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CA 02594196 2007-07-23
Protease Inhibiting Polymer
Field of the Invention
The present invention relates to polymers capable of inhibiting matrix
tissue-degrading proteases.
Background of the Invention
Proteases are enzymes that catalyze protein digestion in a wide range of
physiological and pathophysiological processes (e.g. blood clotting and tumor
invasion). During inflammation, a number of proteases are released from
inflammatory cells that degrade the extracellular matrix (ECM) to remove
necrotic
tissue, promote cell migration and facilitate tissue remodeling. These events
are
necessary for tissue protection and repair processes. However, chronic
stimulation may lead to persistent inflammation that generates high
concentrations
of proteases leading to excessive tissue destruction. This process has been
implicated in a number of inflammatory disorders such as arthritis, emphysema,
and chronic wound healing, as well as cancer metastases.
The Applicant has previously described insoluble polymers containing a
hydroxamate group that bind a specific class of tissue-degrading proteases,
namely matrix metalioproteases (MMPs) (Sefton et al, US Patent Appls.
10/420725 and PCT/CA2004/000975. The hydroxamate functional group has a
strong affinity for the zinc ion present in the active site of this class of
enzymes,
making the polymer-enzyme interaction essentially non-reversible. MMPs
degrade the principle structural protein of the extracellular matrix (ECM),
collagen.
However, it is well known that there are numerous types of proteases
involved in inflammatory processes. In addition to the MMPs, serine proteases
such as neutrophil elastase, cathepsin G and plasmin are released into the
extracellular space during inflammation leading to the degradation of a
variety of
ECM or provisional matrix proteins (e.g. elastin, fibrin). In particular,
neutrophil
elastase activity in chronic wounds is elevated, which impairs the formation
of new
tissue. Therefore, materials able to reduce the general proteolytic activity
in
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persistent inflammatory conditions could limit the associated destructive
tissue
turnover and subsequent sequelae of events.
Summary of the Invention
It is desirable to provide polymer that is able to inhibit multiple families
of
extracellular tissue degrading inflammatory enzymes, in particular matrix
metalloproteases and serine proteases.
Accordingly, the Applicant has now shown that the hydroxamate-containing
polymers described in U.S. Patent Appl. 10/420725 and PCT/CA2004/000975 can
be used not only to bind the MMP family, but can also be used to bind other
tissue-degrading, inflammatory enzyme families, in particular serine proteases
such as elastase and cathepsin G. Not wishing to be bound by theory, it is
possible that the latter enzymes are bound through a charge interaction
between
the anionic, acid-containing polymer and the cationic protease. Since the
polymers contain both carboxylic acid and hydroxamate functional groups of
varying proportions, their net acidity can be tailored to vary enzyme
specificity and
binding capacity.
The polymer should be insoluble in bodily solutions, thus providing a
localized therapeutic effect at the site of application. In addition,
degradable
versions of the polymer may be used that result in their eventual dissolution
and
clearance from the site of application.
The polymer can be used to inhibit tissue degradation at the site of
application. Conversely the polymer can be used to promote the formation of
new
tissue at the site of application.
The inhibition of a range of inflammatory proteases with the polymer
reduces the overall, or at least the tissue destructive component, of the
inflammatory response at the site of application
By varying the relative amounts of carboxylic acid and hydroxamate
groups, the selectivity of the polymer for different cationic proteases and
MMPs is
tailored to the particular application site and local biochemistry.
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Brief Description of the Drawings
Figure 1 is a scanning electron micrograph of a hydroxamate-containing
bead. Scale bar represents 60 pm.
Figure 2 is a graph of absorbance versus time resulting from elastase
substrate digestion by human neutrophil elastase after incubation with
hydroxamate-containing beads (initial neutrophil elastase solution conc. = 250
nM). Bead concentration ranges from 0 to 100 mg/mL.
Figure 3 is a graph of absorbance versus time resulting from elastase
substrate digestion by a human chronic skin wound fluid solution after
incubation
with hydroxamate-containing beads. The wound fluid solution is an extract
obtained from a used diabetic foot ulcer wound dressing and the bead
concentration ranges from 0 to 100 mg/mL. A substrate digestion curve is also
shown for a 250 nM neutrophil elastase solution in buffer for comparison.
