b; 21 a+86b;
21 a+87b; 21 a+88b; 21 a+89b; 21 a+90b; 21 a+91 b; 21 a+92b; 21 a+93b;
21 a+94b; 21 a+95b; 21 a+96b; 21 a+97b; 22a+1 b; 22a + 2b; 22a + 3b; 22a+4b;
22a+5b; 22a+6b; 22a+7b; 22a+8b; 22a+9b; 22a+1 Ob; 22a+11 b; 22a+12b;
22a+13b; 22a+14b; 22a+15b; 22a+16b; 22a+17b; 22a+18b; 22a+19b;
22a+20b; 22a+21 b; 22a+22b; 22a+23b; 22a+24b; 22a+25b; 22a+26b;
22a+27b; 22a+28b; 22a+29b; 22a+30b; 22a+31 b; 22a+32b; 22a+33b;
22a+34b; 22a+35b; 22a+36b; 22a+37b; 22a+38b; 22a+39b; 22a+40b;
22a+41 b; 22a+42b; 22a+43b; 22a+44b; 22a+45b; 22a+46b; 22a+47b;
22a+48b; 22a+49b; 22a+50b; 22a+51 b; 22a+52b; 22a+53b; 22a+54b;
22a+55b; 22a+55b; 22a+57b; 22a+58b; 22a+59b; 22a+60b; 22a+61 b;
269
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
22a+62b; 22a+63b; 22a+64b; 22a+65b; 22a+66b; 22a+67b; 22a+68b;
22a+69b; 22a+70b; 22a+71 b; 22a+72b; 22a+73b; 22a+74b; 22a+75b;
22a+76b; 22a+77b; 22a+78b; 22a+79b; 22a+80b; 22a+81 b; 22a+82b;
1
22a+83b; 22a+84b; 22a+85b; 22a+86b; 22a+87b; 22a+88b; 22a+89b;
22a+90b; 22a+91 b; 22a+92b; 22a+93b; 22a+94b; 22a+95b; 22a+96b;
22a+97b; 23a+1 b; 23a + 2b; 23a + 3b; 23a+4b; 23a+5b; 23a+6b; 23a+7b;
23a+8b; 23a+9b; 23a+10b; 23a+11 b; 23a+12b; 23a+13b; 23a+14b; 23a+15b;
23a+16b; 23a+17b; 23a+18b; 23a+19b; 23a+20b; 23a+21 b; 23a+22b;
23a+23b; 23a+24b; 23a+25b; 23a+26b; 23a+27b; 23a+28b; 23a+29b;
23a+30b; 23a+31 b; 23a+32b; 23a+33b; 23a+34b; 23a+35b; 23a+36b;
23a+37b; 23a+38b; 23a+39b; 23a+40b; 23a+41 b; 23a+42b; 23a+43b;
23a+44b; 23a+45b; 23a+46b; 23a+47b; 23a+48b; 23a+49b; 23a+50b;
23a+51 b; 23a+52b; 23a+53b; 23a+54b; 23a+55b; 23a+55b; 23a+57b;
23a+58b; 23a+59b; 23a+60b; 23a+61 b; 23a+62b; 23a+63b; 23a+64b;
23a+65b; 23a+66b; 23a+67b; 23a+68b; 23a+69b; 23a+70b; 23a+71 b;
23a+72b; 23a+73b; 23a+74b; 23a+75b; 23a+76b; 23a+77b; 23a+78b;
23a+79b; 23a+80b; 23a+81 b; 23a+82b; 23a+83b; 23a+84b; 23a+85b;
23a+86b; 23a+87b; 23a+88b; 23a+89b; 23a+90b; 23a+91 b; 23a+92b;
23a+93b; 23a+94b; 23a+95b; 23a+96b; 23a+97b; 24a+1 b; 24a + 2b; 24a + 3b;
24a+4b; 24a+5b; 24a+6b; 24a+7b; 24a+8b; 24a+9b; 24a+1 Ob; 24a+11 b;
24a+12b; 24a+13b; 24a+14b; 24a+15b; 24a+16b; 24a+17b; 24a+18b; '
24a+19b; 24a+20b; 24a+21 b; 24a+22b; 24a+23b; 24a+24b; 24a+25b;
24a+26b; 24a+27b; 24a+28b; 24a+29b; 24a+30b; 24a+31 b; 24a+32b;
24a+33b; 24a+34b; 24a+35b; 24a+36b; 24a+37b; 24a+38b; 24a+39b;
24a+40b; 24a+41 b; 24a+42b; 24a+43b; 24a+44b; 24a+45b; 24a+46b;
24a+47b; 24a+48b; 24a+49b; 24a+50b; 24a+51 b; 24a+52b; 24a+53b;
24a+54b; 24a+55b; 24a+55b; 24a+57b; 24a+58b; 24a+59b; 24a+60b;
24a+61 b; 24a+62b; 24a+63b; 24a+64b; 24a+65b; 24a+66b; 24a+67b;
24a+68b; 24a+69b; 24a+70b; 24a+71 b; 24a+72b; 24a+73b; 24a+74b;
24a+75b; 24a+76b; 24a+77b; 24a+78b; 24a+79b; 24a+80b; 24a+81 b;
270
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
24a+82b; 24a+83b; 24a+84b; 24a+85b; 24a+86b; 24a+87b; 24a+88b;
24a+89b; 24a+90b; 24a+91 b; 24a+92b; 24a+93b; 24a+94b; 24a+95b;
24a+96b; 24a+97b; 25a+1 b; 25a + 2b; 25a + 3b; 25a+4b; 25a+5b; 25a+6b;
25a+7b; 25a+8b; 25a+9b; 25a+10b; 25a+11 b; 25a+12b; 25a+13b; 25a+14b;
25a+15b; 25a+16b; 25a+17b; 25a+18b; 25a+19b; 25a+20b; 25a+21 b;
25a+22b; 25a+23b; 25a+24b; 25a+25b; 25a+26b; 25a+27b; 25a+28b;
25a+29b; 25a+30b; 25a+31 b; 25a+32b; 25a+33b; 25a+34b; 25a+35b;
25a+36b; 25a+37b; 25a+38b; 25a+39b; 25a+40b; 25a+41 b; 25a+42b;
25a+43b; 25a+44b; 25a+45b; 25a+46b; 25a+47b; 25a+48b; 25a+49b;
25a+50b; 25a+51 b; 25a+52b; 25a+53b; 25a+54b; 25a+55b; 25a+55b;
25a+57b; 25a+58b; 25a+59b; 25a+60b; 25a+61 b; 25a+62b; 25a+63b;
25a+64b; 25a+65b; 25a+66b; 25a+67b; 25a+68b; 25a+69b; 25a+70b;
25a+71 b; 25a+72b; 25a+73b; 25a+74b; 25a+75b; 25a+76b; 25a+77b;
25a+78b; 25a+79b; 25a+80b; 25a+81 b; 25a+82b; 25a+83b; 25a+84b;
25a+85b; 25a+86b; 25a+87b; 25a+88b; 25a+89b; 25a+90b; 25a+91 b;
25a+92b; 25a+93b; 25a+94b; 25a+95b; 25a+96b; 25a+97b; 26a+1 b; 26a + 2b;
26a + 3b; 26a+4b; 26a+5b; 26a+6b; 26a+7b; 26a+8b; 26a+9b; 26a+10b;
26a+11 b; 26a+12b; 26a+13b; 26a+14b; 26a+15b; 26a+16b; 26a+17b;
26a+18b; 26a+19b; 26a+20b; 26a+21 b; 26a+22b; 26a+23b; 26a+24b;
26a+25b; 26a+26b; 26a+27b; 26a+28b; 26a+29b; 26a+30b; 26a+31 b;
26a+32b; 26a+33b; 26a+34b; 26a+35b; 26a+36b; 26a+37b; 26a+38b;
26a+39b; 26a+40b; 26a+41 b; 26a+42b; 26a+43b; 26a+44b; 26a+45b;
26a+46b; 26a+47b; 26a+48b; 26a+49b; 26a+50b; 26a+51 b; 26a+52b;
26a+53b; 26a+54b; 26a+55b; 26a+55b; 26a+57b; 26a+58b; 26a+59b;
26a+60b; 26a+61 b; 26a+62b; 26a+63b; 26a+64b; 26a+65b; 26a+66b;
26a+67b; 26a+68b; 26a+69b; 26a+70b; 26a+71 b; 26a+72b; 26a+73b;
26a+74b; 26a+75b; 26a+76b; 26a+77b; 26a+78b; 26a+79b; 26a+80b;
26a+81 b; 26a+82b; 26a+83b; 26a+84b; 26a+85b; 26a+86b; 26a+87b;
26a+88b; 26a+89b; 26a+90b; 26a+91 b; 26a+92b; 26a+93b; 26a+94b;
26a+95b; 26a+96b; 26a+97b; 27a+1 b; 27a + 2b; 27a + 3b; 27a+4b; 27a+5b;
271
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
27a+6b; 27a+7b; 27a+8b; 27a+9b; 27a+1 Ob; 27a+11 b; 27a+12b; 27a+13b;
27a+14b; 27a+15b; 27a+16b; 27a+17b; 27a+18b; 27a+19b; 27a+20b;
27a+21 b; 27a+22b; 27a+23b; 27a+24b; 27a+25b; 27a+26b; 27a+27b;
27a+28b; 27a+29b; 27a+30b; 27a+31 b; 27a+32b; 27a+33b; 27a+34b;
27a+35b; 27a+36b; 27a+37b; 27a+38b; 27a+39b; 27a+40b; 27a+41 b;
27a+42b; 27a+43b; 27a+44b; 27a+45b; 27a+46b; 27a+47b; 27a+48b;
27a+49b; 27a+50b; 27a+51 b; 27a+52b; 27a+53b; 27a+54b; 27a+55b;
27a+55b; 27a+57b; 27a+58b; 27a+59b; 27a+60b; 27a+61 b; 27a+62b;
27a+63b; 27a+64b; 27a+65b; 27a+66b; 27a+67b; 27a+68b; 27a+69b;
27a+70b; 27a+71 b; 27a+72b; 27a+73b; 27a+74b; 27a+75b; 27a+76b;
27a+77b; 27a+78b; 27a+79b; 27a+80b; 27a+81 b; 27a+82b; 27a+83b;
27a+84b; 27a+85b; 27a+86b; 27a+87b; 27a+88b; 27a+89b; 27a+90b;
27a+91 b; 27a+92b; 27a+93b; 27a+94b; 27a+95b; 27a+96b; 27a+97b; 28a+1 b;
28a + 2b; 28a + 3b; 28a+4b; 28a+5b; 28a+6b; 28a+7b; 28a+8b; 28a+9b;
28a+1 Ob; 28a+11 b; 28a+12b; 28a+13b; 28a+14b; 28a+15b; 28a+16b;
28a+17b; 28a+18b; 28a+19b; 28a+20b; 28a+21 b; 28a+22b; 28a+23b;
28a+24b; 28a+25b; 28a+26b; 28a+27b; 28a+28b; 28a+29b; 28a+30b;
28a+31 b; 28a+32b; 28a+33b; 28a+34b; 28a+35b; 28a+36b; 28a+37b;
28a+38b; 28a+39b; 28a+40b; 28a+41 b; 28a+42b; 28a+43b; 28a+44b;
28a+45b; 28a+46b; 28a+47b; 28a+48b; 28a+49b; 28a+50b; 28a+51 b;
28a+52b; 28a+53b; 28a+54b; 28a+55b; 28a+55b; 28a+57b; 28a+58b;
28a+59b; 28a+60b; 28a+61 b; 28a+62b; 28a+63b; 28a+64b; 28a+65b;
28a+66b; 28a+67b; 28a+68b; 28a+69b; 28a+70b; 28a+71 b; 28a+72b;
28a+73b; 28a+74b; 28a+75b; 28a+76b; 28a+77b; 28a+78b; 28a+79b;
28a+80b; 28a+81 b; 28a+82b; 28a+83b; 28a+84b; 28a+85b; 28a+86b;
28a+87b; 28a+88b; 28a+89b; 28a+90b; 28a+91 b; 28a+92b; 28a+93b;
28a+94b; 28a+95b; 28a+96b; 28a+97b; 29a+1 b; 29a + 2b; 29a + 3b; 29a+4b;
29a+5b; 29a+6b; 29a+7b; 29a+8b; 29a+9b; 29a+1 Ob; 29a+11 b; 29a+12b;
29a+13b; 29a+14b; 29a+15b; 29a+16b; 29a+17b; 29a+18b; 29a+19b;
29a+20b; 29a+21 b; 29a+22b; 29a+23b; 29a+24b; 29a+25b; 29a+26b;
272
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
29a+27b; 29a+28b; 29a+29b; 29a+30b; 29a+31 b; 29a+32b; 29a+33b;
29a+34b; 29a+35b; 29a+36b; 29a+37b; 29a+38b; 29a+39b; 29a+40b;
29a+41 b; 29a+42b; 29a+43b; 29a+44b; 29a+45b; 29a+46b; 29a+47b;
29a+48b; 29a+49b; 29a+50b; 29a+51 b; 29a+52b; 29a+53b; 29a+54b;
29a+55b; 29a+55b; 29a+57b; 29a+58b; 29a+59b; 29a+60b; 29a+61 b;
29a+62b; 29a+63b; 29a+64b; 29a+65b; 29a+66b; 29a+67b; 29a+68b;
29a+69b; 29a+70b; 29a+71 b; 29a+72b; 29a+73b; 29a+74b; 29a+75b;
29a+76b; 29a+77b; 29a+78b; 29a+79b; 29a+80b; 29a+81 b; 29a+82b;
29a+83b; 29a+84b; 29a+85b; 29a+86b; 29a+87b; 29a+88b; 29a+89b;
29a+90b; 29a+91 b; 29a+92b; 29a+93b; 29a+94b; 29a+95b; 29a+96b;
29a+97b; 30a+1 b; 30a + 2b; 30a + 3b; 30a+4b; 30a+5b; 30a+6b; 30a+7b;
30a+8b; 30a+9b; 30a+1 Ob; 30a+11 b; 30a+12b; 30a+13b; 30a+14b; 30a+15b;
30a+16b; 30a+17b; 30a+18b; 30a+19b; 30a+20b; 30a+21 b; 30a+22b;
30a+23b; 30a+24b; 30a+25b; 30a+26b; 30a+27b; 30a+28b; 30a+29b;
30a+30b; 30a+31 b; 30a+32b; 30a+33b; 30a+34b; 30a+35b; 30a+36b;
30a+37b; 30a+38b; 30a+39b; 30a+40b; 30a+41 b; 30a+42b; 30a+43b;
30a+44b; 30a+45b; 30a+46b; 30a+47b; 30a+48b; 30a+49b; 30a+50b;
30a+51 b; 30a+52b; 30a+53b; 30a+54b; 30a+55b; 30a+55b; 30a+57b;
30a+58b; 30a+59b; 30a+60b; 30a+61 b; 30a+62b; 30a+63b; 30a+64b;
30a+65b; 30a+66b; 30a+67b; 30a+68b; 30a+69b; 30a+70b; 30a+71 b;
