U.S. patent application number 14/072299 was filed with the patent office on 2014-02-27 for compositions and methods for promoting the healing of tissue of multicellular organisms.
The applicant listed for this patent is David J. Vachon. Invention is credited to David J. Vachon.
Application Number | 20140056841 14/072299 |
Document ID | / |
Family ID | 42784516 |
Filed Date | 2014-02-27 |
United States Patent
Application |
20140056841 |
Kind Code |
A1 |
Vachon; David J. |
February 27, 2014 |
Compositions and Methods for Promoting the Healing of Tissue of
Multicellular Organisms
Abstract
Compositions are provided for promoting healing of tissue of a
vertebrate organism. The compositions can be for internal
administration of a therapeutically effective amount of
pharmacologically active, protease inhibiting, aqueous media
soluble polysulfonated materials in salt form and associated with a
secondary material to reduce one or more of inflammation, bacterial
proliferation, proteolytic activity, and cancerous cell growth. The
compositions may additionally or alternatively be cross-linked so
as to alter the solubility of these pharmacologically active salts
or slow dissolution by providing biodegradable cross-linkers.
Compositions for healing the tissue of a multicellular organism are
provided that can include a polysulfonated material in a liquid
mixture, as solid particles or constructs that may or may not
biodegrade or deliver a pharmacologically relevant value. Some of
the compositions are also provided for inclusion into a device for
preventing infection, reducing inflammation, and preserving the
activity of a protein or protein drug.
Inventors: |
Vachon; David J.; (Spokane,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vachon; David J. |
Spokane |
WA |
US |
|
|
Family ID: |
42784516 |
Appl. No.: |
14/072299 |
Filed: |
November 5, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12690081 |
Jan 19, 2010 |
|
|
|
14072299 |
|
|
|
|
12162990 |
Jul 31, 2008 |
|
|
|
PCT/US07/02780 |
Jan 31, 2007 |
|
|
|
12690081 |
|
|
|
|
60764033 |
Jan 31, 2006 |
|
|
|
Current U.S.
Class: |
424/78.27 ;
525/333.5 |
Current CPC
Class: |
A61L 2300/80 20130101;
A61P 3/02 20180101; A61P 17/02 20180101; A61K 38/00 20130101; A61K
31/713 20130101; A61K 33/38 20130101; A61K 31/65 20130101; A61K
31/198 20130101; A61P 35/00 20180101; A61L 26/0052 20130101; A61P
29/00 20180101; A61K 31/444 20130101; A61K 31/18 20130101; A61P
31/04 20180101; A61P 43/00 20180101; A61P 7/02 20180101; A61L
26/0066 20130101; A61P 7/04 20180101; A61K 31/167 20130101; A61K
31/795 20130101; A61P 23/00 20180101; A61P 31/00 20180101; A61L
2300/434 20130101; A61K 45/06 20130101; A61K 31/167 20130101; A61K
2300/00 20130101; A61K 31/18 20130101; A61K 2300/00 20130101; A61K
31/198 20130101; A61K 2300/00 20130101; A61K 31/444 20130101; A61K
2300/00 20130101; A61K 31/65 20130101; A61K 2300/00 20130101; A61K
31/713 20130101; A61K 2300/00 20130101; A61K 33/38 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/78.27 ;
525/333.5 |
International
Class: |
A61K 31/795 20060101
A61K031/795 |
Claims
1: A composition for the healing of a multicellular organism, the
composition comprising at least one-polysulfonated material, the
polysulfonated material being insoluble in deionized water and
soluble in ion-comprising aqueous media, wherein the polysulfonated
material comprises a arylenevinylsulfonate, a chondroitin
sulfonate, and/or a polyvinyl sulfonate.
2: The composition of claim 1 wherein the arylenevinylsulfonate
comprises one or more of polyalkylstyrene sulfonate, polystyrene
arginine sulfonate, polystyrene mafenide sulfonate, silver
substituted polystyrene sulfonate, chlorhexidine substituted
polystyrene sulfonate, and/or doxycycline substituted polystyrene
sulfonate.
3: The composition of claim 1 wherein the chondroitin sulfonate
comprises one or more of silver chondroitin-6-sulfate,
sodium/silver chondroitin-6-sulfate, and/or arginine
chondroitin-6-sulfate
4: The composition of claim 1 wherein the polyvinyl sulfonate
comprises one or more of polyarylene vinyl sulfonate, polyvinyl
sulfonate, and, and/or aryl ring-modified polyarylene vinyl
sulfonate.
5: The composition of claim 1 wherein the polysulfonated material
is associated with at least one cation of pharmacologically
therapeutic value.
6: The composition of claim 1 wherein the polysulfonated material
is associated with at least two metal cations.
7: The composition of claim 1 wherein the polysulfonated material
is associated with at least one organic cation.
8: The composition of claim 7 wherein the organic cation is one or
more of tetracycline, doxycycline, minocycline, octenidine,
chlorhexidine, benzalkonium, cetylpyridinium, quaternary ammonium,
arginine, lysine, glutathione, mafenide, albuterol, and
lidocaine.
9: The composition of claim 1 wherein the polysulfonated material
is associated with a mixture of metal and organic cations.
10: The composition of claim 1 wherein the polysulfonated material
is associated with metallic cations of sodium and silver.
11: The composition of claim 1 wherein the polysulfonated material
comprises at least two sulfonate groups modified to sulfonamides,
the sulfonamides configured to cross-link at least one portion of
the polysulfonated material with another portion of the
polysulfonated material.
12: The composition of claim 1 further comprising a solid matrix
material.
13: The composition of claim 12 wherein the matrix material
comprises biodegradable material.
14: The composition of claim 12, wherein the matrix material is a
non-biodegradable biomedical material comprising at least one of a
silicone, a polyurethane, a polyalkylene, a hydrogel, or a
polyester.
15: The composition of claim 1 formulated as a gel, paste, cream,
solution, ointment, or lotion.
16: The composition of claim 1 further comprising collagen or
gelatin.
17: A composition for the healing of a multicellular organism, the
composition comprising a polysulfonated material that is both
insoluble in deionized water and soluble in ion-comprising aqueous
media.
18: A composition for the healing of a multicellular organism, the
composition comprising a polysulfonated salt that is both insoluble
in deionized water and soluble in ion-comprising aqueous media.
19: The composition of claim 18 wherein the cation associated with
the polysulfonated salt is either organic or inorganic.
20: The composition of claim 18 wherein the cation associated with
the polysulfonated salt is silver ion.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/690,081 filed Jan. 19, 2010, which is a
continuation in part of, and claims priority to, U.S. patent
application Ser. No. 12/162,990 filed Jul. 31, 2008 claiming
priority to International Patent Application No. PCT/US07/02780
filed Jan. 31, 2007, both of which claim priority to U.S.
Provisional Patent Application Ser. No. 60/764,033 entitled "Method
For The Reduction of Protease Levels and Delivering Cationic
Therapeutic Agents Using Water-Soluble Polyanionic Oligomers &
Polymers & Their Salts" filed Jan. 31, 2006. The present patent
application hereby incorporates each of these listed patent
applications by reference in their entirety.
TECHNICAL FIELD
[0002] Compositions and methods for promoting the healing of tissue
of multicellular organisms.
BACKGROUND
[0003] The biochemical environment of the non-healing wound (as
well as serious wounds, including infected wounds, and/or chronic
wounds) is different from that of the normal healing wound in ways
that negatively affect multiple aspects of the healing process.
[0004] In each wound, one of the three mechanisms can predominate.
The three mechanisms of wound healing are contraction,
epithelialization, and connective tissue deposition. Contraction is
the method by which wound healing occurs at an amputation site such
as the tip of a finger. Epithelialization can predominate in the
healing of abrasions and connective tissue deposition occurs when
lacerations are sutured closed. The stages of healing include
hemostasis, inflammation, proliferation and remodeling. In each of
these stages, specific components can play a part through several
mediators. In hemostasis, platelets, endothelial cells, and fibrin,
and fibronectin act in concert through mediation by various
biological factors including cytokines. Cytokines are non-antibody
proteins that are released from some cells and act as intracellular
mediators. Cytokines include lymphokines and interleukins.
Inflammation occurs through the action of neutrophils, macrophages
and lymphocytes mediated by growth factors and proteases. Proteases
are enzymes that lyse proteins and are also known as proteinases.
Herein, proteinase and protease can be used interchangeably.
Proliferation occurs through the actions of fibroblasts,
epithelial, and endothelial cells and is largely dependent on
growth factors and collagen deposition. Remodeling is characterized
by collagen cross linking and collagen degradation increasing scar
strength as maturation of scar formation occurs.
[0005] Normal wound healing can be considered a balance of damaged
tissue removal and new tissue formation. Many processes are present
that can regulate the biological processes and pathways associated
with normal wound repair. An alteration in any of these
physiological processes can lead to the formation of a chronic
wound.
[0006] Inflammation and/or innate immunity are related to cancerous
cell growth. Early in the neoplastic process, inflammatory cells
and their released molecular species influence the growth,
migration and differentiation of all cell types in the tumor
microenvironment, whereas later in the tumorigenic process,
neoplastic cells also divert inflammatory mechanisms, such as
proteinase production, and chemokine/cytokine functions in favor of
tumor spreading and metastasis. Human polymorphonuclear neutrophils
(PMN) comprise 50-70% of circulating leukocytes and induce
inflammatory reactions that can be either cytotoxic for tumor cells
or aid in tumor growth and metastasis.
[0007] The present disclosure provides compositions and/or methods
using compositions that can reduce one or both of inflammation and
cancerous cell growth, inhibit bacterial organisms, and/or
generally promote healing and wellness, among other potential
advantages in multicellular organisms.
SUMMARY
[0008] Compositions are provided for promoting the healing of
tissue of a multicellular organism. The compositions include
pharmacologically active, protease inhibiting, aqueous media
soluble polysulfonated materials in salt forms in a liquid or solid
mixture to reduce one or more of inflammation, proteolytic
activity, bacterial proliferation, and cancerous cell growth.
[0009] Compositions are also provided for promoting healing of
tissue of a vertebrate organism. The compositions can be for
internal administration of a therapeutically effective amount of
pharmacologically active, protease inhibiting, aqueous media
soluble polysulfonated materials in salt form and associated with a
solid material to reduce one or both of inflammation and cancerous
cell growth. The polysulfonated materials in salt form may also be
cross-linked so as to alter the solubility of these
pharmacologically active salts.
[0010] Compositions are also provided for inclusion into a device
for preventing infection, reducing inflammation, and preserving the
activity of a protein or protein drug where the device is
constructed as a solid material which is associated with a drug
delivery device or a biosensor.
FIGURES
[0011] FIG. 1 is an example depicting the interaction of
compositions of the disclosure with various proteinases produced by
neutrophils and other cells according to an embodiment of the
disclosure.
[0012] FIG. 2 is an example depicting the interaction of
compositions of the disclosure with tissue fluids including salts,
and enzymes produced by neutrophils and other cells, according to
an embodiment of the disclosure.