Figure 4 is a graph of absorbance versus time resulting from cathepsin G
substrate digestion by cathepsin G after incubation with hydroxamate-
containing
beads (initial cathepsin G solution conc. = 250 nM). Bead concentration ranges
from 0 to 100 mg/mL.
Figure 5 is a graph of absorbance versus time resulting from cathepsin G
substrate digestion by a human chronic skin wound fluid solution after
incubation
with hydroxamate-containing beads. The wound fluid solution is an extract
obtained from a used diabetic foot ulcer wound dressing and the bead
concentration ranges from 0 to 100 mg/mL.
Detailed Description of the Invention
The Protease
The polymer of the invention may be used to inhibit multiple families of
extracellular proteases. Examples of extracellular protease families include
serine
proteases like chymotrypsin, trypsin, and elastase, plasmin, and cathepsin G;
cysteine proteases like cathepsin S; matrix metalloproteases; and other
metailoproteases such as aggrecanase.
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The Polymer
Hydroxamate-containing polymer (HX polymer) is synthesized by surface
modification of cross-linked polymethacrylic acid (PMAA)-co-methyl
methacrylate
(MAA) beads (resulting in a novel composition of PMAA-MMA-HX). In the formula
RIC(O)N(OH)R2, R, represents the polymer main chain and R2 represents
hydrogen. This method results in beads that are not soluble, but are useable
as
such; the surface modification method can be applied to other shapes, but the
materials will need to be in their final form prior to modification.
Polymerizable HX monomer can be used to synthesize an HX
homopolymer or copolymerized with any other suitable comonomers to produce
polymers with a variety of properties. With reference to the formula
RjC(O)N(OH)R2, R, represents CH2=C-CH3 and R2 represents hydrogen.
HX homopolymer synthesized from the HX monomer can also be grafted
onto any derivatizable polymer to produce additional hydroxamate-containing
polymers. With reference to the formula RjC(O)N(OH)R2, R, represents any
chemical group of a derivatizable polymer and R2 represents hydrogen.
Further details about the synthesis of the polymer are described in U.S.
Patent Applns. 10/420725 (Publication No. U.S. 2004-0213758) and 11/714730
(Publication No. U.S. 2007-0160655), as well as PCT/CA2004/000975
(W0/2006/002506), all of which are incorporated by reference herein.
Uses
The polymers of the invention may be used to treat imflammatory
conditions, such as skin reactions, allergic reactions, asthma, lung diseases
or
responses, kidney diseases, acute inflammatory diseases, periodontal diseases,
vascular inflammatory disease, chronic inflammation, atherosclerosis, immune
related diseases, angiopathy, myocarditis, nephritis, Crohn's disease, wound
healing, arthritis, type I or II diabetes and the associated vascular
pathologies, or
aneurysmal disease.
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Examples
Example 1- Synthesis of Hydroxamate-containing Polymer Beads
Crosslinked polymethylmethacylate-co-methacrylic acid beads were
suspended in anhydrous N,N-dimethylformamide (DMF) at approximately 10%
wt/vol and allowed to equilibrate for 30 min at 0 C while stirring. A 100%
molar
excess of N-methyl morpholine and isobutyl chloroformate, relative to the
methacrylic acid content of the beads, was added to the bead suspension. The
reaction proceeded at 0 C for 30 min, the beads were filtered from suspension
and washed with DMF. The beads were transferred to a vessel containing a
100% molar excess of hydroxylamine solution in water and the reaction
proceeded at ambient temperature for at least 1 hr. The beads were then
filtered
and washed with water, 0.1 M HCI and again with water, then dried at 55-60 C.