30a+72b; 30a+73b; 30a+74b; 30a+75b; 30a+76b; 30a+77b; 30a+78b;
30a+79b; 30a+80b; 30a+81 b; 30a+82b; 30a+83b; 30a+84b; 30a+85b;
30a+86b; 30a+87b; 30a+88b; 30a+89b; 30a+90b; 30a+91 b; 30a+92b;
30a+93b; 30a+94b; 30a+95b; 30a+96b; 30a+97b; 31 a+1 b; 31 a + 2b; 31 a + 3b;
31 a+4b; 31 a+5b; 31 a+6b; 31 a+7b; 31 a+8b; 31 a+9b; 31 a+1 Ob; 31 a+11 b;
31 a+12b; 31 a+13b; 31 a+14b; 31 a+15b; 31 a+16b; 31 a+17b; 31 a+18b;
31 a+19b; 31 a+20b; 31 a+21 b; 31 a+22b; 31 a+23b; 31 a+24b; 31 a+25b;
31 a+26b; 31 a+27b; 31 a+28b; 31 a+29b; 31 a+30b; 31 a+31 b; 31 a+32b;
31 a+33b; 31 a+34b; 31 a+35b; 31 a+36b; 31 a+37b; 31 a+38b; 31 a+39b;
31 a+40b; 31 a+41 b; 31 a+42b; 31 a+43b; 31 a+44b; 31 a+45b; 31 a+46b;
273
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
31 a+47b; 31 a+48b; 31 a+49b; 31 a+50b; 31 a+51 b; 31 a+52b; 31 a+53b;
31 a+54b; 31 a+55b; 31 a+55b; 31 a+57b; 31 a+58b; 31 a+59b; 31 a+60b;
31 a+61 b; 31 a+62b; 31 a+63b; 31 a+64b; 31 a+65b; 31 a+66b; 31 a+67b;
31 a+68b; 31 a+69b; 31 a+70b; 31 a+71 b; 31 a+72b; 31 a+73b; 31 a+74b;
31 a+75b; 31 a+76b; 31 a+77b; 31 a+78b; 31 a+79b; 31 a+80b; 31 a+81 b;
31 a+82b; 31 a+83b; 31 a+84b; 31 a+85b; 31 a+86b; 31 a+87b; 31 a+88b;
31 a+89b; 31 a+90b; 31 a+91 b; 31 a+92b; 31 a+93b; 31 a+94b; 31 a+95b;
31 a+96b; 31 a+97b; 32a+1 b; 32a + 2b; 32a + 3b; 32a+4b; 32a+5b; 32a+6b;
32a+7b; 32a+8b; 32a+9b; 32a+10b; 32a+11 b; 32a+12b; 32a+13b; 32a+14b;
32a+15b; 32a+16b; 32a+17b; 32a+18b; 32a+19b; 32a+20b; 32a+21 b;
32a+22b; 32a+23b; 32a+24b; 32a+25b; 32a+26b; 32a+27b; 32a+28b;
32a+29b; 32a+30b; 32a+31 b; 32a+32b; 32a+33b; 32a+34b; 32a+35b;
32a+36b; 32a+37b; 32a+38b; 32a+39b; 32a+40b; 32a+41 b; 32a+42b;
32a+43b; 32a+44b; 32a+45b; 32a+46b; 32a+47b; 32a+48b; 32a+49b;
32a+50b; 32a+51 b; 32a+52b; 32a+53b; 32a+54b; 32a+55b; 32a+55b;
32a+57b; 32a+58b; 32a+59b; 32a+60b; 32a+61 b; 32a+62b; 32a+63b;
32a+64b; 32a+65b; 32a+66b; 32a+67b; 32a+68b; 32a+69b; 32a+70b;
32a+71 b; 32a+72b; 32a+73b; 32a+74b; 32a+75b; 32a+76b; 32a+77b;
32a+78b; 32a+79b; 32a+80b; 32a+81 b; 32a+82b; 32a+83b; 32a+84b;
32a+85b; 32a+86b; 32a+87b; 32a+88b; 32a+89b; 32a+90b; 32a+91 b;
32a+92b; 32a+93b; 32a+94b; 32a+95b; 32a+96b; 32a+97b; 33a+1 b; 33a + 2b;
33a + 3b; 33a+4b; 33a+5b; 33a+6b; 33a+7b; 33a+8b; 33a+9b; 33a+10b;
33a+11 b; 33a+12b; 33a+13b; 33a+14b; 33a+15b; 33a+16b; 33a+17b;
33a+18b; 33a+19b; 33a+20b; 33a+21 b; 33a+22b; 33a+23b; 33a+24b;
33a+25b; 33a+26b; 33a+27b; 33a+28b; 33a+29b; 33a+30b; 33a+31 b;
33a+32b; 33a+33b; 33a+34b; 33a+35b; 33a+36b; 33a+37b; 33a+38b;
33a+39b; 33a+40b; 33a+41 b; 33a+42b; 33a+43b; 33a+44b; 33a+45b;
33a+46b; 33a+47b; 33a+48b; 33a+49b; 33a+50b; 33a+51 b; 33a+52b;
33a+53b; 33a+54b; 33a+55b; 33a+55b; 33a+57b; 33a+58b; 33a+59b;
33a+60b; 33a+61 b; 33a+62b; 33a+63b; 33a+64b; 33a+65b; 33a+66b;
274
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
33a+67b; 33a+68b; 33a+69b; 33a+70b; 33a+71 b; 33a+72b; 33a+73b;
33a+74b; 33a+75b; 33a+76b; 33a+77b; 33a+78b; 33a+79b; 33a+80b;
33a+81 b; 33a+82b; 33a+83b; 33a+84b; 33a+85b; 33a+86b; 33a+87b;
33a+88b; 33a+89b; 33a+90b; 33a+91 b; 33a+92b; 33a+93b; 33a+94b;
33a+95b; 33a+96b; 33a+97b; 34a+1 b; 34a + 2b; 34a + 3b; 34a+4b; 34a+5b;
34a+6b; 34a+7b; 34a+8b; 34a+9b; 34a+10b; 34a+11 b; 34a+12b; 34a+13b;
34a+14b; 34a+15b; 34a+16b; 34a+17b; 34a+18b; 34a+19b; 34a+20b;
34a+21 b; 34a+22b; 34a+23b; 34a+24b; 34a+25b; 34a+26b; 34a+27b;
34a+28b; 34a+29b; 34a+30b; 34a+31 b; 34a+32b; 34a+33b; 34a+34b;
34a+35b; 34a+36b; 34a+37b; 34a+38b; 34a+39b; 34a+40b; 34a+41 b;
34a+42b; 34a+43b; 34a+44b; 34a+45b; 34a+46b; 34a+47b; 34a+48b;
34a+49b; 34a+50b; 34a+51 b; 34a+52b; 34a+53b; 34a+54b; 34a+55b;
34a+55b; 34a+57b; 34a+58b; 34a+59b; 34a+60b; 34a+61 b; 34a+62b;
34a+63b; 34a+64b; 34a+65b; 34a+66b; 34a+67b; 34a+68b; 34a+69b;
34a+70b; 34a+71 b; 34a+72b; 34a+73b; 34a+74b; 34a+75b; 34a+76b;
34a+77b; 34a+78b; 34a+79b; 34a+80b; 34a+81 b; 34a+82b; 34a+83b;
34a+84b; 34a+85b; 34a+86b; 34a+87b; 34a+88b; 34a+89b; 34a+90b;
34a+91 b; 34a+92b; 34a+93b; 34a+94b; 34a+95b; 34a+96b; 34a+97b; 35a+1 b;
35a + 2b; 35a + 3b; 35a+4b; 35a+5b; 35a+6b; 35a+7b; 35a+8b; 35a+9b;
35a+1 Ob; 35a+11 b; 35a+12b; 35a+13b; 35a+14b; 35a+15b; 35a+16b;
35a+17b; 35a+18b; 35a+19b; 35a+20b; 35a+21 b; 35a+22b; 35a+23b;
35a+24b; 35a+25b; 35a+26b; 35a+27b; 35a+28b; 35a+29b; 35a+30b;
35a+31 b; 35a+32b; 35a+33b; 35a+34b; 35a+35b; 35a+36b; 35a+37b;
35a+38b; 35a+39b; 35a+40b; 35a+41 b; 35a+42b; 35a+43b; 35a+44b;
35a+45b; 35a+46b; 35a+47b; 35a+48b; 35a+49b; 35a+50b; 35a+51 b;
35a+52b; 35a+53b; 35a+54b; 35a+55b; 35a+55b; 35a+57b; 35a+58b;
35a+59b; 35a+60b; 35a+61 b; 35a+62b; 35a+63b; 35a+64b; 35a+65b;
35a+66b; 35a+67b; 35a+68b; 35a+69b; 35a+70b; 35a+71 b; 35a+72b;
35a+73b; 35a+74b; 35a+75b; 35a+76b; 35a+77b; 35a+78b; 35a+79b;
35a+80b; 35a+81 b; 35a+82b; 35a+83b; 35a+84b; 35a+85b; 35a+86b;
275
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
35a+87b; 35a+88b; 35a+89b; 35a+90b; 35a+91 b; 35a+92b; 35a+93b;
35a+94b; 35a+95b; 35a+96b; 35a+97b; 36a+1 b; 36a + 2b; 36a + 3b; 36a+4b;
36a+5b; 36a+6b; 36a+7b; 36a+8b; 36a+9b; 36a+1 Ob; 36a+11 b; 36a+12b;
36a+13b; 36a+14b; 36a+15b; 36a+16b; 36a+17b; 36a+18b; 36a+19b;
36a+20b; 36a+21 b; 36a+22b; 36a+23b; 36a+24b; 36a+25b; 36a+26b;
36a+27b; 36a+28b; 36a+29b; 36a+30b; 36a+31 b; 36a+32b; 36a+33b;
36a+34b; 36a+35b; 36a+36b; 36a+37b; 36a+38b; 36a+39b; 36a+40b;
36a+41 b; 36a+42b; 36a+43b; 36a+44b; 36a+45b; 36a+46b; 36a+47b;
36a+48b; 36a+49b; 36a+50b; 36a+51 b; 36a+52b; 36a+53b; 36a+54b;
36a+55b; 36a+55b; 36a+57b; 36a+58b; 36a+59b; 36a+60b; 36a+61 b;
36a+62b; 36a+63b; 36a+64b; 36a+65b; 36a+66b; 36a+67b; 36a+68b;
36a+69b; 36a+70b; 36a+71 b; 36a+72b; 36a+73b; 36a+74b; 36a+75b;
36a+76b; 36a+77b; 36a+78b; 36a+79b; 36a+80b; 36a+81 b; 36a+82b;
36a+83b; 36a+84b; 36a+85b; 36a+86b; 36a+87b; 36a+88b; 36a+89b;
36a+90b; 36a+91 b; 36a+92b; 36a+93b; 36a+94b; 36a+95b; 36a+96b;
36a+97b; 37a+1 b; 37a + 2b; 37a + 3b; 37a+4b; 37a+5b; 37a+6b; 37a+7b;
37a+8b; 37a+9b; 37a+1 Ob; 37a+11 b; 37a+12b; 37a+13b; 37a+14b; 37a+15b;
37a+16b; 37a+17b; 37a+18b; 37a+19b; 37a+20b; 37a+21 b; 37a+22b;
37a+23b; 37a+24b; 37a+25b; 37a+26b; 37a+27b; 37a+28b; 37a+29b;
37a+30b; 37a+31 b; 37a+32b; 37a+33b; 37a+34b; 37a+35b; 37a+36b;
37a+37b; 37a+38b; 37a+39b; 37a+40b; 37a+41 b; 37a+42b; 37a+43b;
37a+44b; 37a+45b; 37a+46b; 37a+47b; 37a+48b; 37a+49b; 37a+50b;
37a+51 b; 37a+52b; 37a+53b; 37a+54b; 37a+55b; 37a+55b; 37a+57b;
37a+58b; 37a+59b; 37a+60b; 37a+61 b; 37a+62b; 37a+63b; 37a+64b;
37a+65b; 37a+66b; 37a+67b; 37a+68b; 37a+69b; 37a+70b; 37a+71 b;
37a+72b; 37a+73b; 37a+74b; 37a+75b; 37a+76b; 37a+77b; 37a+78b;
37a+79b; 37a+80b; 37a+81 b; 37a+82b; 37a+83b; 37a+84b; 37a+85b;
37a+86b; 37a+87b; 37a+88b; 37a+89b; 37a+90b; 37a+91 b; 37a+92b;
37a+93b; 37a+94b; 37a+95b; 37a+96b; 37a+97b; 38a+1 b; 38a + 2b; 38a + 3b;
38a+4b; 38a+5b; 38a+6b; 38a+7b; 38a+8b; 38a+9b; 38a+1 Ob; 38a+11 b;
276
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
38a+12b; 38a+13b; 38a+14b; 38a+15b; 38a+16b; 38a+17b; 38a+18b;
38a+19b; 38a+20b; 38a+21 b; 38a+22b; 38a+23b; 38a+24b; 38a+25b;
38a+26b; 38a+27b; 38a+28b; 38a+29b; 38a+30b; 38a+31 b; 38a+32b;
38a+33b; 38a+34b; 38a+35b; 38a+36b; 38a+37b; 38a+38b; 38a+39b;
38a+40b; 38a+41 b; 38a+42b; 38a+43b; 38a+44b; 38a+45b; 38a+46b;
38a+47b; 38a+48b; 38a+49b; 38a+50b; 38a+51 b; 38a+52b; 38a+53b;
38a+54b; 38a+55b; 38a+55b; 38a+57b; 38a+58b; 38a+59b; 38a+60b;
38a+61 b; 38a+62b; 38a+63b; 38a+64b; 38a+65b; 38a+66b; 38a+67b;
38a+68b; 38a+69b; 38a+70b; 38a+71 b; 38a+72b; 38a+73b; 38a+74b;
38a+75b; 38a+76b; 38a+77b; 38a+78b; 38a+79b; 38a+80b; 38a+81 b;
38a+82b; 38a+83b; 38a+84b; 38a+85b; 38a+86b; 38a+87b; 38a+88b;
38a+89b; 38a+90b; 38a+91 b; 38a+92b; 38a+93b; 38a+94b; 38a+95b;
38a+96b; 38a+97b; 39a+1 b; 39a + 2b; 39a + 3b; 39a+4b; 39a+5b; 39a+6b;
39a+7b; 39a+8b; 39a+9b; 39a+1 Ob; 39a+11 b; 39a+12b; 39a+13b; 39a+14b;
39a+15b; 39a+16b; 39a+17b; 39a+18b; 39a+19b; 39a+20b; 39a+21 b;
39a+22b; 39a+23b; 39a+24b; 39a+25b; 39a+26b; 39a+27b; 39a+28b;
39a+29b; 39a+30b; 39a+31 b; 39a+32b; 39a+33b; 39a+34b; 39a+35b;
39a+36b; 39a+37b; 39a+38b; 39a+39b; 39a+40b; 39a+41 b; 39a+42b;