[0013] FIG. 3 shows example preparations of compositions of the
disclosure according to an embodiment of the disclosure.
[0014] FIG. 4 shows example preparations of compositions of the
disclosure according to an embodiment of the disclosure.
[0015] FIG. 5 is a depiction of an example application according to
an embodiment of the disclosure.
[0016] FIG. 6 is a depiction of an example application according to
an embodiment of the disclosure.
[0017] FIG. 7 is a depiction of example applications according to
an embodiment in the disclosure.
[0018] FIG. 8 refers to a preparation of material 18 (Tecophilic
Polyurethane film containing sodium polystyrene sulfonate/SPSS) and
refers to the inhibition of Elastase by SPSS versus controls.
[0019] FIG. 9 refers to a preparation of material 18
(DJV-188--ointment containing sodium polystyrene sulfonate/SPSS)
and refers to the inhibition of Elastase by SPSS versus
controls.
[0020] FIG. 10 refers to a preparation of material 18
(DJV-188--ointment containing sodium polystyrene sulfonate/SPSS)
and two different molecular weights of pure SPSS and refers to the
inhibition of Elastase by SPSS versus controls (Nuggets &
Gauze).
[0021] FIG. 11 refers to a preparation of material 18
(DJV-188--ointment containing sodium polystyrene sulfonate/SPSS)
and refers to the inhibition of Cathepsin G by SPSS versus
controls.
[0022] FIG. 12 refers to a preparation of material 18
(DJV-188--ointment containing sodium polystyrene sulfonate/SPSS)
and two different molecular weights of pure SPSS and refers to the
inhibition of MMP-9 by SPSS versus controls (Nuggets, unlabeled
gel, Cutinova gel, & Gauze).
[0023] FIG. 13 refers to a preparation of material 18
(DJV-188--ointment containing sodium polystyrene sulfonate/SPSS)
and two different molecular weights of pure SPSS and refers to the
inhibition of MMP-8 by SPSS versus controls (Nuggets, unlabeled
gel, Cutinova gel, & Gauze).
[0024] FIG. 14 refers to a preparation of material 18 (Calcium
alginate film containing (calcium) polystyrene sulfonate of two
different molecular weights) and two different solution
preparations and refers to the inhibition of Elastase by (calcium)
PSS versus controls (gauze).
[0025] FIG. 15 refers to a dose response curve for SPSS in relation
to its ability to inhibit a fixed amount of Elastase.
[0026] FIG. 16 refers to the ability of a mixed silver-sodium
derivative of polystyrene sulfonate (14% silver) to kill three
relevant bacterial organisms.
[0027] FIG. 17 refers to the healing benefit/results from a wound
healing study where an example of material 18 (a mixed
sodium-silver derivative of PSS (14% silver) compounded into an
vehicle/ointment) was compared to a control (vehicle only).
[0028] FIG. 18 refers to the preparation of material 18 (SPSS
compounded into PLGA microspheres).
DESCRIPTION
[0029] Compositions and methods of the present disclosure will be
described with reference to FIGS. 1-18. Referring to FIG. 1, a
general proteinase inhibition scheme 10 is shown that includes a
cell 12 producing a metalloproteinase 14 and a serine, cysteine, or
aspartic acid proteinase 16. Scheme 10 further includes the
inhibition of proteinases 14 and 16 by a polysulfonated material
18. It is understood that several different proteinases may be
inhibited (serine, cysteine, aspartic acid, &
metallo-proteinases) and generally those proteinases (proteins)
with high isoelectric points where at physiologic pH the overall
positive charge on the protein provides the basis for a strong
electrostatic interaction with the highly anionic polysulfonated
material.
[0030] The polysulfonated therapeutic compounds (material 18)
described in this application represent a new class of natural,
semisynthetic, or synthetic polysulfonated proteinase inhibitors
(i.e. drugs) of both high isoelectric point proteinases and
metalloproteinases without the need for additional proteinase
inhibitor compounds. The effectiveness of the polysulfonated
compounds is reflected in the examples and in the case of the
ability of the water-soluble material 18 (Sodium polystyrene
sulfonate, SPSS) to very effectively inhibit MMPs 8 & 9. FIG. 6
of this application describes the assay of water-soluble
polysulfonated materials against MMP-8 and demonstrates the
unexpected results that the water soluble material 18 (SPSS) is
significantly more effective at inhibiting elastase than the
non-water soluble strong cation exchange material (nuggets) and
that the water soluble material 18 (SPSS) is remarkably effective
against MMPs 8 & 9 whereas the non-water soluble strong cation
exchange material is ineffective against MMPs 8 & 9.
Furthermore, it was unexpected that the solubility of the
polysulfonated materials described herein can be tailored to be
completely insoluble in deionized water yet soluble in aqueous
media.
[0031] Polysulfonated material 18 (hereinafter referred to as
"material 18") can be a polysulfonated material, a polysulfated
material and/or a polysulfonic acid salt or a polysulfated material
of acid or salt form thereof. In some instances, the repeating unit
of material 18 may be represented chemically as
[R(SO3.sup.-X.sup.+).sub.m].sub.n with m representing the number of
sulfonates or sulfates within a repeating unit of a macromolecule
and where n is at least one and m is greater than 1. The R group
contains carbon, hydrogen, and may possess other atoms including
heteroatoms such as nitrogen, sulfur, and oxygen. "X" can be one or
more variable cations including metal and organic species and may
comprise mixtures of metal cations, organic cations or mixed metal
cation-organic cation combinations. When the R group of material 18
is a repeating unit of a polymeric material, for example a
polysaccharide, it is understood that the R group possesses an
oxygen atom that is covalently linked between the ring (carbon) of
the sugar and the SO.sub.3.sup.-X.sup.+ functionality. As such, it
is understood that the sulfonate groups of material 18 are
associated with either counter cations or protons (H.sup.+) in
order to maintain nature's law of neutrality.
[0032] Sulfated polysaccharides, such as glycosaminoglycans are
generally in their subsequent sodium salt forms and are generally
highly water-soluble. Given the water solubility of these
compounds, ion exchange to substitute other metal cations or
organic cations for sodium can be readily accomplished using a
variety of methods including for example the use of water-insoluble
weak cation exchange resins.
[0033] While these counter ions are not shown in FIG. 1, it is
understood that they are involved in the molecular architecture.
The R group can be the backbone of an oligomer, such as a dimer
and/or trimer, or a polymer, also known as a macromolecule for
example. In accordance with other implementations, the oligomer or
polymer can comprise monomeric units of arylenevinyl sulfonate,
styrene sulfonate, alkyl styrene sulfonate such as methyl styrene
sulfonate, sulfated saccharides, and/or vinyl sulfonate monomers as
well as nonsulfonated monomers. The oligomer can include repeating
units of the same monomer, or more than one monomer where the
monomer may be chiral, achiral or a racemate. A racemate, or
racemic mixture, is the result of having a mixture of chiral
enantiomers each of which have chiral centers that are in essence
mirror images of each other. In some cases, one enantiomer can have
biological activity and its mirror image will not. In cases where
more than one chiral center is involved, diastereomers can result.
Diastereomerism occurs when two or more stereoisomers of a compound
have different configurations at one or more (but not all) of the
equivalent (related) stereocenters and are not mirror images of
each other. Chirality, optical activity, and thus biological
activity may be associated with the backbone structure, such as
with polysaccharides or chirality may be induced in typically
achiral materials by adding pendant chiral groups, i.e. those
groups that possess typically one connecting point to the backbone
(suspended from) and not critical to the connectivity of the
backbone, or by modifying the aromatic (aryl) ring of an
polyarylenevinyl sulfonate (aryl ring-modified) to include a chiral
group. Chirality is typically measured by an optical activity
(rotation of plane-polarized light) whereby the light is rotated
clockwise or counterclockwise.
[0034] The literature describes polymeric (water insoluble) medical
materials that possess protease inhibition capabilities. In some
instances water-insoluble polycationic ion exchange ligands are
used and in other instances polyanionic ion exchange (hydrogel)
materials are described for aiding in wound healing. The materials
discussed in the literature are configured with a variety of
chemistries and include phosphoryl groups, sulfonate groups, and
quaternary ammonium groups in addition to biopolymers that include
collagen. Several of these materials target neutrophil elastase and
the collagenase MMP-8 and gelatinase MMP-9. Of all of the above
mentioned chemistries, the biopolymers are not true inhibitors of
proteinases and only compete for the analyte in the assays. The
insoluble strongly anionic dressings are effective against elastase
but ineffective against the metalloproteinases (MMP-8 & MMP-9)
unless a tetracycline is used in addition.
[0035] According to an example implementation, the oligomer can be
incorporated into other materials. For example, material 18 can
also be a polymer comprising the oligomer. The oligomer can be
copolymerized with other monomers and/or other oligomers to form a
copolymer. In accordance with embodiments of the disclosure, the
polymer can include repeating oligomeric units, where the
oligomeric units may be comprised of identical monomer units or
combinations of monomer units. For example, material 18 can be
polyarylenevinyl sulfonate, polystyrene-sulfonate,
polymethylstyrene-sulfonate polyvinylsulfonate,
polyantholesulfonate, and/or acrylamidomethyl propane sulfonate
polymer, among others.
[0036] Accordingly, polysulfonated material 18 may be synthesized
by polymerization of one or more sulfonated monomers or by
sulfonation of a synthetic or semi-synthetic polymer or sulfation
of a synthetic, semi-synthetic, or naturally occurring polymer such
as a polysaccharide. The sulfonated or non-sulfonated monomers that
eventually lead to material 18 being sulfonated may be chiral,
achiral (non-chiral), or racemic mixtures or combinations thereof.
In the case of a chiral monomer (either sulfonated or
non-sulfonated) that is polymerized, the resulting polymer can be
chiral (i.e. it does not possess a plane of symmetry or cannot be
superimposed over its mirror image) and can be optically active. In
the case of the non-sulfonated monomer, sulfonation post
polymerization can be carried out. The resulting material may
possess unique properties that allow specific interactions with
biologically active species such as observed for glycosaminoglycans
some of which are known to possess a "sulfation code". This
sulfation code can allow for the polysulfonated material to protect
biological species necessary for good healing and repair. In some
cases, the sulfation code can be designed into a synthetic
polysulfonated compound by the inclusion of chemical
functionalities that allow the material 18 to interact favorably
with biological molecules such as peptides, growth factors,
cytokines and the like.
[0037] In another example, the presence of small amounts of
material 18 can provide protection for protein therapeutic agents,
such as insulin, from being degraded by enzymatic processes which
can render these proteins ineffective and potentially
proinflammatory to the site at which the drug is being delivered.