Example 2 - Neutrophil Elastase Inhibition by Hydroxamate-containing Polymer
Beads
Hydroxamate-containing polymer beads were produced as described
above. The beads were incubated in assay buffer at 50 mg/mL for 1.5 hours at
room temperature to equilibrate. The beads were then incubated in a solution
of
neutrophil elastase (human, Calbiochem) for 1.5 hour. Then an aliquot of each
incubation solution was transferred to a 96-well microplate. The colorimetric
substrate solution (colorimetric elastase substrate 1, Calbiochem) was added
to
each well and the absorbance was read at 410 nm for 20 min. The initial,
linear
slopes were determined for each substrate digestion time curve and used as an
indicator of elastase activity.
Figure 2 shows the substrate digestion curves for solutions of neutrophil
elastase after incubation with varying amounts of hydroxamate-containing
polymer
beads. The beads exhibit a high degree of solution elastase activity for all
bead
amounts tested.
Hydroxamate-containing polymer beads were then incubated with human
chronic wound fluid solution to evaluate their enzyme inhibitory capacity in a
more
physiologic, multi-protein solution. The chronic wound solution was prepared
by
extracting wound exudate in a used foam dressing with phosphate buffered
saline
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(PBS) for 24 h at room temperature. The wound fluid solution was then
centrifuged to remove insoluble particles of dressing and tissue and frozen at
-
70 C until use. The beads were incubated in the human wound fluid solution for
1
h, then an aliquot (40 pL per well) of each solution was transferred to a 96-
well
microplate. The colorimetric substrate solution and assay buffer were added to
each well and the absorbance was read at 410 nm for 30 min. The initial,
linear
slopes were determined for each substrate digestion time curve and used as an
indicator of elastase activity.
Figure 3 shows the elastase substrate digestion curves for human wound
fluid solutions after incubation with varying amounts of hydroxamate-
containing
polymer beads. Increasing bead loadings result in decreasing elastase activity
in
the human wound fluid sample. The degree of inhibition is slightly lower than
in
the model elastase solution (in buffer) indicating some reduction in bead
inhibitory
effect (on this single enzyme) possibly resulting from competitive protein
binding.
Example 3 - Cathepsin G Inhibition by Hydroxamate-containing Polymer Beads
Hydroxamate-containing polymer beads were produced as described
above. The beads were incubated in assay buffer at 50 mg/mL for 1.5 hours at
room temperature to equilibrate. The beads were then incubated in a solution
of
cathepsin G (human, Calbiochem) for 1.5 hour. Then an aliquot of each
incubation solution was transferred to a 96-well microplate. The colorimetric
substrate solution (colorimetric cathepsin G substrate 2, Calbiochem) was
added
to each well and the absorbance was read at 410 nm for 40 min. The initial,
linear
slopes were determined for each substrate digestion time curve and used as an
indicator of cathepsin activity.
Figure 4 shows the substrate digestion curves for solutions of cathepsin G
after incubation with varying amounts of hydroxamate-containing polymer beads.
Increasing bead amounts lead to decreasing cathepsin G activity (i.e.
increasing
inhibition).
Hydroxamate-containing polymer beads were then incubated with human
chronic wound fluid solution to evaluate their enzyme inhibitory capacity in a
more
physiologic, multi-protein solution. The beads were incubated in the human
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wound fluid solution for 1 h at room temperature, then an aliquot (40 pL per
well)
of each solution was transferred to a 96-well microplate. The colorimetric
substrate solution and assay buffer were added to each well and the absorbance
was read at 410 nm for 30 min. The initial, linear slopes were determined for
each
substrate digestion time curve and used as an indicator of cathepsin G
activity.
Figure 5 shows the cathepsin G substrate digestion curves for human
wound fluid solutions after incubation with varying amounts of hydroxamate-
containing polymer beads. Increasing bead loadings result in decreasing
cathepsin G activity in the human wound fluid sample, indicating that the
beads
retained their inhibitory effect in the multi-component protein wound fluid
solution.
Example 4 - Inhibitor of MMPs by Hydroxamate Polymer Beads
MMPs have been inhibited by hydroxamate polymer beads as described in
U.S. Patent Applns. 10/420725 (Publication No. U.S. 2004-0213758) and
11/714730 (Publication No. U.S. 2007-0160655), as well as PCT/CA2004/000975
(WO/2006/002506), all of which are incorporated by reference herein.
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