39a+43b; 39a+44b; 39a+45b; 39a+46b; 39a+47b; 39a+48b; 39a+49b;
39a+50b; 39a+51 b; 39a+52b; 39a+53b; 39a+54b; 39a+55b; 39a+55b;
39a+57b; 39a+58b; 39a+59b; 39a+60b; 39a+61 b; 39a+62b; 39a+63b;
39a+64b; 39a+65b; 39a+66b; 39a+67b; 39a+68b; 39a+69b; 39a+70b;
39a+71 b; 39a+72b; 39a+73b; 39a+74b; 39a+75b; 39a+76b; 39a+77b;
39a+78b; 39a+79b; 39a+80b; 39a+81 b; 39a+82b; 39a+83b; 39a+84b;
39a+85b; 39a+86b; 39a+87b; 39a+88b; 39a+89b; 39a+90b; 39a+91 b;
39a+92b; 39a+93b; 39a+94b; 39a+95b; 39a+96b; 39a+97b; 40a+1 b; 40a + 2b;
40a + 3b; 40a+4b; 40a+5b; 40a+6b; 40a+7b; 40a+8b; 40a+9b; 40a+10b;
40a+11 b; 40a+12b; 40a+13b; 40a+14b; 40a+15b; 40a+16b; 40a+17b;
40a+18b; 40a+19b; 40a+20b; 40a+21 b; 40a+22b; 40a+23b; 40a+24b;
40a+25b; 40a+26b; 40a+27b; 40a+28b; 40a+29b; 40a+30b; 40a+31 b;
277
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
40a+32b; 40a+33b; 40a+34b; 40a+35b; 40a+36b; 40a+37b; 40a+38b;
40a+39b; 40a+40b; 40a+41 b; 40a+42b; 40a+43b; 40a+44b; 40a+45b;
40a+46b; 40a+47b; 40a+48b; 40a+49b; 40a+50b; 40a+51 b; 40a+52b;
40a+53b; 40a+54b; 40a+55b; 40a+55b; 40a+57b; 40a+58b; 40a+59b;
40a+60b; 40a+61 b; 40a+62b; 40a+63b; 40a+64b; 40a+65b; 40a+66b;
40a+67b; 40a+68b; 40a+69b; 40a+70b; 40a+71 b; 40a+72b; 40a+73b;
40a+74b; 40a+75b; 40a+76b; 40a+77b; 40a+78b; 40a+79b; 40a+80b;
40a+81 b; 40a+82b; 40a+83b; 40a+84b; 40a+85b; 40a+86b; 40a+87b;
40a+88b; 40a+89b; 40a+90b; 40a+91 b; 40a+92b; 40a+93b; 40a+94b;
40a+95b; 40a+96b; 40a+97b; 41 a+1 b; 41 a + 2b; 41 a + 3b; 41 a+4b; 41 a+5b;
41 a+6b; 41 a+7b; 41 a+8b; 41 a+9b; 41 a+1 Ob; 41 a+11 b; 41 a+12b; 41 a+13b;
41 a+14b; 41 a+15b; 41 a+16b; 41 a+17b; 41 a+18b; 41 a+19b; 41 a+20b;
41 a+21 b; 41 a+22b; 41 a+23b; 41 a+24b; 41 a+25b; 41 a+26b; 41 a+27b;
41 a+28b; 41 a+29b; 41 a+30b; 41 a+31 b; 41 a+32b; 41 a+33b; 41 a+34b;
41 a+35b; 41 a+36b; 41 a+37b; 41 a+38b; 41 a+39b; 41 a+40b; 41 a+41 b;
41 a+42b; 41 a+43b; 41 a+44b; 41 a+45b; 41 a+46b; 41 a+47b; 41 a+48b;
41 a+49b; 41 a+50b; 41 a+51 b; 41 a+52b; 41 a+53b; 41 a+54b; 41 a+55b;
41 a+55b; 41 a+57b; 41 a+58b; 41 a+59b; 41 a+60b; 41 a+61 b; 41 a+62b;
41 a+63b; 41 a+64b; 41 a+65b; 41 a+66b; 41 a+67b; 41 a+68b; 41 a+69b;
41 a+70b; 41 a+71 b; 41 a+72b; 41 a+73b; 41 a+74b; 41 a+75b; 41 a+76b;
41 a+77b; 41 a+78b; 41 a+79b; 41 a+80b; 41 a+81 b; 41 a+82b; 41 a+83b;
41 a+84b; 41 a+85b; 41 a+86b; 41 a+87b; 41 a+88b; 41 a+89b; 41 a+90b;
41 a+91 b; 41 a+92b; 41 a+93b; 41 a+94b; 41 a+95b; 41 a+96b; 41 a+97b; 42a+1
b;
42a + 2b; 42a + 3b; 42a+4b; 42a+5b; 42a+6b; 42a+7b; 42a+8b; 42a+9b;
42a+1 Ob; 42a+11 b; 42a+12b; 42a+13b; 42a+14b; 42a+15b; 42a+16b;
42a+17b; 42a+18b; 42a+19b; 42a+20b; 42a+21 b; 42a+22b; 42a+23b;
42a+24b; 42a+25b; 42a+26b; 42a+27b; 42a+28b; 42a+29b; 42a+30b;
42a+31 b; 42a+32b; 42a+33b; 42a+34b; 42a+35b; 42a+36b; 42a+37b;
42a+38b; 42a+39b; 42a+40b; 42a+41 b; 42a+42b; 42a+43b; 42a+44b;
42a+45b; 42a+46b; 42a+47b; 42a+48b; 42a+49b; 42a+50b; 42a+51 b;
278
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
42a+52b; 42a+53b; 42a+54b; 42a+55b; 42a+55b; 42a+57b; 42a+58b;
42a+59b; 42a+60b; 42a+61 b; 42a+62b; 42a+63b; 42a+64b; 42a+65b;
42a+66b; 42a+67b; 42a+68b; 42a+69b; 42a+70b; 42a+71 b; 42a+72b;
42a+73b; 42a+74b; 42a+75b; 42a+76b; 42a+77b; 42a+78b; 42a+79b;
42a+80b; 42a+81 b; 42a+82b; 42a+83b; 42a+84b; 42a+85b; 42a+86b;
42a+87b; 42a+88b; 42a+89b; 42a+90b; 42a+91 b; 42a+92b; 42a+93b;
42a+94b; 42a+95b; 42a+96b; 42a+97b; 43a+1 b; 43a + 2b; 43a + 3b; 43a+4b;
43a+5b; 43a+6b; 43a+7b; 43a+8b; 43a+9b; 43a+1 Ob; 43a+11 b; 43a+12b;
43a+13b; 43a+14b; 43a+15b; 43a+16b; 43a+17b; 43a+18b; 43a+19b;
43a+20b; 43a+21 b; 43a+22b; 43a+23b; 43a+24b; 43a+25b; 43a+26b;
43a+27b; 43a+28b; 43a+29b; 43a+30b; 43a+31 b; 43a+32b; 43a+33b;
43a+34b; 43a+35b; 43a+36b; 43a+37b; 43a+38b; 43a+39b; 43a+40b;
43a+41 b; 43a+42b; 43a+43b; 43a+44b; 43a+45b; 43a+46b; 43a+47b;
43a+48b; 43a+49b; 43a+50b; 43a+51 b; 43a+52b; 43a+53b; 43a+54b;
43a+55b; 43a+55b; 43a+57b; 43a+58b; 43a+59b; 43a+60b; 43a+61 b;
43a+62b; 43a+63b; 43a+64b; 43a+65b; 43a+66b; 43a+67b; 43a+68b;
43a+69b; 43a+70b; 43a+71 b; 43a+72b; 43a+73b; 43a+74b; 43a+75b;
43a+76b; 43a+77b; 43a+78b; 43a+79b; 43a+80b; 43a+81 b; 43a+82b;
43a+83b; 43a+84b; 43a+85b; 43a+86b; 43a+87b; 43a+88b; 43a+89b;
43a+90b; 43a+91 b; 43a+92b; 43a+93b; 43a+94b; 43a+95b; 43a+96b;
43a+97b; 44a+1 b; 44a + 2b; 44a + 3b; 44a+4b; 44a+5b; 44a+6b; 44a+7b;
44a+8b; 44a+9b; 44a+1 Ob; 44a+11 b; 44a+12b; 44a+13b; 44a+14b; 44a+15b;
44a+16b; 44a+17b; 44a+18b; 44a+19b; 44a+20b; 44a+21 b; 44a+22b;
44a+23b; 44a+24b; 44a+25b; 44a+26b; 44a+27b; 44a+28b; 44a+29b;
44a+30b; 44a+31 b; 44a+32b; 44a+33b; 44a+34b; 44a+35b; 44a+36b;
44a+37b; 44a+38b; 44a+39b; 44a+40b; 44a+41 b; 44a+42b; 44a+43b;
44a+44b; 44a+45b; 44a+46b; 44a+47b; 44a+48b; 44a+49b; 44a+50b;
44a+51 b; 44a+52b; 44a+53b; 44a+54b; 44a+55b; 44a+55b; 44a+57b;
44a+58b; 44a+59b; 44a+60b; 44a+61 b; 44a+62b; 44a+63b; 44a+64b;
44a+65b; 44a+66b; 44a+67b; 44a+68b; 44a+69b; 44a+70b; 44a+71 b;
279
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
44a+72b; 44a+73b; 44a+74b; 44a+75b; 44a+76b; 44a+77b; 44a+78b;
44a+79b; 44a+80b; 44a+81 b; 44a+82b; 44a+83b; 44a+84b; 44a+85b;
44a+86b; 44a+87b; 44a+88b; 44a+89b; 44a+90b; 44a+91 b; 44a+92b;
44a+93b; 44a+94b; 44a+95b; 44a+96b; 44a+97b; 45a+1 b; 45a + 2b; 45a + 3b;
45a+4b; 45a+5b; 45a+6b; 45a+7b; 45a+8b; 45a+9b; 45a+1 Ob; 45a+11 b;
45a+12b; 45a+13b; 45a+14b; 45a+15b; 45a+16b; 45a+17b; 45a+18b;
45a+19b; 45a+20b; 45a+21 b; 45a+22b; 45a+23b; 45a+24b; 45a+25b;
45a+26b; 45a+27b; 45a+28b; 45a+29b; 45a+30b; 45a+31 b; 45a+32b;
45a+33b; 45a+34b; 45a+35b; 45a+36b; 45a+37b; 45a+38b; 45a+39b;
45a+40b; 45a+41 b; 45a+42b; 45a+43b; 45a+44b; 45a+45b; 45a+46b;
45a+47b; 45a+48b; 45a+49b; 45a+50b; 45a+51 b; 45a+52b; 45a+53b;
45a+54b; 45a+55b; 45a+55b; 45a+57b; 45a+58b; 45a+59b; 45a+60b;
45a+61 b; 45a+62b; 45a+63b; 45a+64b; 45a+65b; 45a+66b; 45a+67b;
45a+68b; 45a+69b; 45a+70b; 45a+71 b; 45a+72b; 45a+73b; 45a+74b;
45a+75b; 45a+76b; 45a+77b; 45a+78b; 45a+79b; 45a+80b; 45a+81 b;
45a+82b; 45a+83b; 45a+84b; 45a+85b; 45a+86b; 45a+87b; 45a+88b;
45a+89b; 45a+90b; 45a+91 b; 45a+92b; 45a+93b; 45a+94b; 45a+95b;
45a+96b; 45a+97b; 46a+1 b; 46a + 2b; 46a + 3b; 46a+4b; 46a+5b; 46a+6b;
46a+7b; 46a+8b; 46a+9b; 46a+1 Ob; 46a+11 b; 46a+12b; 46a+13b; 46a+14b;
46a+15b; 46a+16b; 46a+17b; 46a+18b; 46a+19b; 46a+20b; 46a+21 b;
46a+22b; 46a+23b; 46a+24b; 46a+25b; 46a+26b; 46a+27b; 46a+28b;
46a+29b; 46a+30b; 46a+31 b; 46a+32b; 46a+33b; 46a+34b; 46a+35b;
46a+36b; 46a+37b; 46a+38b; 46a+39b; 46a+40b; 46a+41 b; 46a+42b;
46a+43b; 46a+44b; 46a+45b; 46a+46b; 46a+47b; 46a+48b; 46a+49b;
46a+50b; 46a+51 b; 46a+52b; 46a+53b; 46a+54b; 46a+55b; 46a+55b;
46a+57b; 46a+58b; 46a+59b; 46a+60b; 46a+61 b; 46a+62b; 46a+63b;
46a+64b; 46a+65b; 46a+66b; 46a+67b; 46a+68b; 46a+69b; 46a+70b;
46a+71 b; 46a+72b; 46a+73b; 46a+74b; 46a+75b; 46a+76b; 46a+77b;
46a+78b; 46a+79b; 46a+80b; 46a+81 b; 46a+82b; 46a+83b; 46a+84b;
46a+85b; 46a+86b; 46a+87b; 46a+88b; 46a+89b; 46a+90b; 46a+91 b;
280
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
46a+92b; 46a+93b; 46a+94b; 46a+95b; 46a+96b; 46a+97b; 47a+1 b; 47a + 2b;
47a + 3b; 47a+4b; 47a+5b; 47a+6b; 47a+7b; 47a+8b; 47a+9b; 47a+10b;
47a+11 b; 47a+12b; 47a+13b; 47a+14b; 47a+15b; 47a+16b; 47a+17b;
47a+18b; 47a+19b; 47a+20b; 47a+21 b; 47a+22b; 47a+23b; 47a+24b;
47a+25b; 47a+26b; 47a+27b; 47a+28b; 47a+29b; 47a+30b; 47a+31 b;
47a+32b; 47a+33b; 47a+34b; 47a+35b; 47a+36b; 47a+37b; 47a+38b;
47a+39b; 47a+40b; 47a+41 b; 47a+42b; 47a+43b; 47a+44b; 47a+45b;
47a+46b; 47a+47b; 47a+48b; 47a+49b; 47a+50b; 47a+51 b; 47a+52b;
47a+53b; 47a+54b; 47a+55b; 47a+55b; 47a+57b; 47a+58b; 47a+59b;
47a+60b; 47a+61 b; 47a+62b; 47a+63b; 47a+64b; 47a+65b; 47a+66b;
47a+67b; 47a+68b; 47a+69b; 47a+70b; 47a+71 b; 47a+72b; 47a+73b;
47a+74b; 47a+75b; 47a+76b; 47a+77b; 47a+78b; 47a+79b; 47a+80b;
47a+81 b; 47a+82b; 47a+83b; 47a+84b; 47a+85b; 47a+86b; 47a+87b;
47a+88b; 47a+89b; 47a+90b; 47a+91 b; 47a+92b; 47a+93b; 47a+94b;
47a+95b; 47a+96b; 47a+97b; 48a+1 b; 48a + 2b; 48a + 3b; 48a+4b; 48a+5b;
48a+6b; 48a+7b; 48a+8b; 48a+9b; 48a+10b; 48a+11 b; 48a+12b; 48a+13b;
48a+14b; 48a+15b; 48a+16b; 48a+17b; 48a+18b; 48a+19b; 48a+20b;