In one example, a solid polymeric sheet containing polysulfonated
material 18 is fabricated from a biomedical material such as a
silicone gel and the solid sheet is positioned to surround a
transcutaneous access point through which a medical device such as
an infusion set makes contact with a patient's tissue. The solid
polymeric sheet, for example, can be formulated with an
antibacterial, protease inhibiting, aqueous media soluble,
polysulfonated material in salt form in order to protect the wound
from invading microorganisms, thus preserving the viability of the
subcutaneous tissue for the uptake of drug, and to preserve the
drug by preventing degradation of the protein by proteases such a
neutrophil elastase. Neutrophil elastase is known to lyse proteins
at alanine-valine peptide bonds, among others. The alanine-valine
peptide linkage is present in insulin thus rendering it susceptible
to degradation. The protease inhibiting characteristics of material
18 can provide protection against this kind of degradation for
example.
[0038] It is important to note that the use of an aqueous
media-soluble polysulfonated material to prevent infection is not
limited to a solid sheet configuration and may be formulated as a
coating that may be provided directly on a medical device such as a
transcutaneous infusion set or a glucose sensor. It is further
understood that an antimicrobial agent alone, such as
chlorhexidine, in such a device alone without a formulation to
include a polysulfonated material may provide protection against
invading pathogens and as such may increase the useful lifetime of
use devices such as glucose sensors and infusion sets.
[0039] Furthermore, the ability of material 18 in its sulfonated
form to inhibit proteases (enzymatic species that can include other
proteins that degrade proteins) can prevent certain biological
species from degradation in environments where proteolytic activity
is high. The charge on these enzymes when their overall charge is
neutral (isoelectric point) and whether or not the activity of
these enzymes is related to an active site containing metal cation
such as Zn.sup.++ are relevant to this application.
[0040] The isoelectric point, sometimes abbreviated to IEP, is the
pH at which a particular molecule or surface is absent of any net
electrical charge. Amphoteric molecules called zwitterions contain
both positive and negative charges depending on the functional
groups present in the molecule. The net charge on the molecule is
affected by the pH of their surrounding environment and can become
more positively or negatively charged due to the loss or gain of
protons (H+). The IEP is the pH value at which the molecule carries
no electrical charge, where the negative and positive charges are
in balance. Surfaces naturally charge to form a double layer. In
biological systems, the surface charge-determining ions are
hydronium (H.sub.3O.sup.+), and hydroxide (OH.sup.-), counter ions
that can include chloride (Cl.sup.-), as well as metal cations that
include sodium (Na.sup.+) and potassium (K.sup.+) for example. The
net surface charge is affected by the pH of the medium/liquid
surrounding the species of interest. The IEP value can affect the
solubility of a molecule at a given pH. Such molecules have minimum
solubility in water or salt solutions at the pH which corresponds
to their IEP and often precipitate out of solution. Biological
amphoteric molecules like proteins (polyaminoacids also known as
polypeptides) contain both acidic and basic functional groups. The
amino acids which make up proteins may be positive, negative,
neutral or polar in nature, and together give a protein its overall
charge.
[0041] At pH values below their IEP, proteins carry a net positive
charge; above their isoelectric point the proteins are deprotonated
and carry a negative charge. Proteins (including proteases) that
have high isoelectric points (>7.5) are to some degree cationic
(positively charged) at physiologic (neutral) pH (7.0). The higher
the IEP, the more positive character the protein will possess at
pH=7.0.
[0042] For the purposes of this application, examples of
proteinases that are of merit and interest to inhibit can include
aspartic acid, cysteine, metallo-, and serine proteinases, among
others.
[0043] Sulfonate groups have a high affinity for cations, including
multivalent cations such as Zn.sup.++, Ca.sup.++, and Mn.sup.++ for
example. In the case of a multivalent cation (.gtoreq. charge of
2+), in order to satisfy the law of neutrality more than one
sulfonate/sulfate group can interact with the cation or the cation
may have other anions associated with it in addition to the
sulfonate/sulfate groups.
[0044] The sulfonic acid group, in its acid form is a very
effective cation exchange group and can exchange metal cations, for
example, for protons. Thus, the SO.sub.3H moiety can interact with
NaCl to form --SO.sub.3Na and HCl for example.
[0045] Material 18 can also include other sulfonated compounds such
as, but not limited to, polymers of sulfated saccharides or
polysulfated polysaccharides, such as dextrin sulfate, dextran
sulfate, chitosan sulfate, or cellulose sulfate, among others. The
sulfonate group of material 18 can be coupled directly to a
structural unit depicted by --OR, with the R group representing the
remainder of material 18, and the coupling to an oxygen atom {O}
forming what is referred to as a sulfate group
(--OSO.sub.3.sup.-X.sup.+). Accordingly, sulfate groups contain
sulfonate groups (--SO.sub.3.sup.-X.sup.+). As such, material 18
can include polysulfonates including sulfonic acids, sulfonic acid
salts, and polysulfated compounds, among others. The polysulfated
compounds can include synthetic, semi-synthetic, and/or naturally
occurring polysulfated polysaccharides that include chondroitin
sulfate, heparan sulfate, heparin, or dextran sulfate given as an
example above, as well as the sulfated semisynthetic polysaccharide
pentosan polysulfate, for example. For instance, Sulfated
polysaccharides, also known as glycosaminoglycans or GAGs, are
efficient ion exchange materials by virtue of the sulfonate group
present.
[0046] In some cases, material 18 can have a molecular weight of
from about 600 grams/mole to about 1,000,000 grams/mole but may be
in excess of 2,000,000 g/mole. As an example, material 18 can be a
polymer or copolymer having a molecular weight of at least about
70,000 Daltons but may be less or more depending upon the
application. Material 18 can also include polysulfonated material
blended with another material. For example, polysulfonated
materials such as polystyrenesulfonate can be blended with
materials such as hydrogel(s). Hydrogels can include, but are not
limited to, alginates, polyacrylates, polyalkylene oxides, and/or
poly (N-vinyl pyrrolidone). The hydrogel may also be amorphous,
i.e. a viscous gel as opposed to a solid such as a formulation of
carboxymethylcellulose containing a humectant such as propylene
glycol or glycerin. It is also possible to combine the components
of material 18 in their separate forms into a liquid mixture for
example allowing for the association to occur in the liquid or
semisolid mixture such as an ointment comprising Petrolatum, Fatty
Alcohol (stearyl), Emollient (isopropyl myristate), Emulsifying
Agent (polyoxy (40) stearate, sorbitan monooleate), Humectant
(propylene glycol), and sterile deionized water, among others. In
this method the resulting association may result in a homogenous or
heterogeneous formulation. Material 18 can also be blended with
polyurethanes, for example. Material 18 can also be blended with
naturally occurring polymers that include chitosan, hyaluronic
acid, and starch, among others.
[0047] Material 18 may additionally be covalently cross-linked with
a diamine and a coupling agent. For example this may form a
sulfonamide at some or all junctions where the diamine links to the
sulfonic acid groups of material 18. In some cases, the
cross-linking of the polysulfonated material can serve to alter the
solubility of the polysulfonated material. The solubility is
dependent upon the number of cross-link points introduced. Examples
of cross-linkers can include peptides, aromatic or aliphatic
diamines, diaminosaccharide and the like. Coupling agents can
include 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluoro phosphate (HATU), or
O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate
(HBTU), for example but may include pretreatment of the acid form
of the polymer with thionyl chloride in order to yield the sulfonyl
chloride which will react with an amine to form a sulfonamide.
Furthermore, in cases where the cross-linker is a peptide sequence,
one or more of the peptide bonds within the peptide may be designed
to be susceptible to proteolytic cleavage. Peptides with the
alanine-valine linkage would for example be susceptible to elastase
cleavage. It is understood that MMPs such as MMPs 8 & 9 can
cleave peptides at different peptide bonds. For example MMP-9 is
known to cleave proline-proline and lysine-lysine bonds for
example. As such, it is understood that these linkages can be built
into the cross-linker in order to ensure that enzymatic degradation
occurs thus leading to dissolution and essentially biodegradation
of material 18. This feature would enable solubilization of the
cross-linked material as the cross-links are cleaved (i.e.
degradation of the linkage). Solubilization of the material can
allow for the polysulfonated material 18 to function as a drug
delivery vehicle or a gene delivery vehicle for example while
providing some protease inhibitor characteristics. It is worth
noting that if an amino compound with only one available amino
group is added in the same fashion, partially modified sulfonamides
can result. If the amine is a peptide, for example, a protease
inhibitor with additional biological activity can result.
[0048] The SO.sub.3.sup.- group can be referred to as a sulfonate
group. The sulfonate group can be a terminal sulfonate group, and
material 18 can include at least one terminal sulfonate group. In
accordance with embodiments of the disclosure, the SO.sub.3.sup.-
groups of the polysulfonated material can extend from the oligomer
backbone, such as a polymer or copolymer backbone.
[0049] The sulfonate group can take the form of an acid, for
example. As an acid, the sulfonate group can be protonated, such as
SO.sub.3H. Material 18 can include many sulfonate groups and these
sulfonate groups may all be protonated or some may be protonated
while others are unprotonated depending upon the degree of
substitution. According to another embodiment of the disclosure,
the sulfonate groups of material 18 may be a component of a salt,
such as a metal or organic salt. According to embodiments of this
configuration, material 18 can be referred to as a polyanionic
salt, such as polymetallosulfonate and/or a polyorganosulfonate.
The sulfonate group of material 18 can be associated with either or
both of an inorganic or organic element or compound that may be of
pharmacologically relevant value. Pharmacologically relevant metal
ions and organic ions are understood to be of value in treating at
least one element of a medical condition. One such example is a
bacterial infection which may be treated by an antibiotic compound
such as a tetracycline, or a metal ion such as silver ion. As such
it is understood that a polysulfonate may be substituted with more
than one individual cationic species and may be either organic,
inorganic (metal), or mixed organic-inorganic varieties.
[0050] Material 18 may also comprise a synthetic, semisynthetic, or
biological polymer having at least one sulfonyl group extending
from the polymer backbone. In one embodiment, a sulfonyl group is
terminal. Examples include association of this sulfonyl group with
an amino acid or as a component of a salt wherein the salt is a
combination of the polyanionic sulfonate combined with an organic
cation, where the organic cation can be for example one or more,
but not limited to tetracycline, doxycycline, minocycline,
gentamicin, arginine, linear and cyclic peptides, lysine,
carnosine, glutathione, chlorhexidine, polyhexamethylenebiguanide,
the cytotoxic chemotherapeutic agent doxorubicin, or lidocaine,
among others. Thus, material 18 may be a polyanionic salt. For
example, as a polyanionic salt material 18 can be one or more of a
polyanionic metal salt, a polyanionic organic salt, or a mixed
polyorganic-polyanionic inorganic (metal) salt, among others.
[0051] In accordance with an implementation, the sulfonate group
can be associated with a complimentary cation. As an example, the
sulfonate group can be associated with an inorganic species such as
one or more of a positively charged metal such as Na.sup.+,
Ag.sup.+, K.sup.+, Li.sup.+, Au.sup.+, Ca.sup.++, Zn.sup.++,
Mn.sup.++, Mg.sup.++, Fe.sup.++/Fe.sup.+++, and/or Ce.sup.+++. The
sulfonate can also be associated with NH.sub.4.sup.+ or
NR.sub.4.sup.+ where R represents an alkyl, aryl or alkyl-aryl
substituent for example. According to another example, the
sulfonate group can be associated with one or more organic species.
Examples of such organic species can include nitrogen containing
organic species such as, an amino acid, a tetracycline,
doxycycline, arginine, lysine, glutathione, lidocaine, albuterol,
and/or alkyl/benzylammonium, among others.
[0052] In accordance with an example implementation, material 18
can be sodium polystyrene sulfonate (SPSS), a neutralized
derivative of the corresponding polystyrene sulfonic acid. Sodium
polystyrene sulfonate (SPSS) is a pharmacologically active compound
used in the treatment of hyperkalemia. Sodium polystyrene
sulfonate, (SPSS, sold as Kayexalate, Kalexate, or Kionex powder),
is an anionic polymer commonly used in the treatment of
hyperkalemia (high levels of potassium in the blood) and
occasionally used in pharmaceutical formulations as an inert
ingredient. As a treatment for hyperkalemia, PSS is taken either by
mouth or enema and its action as a strong cation-exchange resin
(highly anionic) removes excess potassium in these patients by
exchange for the available sodium (via a metathesis exchange
reaction of sodium for potassium) from the blood across the highly
vascularized large intestine. The same result can be achieved with
calcium polystyrene sulfonate as well. As such, both sodium and
calcium are considered pharmacologically active components of the
molecule. This polymetallosulfonate (SPSS or sodium heparin for
example) may be further exchanged with any variety of metal cations
to prepare mono, di, tri, and even tetravalent metal salt
derivatives. Similarly, the polymetallosulfonate, such as SPSS or
any sulfated polysaccharide such as heparan sulfate, may be
converted to a polyorganosulfonate derivative by exchange of sodium
cation for a protonated nitrogen atom containing salt of interest.
Additionally, the sulfated polysaccharide may be a proteoglycan
with main chain components that include adermatan sulfate, or
keratan sulfate, among others. Proteoglycans may include aggrecan,
versican or smaller sized species that include decorin, biglycan,
fibromodulin, keratocan, osteoglycin, and lumican, among
others.
[0053] Generally, sulfated saccharides (polysulfonates) are found
in their sodium salt forms. However, these compounds may be
modified to include nitrogen atom containing salts, among others.
In such salts, compounds containing protonatable nitrogen atoms,
can include, but are not limited to, amines, amidines, imines,
thiazoles, imidazoles, and/or pyridines, among others.
Additionally, ammonium salt derivatives may be prepared by the
exposure of an amino compound to the acid form of the
polysulfonated material, i.e. the sulfonic acid form. In some
embodiments, derivatives of material 18 can be produced by
chemically or biochemically modifying material 18. In one such
example, the cation of material 18 can be modified to include the
oligodynamic silver cation (Ag.sup.+) by substituting/exchanging
Ag.sup.+ for sodium (Na.sup.+) by the use of a soluble or insoluble
silver salt. Examples of silver salts for exchange of silver to
material 18 include soluble silver nitrate, or silver acetate which
has very low solubility in deionized water or Dowex silver ion
modified exchange resins (strong and weak cation exchangers) which
have very little if any solubility in deionized water or other
aqueous media. Examples of silver ion modified material 18 may
include silver polystyrene sulfonate, heparin silver (Ag.sup.+),
heparan (Ag.sup.+) sulfate, or chondroitin (Ag.sup.+) sulfate where
silver cations are exchanged onto the polysulfated saccharide using
a cation resin exchange methodology identical or similar to that
described in examples 14 & 15 among others. When polysulfated
compounds (material 18) are to be prepared, the type of ion
exchange resin that provides good substitution is a weak ion cation
exchange resin such as Dowex Mac-3. Silver salts with low deionized
water solubility such as silver citrate and the like may be
utilized as well. Because of low deionized water solubility, sodium
for silver exchange leads to the formation of the exchange product
sodium citrate and the consumption of a (molar) equivalent amount
of silver citrate to that of the sulfonates present in
solution.
[0054] Silver has been utilized as an antimicrobial agent
especially in the treatment of burns. Although metallic silver
develops a surface layer of oxide which tends to passivate the
material, in aqueous environments silver ions are readily released
and its antimicrobial activity stems from an intracellular
accumulation of silver ions. Silver ions readily bind to negatively
charged components in proteins and nucleic acids, thereby effecting
structural changes in bacterial cell walls, membranes and nucleic
acids that affect viability. In particular, it is speculated that
silver ions interact with thiol and other groups so that multiple
events simultaneously interfere with microbial processes. It is
believed that silver ions can bind to DNA to block transcription,
bind to cell surface components to interrupt bacterial respiration
and interfere with ATP synthesis.
[0055] The complex issues concerning the toxicity of silver to
mammalian systems have prevented more wide-spread use. Skin
discoloration and irritation associated with the use of silver
nitrate is well documented; absorption of silver, systemic
distribution and excretion in urine has also been reported.
[0056] In wound care, silver has been utilized in several products.
Silver nitrate is not widely used anymore, but silver sulfadiazine
(SSD) and silver releasing dressings have become popular. One
unfavorable characteristic of SSD is its low aqueous media
solubility thus limiting its penetration through the skin whereas
many of the silver salts of polysulfonated material 18 are freely
soluble in aqueous media. It is important to note that substitution
may be partial or complete. In other words, a metal cation such as
silver cation or a molecular cation such as gentamicin may be
substituted for sodium or some other relevant cation in varying
percentages between 0 and 100%. In one example sodium polystyrene
sulfonate (SPSS) may be substituted with silver cation (Ag.sup.+)
using the method described in example 3 to prepare a mixed
sodium-silver polystyrene sulfonate of 14% Ag.sup.+ substitution
that is completely soluble in deionized water as well as other
aqueous media. In another example, a tetracycline as a salt (e.g.
hydrochloride) can be substituted for sodium of material 18 via
cation exchange. Further, the sulfonic acid derivative of material
18 may be used as a proton source during an acid-base reaction by
treatment with, for example, an amino acid such as arginine, or a
biogenic amino compound such as tyramine or dopamine. Similarly,
the polyanion or polysulfonic acid (anion) of material 18 can be
exchanged with a polycation or polyamine, such as a strongly basic
ion exchange resin, for example, poly-L-lysine or the poly cationic
species polyhexamethylene biguanide.
[0057] It is worth noting at this point that just as the
cation-exchange modifications to material 18 need not be all
(100%), the cross-linking substitutions introduced to alter the
solubility of material 18 as described above need not be all (100%)
or nothing (0%) and may be engineered to something in between.
Instead, some implementations, can specifically utilize partial
modifications to achieve useful resultant or derivative materials.
In fact, partial cross-linking substitution leaves available
sulfonate groups for cation binding and can produce derivative
materials that have characteristics that are different from those
of the unmodified material 18 and any of the fully substituted
derivative materials. For instance with the cation-exchange
modified silver analogs described above, where Ag.sup.+ for
Na.sup.+ substitution is carried out, a substitution of less than
100% can result in a material that retains good water solubility
and has lower toxicity than the fully substituted counterpart. In
one such example, about 14% Ag.sup.+ substitution (i.e., .about.14
out of every 100 Na.sup.+ are substituted by Ag.sup.+) can be
utilized. In another example, about 34% Ag.sup.+ substitution can
be utilized. In a further example, about 79% Ag.sup.+ substitution
can be utilized. The amount of substitution can affect several
properties of interest of the derivative material. For instance,
the amount of substitution can affect the water solubility of the
derivative material. In application where water solubility of the
derivative material is desired, a relatively lower percentage of
Ag.sup.+ for Na.sup.+ substitution can be utilized while still
offering the beneficial antibacterial properties of silver cation
with complete water solubility. For example, the listed 14% and 34%
substituted derivative materials are water-soluble yet offer
antimicrobial properties against Staphylococcus aureus,
Acinetobacter baumannii, and/or Pseudomonas aeruginosa, among
others, for human patients. Similarly, a relatively higher
percentage of Ag.sup.+ for Na.sup.+ substitution, such as the
listed 79% example, can be utilized where lower solubility is
desired. When Material 18, for example sodium polystyrene sulfonate
(SPSS, Material 18-sod) is 100% substituted/exchanged with a cation
that does not prevent its dissolution into deionized water, for
example with the antibiotic mafenide (Material 18-maf), the
derivatized material, Material 18-maf, may be combined with an
equimolar amount of Material 18-sod in DI water in order to yield
Material 18-maf/sod, where mafenide cations and sodium cations each
occupy approximately equal numbers of sulfonate group sites in the
polymer. Such a mixed complex can be isolated by lyophilizing the
solution to yield a solid mixed salt material 18. This approach
works well for repeatedly achieving substitutions where some amount
less than 100% are desired and the materials are soluble in water.
This methodology makes it relatively straightforward to synthesize
a sulfonated material 18 with multiple substitutions at calculated
and defined target levels as long as the starting materials are
soluble in water and characterizable in terms of their individual
substitutions. As such, substitutions where at least two metal
cations are included can be prepared at any ratio desired. The same
applies to organic cation substitutions and mixed organic-metal
cation substitutions as well.
[0058] It is also worth noting that while specific substitutions
are described in isolation, it is recognized that two or more
partial substitutions can be combined to produce derivative
materials that have potentially advantageous properties. For
instance, partial Ag.sup.+ for Na.sup.+ substitution can be
advantageous for at least two reasons: 1. Silver cation is
cytotoxic and the higher the concentration of silver cation the
greater the toxicity of the salt to living systems, and 2. As the
substitution of silver is increased, the water solubility of the
salt generally decreases. Because silver-based antibacterials are
generally slow to kill bacterial pathogens, lower water solubility
may decrease the effectiveness of such an agent. Although the
incorporation of silver cation at 100% of theoretical substitution
(for example of Ag.sup.+ for Na.sup.+) may be a very potent
antimicrobial agent, it will be more toxic to fibroblasts,
keratinocytes, and other cells than a formulation with a lower
level of substitution. For example, Polystyrene sulfonate-Na/Ag
(mixed sodium cation/silver cation) at various levels of silver
cation substitution (14, 34, and 78 mol %) have been shown to be
effective at killing a variety of bacterial organisms. the higher
the level of silver substitution, the lower the concentration of
Material 18 that is required to kill bacterial pathogens. Material
18 is effective in the micromolar range for 14% Ag substitution, in
the nanomolar range for 34% substitution, and in the subnanomolar
range for 78% substitution. However, as the silver cation
substitution increases the cytotoxicity to neonatal fibroblasts was
observed to increase as well.