48a+21 b; 48a+22b; 48a+23b; 48a+24b; 48a+25b; 48a+26b; 48a+27b;
48a+28b; 48a+29b; 48a+30b; 48a+31 b; 48a+32b; 48a+33b; 48a+34b;
48a+35b; 48a+36b; 48a+37b; 48a+38b; 48a+39b; 48a+40b; 48a+41 b;
48a+42b; 48a+43b; 48a+44b; 48a+45b; 48a+46b; 48a+47b; 48a+48b;
48a+49b; 48a+50b; 48a+51 b; 48a+52b; 48a+53b; 48a+54b; 48a+55b;
48a+55b; 48a+57b; 48a+58b; 48a+59b; 48a+60b; 48a+61 b; 48a+62b;
48a+63b; 48a+64b; 48a+65b; 48a+66b; 48a+67b; 48a+68b; 48a+69b;
48a+70b; 48a+71 b; 48a+72b; 48a+73b; 48a+74b; 48a+75b; 48a+76b;
48a+77b; 48a+78b; 48a+79b; 48a+80b; 48a+81 b; 48a+82b; 48a+83b;
48a+84b; 48a+85b; 48a+86b; 48a+87b; 48a+88b; 48a+89b; 48a+90b;
48a+91 b; 48a+92b; 48a+93b; 48a+94b; 48a+95b; 48a+96b; 48a+97b; 49a+1 b;
49a + 2b; 49a + 3b; 49a+4b; 49a+5b; 49a+6b; 49a+7b; 49a+8b; 49a+9b;
49a+1 Ob; 49a+11 b; 49a+12b; 49a+13b; 49a+14b; 49a+15b; 49a+16b;
281
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
49a+17b; 49a+18b; 49a+19b; 49a+20b; 49a+21 b; 49a+22b; 49a+23b;
49a+24b; 49a+25b; 49a+26b; 49a+27b; 49a+28b; 49a+29b; 49a+30b;
49a+31 b; 49a+32b; 49a+33b; 49a+34b; 49a+35b; 49a+36b; 49a+37b;
49a+38b; 49a+39b; 49a+40b; 49a+41 b; 49a+42b; 49a+43b; 49a+44b;
49a+45b; 49a+46b; 49a+47b; 49a+48b; 49a+49b; 49a+50b; 49a+51 b;
49a+52b; 49a+53b; 49a+54b; 49a+55b; 49a+55b; 49a+57b; 49a+58b;
49a+59b; 49a+60b; 49a+61 b; 49a+62b; 49a+63b; 49a+64b; 49a+65b;
49a+66b; 49a+67b; 49a+68b; 49a+69b; 49a+70b; 49a+71 b; 49a+72b;
49a+73b; 49a+74b; 49a+75b; 49a+76b; 49a+77b; 49a+78b; 49a+79b;
49a+80b; 49a+81 b; 49a+82b; 49a+83b; 49a+84b; 49a+85b; 49a+86b;
49a+87b; 49a+88b; 49a+89b; 49a+90b; 49a+91 b; 49a+92b; 49a+93b;
49a+94b; 49a+95b; 49a+96b; 49a+97b; 50a+1 b; 50a + 2b; 50a + 3b; 50a+4b;
50a+5b; 50a+6b; 50a+7b; 50a+8b; 50a+9b; 50a+1 Ob; 50a+11 b; 50a+12b;
50a+13b; 50a+14b; 50a+15b; 50a+16b; 50a+17b; 50a+18b; 50a+19b;
50a+20b; 50a+21 b; 50a+22b; 50a+23b; 50a+24b; 50a+25b; 50a+26b;
50a+27b; 50a+28b; 50a+29b; 50a+30b; 50a+31 b; 50a+32b; 50a+33b;
50a+34b; 50a+35b; 50a+36b; 50a+37b; 50a+38b; 50a+39b; 50a+40b;
50a+41 b; 50a+42b; 50a+43b; 50a+44b; 50a+45b; 50a+46b; 50a+47b;
50a+48b; 50a+49b; 50a+50b; 50a+51 b; 50a+52b; 50a+53b; 50a+54b;
50a+55b; 50a+55b; 50a+57b; 50a+58b; 50a+59b; 50a+60b; 50a+61 b;
50a+62b; 50a+63b; 50a+64b; 50a+65b; 50a+66b; 50a+67b; 50a+68b;
50a+69b; 50a+70b; 50a+71 b; 50a+72b; 50a+73b; 50a+74b; 50a+75b;
50a+76b; 50a+77b; 50a+78b; 50a+79b; 50a+80b; 50a+81 b; 50a+82b;
50a+83b; 50a+84b; 50a+85b; 50a+86b; 50a+87b; 50a+88b; 50a+89b;
50a+90b; 50a+91 b; 50a+92b; 50a+93b; 50a+94b; 50a+95b; 50a+96b;
50a+97b; 51 a+1 b; 51 a + 2b; 51 a + 3b; 51 a+4b; 51 a+5b; 51 a+6b; 51 a+7b;
51 a+8b; 51 a+9b; 51 a+1 Ob; 51 a+11 b; 51 a+12b; 51 a+13b; 51 a+14b; 51
a+15b;
51 a+16b; 51 a+17b; 51 a+18b; 51 a+19b; 51 a+20b; 51 a+21 b; 51 a+22b;
51 a+23b; 51 a+24b; 51 a+25b; 51 a+26b; 51 a+27b; 51 a+28b; 51 a+29b;
51 a+30b; 51 a+31 b; 51 a+32b; 51 a+33b; 51 a+34b; 51 a+35b; 51 a+36b;
282
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
51 a+37b; 51 a+38b; 51 a+39b; 51 a+40b; 51 a+41 b; 51 a+42b; 51 a+43b;
51 a+44b; 51 a+45b; 51 a+46b; 51 a+47b; 51 a+48b; 51 a+49b; 51 a+50b;
51 a+51 b; 51 a+52b; 51 a+53b; 51 a+54b; 51 a+55b; 51 a+55b; 51 a+57b;
51 a+58b; 51 a+59b; 51 a+60b; 51 a+61 b; 51 a+62b; 51 a+63b; 51 a+64b;
51 a+65b; 51 a+66b; 51 a+67b; 51 a+68b; 51 a+69b; 51 a+70b; 51 a+71 b;
51 a+72b; 51 a+73b; 51 a+74b; 51 a+75b; 51 a+76b; 51 a+77b; 51 a+78b;
51 a+79b; 51 a+80b; 51 a+81 b; 51 a+82b; 51 a+83b; 51 a+84b; 51 a+85b;
51 a+86b; 51 a+87b; 51 a+88b; 51 a+89b; 51 a+90b; 51 a+91 b; 51 a+92b;
51 a+93b; 51 a+94b; 51 a+95b; 51 a+96b; 51 a+97b; 52a+1 b; 52a + 2b; 52a + 3b;
52a+4b; 52a+5b; 52a+6b; 52a+7b; 52a+8b; 52a+9b; 52a+1 Ob; 52a+11 b;
52a+12b; 52a+13b; 52a+14b; 52a+15b; 52a+16b; 52a+17b; 52a+18b;
52a+19b; 52a+20b; 52a+21 b; 52a+22b; 52a+23b; 52a+24b; 52a+25b;
52a+26b; 52a+27b; 52a+28b; 52a+29b; 52a+30b; 52a+31 b; 52a+32b;
52a+33b; 52a+34b; 52a+35b; 52a+36b; 52a+37b; 52a+38b; 52a+39b;
52a+40b; 52a+41 b; 52a+42b; 52a+43b; 52a+44b; 52a+45b; 52a+46b;
52a+47b; 52a+48b; 52a+49b; 52a+50b; 52a+51 b; 52a+52b; 52a+53b;
52a+54b; 52a+55b; 52a+55b; 52a+57b; 52a+58b; 52a+59b; 52a+60b;
52a+61 b; 52a+62b; 52a+63b; 52a+64b; 52a+65b; 52a+66b; 52a+67b;
52a+68b; 52a+69b; 52a+70b; 52a+71 b; 52a+72b; 52a+73b; 52a+74b;
52a+75b; 52a+76b; 52a+77b; 52a+78b; 52a+79b; 52a+80b; 52a+81 b;
52a+82b; 52a+83b; 52a+84b; 52a+85b; 52a+86b; 52a+87b; 52a+88b;
52a+89b; 52a+90b; 52a+91 b; 52a+92b; 52a+93b; 52a+94b; 52a+95b;
52a+96b; 52a+97b; 53a+1 b; 53a + 2b; 53a + 3b; 53a+4b; 53a+5b; 53a+6b;
53a+7b; 53a+8b; 53a+9b; 53a+10b; 53a+11 b; 53a+12b; 53a+13b; 53a+14b;
53a+15b; 53a+16b; 53a+17b; 53a+18b; 53a+19b; 53a+20b; 53a+21 b;
53a+22b; 53a+23b; 53a+24b; 53a+25b; 53a+26b; 53a+27b; 53a+28b;
53a+29b; 53a+30b; 53a+31 b; 53a+32b; 53a+33b; 53a+34b; 53a+35b;
53a+36b; 53a+37b; 53a+38b; 53a+39b; 53a+40b; 53a+41 b; 53a+42b;
53a+43b; 53a+44b; 53a+45b; 53a+46b; 53a+47b; 53a+48b; 53a+49b;
53a+50b; 53a+51 b; 53a+52b; 53a+53b; 53a+54b; 53a+55b; 53a+55b;
283
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
53a+57b; 53a+58b; 53a+59b; 53a+60b; 53a+61 b; 53a+62b; 53a+63b;
53a+64b; 53a+65b; 53a+66b; 53a+67b; 53a+68b; 53a+69b; 53a+70b;
53a+71 b; 53a+72b; 53a+73b; 53a+74b; 53a+75b; 53a+76b; 53a+77b;
53a+78b; 53a+79b; 53a+80b; 53a+81 b; 53a+82b; 53a+83b; 53a+84b;
53a+85b; 53a+86b; 53a+87b; 53a+88b; 53a+89b; 53a+90b; 53a+91 b;
53a+92b; 53a+93b; 53a+94b; 53a+95b; 53a+96b; 53a+97b; 54a+1 b; 54a + 2b;
54a + 3b; 54a+4b; 54a+5b; 54a+6b; 54a+7b; 54a+8b; 54a+9b; 54a+10b;
54a+11 b; 54a+12b; 54a+13b; 54a+14b; 54a+15b; 54a+16b; 54a+17b;
54a+18b; 54a+19b; 54a+20b; 54a+21 b; 54a+22b; 54a+23b; 54a+24b;
54a+25b; 54a+26b; 54a+27b; 54a+28b; 54a+29b; 54a+30b; 54a+31 b;
54a+32b; 54a+33b; 54a+34b; 54a+35b; 54a+36b; 54a+37b; 54a+38b;
54a+39b; 54a+40b; 54a+41 b; 54a+42b; 54a+43b; 54a+44b; 54a+45b;
54a+46b; 54a+47b; 54a+48b; 54a+49b; 54a+50b; 54a+51 b; 54a+52b;
54a+53b; 54a+54b; 54a+55b; 54a+55b; 54a+57b; 54a+58b; 54a+59b;
54a+60b; 54a+61 b; 54a+62b; 54a+63b; 54a+64b; 54a+65b; 54a+66b;
54a+67b; 54a+68b; 54a+69b; 54a+70b; 54a+71 b; 54a+72b; 54a+73b;
54a+74b; 54a+75b; 54a+76b; 54a+77b; 54a+78b; 54a+79b; 54a+80b;
54a+81 b; 54a+82b; 54a+83b; 54a+84b; 54a+85b; 54a+86b; 54a+87b;
54a+88b; 54a+89b; 54a+90b; 54a+91 b; 54a+92b; 54a+93b; 54a+94b;
54a+95b; 54a+96b; 54a+97b; 55a+1 b; 55a + 2b; 55a + 3b; 55a+4b; 55a+5b;
55a+6b; 55a+7b; 55a+8b; 55a+9b; 55a+1 Ob; 55a+11 b; 55a+12b; 55a+13b;
55a+14b; 55a+15b; 55a+16b; 55a+17b; 55a+18b; 55a+19b; 55a+20b;
55a+21 b; 55a+22b; 55a+23b; 55a+24b; 55a+25b; 55a+26b; 55a+27b;
55a+28b; 55a+29b; 55a+30b; 55a+31 b; 55a+32b; 55a+33b; 55a+34b;
55a+35b; 55a+36b; 55a+37b; 55a+38b; 55a+39b; 55a+40b; 55a+41 b;
55a+42b; 55a+43b; 55a+44b; 55a+45b; 55a+46b; 55a+47b; 55a+48b;
55a+49b; 55a+50b; 55a+51 b; 55a+52b; 55a+53b; 55a+54b; 55a+55b;
55a+55b; 55a+57b; 55a+58b; 55a+59b; 55a+60b; 55a+61 b; 55a+62b;
55a+63b; 55a+64b; 55a+65b; 55a+66b; 55a+67b; 55a+68b; 55a+69b;
55a+70b; 55a+71 b; 55a+72b; 55a+73b; 55a+74b; 55a+75b; 55a+76b;
284
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
55a+77b; 55a+78b; 55a+79b; 55a+80b; 55a+81 b; 55a+82b; 55a+83b;
55a+84b; 55a+85b; 55a+86b; 55a+87b; 55a+88b; 55a+89b; 55a+90b;
55a+91 b; 55a+92b; 55a+93b; 55a+94b; 55a+95b; 55a+96b; 55a+97b; 56a+1 b;
56a + 2b; 56a + 3b; 56a+4b; 56a+5b; 56a+6b; 56a+7b; 56a+8b; 56a+9b;
56a+1 Ob; 56a+11 b; 56a+12b; 56a+13b; 56a+14b; 56a+15b; 56a+16b;
56a+17b; 56a+18b; 56a+19b; 56a+20b; 56a+21 b; 56a+22b; 56a+23b;
56a+24b; 56a+25b; 56a+26b; 56a+27b; 56a+28b; 56a+29b; 56a+30b;
56a+31 b; 56a+32b; 56a+33b; 56a+34b; 56a+35b; 56a+36b; 56a+37b;
56a+38b; 56a+39b; 56a+40b; 56a+41 b; 56a+42b; 56a+43b; 56a+44b;
56a+45b; 56a+46b; 56a+47b; 56a+48b; 56a+49b; 56a+50b; 56a+51 b;
56a+52b; 56a+53b; 56a+54b; 56a+55b; 56a+55b; 56a+57b; 56a+58b;
56a+59b; 56a+60b; 56a+61 b; 56a+62b; 56a+63b; 56a+64b; 56a+65b;
56a+66b; 56a+67b; 56a+68b; 56a+69b; 56a+70b; 56a+71 b; 56a+72b;
56a+73b; 56a+74b; 56a+75b; 56a+76b; 56a+77b; 56a+78b; 56a+79b;
56a+80b; 56a+81 b; 56a+82b; 56a+83b; 56a+84b; 56a+85b; 56a+86b;