[0059] In other embodiments, Material 18 can be formulated as a
salt with three or more cations. For example, material 18 may
include partial antibiotic incorporation (such as mafenide),
partial for Na.sup.+ substitution, and partial Ag.sup.+
substitution to produce a material that has a combination of
Ag.sup.+, Na.sup.+, and mafenide cation incorporated. It is also
possible to achieve this combination (cation) salt by combining
appropriate amounts of a sodium salt of material 18, a mafenide
salt of material 18, and a 100% silver salt or mixed sodium-silver
salt of material 18, dissolving all three into DI water to achieve
a solution and subsequently lyophilizing the solution to a solid.
In solution, the salts which are all substituted at 100% of their
respective cations, exchange cations rapidly reaching a mixed
cation polysulfonated material.
[0060] To summarize, material 18 can be associated with numerous
elemental cations and cations of compounds that have
pharmacologically therapeutic value, either singularly (100% of one
cation type) or in combination (two or more cations to make 100%).
For example, material 18 can be associated with paramagnetic ions
such as Mn.sup.+2; Gd.sup.+2, Fe.sup.+3, as well as radio-opaque
metal ions of barium, tungsten, and radioactive ions of strontium,
rhenium, yttrium, divalent metal cations Ca.sup.+2, Zn.sup.+2,
Cu.sup.+2, Mg.sup.+2, Co.sup.+2, monovalent metal cations Na.sup.+,
Ag.sup.+; Au.sup.+, Li.sup.+, K.sup.+, as well as a wide variety of
organic cations that includes various antibiotics, among others.
Generally, silver cation is antibacterial and exchange onto the
polysulfonate yields material 18 with antibacterial properties.
Surprisingly, partial silver substitution produces a material 18
that possesses good antibacterial properties, minimal toxicity, and
excellent solubility. Example 3 describes the preparation of a
partially substituted SPSS-Ag salt (mixed sodium-silver
substitution) with excellent solubility. Example 11 describes the
materials antimicrobial effectiveness against a variety of
bacterial pathogens and example 12 provides an indication of the in
vivo effectiveness of a salve formulation containing the partially
silver substituted material 18. The preservation of tissue was
shown to be statistically better than the control treatment without
overt toxicity.
[0061] Referring to FIG. 2, scheme 10A is shown with material 18
being associated with at least a portion of therapeutic agent
R.sup.+. Example agents R.sup.+ associated and/or coupled to
material 18 are provided herein. When provided to inhibit
inflammation or cancerous cell growth, the portion of therapeutic
agent R.sup.+ can be released from material 18 and form therapeutic
agent R.sup.+X.sup.-, via ionic exchange, for example. According to
an implementation, material 18 can simultaneously provide
proteinase inhibition in addition to a treatment related to
therapeutic agent R.sup.+X.sup.-. In some cases, the proteinase
inhibition capability and the action provided by an ionically bound
therapeutic agent can be provided simultaneously.
[0062] Referring to FIGS. 3 and 4, preparations of material 18 are
shown in both liquid (FIG. 3) and solid (FIG. 4) form. Referring to
FIG. 3, preparation 20 includes a mixture 22 within container 24.
Mixture 22 may include at least two components with at least one of
the two components being material 18 including a cross-linked form
of the polysulfonated material. According to an embodiment of the
disclosure, mixture 22 can be a liquid mixture. Material 18 can be
present in mixture 22 in the form of a soluble component, for
example, or in the form of an insoluble component, as another
example. Mixture 22 can include a hydrophilic solvent, such as
water, or a hydrophobic component such as petrolatum along with
material 18. In one implementation, material 18 can be formulated
to be insoluble in deionized water, but soluble in ionic solutions
with solubility mediated by the ionic strength. Thus dissolution of
the salt and subsequent release of each of the two or more species
present in the salt can be modulated by the ionic concentrations,
for example, found in biological fluids. Such a occurrence can
allow material 18 to be prepared as an insoluble salt in deionized
water where two aqueous solutions (for example one of sodium
polystyrene sulfonate (SPSS) and one of doxycycline hydrochloride)
are combined to yield a deionized water-insoluble polystyrene
sulfonate salt of doxycycline (PSS-Dox) which forms as a
precipitate that settles out following the combination of the two
deionized water solutions. Material 18, in some cases, can be
obtained in a relatively pure form by simple filtration. The
deionized water-insoluble salt can be slowly dissolved into an
isotonic aqueous saline solution, or biologically equivalent
solution with sodium, potassium, and/or calcium ions where these
ions can exchange with doxycycline ions in order to yield SPSS (or
the calcium or potassium salt of PSS) and doxycycline
hydrochloride. As used herein, aqueous solutions containing sodium,
potassium, and/or calcium ions (simple and relevant biologically
relevant salts), as well as amino acids, proteins, peptides or the
like are referred to as "aqueous media". Such solutions may include
but are not limited to phosphate buffered saline solution (PBS),
saline, serum (including fetal bovine serum and human serum), and
other biological media. Thus, these deionized water salts can be
effective "controlled-release" compounds when placed into or onto
biological systems where biological fluids provide the exchange
medium for dissolution. Thus, the deionized water-insoluble salts
are, in fact, water soluble as long as ions are present and the
timescale of dissolution is driven by the cation concentration and
the rate of cation exchange. In one example, this form can be
administered to a patient directly as a micronized powder that can
be placed directly into the periodontal pocket for the treatment of
periodontitis, for example. Similarly, the polystyrene sulfonate
salt formed by the reaction of sodium polystyrene sulfonate (SPSS)
with chlorhexidine digluconate is insoluble in water. Salts that
are insoluble in deionized water can interact with the patient's
bodily fluids which facilitate the slow dissolution (and subsequent
ionization) of material 18 by cation exchange thus leading to
protease inhibition and bacterial organism control, for example. In
other cases, material 18 can remain completely water-soluble even
when 100% exchange of the replacement cation is carried out. For
example, a combination of sodium polystyrene sulfonate (SPSS) and
mafenide acetate (4-(aminomethyl)benzenesulfonamide acetate salt)
in a 1:1 molar combination results in a clear solution that when
lyophilized yields a white powder mixture consisting of mafenide
polystyrene sulfonate and sodium acetate. The sodium acetate may be
dialyzed away using cutoff filters or using dialysis tubing. Other
examples of water-soluble salts include those of arginine,
carnosine, glutathione, Ca.sup.++, Ag.sup.++, and Co.sup.++.
Mixture 22 can also be hydrophobic, such as would be obtained by
emulsifying material 18 into a hydrocarbon base along with
appropriate additional constituents. Material 18 can be synthesized
to be hydrophobic or hydrophilic depending on the choice of
cation.
[0063] Mixture 22 can include additional components as well as
material 18. The additional components may include but are not
limited to detergents, emulsifying agents, emollients,
antioxidants, excipients, wetting agents, essential oils,
flavoring, preservatives, viscosity modifiers, lubricants,
petrolatum products, and a skin permeation enhancer.
[0064] Detergents can include Tween 80 (Polysorbate 80), for
example. The skin permeation enhancers can include one or more of a
linoleic acid, an alpha-linoleic acid, an oleic acid, cod liver
oil, menthol derivatives, squalene, glycerol derivatives, herbal
ingredients, and senkyu ether extract. Mixture 22 can be a neutral,
hydrophilic matrix cream, lotion, ointment, solution, or gel, and
material 18 can be solubilized or dispersed into this mixture.
Collectively these kinds of formulations may be referred to as a
salve. The gel, for example, can include any variety of largely
aqueous based amorphous hydrogel formulations that include, but are
not limited to, a hydrophilic water-soluble polymer such as
carboxymethylcellulose (CMC), or material 18 can be combined into
an ointment which may be a more complex mixture including
emulsified formulations. Such an emulsified formulation can include
White Petrolatum, Fatty Alcohol (stearyl), Emollient (isopropyl
myristate), Emulsifying Agent (polyoxy (40) stearate, sorbitan
monooleate), Humectant (propylene glycol), and sterile DI
water.
[0065] In accordance with an example implementation, mixture 22 can
include water and material 18, with material 18 being a
polysulfonated salt. Mixture 22 may be buffered to a pH of from
about 3.5 to about 8.0 as required to keep the sulfonate groups of
material 18 in the desired state.
[0066] In accordance with another example implementation, mixture
22 can be homogeneous or heterogeneous. For example, mixture 22 can
be a homogeneous mixture of water, water-soluble additives, and
water-soluble polysulfonated material, as for example in a
mouthwash or homogeneous wound gel that may comprise carboxymethyl
cellulose for example. As another example, mixture 22 can be a
heterogeneous mixture, such as an emulsion, similar to that found
in a wound gel or toothpaste. In one such implementation mixture 22
can be in the form of a gel, cream, paste, or lotion, for example.
The paste may also include that of a tooth paste for example.
[0067] Material 18 may be associated and/or provided with any
variety of pharmacologically active cations that can include
chemotherapeutic agents for the treatment of a variety of symptoms
including: methotrexate; fluorouracil; adriamycin; ansamitocin;
cytosine arabinoside; arabinosyl adenine; mercaptopolylysine; PAM;
L-PAM (phenylalanine mustard); mercaptopurine; mitotane;
procarbazine dactinomycin (actinomycin D); mitomycin; plicamycin
(mithramycin); aminoglutethimide; estramustine; flutamide;
leuprolide; megestrol; tamoxifen; amsacrine (m-AMSA); asparaginase
(L-asparaginase) Erwina asparaginase); etoposide (VP-16);
interferon .alpha.-2a; interferon .alpha.-2b; teniposide (VM-26);
adriamycin; arabinosyl; procarbazine; and dacarbazine. In
accordance with additional embodiments of the disclosure, material
18 may also associated with and/or provided in a preparation with
Nitrogen mustards: (Chlorambucil, Chlormethine, Cyclophosphamide,
Ifosfamide, Melphalan). Nitrosoureas: (Carmustine, Fotemustine,
Lomustine, Streptozocin). Platinum: (Carboplatin, Cisplatin,
Oxaliplatin, BBR3464). Busulfan, Dacarbazine, Mechlorethamine,
Procarbazine, Temozolomide, ThioTEPA, Uramustine; Antimetabolites:
Folic acid: (Methotrexate, Pemetrexed, Raltitrexed). Purine:
(Cladribine, Clofarabine, Fludarabine, Mercaptopurine,
Thioguanine). Pyrimidine: (Capecitabine). Cytarabine, Fluorouracil,
Gemcitabine; Vincaalkaloids: (Vinblastine, Vincristine, Vindesine,
Vinorelbine); Cytotoxic/antitumor antibiotics: Anthracycline
family: (Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,
Mitoxantrone, Valrubicin). Bleomycin, Mitomycin; Topoisomerase
inhibitors: Topotecan, Irinotecan; Monoclonal antibodies:
Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab, Panitumumab,
Rituximab, Infliximab, Tositumomab, Trastuzumab, Etanercept;
Photosensitizers: Aminolevulinic acid, Methyl aminolevulinate,
Porfimer sodium, Verteporfin; Kinase Inhibitors: Dasatinib,
Erlotinib, Gefitinib, Imatinib, Lapatinib, Nilotinib, Sorafenib,
Sunitinib, Vandetanib (ZD6474).
[0068] Additional compounds that may be provided and/or associated
with material 18, include: Altretamine, Anagrelide, Bortezomib,
Denileukin diftitox, Estramustine, Pentostatin, Pegaspargase,
Alagebrium (3-phenacyl-4,5-dimethylthiazolium, anti-helmintics;
antitoxins; antivenins; aminoglycosides; theophylline;
aminophylline; hemin; hematoporphyrins; muramyldipeptide;
muramyltripeptide; lymphokines; macrophage activation factor;
N-acetyl-muramyl-L-alanyl-D-isoglutamine; ketoconazole; nystatin;
griseofulvin; flucytosine (5-fc); miconazole; amphotericin B;
ricin; cyclosporins; sulfazecin; growth hormone, melanocyte
stimulating hormone; triamcinolone; fludrocortisone; oxytocin;
vassopressin; cyanocobalamin; super oxide dismutase; alkaline
phosphatase; amelexanox; glutathione; carnosine; p-aminosalicylic
acid; isoniazid; capreomycin; cycloserine; ethambutol; ethionamide;
pyrazinamide; rifampin; and streptomycin; acyclovir; amantadine
azidothymidine; ribavirin and vidarabine; diltiazem; nifedipine;
verapamil; dapsone; octenidine; chloramphenicol; neomycin;
cefaclor; cefadroxil; cephalexin; erythromycin; clindamycin;
lincomycin; bacampicillin; carbenicillin; dicloxacillin;
cyclacillin; picloxacillin; hetacillin; methicillin; nafcillin;
oxacillin; penicillins (G&V); ticarcillin; rifampin;
doxycycline; minocycline, mefenamic acid; oxyphenbutazone;
phenylbutazone; piroxicam; sulindac; tolmetin; chloroquine;
hydroxychloroquine; metronidazole; quinine; quinidine; meglumine;
penicillamine; paregoric; codeine; heroin; methadone; morphine;
opium; and papaverine; noscapine; deslanoside; atracurium;
gallamine; metocurine; pancuronium; succinylcholine
(suxamethonium); tubocurarine; vecuronium; ethchlorvynol;
flurazepam; glutethimide; methotrimeprazine; methyprylon;
midazolam; temazepam; triazolam; bupivacaine; chloroprocaine;
etidocaine; lidocaine; mepivacaine; procaine; marcaine; tetracaine;
droperidol; etomidate; fentanyl; ketamine; benzyl trimethyl
ammonium, chlorhexidine; amino acids (natural & synthetic);
nicotinic acid; nicotinamide, pyridoxine; nucleosides (purines);
thiamine; coenzyme A; pentoxifylline; 3-amino-4-hydroxybutyric
acid; 6-diazo-5-oxo-L-norleucine; aceclofenac; acediasulfone;
alminoprofen; amfenac; amoxicillin; ampicillin; apalcillin;
apicycline; aspoxicillin; azaserine; aztreonam; bambermycin(s);
biapenem; bromfenac; bucillamine; bumadizon; candicidin(s);
carbenicillin; carprofen; carumonam; carzinophillin A; cefamandole;
cefatrizine; cefbuperazone; cefclidin; cefdinir; cefditoren;
cefepime; cefetamet; cefixime; cefmenoxime; cefminox; cefodizime;
cefonicid; cefoperazone; ceforanide; cefotaxime; cefotetan;
cefotiam; cefozopran; cefpimizole; cefpiramide; cefpirome;
cefprozil; cefroxadine; ceftazidime; cefteram; ceftibuten;
ceftriaxone; cefuzonam; cephaloglycin; cephalosporin C; cephradine;
ciprofloxacin; clinafloxacin; cyclacillin; denopterin; diclofenac;
edatrexate; enfenamic acid; enoxacin; epicillin; etodolac;
flomoxef; flufenamic acid; grepafloxacin; hetacillin; imipenem;
lomefloxacin; lymecycline; meclofenamic acid; melphalan; meropenem;
moxalactam; mupirocin; mycophenolic acid; nadifloxacin; niflumic
acid; norfloxacin; oxaceprol; panipenem; pazufloxacin; penicillin
N; pipemidic acid; podophyllinic acid 2-ethylhydrazide;
procodazole; pseudoephedrine; pteropterin; quinacillin; ritipenem;
romurtide; S-adenosylmethionine; salazosulfadimidine; sparfloxacin;
streptonigrin; succisulfone; sulfachrysoidine; sulfaloxic acid;
teicoplanin; temafloxacin; temocillin; tetracycline; tolfenamic
acid;
(N-((5-(((1;4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-
-thienyl)carbonyl)-L-glutamic acid); tosufloxacin; trovafloxacin;
doxyxycline; mafenide; minicycline; tigemonam; or vancomycin;
lucensomycin; natamycin or; 6-diazo-5-oxo-L-norleucine; denopterin;
edatrexate; eflomithine;
(N-((5-(((1;4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-
-thienyl)carbonyl)-L-glutamic acid)-ubenimex. In accordance with
yet another example, material 18 can be associated and/or provided
with albuterol, terbutaline, and/or ephedrines, as well as natural
or synthetic peptides.
[0069] Mixture 22 can be provided to an application apparatus such
as application apparatus 26. In the depicted embodiment, apparatus
26 is a collapsible tube. Mixture 22 can take the form of a lotion
or gel which can be extruded from apparatus 26 upon application of
force. In accordance with another embodiment, mixture 22 can be
provided to a container configured for pressurization such as an
aerosol can or an inhaler. In one implementation, mixture 22 can
include a propellant and material 18. Under pressure in a confined
container, mixture 22 can be expelled from the pressurized
container in aerosol form. Mixture 22 may be provided from a
nebulizer or inhaler as well.
[0070] Referring to FIG. 4, preparation 30 is shown that includes
particles 32 within container 34. Particles 32 can be solid and
include polysulfonated material 18, for example. In accordance with
an example configuration, individual ones of particles 32 can be
hydrogel beads. The hydrogel of the hydrogel beads can be
manipulated to include material 18. For instance, the hydrogel can
be cross-linked in the presence of, and/or blended with, material
18 to form a solid blend. The hydrogel can polyethylene
glycol-based and/or polyvinyl alcohol-based, for example. In
accordance with other embodiments of the disclosure, material 18
can be dispersed into a solid matrix of cross-linked acrylic
acid-based polymer such as methacrylic acid or any of its esters
including poly(2-hydroxy ethyl methacrylate) (HEMA), polypropylene
oxide, polyethylene oxide, polyvinyl alcohol, a polyurethane, a
polyester, alginate, silicone, hydrocolloid, and/or other
hydrogels, or an alkylene polymer (polyalkylene) such as
polypropylene or polyethylene. Further, individual ones of
particles 32 can include poly(N-vinyl pyrrolidone), poly(vinyl
alcohol), poly(acrylic acid), polyacrylamide including
poly(N-isopropylacrylamide), poly(ethylene-co-vinyl acetate),
poly(ethylene glycol)/polyethylene oxide, poly(methacrylic acid),
polyurethanes, and silicones, among others.
[0071] In accordance with another implementation, individual ones
of particles 32 can include material 18 as a biodegradable polymer
or material 18 associated with a biodegradable polymer. Example
biodegradable polymers include, but are not limited to,
lactide/glycolides, polyglycolides, polyorthoesters, and/or
polylactides, polycaprolactones, polydioxanones, starches,
cellulose, chitosan, and cross-linked natural polymers such as
collagen, gelatin or elastin.
[0072] In some implementations, individual ones of the particles
can be microspheres that include material 18. In accordance with
another implementation, individual ones of particles 32 can include
a degradable substrate, such as collagen, for example. Individual
ones of particles 32 can also include gelatin or the
heterosaccharide pectin.
[0073] Individual ones of the particles 32 can be microspheres that
include material 18. In accordance with another implementation,
individual ones of particles 32 can include a degradable substrate,
such as collagen, for example. Individual ones of particles 32 can
also include gelatin or the heterosaccharide pectin.
[0074] As an example, apparatus 36 can be used to apply particles
32. An example of apparatus 36 includes a syringe; however
additional applicators may be utilized, such as gauze and/or
collapsible tubing. In accordance with an example embodiment,
particles 32 may be provided to apparatus 36 in the form of an
injectable mixture. Particles 32 within the injectable mixture may
or may not be dissolved once injected. In accordance with another
implementation, particles 32 can be material 18 of mixture 22. As a
component of mixture 22, particles 32 may be provided as material
18 according to example embodiments.
[0075] Referring to FIGS. 3 and 4, preparations 20 and 30,
respectively are not mutually exclusive. Compositions that may be
included within mixture 22 may also be incorporated into particles
32. Likewise, compositions that may be included within particles 32
may also be incorporated into mixture 22. According to example
implementations, preparations 20 and/or 30 may include biologically
active material. Example biologically active materials can include,
but are not limited to, one or more of peptides, proteins,
cytokines, healing factors, antibiotics, cytotoxins, VEGF, PDGF,
EGF or other relevant growth factors, including, but not limited to
exogenous growth factors. Preparations 20 and/or 30 may also
include one or more of an angiogenesis stimulant, antibacterial,
antibiotic agent, or antiangiogenic agent. According to an example
implementation, material 18 may inhibit the degradation of
exogenous and/or endogenous factors. For example, material 18 may
be provided along with an exogenous material of an organism.
Material 18 may prevent the degradation of the exogenous material
providing for the retained therapeutic activity of the exogenous
material. Material 18 and the exogenous and/or endogenous materials
may be provided simultaneously to the organism.
[0076] Material 18 may also be associated with a solid and
fabricated into the form of a sheet or a coating. For example,
Material 18 may be prepared to have antimicrobial properties and
may be fashioned into a solid formulation for the treatment of
burns or infected wounds as with a dressing, as inclusion into a
coating for medical device or into a solid sheet protective
component around a device where a breach in the skin may increase
the likelihood of infection. As a component of a wound dressing,
material 18 may be associated with, but not limited to, natural
biopolymers such collagen, gelatin, or biomedical materials that
include polyurethanes, silicones, and hydrogels for example.
[0077] Preparations 20 and/or 30 may have a concentration of
material 18 of about 1 mg/ml, although higher or lower
concentrations can be used if desired. For example, concentrations
as low as about 0.01 .mu.g/ml, or as high as the limit of
solubility of material 18 in mixture 22 and/or particles 32, may be
used in a formulation such as amorphous gel or solid dressing such
as a those fabricated of calcium alginate or a hydrogel.
Preparations 20 and/or 30 may contain a concentration of material
18 of from about 0.00001 to about 500 mg/ml depending upon the
toxicity of the material to the living organism.
[0078] Preparations 20 and/or 30 may be applied via short or long
term application. Preparations including vehicles such as sterile
phosphate buffered saline (PBS) or sterile deionized water are
suitable for short term application of material 18. For longer term
application, use of a slow release vehicle may be utilized. For
example, a gel formulation preparation can be used for effective
delivery of material 18.