56a+87b; 56a+88b; 56a+89b; 56a+90b; 56a+91 b; 56a+92b; 56a+93b;
56a+94b; 56a+95b; 56a+96b; 56a+97b; 57a+1 b; 57a + 2b; 57a + 3b; 57a+4b;
57a+5b; 57a+6b; 57a+7b; 57a+8b; 57a+9b; 57a+1 Ob; 57a+11 b; 57a+12b;
57a+13b; 57a+14b; 57a+15b; 57a+16b; 57a+17b; 57a+18b; 57a+19b;
57a+20b; 57a+21 b; 57a+22b; 57a+23b; 57a+24b; 57a+25b; 57a+26b;
57a+27b; 57a+28b; 57a+29b; 57a+30b; 57a+31 b; 57a+32b; 57a+33b;
57a+34b; 57a+35b; 57a+36b; 57a+37b; 57a+38b; 57a+39b; 57a+40b;
57a+41 b; 57a+42b; 57a+43b; 57a+44b; 57a+45b; 57a+46b; 57a+47b;
57a+48b; 57a+49b; 57a+50b; 57a+51 b; 57a+52b; 57a+53b; 57a+54b;
57a+55b; 57a+55b; 57a+57b; 57a+58b; 57a+59b; 57a+60b; 57a+61 b;
57a+62b; 57a+63b; 57a+64b; 57a+65b; 57a+66b; 57a+67b; 57a+68b;
57a+69b; 57a+70b; 57a+71 b; 57a+72b; 57a+73b; 57a+74b; 57a+75b;
57a+76b; 57a+77b; 57a+78b; 57a+79b; 57a+80b; 57a+81 b; 57a+82b;
57a+83b; 57a+84b; 57a+85b; 57a+86b; 57a+87b; 57a+88b; 57a+89b;
57a+90b; 57a+91 b; 57a+92b; 57a+93b; 57a+94b; 57a+95b; 57a+96b;
285
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
57a+97b; 58a+1 b; 58a + 2b; 58a + 3b; 58a+4b; 58a+5b; 58a+6b; 58a+7b;
58a+8b; 58a+9b; 58a+1 Ob; 58a+11 b; 58a+12b; 58a+13b; 58a+14b; 58a+15b;
58a+16b; 58a+17b; 58a+18b; 58a+19b; 58a+20b; 58a+21 b; 58a+22b;
58a+23b; 58a+24b; 58a+25b; 58a+26b; 58a+27b; 58a+28b; 58a+29b;
58a+30b; 58a+31 b; 58a+32b; 58a+33b; 58a+34b; 58a+35b; 58a+36b;
58a+37b; 58a+38b; 58a+39b; 58a+40b; 58a+41 b; 58a+42b; 58a+43b;
58a+44b; 58a+45b; 58a+46b; 58a+47b; 58a+48b; 58a+49b; 58a+50b;
58a+51 b; 58a+52b; 58a+53b; 58a+54b; 58a+55b; 58a+55b; 58a+57b;
58a+58b; 58a+59b; 58a+60b; 58a+61 b; 58a+62b; 58a+63b; 58a+64b;
58a+65b; 58a+66b; 58a+67b; 58a+68b; 58a+69b; 58a+70b; 58a+71 b;
58a+72b; 58a+73b; 58a+74b; 58a+75b; 58a+76b; 58a+77b; 58a+78b;
58a+79b; 58a+80b; 58a+81 b; 58a+82b; 58a+83b; 58a+84b; 58a+85b;
58a+86b; 58a+87b; 58a+88b; 58a+89b; 58a+90b; 58a+91 b; 58a+92b;
58a+93b; 58a+94b; 58a+95b; 58a+96b; 58a+97b; 59a+1 b; 59a + 2b; 59a + 3b;
59a+4b; 59a+5b; 59a+6b; 59a+7b; 59a+8b; 59a+9b; 59a+1 Ob; 59a+11 b;
59a+12b; 59a+13b; 59a+14b; 59a+15b; 59a+16b; 59a+17b; 59a+18b;
59a+19b; 59a+20b; 59a+21 b; 59a+22b; 59a+23b; 59a+24b; 59a+25b;
59a+26b; 59a+27b; 59a+28b; 59a+29b; 59a+30b; 59a+31 b; 59a+32b;
59a+33b; 59a+34b; 59a+35b; 59a+36b; 59a+37b; 59a+38b; 59a+39b;
59a+40b; 59a+41 b; 59a+42b; 59a+43b; 59a+44b; 59a+45b; 59a+46b;
59a+47b; 59a+48b; 59a+49b; 59a+50b; 59a+51 b; 59a+52b; 59a+53b;
59a+54b; 59a+55b; 59a+55b; 59a+57b; 59a+58b; 59a+59b; 59a+60b;
59a+61 b; 59a+62b; 59a+63b; 59a+64b; 59a+65b; 59a+66b; 59a+67b;
59a+68b; 59a+69b; 59a+70b; 59a+71 b; 59a+72b; 59a+73b; 59a+74b;
59a+75b; 59a+76b; 59a+77b; 59a+78b; 59a+79b; 59a+80b; 59a+81 b;
59a+82b; 59a+83b; 59a+84b; 59a+85b; 59a+86b; 59a+87b; 59a+88b;
59a+89b; 59a+90b; 59a+91 b; 59a+92b; 59a+93b; 59a+94b; 59a+95b;
59a+96b; 59a+97b; 60a+1 b; 60a + 2b; 60a + 3b; 60a+4b; 60a+5b; 60a+6b;
60a+7b; 60a+8b; 60a+9b; 60a+10b; 60a+11 b; 60a+12b; 60a+13b; 60a+14b;
60a+15b; 60a+16b; 60a+17b; 60a+18b; 60a+19b; 60a+20b; 60a+21 b;
286
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
60a+22b; 60a+23b; 60a+24b; 60a+25b; 60a+26b; 60a+27b; 60a+28b;
60a+29b; 60a+30b; 60a+31 b; 60a+32b; 60a+33b; 60a+34b; 60a+35b;
60a+36b; 60a+37b; 60a+38b; 60a+39b; 60a+40b; 60a+41 b; 60a+42b;
60a+43b; 60a+44b; 60a+45b; 60a+46b; 60a+47b; 60a+48b; 60a+49b;
60a+50b; 60a+51 b; 60a+52b; 60a+53b; 60a+54b; 60a+55b; 60a+55b;
60a+57b; 60a+58b; 60a+59b; 60a+60b; 60a+61 b; 60a+62b; 60a+63b;
60a+64b; 60a+65b; 60a+66b; 60a+67b; 60a+68b; 60a+69b; 60a+70b;
60a+71 b; 60a+72b; 60a+73b; 60a+74b; 60a+75b; 60a+76b; 60a+77b;
60a+78b; 60a+79b; 60a+80b; 60a+81 b; 60a+82b; 60a+83b; 60a+84b;
60a+85b; 60a+86b; 60a+87b; 60a+88b; 60a+89b; 60a+90b; 60a+91 b;
60a+92b; 60a+93b; 60a+94b; 60a+95b; 60a+96b; 60a+97b; etc.
Within certain embodiments of the invention, the therapeutic
composition can also comprise radio-opaque, echogenic materials and
magnetic resonance imaging (MRI) responsive materials (i.e., MRI contrast
agents) to aid in visualization of the composition under ultrasound,
fluoroscopy
and/or MRI. For example, a composition may be echogenic or radiopaque
(e.g., made with echogenic or radiopaque with materials such as powdered
tantalum, tungsten, barium carbonate, bismuth oxide, barium sulfate,
metrazimide, iopamidol, iohexol, iopromide, iobitridol, iomeprol, iopentol,
ioversol, ioxilan, iodixanol, iotrolan, acetrizoic acid derivatives,
diatrizoic acid
derivatives, iothalamic acid derivatives, ioxithalamic acid derivatives,
metrizoic
acid derivatives, iodamide, lypophylic agents, iodipamide and ioglycamic acid
or, by the addition of microspheres or bubbles which present an acoustic
interface). For visualization under MRI, contrast agents (e.g., gadolinium
(III)
chelates or iron oxide compounds) may be incorporated into the composition..
The compositions may, alternatively, or in addition, be visualized
under visible light, using fluorescence, or by other spectroscopic means.
Visualization agents that can be included for this purpose include dyes,
pigments, and other colored agents. In one aspect, the composition may
287
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
further include a colorant to improve visualization of the composition in vivo
andlor ex vivo. Frequently, compositions can be difficult to visualize upon
delivery into a host, especially at the margins of an implant or tissue. A
coloring
agent can be incorporated into a composition to reduce or eliminate the
incidence or severity of this problem. The coloring agent provides a unique
color, increased contrast, or unique fluorescence characteristics to the
composition. In one aspect, a composition is provided that includes a colorant
such that it is readily visible (under visible light or using a fluorescence
technique) and easily differentiated from its implant site. In another aspect,
a
colorant can be included in a liquid or semi-solid composition. For example, a
single component of a two component mixture may be colored, such that when
combined ex-vivo or in-vivo, the mixture is sufficiently colored.
The coloring agent may be, for example, an endogenous
compound (e.g., an amino acid or vitamin) or a nutrient or food material and
may be a hydrophobic or a hydrophilic compound. Preferably, the colorant has
a very low or no toxicity at the concentration used. Also preferred are
colorants
that are safe and normally enter the body through absorption such as ~-
carotene. Representative examples of colored nutrients (under visible light)
include fat soluble vitamins such as Vitamin A (yellow); water soluble
vitamins
such as Vitamin B12 (pink-red) and folic acid (yellow-orange); carotenoids
such
as ~3-carotene (yellow-purple) and lycopene (red). Other examples of coloring
agents include natural product (berry and fruit) extracts such as anthrocyanin
(purple) and saffron extract (dark red). The coloring agent may be a
fluorescent
or phosphorescent compound such as a-tocopherolquinol (a Vitamin E
derivative) or L-tryptophan.
In one aspect, the compositions of the present invention include
one or more coloring agents, also referred to as dyestuffs, which will be
present
in an effective amount to impart observable coloration to the composition,
e.g.,
the gel. Examples of coloring agents include dyes suitable for food such as
those known as F. D. & C. dyes and natural coloring agents such as grape skin
288
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
extract, beet red powder, beta carotene, annato, carmine, turmeric, paprika,
and so forth. Derivatives, analogues, and isomers of any of the above colored
compound also may be used. The method for incorporating a colorant into an
implant or therapeutic composition may be varied depending on the properties
of and the desired location for the colorant. For example, a hydrophobic
colorant may be selected for hydrophobic matrices. The colorant may be
incorporated into a carrier matrix, such as micelles. Further, the pH of the
environment may be controlled to further control the color and intensity.