[0079] Referring to FIGS. 5 and 6, example methods for applying
preparations 20 and 30 are depicted. In accordance with example
embodiments, these methods can promote the healing of tissue of a
multicellular organism, including but not limited to vertebrate
organisms. In accordance with example implementations, a
therapeutically effective amount of material 18 can be administered
to the organism to reduce one or both of inflammation and cancerous
cell growth.
[0080] In other embodiments, a porous collagen substrate or
construct, for example a sponge, or an electrostatically processed
(electrospun or electrosprayed) collagen substrate or construct is
formulated to include one or more of material 18, a growth factor,
an angiogenic agent, a nutrient, a nitric oxide donor or precursor,
and optionally mammalian cells. The combination of the
biodegradable substrate, for example a collagen or gelatin, and a
growth factor along with material 18 provides a basis for dermal
and subdermal healing, i.e. a dermal substitute. Material 18 is
combined with the growth factor in order to protect the growth
factor from degradation that may result as a consequence of the
presence of inflammatory cells and hence proteases. Dermal
substitutes are born from the idea of re-engineering tissue and its
components, i.e. "tissue engineering". In the aforementioned
examples a substitute may be cellular or acellular in nature.
Acellular substitutes are attractive because they are less costly
and handling/manufacturing is simpler as the presence of living
cells is restrictive when sterilization and shelf life are
concerned. An acellular substitute may have application in the
repair of skin, for example, where a cancerous growth has been
removed, where a patient has had a prolonged open wound, or where a
patient has been severely burned and requires rapid grafting but
lacks sufficient tissue to provide a "homograft". Of course a
cellularized substitute may also be used, however the cost for such
a substitute will be significantly higher.
[0081] In electrospinning, polymer solutions, or melts, are
deposited as overlapping (continuous) fibers thus creating mats
rather than droplets as a consequence of the advantageous
entanglement of chains within melts or solutions at sufficiently
high polymer concentrations. The fibers are derived by charging a
liquid typically to 5-30 kV vs. a ground a short distance away,
which leads to charge injection into the liquid from the electrode.
The sign of the injected charge depends upon the polarity of the
electrode; a negative electrode produces a negatively charged
liquid. The charged liquid is attracted to the ground electrode of
opposite polarity, forming a so-called Taylor cone at the nozzle
tip and, eventually, a fiber jet as the electric field strength
exceeds the surface tension of the solution. The continuous fiber
is collected at the opposite electrode in a form that can be
controlled by the geometry and movement of the ejection electrode
and the collecting electrode.
[0082] In general, chronic wounds can be characterized by a
prolonged inflammatory phase, which ultimately can result in
elevated protease activity and the subsequent degradation of growth
factors and other positive wound healing factors, with the overall
effect being impaired healing. Chronic wounds can be considered an
imbalance between tissue deposition, stimulated by growth factors,
and tissue destruction mediated by proteases. Chronic wounds of
diverse etiologies can have elevated levels of a specific class of
proteolytic enzymes known as the matrix metalloproteases (MMPs).
The effects of these high levels of MMPs in the wound environment
may include local destruction of growth factors and their receptors
as well as degradation of granulation tissue components.
[0083] While the overall goal of wound healing is to synthesize and
deposit new tissue so as to reestablish continuity and function, it
can be noted that controlled tissue degradation is a normal part of
the wound healing process. Much of the tissue degradation related
to wound healing is performed by MMPs. The MMPs are a family of
structurally related, protein-degrading enzymes that require
calcium ions for structural conformation and zinc ions in their
active site for function. About 20 different members of the family
have been identified, and they share similar structure (about 40%
amino acid homology). Multiple cell types, including macrophages,
fibroblasts, neutrophils, epithelial cells, and endothelial cells,
synthesize MMPs in the presence of specific biochemical signals,
such as inflammatory cytokines (e.g., TNF*, IL-1b). MMPs play a
role in many normal physiological processes, such as wound healing,
embryonic development, and menstruation. An individual MMP may have
one or multiple protein substrates that it degrades. Certain MMPs
are very specific in their function (e.g., the collagenases only
degrade collagen). Specifically, they cleave the collagen triple
helix at a single point. This cleavage then allows the rigid triple
helix to relax and unravel, resulting in two gelatin fragments.
Other MMPs have multiple substrates; some redundancy of substrates
between MMPs is evident. When redundancy exists, usually one MMP
degrades a particular substrate preferentially.
[0084] Collectively, the MMP family of enzymes is capable of
digesting almost all of the components of the extracellular matrix.
In order for healing to progress and result in repair, a balance
may exist between the protein-degrading activities of MMPs and
other cellular activity directed towards the synthesis and
deposition of the protein components of granulation tissue. The
proteolytic activity of MMPs is controlled by various mechanisms,
including gene transcription, production of the enzyme in an
inactive form (called a zymogen) that requires extracellular
activation, and by local secretion of endogenous enzyme inhibitors
called tissue inhibitors of metalloproteases (or TIMPs). The same
cells that produce MMPs can synthesize TIMPs. Four different TIMPs
have been identified in tissues (TIMP-1, TIMP-2, TIMP-3, and
TIMP-4). These TIMPs can inhibit all of the MMPs by binding to the
zinc-containing active site of the enzyme. TIMPs do not bind to the
zymogen form of the enzyme. During normal wound repair, a delicate
balance can exist between the MMP and TIMP activity levels. If the
balance is disturbed, high levels of MMPs may result in excessive
tissue degradation or destruction of other protein components in
the extracellular matrix (ECM), such as growth factors, cell
surface receptors, and even the TIMPs themselves.
[0085] At least one other characteristic of some chronic wounds is
the excess of proteases that are detected in the extracellular
space. While controlled degradation can occur during normal wound
healing, excess or prolonged proteolytic activity is considered
detrimental and thought to contribute to the lag in healing of the
wound. In some instances, excess bacterial loading in wounds can
postpone healing. Material 18 can be effective at addressing
bacterial loading and excessive proteolysis of chronic wounds
leading to faster and better healing.
[0086] With regard to cancerous cell growth, some of the neutrophil
and other cell-induced tumor-promoting effects are attributed to
their abilities to express and release proteases. Neutrophil
degranulation results in the release of serine proteases, such as
elastase, cathepsin G and protease-3, which may contribute to the
activation of MMPs that mediate tumor cell invasiveness.
[0087] Tumorigenesis involves not only tumor cells that become
transformed but also the tumor stroma which reacts by inducing
inflammatory and angiogenic responses. Angiogenesis, the formation
of new capillaries from preexisting vessels, is typically required
for tumor growth and metastasis. During angiogenesis, quiescent
endothelial cells are activated and they initiate migration by
degrading the basement membranes through the action of specific
(expressed) proteins, in particular, MMPs.
[0088] MMPs promote tumor progression not only through ECM
degradation but also through signaling functions. MMPs counter
apoptosis, orchestrate angiogenesis, regulate innate immunity, and
promote metastasis and tumor growth. Stromal and immune-defense
responses can eventually fail, resulting in immune-cell evasion,
phenotypic evolution of metastases, chemotherapeutic resistance and
further tumor dissemination. MMP binding to cell-surface proteins
may have an effect on intracellular signaling, facilitate proenzyme
localization and activation, mediate cell motility by disruption of
cell contacts with the ECM, and promote internalization of the
enzyme. For example, integrins are shown to act as receptors for
several proteases, including MMPs. Such interactions have been
detected in caveolae, invadopodia, and at the leading edge of
migrating cells, where directed proteolytic activity is likely to
be needed. The first interaction between an integrin and an MMP
(MMP-2) was identified on the surface of melanoma cells and
angiogenic blood vessels. Furthermore, MT1-MMP was shown to
activate the integrin, .alpha.V.beta.3, through proteolytic
cleavage. Additionally, .alpha.V.beta.3-integrin may have
modulatory properties on MMP-2 activity by binding to its
C-terminal domain.
[0089] Further, CD44, which is the principal receptor for
hyaluronan, can also serve as a MMP-9-docking molecule. Interaction
of MMPs with the cell surface not only may be needed for proenzyme
activation and targeting at specific sites for degradation of
cell-surface substrates, but also could promote intracellular
degradation via receptor-mediated endocytosis.
[0090] Leukocyte elastase (LE) is a serine protease, expressed by
polymorphonuclear (PMN) leukocytes, mainly neutrophils. LE acts
both at the intra-cellular level to kill engulfed pathogens, and at
the extra-cellular level as mediator of coagulation, immune
responses, and wound debridement. Since LE has the potential to
degrade some structural proteins of the extra-cellular matrix
(ECM), such as elastin, fibronectin and collagens, production of
excess amounts of active LE has been identified in a number of
pathological conditions leading to impairment of ECM organization
that include rheumatoid arthritis, emphysema, chronic obstructive
pulmonary disease (C.O.P.D.), cystic fibrosis, some chronic wounds,
inflammatory bowel diseases, and tumor progression for example. LE
also activates the pro-enzymatic form of matrix metalloproteinase-9
(MMP-9) massively released by the PMNs, and instrumental to their
extravasation. Human tissues are normally protected from excessive
LE activity by endogenous inhibitors such as
.alpha.1-protease-inhibitor (.alpha.1-PI), .alpha.2-macroglobulin,
and secretory leukoprotease inhibitor (SLPI). An enzyme/inhibitor
imbalance may lead to increased lysis of ECM macromolecules and
thus an increased risk of tissue injury in areas infiltrated by
activated PMNs. Furthermore, given the ability of LE to degrade
multiple cytokines, receptors, and complement components, a
negative modulation of the inflammatory response may favor antigen
persistence, leading to chronic inflammation. As for the
possibility of using exogenous LE-inhibitors for therapeutic
purposes, to date many of the inhibitors that have been developed
present side effects that make them less than ideally suitable for
human use. However, material 18 may be provided to the organism in
an effort to protect both exogenous and endogenous factors.
[0091] Referring to FIG. 5, organism 40 may have a wound 42, such
as an epidermal wound. Example wounds include, but are not limited
to, burns (thermal and chemical) and chronic ulcers, such as
pressure ulcers, diabetic ulcers, venous leg ulcers, and
periodontitis. Wound 42 can also include atopic dermatitis, a
common form of inflammation of the skin and characterized by
elevated tissue levels of cathepsin G. Atopic dermatitis is a
chronic skin disorder characterized by pruritus, dry skin, and
excoriation, which may be localized to a few patches or involve
large portions of the body. Wound 42 can also be surgical or the
result of trauma such as abrasions, skin tears, and/or
blisters.
[0092] In accordance with the embodiment depicted in FIG. 5,
mixture 22 including material 18 can be administered topically to
wound 42. As described above, mixture 22 can be a liquid such as a
gel, cream, or lotion. In accordance with another implementation,
mixture 22 including material 18 can be applied to a substrate such
as a gauze or sponge, and the substrate can be applied to wound
42.