In one aspect, the compositions of the present invention include
one or more preservatives or bacteriostatic agents present in an effective
amount to preserve the composition and/or inhibit bacterial growth in the
composition, for example, bismuth tribromophenate, methyl hydroxybenzoate,
bacitracin, ethyl hydroxybenzoate, propyl hydroxybenzoate, erythromycin,
chlorocresol, benzalkonium chlorides, and the like. Examples of the
preservative include paraoxybenzoic acid esters, chlorobutanol, benzylalcohol,
phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. In one aspect, the
compositions of the present invention include one or more bactericidal (also
known as bacteriacidal) agents. ,
In one aspect, the compositions of the present invention include
one or more antioxidants, present in an effective amount. Examples of the
antioxidant include sulfites, alpha-tocopherol, beta-carotene and ascorbic
acid.
Further, therapeutic compositions of the present invention should
preferably be have a stable shelf-life of at least several months and capable
of
being produced and maintained under sterile conditions. The composition may
be sterile either by preparing them under aseptic environment and/or they may
be terminally sterilized using methods known in the art. A combination of both
of these methods may also be used to prepare the composition in the sterile
form. Sterilization may also occur by terminally using gamma radiation or
electron beam sterilization methods.
289
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In one aspect, the compounds and compositions of the present
invention are sterile. Many pharmaceuticals are manufactured to be sterile and
this criterion is defined by the USP XXII <1211 >. The term "USP" refers to
U.S.
Pharmacopeia (see www.usp.org, Rockville, MD). Sterilization in this
embodiment may be accomplished by a number of means accepted in the
industry and listed in the USP XXII <1211 >, including gas sterilization,
ionizing
radiation or, when appropriate, filtration. Sterilization may be maintained by
what is termed asceptic processing, defined also in USP XXII <1211 >.
Acceptable gases used for gas sterilization include ethylene oxide. Acceptable
radiation types used for ionizing radiation methods include gamma, for
instance
from a cobalt 60 source and electron beam. A typical dose of gamma radiation
is 2.5 MRad. Filtration may be accomplished using a filter with suitable pore
size, for example 0.22 ~,m and of a suitable material, for instance
polytetrafluoroethylene (e.g., TEFLON from E. I. DuPont De Nemours and
Company, Wilmington, DE).
In another aspect, the compositions of the present invention are
contained in a container that allows them to be used for their intended
purpose,
i.e., as a pharmaceutical composition. Properties of the container that are
important are a volume of empty space to allow for the addition of a
constitution
medium, such as water or other aqueous medium, e.g., saline, acceptable light
transmission characteristics in order to prevent light energy from damaging
the
composition in the container (refer to USP XXI I <661 >), an acceptable limit
of
extractables within the container material (refer to USP XXII), an acceptable
barrier capacity for moisture (refer to USP XXI I <671 >) or oxygen. In the
case
of oxygen penetration, this may be controlled by including in the container, a
positive pressure of an inert gas, such as high purity nitrogen, or a noble
gas,
such as argon.
Typical materials used to make containers for pharmaceuticals
include USP Type I through III and Type NP glass (refer to USP XXII <661 >),
290
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polyethylene, TEFLON, silicone, and gray-butyl rubber. For parenterals, USP
Types I to III glass and polyethylene are preferred.
E. Methods for Utilizing Compositions
The compositions of the present invention can be used in a
variety of different applications. For example, the compositions may be used
for (a) preventing tissue adhesions; (b) treating or preventing inflammatory
arthritis; (c) prevention of cartilage loss; (d) treating or preventing
hypertrophic
scars/keloids; (e) treating or preventing vascular disease; and (f) coating
medical implants and devices. A more detailed description of several specific
applications is given below.
Adhesion Prevention
The present invention provides compositions for use in the
prevention of adhesions (e.g., surgical adhesions). The polymeric compositions
may include one or more therapeutically active agents (e.g., anti-scarring
agents), which provide pharmacological alteration of cellular and/ or non-
cellular processes involved in the development and/or progression of surgical
adhesions. Therapeutically active agents are described that can reduce
surgical adhesions by inhibiting the formation of fibrous or scar tissue. In
another aspect, the present invention provides surgical adhesion barriers that
include an anti-scarring agent or a composition that includes an anti-scarring
agent.
Surgical adhesions are abnormal, fibrous bands of scar tissue that
can form inside the body as a result of the healing process that follows any
open or minimally invasive surgical procedure including abdominal,
gynecologic, cardiothoracic, spinal, plastic, vascular, ENT, ophthalmologic,
urologic, neuro, or orthopedic surgery. Surgical adhesions are typically
connective tissue structures that form between adjacent injured areas within
the
body. Briefly, localized areas of injury trigger an inflammatory and healing
291
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
response that culminates in healing and scar tissue formation. If scarring
results in the formation of fibrous tissue bands or adherence of adjacent
anatomical structures (that should be separate), surgical adhesion formation
is
said to have occurred. Adhesions can range from flimsy, easily separable
structures to dense, tenacious fibrous structures that can only be separated
by
surgical dissection. While many adhesions are benign, some can cause
significant clinical problems and are a leading cause of repeat surgical
intervention. Surgery to breakdown adhesions (adhesiolysis) often results in
failure and recurrence because the surgical trauma involved in breaking down
the adhesion triggers the entire process to repeat itself. Surgical breakdown
of
adhesions is a significant clinical problem and it is estimated that there
were
473,000 adhesiolysis procedures in the US in 2002. According to the
Diagnosis-Related Groups (DRGs), the total hospital charges for these
procedures is likely to be at least US $10 billion annually.
Since all interventions involve a certain degree of trauma to the
operative tissues, virtually any procedure (no matter how well executed) has
the
potential to result in the formation of clinically significant adhesion
formation.
Adhesions can be triggered by surgical trauma such as cutting, manipulation,
retraction or suturing, as well as from inflammation, infection (e.g., fungal
or
mycobacterium), bleeding or the presence of a foreign body. Surgical trauma
may also result from tissue drying, ischemia, or thermal injury. Due to the
diverse etiology of surgical adhesions, the potential for formation exists
regardless of whether the surgery is done in a so-called minimally invasive
fashion (e.g., catheter-based therapies, laparoscopy) or in a standard open
technique involving one or more relatively large incisions. Although a
potential
complication of any surgical intervention, surgical adhesions are particularly
problematic in GI surgery (causing bowel obstruction), gynecological surgery
(causing pain and/or infertility), tendon repairs (causing shortening and
flexion
deformities), joint capsule procedures (causing capsular contractures), and
nerve and muscle repair procedures (causing diminished or lost function).
292
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Surgical adhesions may cause various, often serious and
unpredictable clinical complications; some of which manifest themselves only
years after the original procedure was completed. Complications from surgical
adhesions are a major cause of failed surgical therapy and are the leading
cause of bowel obstruction and infertility. Other adhesion-related
complications
include chronic back or pelvic pain, intestinal obstruction, urethral
obstruction
and voiding dysfunction. Relieving the post-surgical complications caused by
adhesions generally requires another surgery. However, the subsequent
surgery is further complicated by adhesions formed as a result of the previous
surgery. In addition, the second surgery is likely to result in further
adhesions
and a continuing cycle of additional surgical complications.
The placement of medical devices and implants also increases
the risk that surgical adhesions will occur. In addition to the above
mechanisms, an implanted device can trigger a "foreign body" response where
the immune system recognizes the implant as foreign and triggers an
inflammatory reaction that ultimately leads to scar tissue formation. A
specific
form of foreign body reaction in response to medical device placement is
complete enclosure ("walling off') of the implant in a capsule of scar tissue
(encapsulation). Fibrous encapsulation of implanted devices and implants can
complicate any procedure, but breast augmentation and reconstruction surgery,
joint replacement surgery, hernia repair surgery, artificial vascular graft
surgery,
stent placement, and neurosurgery are particularly prone to this complication.
In each case, the implant becomes encapsulated by a fibrous connective tissue
capsule which compromises or impairs the function of the surgical implant
(e.g.,
breast implant, artificial joint, surgical mesh, vascular graft, stent or
dural
patch).
Adhesions generally begin to form within the first several days
after surgery. Generally, adhesion formation is an inflammatory reaction in
which factors are released, increasing vascular permeability and resulting in
fibrinogen influx and fibrin deposition. This deposition forms a matrix that
293
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
bridges the abutting tissues. Fibroblasts accumulate, attach to the matrix,
deposit collagen and induce angiogenesis. If this cascade of events can be
prevented within 4 to 5 days following surgery, then adhesion formation may be
inhibited.
Various modes of adhesion prevention have been examined,
including (1 ) prevention of fibrin deposition, (2) reduction of local tissue
inflammation and (3) removal of fibrin deposits. Fibrin deposition is
prevented
through the use of physical barriers that are either mechanical or comprised
of
viscous solutions. Barriers have the added advantage of physically preventing
adjacent tissues from contacting each other and thereby reducing the
probability that they will scar together. Although many investigators and
commercial products utilize adhesion prevention barriers, a number of
technical
difficulties exist and significant failure rates have been reported.
Inflammation
is reduced by the administration of drugs such as corticosteroids and non-
steroidal anti-inflammatory drugs. However, the results from the use of these
drugs in animal models have not been encouraging due to the extent of the
inflammatory response and dose restriction due to systemic side effects.
Finally, the removal of fibrin deposits has been investigated using
proteolytic
and fibrinolytic enzymes. A potential complication to the clinical use of
these
enzymes is the possibility for post-surgical excessive bleeding (surgical
hemostasis is critical for procedural success).
Numerous polymeric compositions for use in the prevention of
surgical adhesions (e.g., surgical adhesion barriers) may be used in the
practice of the invention, either alone, or in combination with one or more
anti-
scarring agents. It should be noted that certain polymeric compositions can
themselves help prevent the formation of fibrous tissue at a surgical site. In
certain embodiments, the polymer composition can form a barrier between the
tissue surfaces or organs.
For example, the surgical adhesion barrier may be coated onto
tissue surfaces and may be composed of an aqueous solution of a hydrophilic,
294
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polymeric material (e.g., polypeptides or polysaccharide) having greater than
50,000 molecular weight and a concentration range of 0.01 % to 15% by weight.
See e.g., U.S. Patent No. 6,464,970. The surgical adhesion barrier may be a
crosslinkable system with at least three reactive compounds each having a
polymeric molecular core with at least one functional group. See e.g., U.S.
Patent No. 6,458,889. The surgical adhesions barrier may be composed of a
non-gelling polyoxyalkylene composition with or without a therapeutic agent.
See e.g., U.S. Patent No. 6,436,425. The surgical adhesions barrier may be
composed of an anionic polymer having an acid sulfate and sulfur content
greater than 5% which acts to inhibit monocyte or macrophage invasion. See
e.g., U.S. Patent No. 6,417,173. The surgical adhesions barrier may be an
aqueous composition including a surfactant, pentoxifylline and a
polyoxyalkylene polyether. See e.g., U.S. Patent No. 6,399,624. The surgical
adhesions barrier may be composed by crosslinking two synthetic polymers,
one having nucleophilic groups and the other having electrophilic groups, such
that they form a matrix that may be used to incorporate a biologically active
compound. See e.g., U.S. Patent Nos. 6,323,278; 6,166,130; 6,051,648 and
5,874,500. The surgical adhesion barrier may be composed of hyaluronic acid
compositions such as those described in U.S. Patents Nos. 6,723,709;
6,531,147; and 6,464,970. The surgical adhesions barrier may be a polymeric
tissue coating which is formed by applying a polymerization initiator to the
tissue and then covering it with a water-soluble macromer that is
polymerizable
using free radical initiators under the influence of UV light. See e.g., U.S.
Patent Nos. 6,177,095 and 6,083,524. The surgical adhesions barrier may be
composed of fluent prepolymeric material that is emitted to the tissue surface
and then exposed to activating energy in situ to initiate conversion of the
applied material to non-fluent polymeric form. See e.g., U.S. Patent Nos.
6,004,547 and 5,612,050. The surgical adhesions barrier may be a hydrogel-
forming, self-solvating, absorbable polyester copolymers capable of selective,
segmental association into compliant hydrogels mass upon contact with an
295
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
aqueous environment. See e.g., U.S. Patent No. 5,612,052. The surgical
adhesions barrier may be an anionic polymer effective to inhibit cell invasion
or
fibrosis (e.g., dermatan sulfate, dextran sulfate, pentosan polysulfate, or
alginate), and a pharmaceutically effective carrier, in which the carrier may
be
semi-solid. See e.g., U.S. Patent Nos. 6,756,362; 6,127,348 and 5,994,325.
The surgical adhesions barrier may be an acidified hydrogel comprising a
carboxypolysaccharide and a polyether having a pH in the range of about 2.0 to
about 6Ø See e.g., U.S. Patent No. 6,017,301. The surgical adhesions barrier
may be composed of dextran sulfate having a molecular weight about 40,000 to
500,000 Daltons which is used to inhibit neurite outgrowth. See e.g., U.S.
Patent No. 5,705,178. The surgical adhesions barrier may be a fragmented
biocompatible hydrogel which is at least partially hydrated and is
substantially
free from an aqueous phase, wherein said hydrogel comprises gelatin and will
absorb water when delivered to a moist tissue target site. See e.g., U.S.
Patent
No. 6,066,325. The surgical adhesions barrier may be a water-soluble,
degradable macromer that is composed of at least two-crosslinkable
substituents that may crosslink to other macromers at a localized site when
under the influence of a polymerization initiator. See e.g., U.S. Patent No.
6,465,001. The surgical adhesions barrier may be a biocompatible adhesive
composition comprising at least one alkyl ester cyanoacrylate monomer and a
polymerization initiator or accelerator. See e.g., U.S. Patent No. 6,620,846.
In one embodiment, the polymers that can form a covalent bond
with the tissue to which it is applied may be used. Polymers containing and/or
terminated with electrophilic groups such as succinimidyl, aldehyde, epoxide,
isocyanate, vinyl, vinyl sulfone, maleimide, -S-S-(C5H4N) or activated esters,
such as are used in peptide synthesis may be used as the reagents. For
example, a 4 armed NHS-derivatized polyethylene glycol (e.g., pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate) may be applied to the
tissue in the solid form or in a solution form. In this embodiment, the 4
armed
NHS-derivatized polyethylene glycol is dissolved in an acidic solution (pH
about
296
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
2-3) and is then co-applied to the tissue using a basic buffer (pH > about 8).
The fibrosis-inhibiting agents) may be incorporated directly into either the 4
armed NHS-derivatized polyethylene glycol, the acidic solution or the basic
buffer. In another embodiment, the fibrosis-inhibiting agent may be
incorporated
into a secondary carrier that may then be incorporated into the 4 armed NHS-
derivatized polyethylene glycol, the acidic solution and/or the basic buffer.
Secondary carriers may include microparticles and/or microspheres which are
made from degradable polymers. Degradable polymers may include polyesters,
where the polyester may comprise the residues of one or more of the
monomers selected from lactide, lactic acid, glycolide, glycolic acid, e-
caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone, and block copolymers of the form X-Y, Y-X-Y, R-(Y-X)", R-
(X-Y)" and X-Y-X (where X in a polyalkylene oxide (e.g., poly(ethylene glycol,
polypropylene glycol) and block copolymers of polyethylene oxide) and
polypropylene oxide) (e.g., PLURONIC and PLURONIC R series of polymers
from BASF Corporation, Mount Olive, NJ) and Y is a biodegradable polyester,
where the polyester may comprise the residues of one or more of the
monomers selected from lactide, lactic acid, glycolide, glycolic acid, e-
caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone (e.g., PLG-PEG-PLG) and R is a multifunctional initiator).
In another embodiment, the tissue reactive polymer may be
applied initially and then the fibrosis-inhibiting agent may then be applied
to the
coated tissue. The fibrosis-inhibiting agent may be applied directly to the
tissue
or it may be incorporated into a secondary carrier. Secondary carriers may
include microspheres (as described above), microparticles (as described
above), gels (e.g., hyaluronic acid, carboxymethyl cellulose, dextran,
297
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
polyethylene oxide) - polypropylene oxide) block copolymers as well as
blends, association complexes and crosslinked compositions thereof) and films
(degradable polyesters, where the polyester may comprise the residues of one
or more of the monomers selected from lactide, lactic acid, glycolide,
glycolic
acid, e-caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric
acid, beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone, and block copolymers of the form X-Y, Y-X-Y, R-(Y-X)~, R-
(X-Y)" and X-Y-X (where X in a polyalkylene oxide (e.g., poly(ethylene glycol,
polypropylene glycol) and block copolymers of polyethylene oxide) and
polypropylene oxide) (e.g., PLURONIC and PLURONIC R series of polymers
from BASF Corporation, Mount Olive, NJ) and Y is a biodegradable polyester,
where the polyester may comprise the residues of one or more of the
monomers selected from lactide, lactic acid, glycolide, glycolic acid, e-
caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, b-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone (e.g., PLG-PEG-PLG) and R is a multifunctional initiator,
hyaluronic acid, carboxymethyl cellulose, dextran, polyethylene oxide) -
polypropylene oxide) block copolymers as well as blends, association
complexes and crosslinked compositions thereof.
A preferred polymeric matrix which can be used to help prevent
the formation of fibrous tissue, either alone or in combination with a
fibrosis
inhibiting agent/composition, is formed from reactants comprising either one
or
both of pentaerythritol polyethylene glycol)ether tetra-sulfhydryl] (4-armed
thiol
PEG, which includes structures having a linking groups) between a sulfhydryl
groups) and the terminus of the polyethylene glycol backbone) and
pentaerythritol polyethylene glycol)ether tetra-succinimidyl glutarate] (4-
armed
NHS PEG, which again includes structures having a linking groups) between a
NHS groups) and the terminus of the polyethylene glycol backbone) as
293
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
reactive reagents. Another preferred composition comprises either one or both
of pentaerythritol polyethylene glycol)ether tetra-amino] (4-armed amino PEG,
which includes structures having a linking groups) between an amino groups)
and the terminus of the polyethylene glycol backbone) and pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate] (4-armed NHS PEG,
which again includes structures having a linking groups) between a NHS
groups) and the terminus of the polyethylene glycol backbone) as reactive
reagents. Chemical structures for these reactants are shown in, e.g., U.S.
Patent 5,874,500. Optionally, collagen or a collagen derivative (e.g.,
methylated collagen) is added to the polyethylene glycol)-containing
reactants) to form a preferred crosslinked matrix that can serve as a
polymeric
carrier for a therapeutic agent or a stand-alone composition to help prevent
the
formation of fibrous tissue.
Surgical adhesion barriers, which may be combined with one or
more anti-scarring agents according to the present invention, also include
commercially available products. Examples of surgical adhesion barrier
compositions into which a fibrosis agent can be incorporated include: (a)
sprayable collagen-containing formulations such as COSTASIS or CT3
(Angiotech Pharmaceuticals, Inc., Canada); (b) sprayable PEG-containing
formulations such as COSEAL or ADHIBIT (Angiotech Pharmaceuticals, Inc.),
SPRAYGEL or DURASEAL (both from Confluent Surgical, Inc., Boston, MA) or
FOCALSEAL (Genzyme Corporation, Cambridge, MA); (c) hyaluronic acid-
containing formulations such as RESTYLANE or PERLANE (both from Q-Med
AB, Sweden), HYLAFORM (Inamed Corporation, Santa Barbara, CA),
SYNVISC (Biomatrix, Inc., Ridgefield, NJ), SEPRAFILM or SEPRACOAT (both
from Genzyme Corporation), (d) fibrinogen-containing formulations such as
FLOSEAL or TISSEAL (both from Baxter Healthcare Corporation, Fremont,
CA); (e) polymeric gels such as REPEL (Life Medical Sciences, Inc., Princeton,
NJ) or FLOWGEL (Baxter Healthcare Corporation, Deerfield, IL), (f) surgical
adhesives containing cyanoacrylates such as DERMABOND (Johnson &
299
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Johnson, Inc., New Brunswick, NJ), INDERMIL (U.S. Surgical Company,
Norwalk, CT), GLUSTITCH (Blacklock Medical Products Inc., Canada),
TISSUMEND (Veterinary Products Laboratories, Phoenix, AZ), VETBOND (3M
Company, St. Paul, MN), HISTOACRYL BLUE (Davis & Geck, St. Louis, MO)
and ORABASE SOOTHE-N-SEAL LIQUID PROTECTANT (Colgate-Palmolive
Company, New York, NY); (g) dextran sulfate gels such as the ADCON range
of products (available from Wright Medical Technology, Inc. Arlington, TN),
(h)
lipid based compositions such as ADSURF (Britannia Pharmaceuticals Ltd.,
United Kingdom) and (j) film compositions such as INTERCEED (Ethicon, Inc.,
Somerville, NJ) and HYDROSORB (MacroPore Biosurgery, Inc., San Diego,
CA /Medtronic Sofamor Danek, Memphis, TN).
For greater clarity, several specific applications and treatments
will be described in greater detail including:
i) Adhesion Prevention in Spinal and Neurosurgical
Procedures
Back pain is the number one cause of healthcare expenditures in
the United States and accounts for over $50 billion in costs annually ($100
billion worldwide). Over 12 million people in the U.S. have some form of
degenerative disc disease (DDD) and 10% of them (1.2 million) will require
surgery to correct their problem.
In healthy individuals, the vertebral column is composed of
vertebral bone plates separated by intervertebral discs that form strong
joints
and absorb spinal compression during movement. The intervertebral disc is
comprised of an inner gel-like substance called the nucleus pulposus which is
surrounded by a tough fibrocartilagenous capsule called the annulus fibrosis.
The nucleus pulposus is composed of a loose framework of collagen fibrils and
connective tissue cells (resembling fibroblasts and chondrocytes) embedded in
a gelatinous matrix of glycosaminoglycans and water. The annulus fibrosus is
composed of numerous concentric rings of fibrocartilage that anchor into the
300
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
vertebral bodies. The most common cause of DDD occurs when tears in the
annulus fibrosis create an area of localized weakness that allow bulging,
herniation or sequestration of the nucleus pulposis and annulus fibrosis into
the
spinal canal and/or spinal foramena. The bulging or herniated disc often
compresses nerve tissue such as spinal cord fibers or spinal cord nerve root
fibers. Pressure on the spinal cord or nerve roots from the damaged
intervertebral disc results in neuronal dysfunction (numbness, weakness,
tingling), crippling pain, bowel or bladder disturbances and can frequently
cause
long-term disability. Although many cases of DDD will spontaneously resolve, a
significant number of patients will require surgical intervention in the form
of
minimally invasive procedures, microdiscectomy, major surgical resection of
the
disc, spinal fusion (fusion of adjacent vertebral bone plates using various
techniques and devices), and/or implantation of an artificial disc. The
present
invention provides for the application of an anti-adhesion or anti-fibrosis
agent
in the surgical management of DDD.
Spinal disc removal is mandatory and urgent in cauda equine
syndrome when there is a significant neurological deficit; particularly bowel
or
bladder dysfunction. It is also performed electively to relieve pain and
eliminate
lesser neurological symptoms. The spinal nerve roots exit the spinal canal
through bony spinal foramena (a bony opening between the vertebra above and
the vertebra below) that is a common site of nerve entrapment. To gain access
to the spinal foramen during back surgeries, vertebral bone tissue is often
resected; a process known as laminectomy.
In open surgical resection of a ruptured lumbar disc or entrapped
spinal nerve root (laminectomy) the patient is placed in a modified kneeling
position under general anesthesia. An incision is made in the posterior
midline
and the tissue is dissected away to expose the appropriate interspace; the
ligamentum flavum is dissected and in some cases portions of the bony lamina
are removed to allow adequate visualization. The nerve root is carefully
retracted away to expose the herniated fragment and the defect in the annulus.
301
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
Typically, the cavity of the disc is entered from the tear in the annulus and
the
loose fragments of the nucleus pulposus are removed with pituitary forceps.
Any additional fragments of disc sequestered inside or outside of the disc
space
are also carefully removed and the disc space is forcefully irrigated to
remove
to remove any residual fragments. If tears are present in the dura, the dura
is
closed with sutures that are often augmented with fibrin glue. The tissue is
then
closed with absorbable sutures.
Microlumbar disc excision (microdiscectomy) can be performed as
an outpatient procedure and has largely replaced laminectomy as the
intervention of choice for herniated discs or root entrapment. A one inch
incision is made from the spinous process above the disc affected to the
spinous process below. Using an operating microscope, the tissue is dissected
down to the ligamentum flavum and bone is removed from the lamina until the
nerve root can be clearly identified. The nerve root is carefully retracted
and
the tears in the annulus are visualized under magnification. Microdisc forceps
are used to remove disc fragments through the annular tear and any
sequestered disc fragments are also removed. As with laminectomy, the disc
space is irrigated to remove any disc fragments, any dural tears are repaired
and the tissue is closed with absorbable sutures. It should be noted that
anterior (abdominal) approaches can also be used for both open and
endoscopic lumbar disc excision. Cervical and thoracic disc excisions are
similar to lumbar procedures and can also be performed from a posterior
approach (with laminectomy) or as an anterior discectomy with fusion.
Back surgeries, such as laminectomies, discectomies and
microdiscectomies, often leave the spinal dura exposed and unprotected. As a
result, scar tissue frequently forms between the dura and the surrounding
tissue. This scar is formed from the damaged erector spinae muscles that
overlay the laminectomy site. The result is adhesion development between the
muscle tissue and the fragile dura, thereby, reducing mobility of the spine
and
the nerve roots that exit from it, leading to pain, persistent neurological
302
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
symptoms and slow post-operative recovery. Similarly, adhesions that occur in
the epidural and dural tissue cause complications in spinal injury (e.g.,
compression and crush injuries) cases. In addition, scar and adhesion
formation within the dura and around nerve roots has been implicated in
rendering subsequent (revision and repeat) spine operations technically more
difficult to perform.
To circumvent adhesion development, a scar-reducing barrier
may be inserted between the dural sleeve and the paravertebral musculature
post-laminectomy. Alternatively (or in addition to this), the adhesion
barrier,
either alone or containing a fibrosis-inhibiting agent, can be coated on (or
infiltrated into the tissues around) the spinal nerve as it exits the spinal
canal
and traverses the space between the bony vertebra (i.e., the laminectomy
site).
This reduces cellular and vascular invasion into the epidural space from the
overlying muscle and exposed cancellous bone and thus, reduces the
complications associated with scarring of the canal housing, spinal chord
and/or
nerve roots. In microdiscectomy procedures it is important that the barrier be
deliverable as a spray, gel or fluid material that can be administered via the
delivery port of an endoscope. Once again, the adhesion barrier, either alone
or containing a fibrosis-inhibiting agent, can be sprayed onto the spinal
nerve
(or infiltrated into the tissues~around it) as it exits the spinal canal and
traverses
the space between the bony vertebra (i.e., the laminectomy site). The present
invention discloses barrier compositions, used either alone or combined with a
fibrosis-inhibiting agent, that can be delivered during surgical disc
resection and
microdiscectomy either directly, using specialized delivery catheters, via an
endoscope, or through a needle or other applicator. When dural defects are
present, the fibrosis-inhibiting agent will assist in the healing of the dura
and
prevent complications such as blockage of CSF flow.
In another aspect, adhesion formation may be associated with a
neurosurgical (brain) procedure. Neurosurgical procedures are fraught with
potentially severe post-operative complications that are often attributed to
303
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
surgical trauma and unwanted fibrosis or gliosis (gliosis is scar tissue
formation
in the brain as a result of glial cell activity). Increased intracranial
bleeding,
infection, cerebrospinal fluid leakage and pain are but some complications
resulting from adhesions following neurosurgery. For example, if scar tissue
interrupts the normal circulation of cerebrospinal fluid (CSF) following brain
or
spinal surgery, the fluid can accumulate and exert pressure on surrounding
tissues (causing increased intracranial pressure) leading to severe
complications (such as uncal herneation, brain damage and/or death). Here
the adhesion barrier alone, or combined with a fibrosis-inhibiting agent, can
be
used to prevent excessive dural scarring and adhesion formation in a variety
of
neurosurgical procedures.
There are numerous compositions that may be used alone or
loaded with a therapeutic agent (e.g., a fibrosis-inhibiting agent or an anti-
infective agent), applied to a spinal or neurosurgical site (or to an implant
surface placed in the spine - such as an artificial disc, rods, screws, spinal
cages, drug-delivery pumps, neurostimulation devices; or to an implant placed
in the brain - such as drains, shunts, drug-delivery pumps, neurostimulation
devices) for the prevention of surgical adhesions in neurosurgical procedures.
It should be noted that certain polymeric compositions can themselves help
prevent the formation of fibrous tissue at a spinal or neurosurgical site.
These
compositions are particularly useful for the practice of this embodiment,
either
alone, or in combination with a fibrosis-inhibiting composition.
Various polymeric compositions can be infiltrated into the spinal or
neurosurgical site (e.g., onto tissue at the surgical site or in the vicinity
of the
implant-tissue interface) with or without an additional therapeutic agent for
the
prevention of surgical adhesions.
In one embodiment, the polymers that can form a covalent bond
with the tissue to which it is applied may be used. Polymers containing and/or
terminated with electrophilic groups such as succinimidyl, aldehyde, epoxide,
isocyanate, vinyl, vinyl sulfone, maleimide, -S-S-(C5H4N) or activated esters,
304
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
such as are used in peptide synthesis may be used as the reagents. For
example, a 4 armed NHS-derivatized polyethylene glycol (e.g., pentaerythritol
polyethylene glycol)ether tetra-succinimidyl glutarate) may be applied to the
tissue in the solid form or in a solution form. In this embodiment, the 4
armed
NHS-derivatized polyethylene glycol is dissolved in an acidic solution (pH
about
2-3) and is then co-applied to the tissue using a basic buffer (pH > about 8).
The antifibrosisfibrosis-inhibiting agents) may be incorporated directly into
either the 4 armed NHS-derivatized polyethylene glycol, the acidic solution or
the basic buffer.
In another embodiment, the fibrosis-inhibiting agent may be
incorporated into a secondary carrier that may then be incorporated into the 4
armed NHS-derivatized polyethylene glycol, the acidic solution and/or the
basic
buffer. The secondary carriers may include microparticles and/or microspheres
which are made from degradable polymers. The degradable polymers may
include polyesters, where the polyester may comprise the residues of one or
more of the monomers selected from lactide, lactic acid, glycolide, glycolic
acid,
e-caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, b-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone, and block copolymers of the form X-Y, Y-X-Y, R-(Y-X)", R-
(X-Y)" and X-Y-X (where X in a polyalkylene oxide (e.g., poly(ethylene glycol,
polypropylene glycol) and block copolymers of polyethylene oxide) and
polypropylene oxide) (e.g., PLURONIC and PLURONIC R series of polymers
from BASF Corporation, Mount Olive, NJ) and Y is a biodegradable polyester,
where the polyester may comprise the residues of one or more of the
monomers selected from lactide, lactic acid, glycolide, glycolic acid, e-
caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone (e.g., PLG-PEG-PLG) and R is a multifunctional initiator).
305
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
In another embodiment, the tissue reactive polymer may be
applied initially and then the fibrosis-inhibiting agent may then be applied
to the
coated tissue. The fibrosis-inhibiting agent may be applied directly to the
tissue
or it may be incorporated into a secondary carrier. The secondary carriers may
include microspheres (as described above), microparticles (as described
above), gels (e.g., hyaluronic acid, carboxymethyl cellulose, dextran,
polyethylene oxide) - polypropylene oxide) block copolymers as well as
blends, association complexes and crosslinked compositions thereof) and films
(degradable polyesters, where the polyester may comprise the residues of one
or more of the monomers selected from lactide, lactic acid, glycolide,
glycolic
acid, e-caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric
acid, beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone, and block copolymers of the form X-Y, Y-X-Y, R-(Y-X)", R-
(X-Y)" and X-Y-X where X in a polyalkylene oxide (e.g., poly(ethylene glycol,
polypropylene glycol) and block copolymers of polyethylene oxide) and
polypropylene oxide) (e.g., PLURONIC and PLURONIC R series of polymers
from SASF Corporation, Mount Olive, NJ) and Y is a biodegradable polyester,
where the polyester may comprise the residues of one or more of the
monomers selected from lactide, lactic acid, glycolide, glycolic acid, e-
caprolactone, gamma-caprolactone, hydroxyvaleric acid, hydroxybutyric acid,
beta-butyrolactone, gamma-butyrolactone, gamma-valerolactone, y-
decanolactone, ~-decanolactone, trimethylene carbonate, 1,4-dioxane-2-one or
1,5-dioxepan-Zone (e.g., PLG-PEG-PLG) and R is a multifunctional initiator,
hyaluronic acid, carboxymethyl cellulose, dextran, polyethylene oxide) -
poly(propylene oxide) block copolymers as well as blends, association
complexes and crosslinked compositions thereof.
A preferred polymeric matrix which can be used to help prevent
the formation of fibrous tissue that leads to surgical adhesions, either alone
or
in combination with a fibrosis inhibiting agent/composition, is formed from
306
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
reactants comprising either one or both of pentaerythritol polyethylene
glycol)ether tetra-sulfhydryl] (4-armed thiol PEG, which includes structures
having a linking groups) between a sulfhydryl groups) and the terminus of the
polyethylene glycol backbone) and pentaerythritol polyethylene glycol)ether
tetra-succinimidyl glutarate] (4-armed NHS PEG, which again includes
structures having a linking groups) between a NHS groups) and the terminus
of the polyethylene glycol backbone) as reactive reagents. Another preferred
composition comprises either one or both of pentaerythritol polyethylene
glycol)ether tetra-amino] (4-armed amino PEG, which includes structures
having a linking groups) between an amino groups) and the terminus of the
polyethylene glycol backbone) and pentaerythritol polyethylene glycol)ether
tetra-succinimidyl glutarate] (4-armed NHS PEG, which again includes
structures having a linking groups) between a NHS groups) and the terminus
of the polyethylene glycol backbone) as reactive reagents. Chemical structures
for these reactants are shown in, e.g., U.S. Patent 5,874,500. Optionally,
collagen or a collagen derivative (e.g., methylated collagen) is added to the
polyethylene glycol)-containing reactants) to form a preferred crosslinked
matrix that can serve as a polymeric carrier for a therapeutic agent or a
stand-
alone composition to help prevent the formation of fibrous tissue.
Other examples of polymeric compositions that can be infiltrated
into the spinal or neurosurgical site (e.g., onto tissue at the surgical site
or in
the vicinity of the implant-tissue interface) with or without an additional
fibrosis-
inhibiting (and/or an anti-infective) therapeutic agent for the prevention of
surgical adhesions, include a variety of commercial products. For example,
Confluent Surgical, Inc. makes their DURASEAL which is a synthetic hydrogel
designed to augment sutured dura closures following cranial surgical
procedures. Products that are being developed by Confluent Surgical, Inc. are
described in, for example, U.S. Patent No. 6,379,373. FzioMed, Inc. (San Luis
Obispo, CA) makes OXIPLEX/SP Gel which is being sold as an adhesion
barrier for spine surgery. OXIPLEXISP Gel is being used for the reduction of
307
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
pain and radiculopathy in laminectomy, laminotomy and discectomy surgeries.
Products being developed by FzioMed, Inc, are described in, for example, U.S.
Patent Nos. 6,566,345 and 6,017,301. Anika Therapeutics, Inc. (Woburn, MA)
is developing INCERT-S for the prevention of internal adhesions or scarring
following spinal surgery. INCERT-S is part of a potential family of
bioabsorbable, chemically modified hyaluronic acid therapies. Products being
developed by Anika Therapeutics, Inc. are described in, for example, U.S.
Patent Nos. 6,548,081; 6,537,979; 6,096,727; 6,013,679; 5,502,081 and
5,356,883. Life Medical Sciences, Inc. (Little Silver, NJ) is developing
RELIEVE as a bio-resorbable polymer designed to prevent or reduce the
formation of adhesions that can follow spinal surgery. Products being
developed by Life Medical Sciences, Inc. are described in, for example, U.S.
Patent Nos. 6,696,499; 6,399,624; 6,211,249; 6,136,333 and 5,711,958.
Wright Medical Technology, Inc. is selling the ADCON range of products which
are dextran sulfate gels originally developed by Gliatech, Inc. (Beachwood,
OH)
to inhibit postsurgical peridural fibrosis that occurs in posterior lumbar
laminectomy or laminotomy procedures where nerve routes are exposed.
ADCON provides a barrier between the spinal cord and nerve roots and the
surrounding muscle and bone following lumbar spine surgeries. The ADCON
range of products may be described in, for example, U.S. Patent Nos.
6,417,173; 6,127,348; 6,083,930; 5,994,325 and 5,705,178.
Other commercially available materials that may be used alone or
loaded with a therapeutic agent (e.g., a fibrosis-inhibiting agent and/or an
anti
infective agent), applied to or infiltrated into a spinal or neurosurgical
site (or to
an implant surface) for the prevention of adhesions include: (a) sprayable
collagen-containing formulations such as COSTASIS or CT3; (b) sprayable
PEG-containing formulations such as COSEAL, ADHIBIT, FOCALSEAL, or
SPRAYGEL; (c) fibrinogen-containing formulations such as FLOSEAL or
TISSEAL (both from Baxter Healthcare Corporation, Fremont, CA); (d)
hyaluronic acid-containing formulations such as RESTYLANE, PERLANE,
308
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
HYLAFORM, SYNVISC, SEPRAFILM or SEPRACOAT; (e) polymeric gels for
surgical implantation such as REPEL or FLOWGEL; (f) surgical adhesives
containing cyanoacrylates such as DERMABOND, INDERMIL, GLUSTITCH,
TISSUMEND, VETBOND, HISTOACRYL BLUE and ORABASE SOOTHE-N-
SEAL LIQUID PROTECTANT; (h) lipid based compositions such as ADSURF,
and (j) film compositions such as INTERCEED (Ethicon, Inc., Somerville, NJ)
and HYDROSORB (MacroPore Biosurgery, Inc., San Diego, CA /Medtronic
Sofamor Danek, Memphis, TN). It should be obvious to one of skill in the art
that commercial compositions not specifically cited above as well as next-
generation and/or subsequently-developed commercial products are to be
anticipated and are suitable for use under the present invention.
As described above, the compositions for the prevention of
surgical adhesions can be applied directly or indirectly to the tissue in a
spinal
or neurosurgical site. The polymeric compositions (either with or without a
therapeutic agent) can be administered in any manner described herein.
Exemplary methods include either direct application at the time of surgery,
with
endoscopic, ultrasound, CT, MRI, or fluoroscopic guidance, and/or in
conjunction with the placement of a device or implant at the surgical site.
Representative examples of devices or implants for use in spinal and
neurosurgical procedures includes, without limitation, dural patches, spinal
prostheses (e.g., artificial discs, injectable filling or bulking agents for
discs,
spinal grafts, spinal nucleus implants, intervertebral disc spacers), fusion
cages,
neurostimulation devices, implantable drug-delivery pumps, shunts, drains,
electrodes, and bone fixation devices (e.g., anchoring plates and bone
screws).
The polymeric composition, with or without a fibrosis-inhibiting
agent, may be applied during open or endoscopic procedures: (a) to the surface
of the operative site (e.g., as an injectable, solution, paste, gel, in situ
forming
gel or mesh) before, during, or after the surgical procedure; (b) to the
surface of
the tissue surrounding the operative site (e.g., as an injectable, solution,
paste,
gel, in situ forming gel or mesh) before, during or after the surgical
procedure;
309
CA 02536181 2006-02-15
WO 2005/051452 PCT/US2004/039389
(c) by topical application of the composition into an anatomical space (such
as
the subdural space or intrathecally) at the surgical site (particularly useful
for
this embodiment is the use of polymeric carriers which release the fibrosis-
inhibiting agent over a period ranging from several hours to several weeks -
fluids, suspensions, emulsions, microemulsions, microspheres, pastes, gels,
microparticulates, sprays, aerosols, solid implants and other formulations
which
release the agent and can be delivered into the region where the device will
be
inserted); (d) via percutaneous injection into the tissue in and around the
operative site as a solution, as an infusate, or as a sustained release
preparation; and/or (e) by any combination of the aforementioned methods.
Combination therapies (i.e., combinations of therapeutic agents and
combinations with antithrombotic, anti-infective, and/or antiplatelet agents)
can
also be used.
In certain applications involving the placement of a medical device
or implant, it may be desirable to apply the anti-fibrosis (and/or anti-
infective)
composition at a site that is adjacent to an implant (preferably near the
implant-
tissue interface). This can be accomplished during open or endoscopic
procedures by applying the polymeric composition, with or without a fibrosis-
inhibiting agent: (a) to the implant surface (e.g., as an injectable,
solution,
paste, gel, in situ forming gel, or mesh) before, during, or after the
implantation
procedure; (b) to the surface of the adjacent tissue (e.g., as an injectable,
solution, paste, gel, in situ forming gel, or mesh) immediately prior to,
during, or
after implantation of the implant; (c) to the surface of the implant and the
tissue
surrounding the implant (e.g., as an injectable, solution, paste, gel, in situ
forming gel or mesh) before, during, or after implantation of the implant; (d)
by
topical application of the composition into the anatomical space (such as the
sudural space or intrathecally) where the implant will be placed (particularly
useful for this embodiment is the use of polymeric carriers which release the
fibrosis-inhibiting agent over a period ranging from several hours to several
weeks - fluids, suspensions, emulsions, microemulsions, microspheres, pastes,
310
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 4
CONTENANT LES PAGES 1 A 310
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 4
CONTAINING PAGES 1 TO 310
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