[0093] Upon administration of material 18, protein degradation of
the tissue of organism 40 can be inhibited. In accordance with
example implementations, protein degradation can be prevented via
the inhibition of proteases including metalloproteinases, such as
collagenase (MMP-8) and gelatinase (MMP-9) for example. Inhibition
of cysteine, aspartic acid, serine proteases and metalloproteinases
can also be accomplished. The serine proteases inhibited can
include one or both of elastase and cathepsin G. The cysteine
proteinases that may be inhibited include cathepsins S & K both
of which have isoelectric points in excess of 8.0.
[0094] Mixture 22 may be applied to wound 42 daily, or more or less
frequently as required. A typical daily dosage of material 18 will
be 20 millunits/g/cm.sup.2 of the wound or ulcer, although it will
be recognized that this amount may be varied, and concentrations of
0.1-2000 mu/g/cm.sup.2 advantageously may be used. For example,
ulcers of long duration (such as one year or longer) may require
concentrations of 500 mu/g/cm.sup.2 applied multiple times per day,
such as, for example, 2, 3, or 4 times daily. For ulcers of lesser
duration, or those that are responding well to higher doses, the
dose may be lowered. For example, the protease inhibitor dose may
be lowered sequentially to, for example, 100, 10, 1, or 0.1
mu/g/cm.sup.2. In addition, the application of the inhibitor may be
made less frequently, such as from 4 to 1 times daily.
[0095] Referring to FIG. 6, tissue 52 is shown having composition
38 applied thereto. Tissue 52 includes both cancerous cells 56 and
non-cancerous cells 54. In accordance with an example embodiment, a
therapeutically effective amount of composition 38 including
material 18 associated with a solid material, such as a
microspheres or bead, can be administered internally to reduce one
or both of inflammation and cancerous cell growth.
[0096] The solid components or particles comprising material 18,
including some of its cross-linked forms, may be formulated into
mixture 22 that may be configured as a biodegradable mass such as a
microsphere or wafer incorporating an anti-tumor agent. Anti-tumor
agents may include but are not limited to angiogenesis inhibitors,
DNA intercalators & cross-linkers, DNA synthesis inhibitors,
DNA-RNA transcription regulators, enzyme inhibitors, gene
regulation compounds, microtubule inhibitors, as well as other
agents that address different biological processes or biological
process inhibitors. Such a wafer or microspherical particle
formulation may be implanted at the site of a tumor for example. In
such an example, the controlled degradation of the particles will
slowly release the polysulfonated material and its ionically bound
therapeutic agents thus addressing proteases and cell growth
directly at the site of implantation. The materials that are known
to be biodegradable that may be combined with material 18 to form a
Mixture 22 include but are not limited to lactide-glycolide
copolymers, polyorthoesters, polycaprolactones, polydioxanones,
starches, cellulose, chitosan, and cross-linked natural polymers
such as collagen or elastin.
[0097] Referring to FIG. 7, a solid sheet of biomedical polymer
such as a silicone gel is formulated to include a material 18, such
as PSS-chlorhexidine and fashioned into a collar 60 that is placed
at the base of a cannula 63 as a component of a subcutaneous
infusion set 58 or at the entry point of venous access line 65 of a
PICC (peripherally inserted central catheter) line both of which
can be associated with a drug delivery system. Collectively these
are referred to as transcutaneous access devices. These devices are
for the delivery of liquid substances into the human body or the
removal of, or access to analytes within the human body such with a
biosensor. All of these devices possess an entry point herein
referred to as a transcutaneous access point. It is understood that
the collar 60 may be formulated from a variety of different
biomedical polymers in addition to silicone gel and that the collar
may be fabricated into a variety of shapes such as that resembling
but not limited to a doughnut 61 or a half moon 62 for example and
may be applied to any variety of transcutaneous access devices.
[0098] Other antimicrobial PSS formulations may also be used such
as PSS-doxycycline, PSS-polyhexamethylene biguanide, or
PSS-octenidine. Such a system includes insulin delivery systems
such as insulin pumps/infusion sets, central venous lines like PICC
lines, as well as implanted access ports such as a MediPort or
Port-a-Cath. Other antimicrobial formulations that do not include a
polysulfonated material 18 may also be incorporated in such a
collar 60 as described. For example, chlorhexidine diacetate or
other antimicrobial agent alone or in combination can be
incorporated into a collar 60 as described. Furthermore, it is
understood that other healing aids may be added to the collar.
Examples of healing aids include but are not limited to
antioxidants, vitamins, growth factors, peptides, amino acids, and
cytokines.
[0099] It is understood that such a system may have additional
benefits beyond preventing infection. The benefit includes
providing a protease inhibitor to a site where the open wound may
facilitate degradation of the protein drug for example. Degradation
of a protein drug as a consequence of inflammation and
consequential proteolytic activity in the (subcutaneous wound)
space where the drug is delivered may reduce the effective dose of
the therapeutic compound, and in addition the fragments of the
protein that result from proteolytic degradation of the drug can be
proinflammatory components thus leading to an exacerbated
inflammatory response at the site of delivery. This continuous
inflammation can lead to the inability of the subcutaneous space,
where the drug cannula 60 resides, to effectively absorb the
drug.
[0100] Example embodiments of the disclosure are provided
below.
Example 1
Preparation of Material 18 (PSS-Arginine)
[0101] Sodium Polystyrene Sulfonate (SPSS, 70,000 mw) acquired from
Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES
and purified by precipitation from a 20-25% solution in deionized
water into isopropanol, can be dissolved into deionized water to
yield a 10-25% solids solution. Separately, arginine base in
deionized water (10 mg/mL) is added to account for an equimolar
quantity of sodium polystyrene sulfonate (SPSS), and the solution
stirred for 1 hour at room temperature. The solution is transferred
into a lyophilizer container and frozen. The frozen mass is placed
under vacuum of the lyophilizer and the solid polystyrene arginine
sulfonate is isolated as a flocculent off-white solid.
Example 2
Preparation of Material 18 (PSS-Mafenide)
[0102] Sodium Polystyrene Sulfonate (SPSS, 70,000 mw) acquired from
Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES
and purified as described in Example 1 can be dissolved into
deionized water to yield a 10-25% solids solution. Separately,
4-Aminomethylbenzenesulfonamide acetate is dissolved into deionized
water (10 mg/mL) and is added to account for an equimolar quantity
of sodium polystyrene sulfonate (SPSS), and the solution stirred
for 1 hour at room temperature. The solution is transferred into a
lyophilizer container and frozen. The frozen mass is placed under
vacuum of the lyophilizer and the solid polystyrene mafenide
sulfonate is isolated as a flocculent off-white solid.
Example 3
Preparation of Material 18 (PSS-Ag)
[0103] Sodium Polystyrene Sulfonate (SPSS, 70,000 mw) acquired from
Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES
and purified as described in Example 1 can be dissolved into
deionized water to yield a 10-20% solids solution. Separately,
silver acetate (Fluka) can be suspended into deionized water,
stirred, and the mixture heated to 60.degree. C. while maintaining
stirring. To the stirring mixture, 100 g of Dowex.RTM. Marathon
strong cation exchange resin (Dow Chemical) can be added to the
mixture and dissolution/disappearance of the silver acetate
immediately follows. The Ag.sup.+ modified Dowex.RTM. Marathon
strong cation exchange resin, which is in fact a water-insoluble
salt, can be isolated, washed with deionized water and dried. The
Ag.sup.+ modified cation-exchange resin is photo sensitive and
should be stored away from room light. The dried, Ag.sup.+ modified
Dowex.RTM. Marathon strong cation exchange resin is added to the
10-20% solution of sodium polystyrene sulfonate (SPSS) in a jar
with a PTFE lined cap, the jar is sealed and placed onto a roller
mill for about one hour. Subsequently, the Dowex.RTM. resin is
filtered from the solution, washed with deionized water and the
resulting solution placed into a lyophilizer vessel, the solution
is frozen, and the lyophilizer vessel containing the frozen
material is connected onto the lyophilizer until only a powder
remains. The percentage of silver incorporation onto the PSS
backbone is dependent upon the relative excess of silver-modified
Dowex.RTM. resin utilized. Adjustment (lowering) of the ratio of
silver to sodium in the silver-modified PSS can be accomplished by
blending solutions of sodium polystyrene sulfonate (SPSS) and
PSS-Ag (with Ag substitution up to 100%) and lyophilizing the
equilibrated solution. Note: A 20% substituted PSS-Ag as determined
by atomic absorption (20% Ag, 80% Na) can be combined with an
equimolar amount of SPSS in solution and the solution lyophilized
to yield a 10% Ag substituted PSS.
Example 4
Preparation of Material 18 (Tecophilic Polyurethane Film Containing
Sodium Polystyrene Sulfonate)
[0104] Sodium Polystyrene Sulfonate, 5 grams, (SPSS, 70,000 mw)
acquired from Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178,
UNITED STATES and purified as described in Example 1 can be
combined with 45 g of hydrophilic polyurethane (SPSS formulation)
and the mixture dissolved into a 95:5 mixture of ethanol-deionized
water to yield a 10% solids solution. The solution can be cast in
to a film, air dried and vacuum dried to yield a flexible material.
The polymer-SPSS formulation can be used to evaluate the effect of
SPSS against elastase. The results are detailed in FIG. 8. Note
that the SPSS formulation (1) has reduced the elastase from 30
milliunits to approximately 6 milliunits reflecting a roughly 80%
decrease in activity as depicted in the example.
Example 5
Preparation of Material 18 (Amorphous Hydrogel Gel Containing
Sodium Polystyrene Sulfonate
[0105] About 15 g of Cutinova amorphous hydrogel (Beiersdorf AG,
UnnastraBe 48, D-20245, Hamburg, Germany) can be transferred to a
vial and 1.67 g of sodium polystyrene sulfonate (SPSS) (mw=70,000)
(Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES)
added and the SPSS stirred into the gel using a glass rod to yield
about a 10% (wt./wt.) solids composition (188-DJV). The data are
presented for the SPSS formulation in the Cutinova gel (188-DJV).
The bar graph (FIG. 9) reveals that nearly 80% of the serine
protease elastase is removed from the test sample (human wound
fluid).
Example 6
Application of Material 18
[0106] The elastase inhibiting capacity of the sample from example
5 above (DJV-188) can be compared against Sodium Polystyrene
Sulfonates (SPSS 70K and 1000K) dissolved in buffer. These
identifiers are molecular weights of these materials. These
SPSS-containing formulations can also be compared to Cutinova gel
(same as unlabeled gel), a water-insoluble strong cation-exchange
polymer (nuggets), the 70K and 1000K molecular weights of SPSS, and
gauze. These data reveal that the gel formulation and the solid
SPSS materials are effective inhibitors of the serine proteases
elastase & cathepsin G, and the metalloproteases MMP-8 and
MMP-9 (FIGS. 10, 11, 12, & 13).
Example 7
[0107] Application of Material 18. About 25 grams of sodium
alginate (Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED
STATES) can be combined with 250 mL of sterile deionized water (as
by autoclave sterilization) and the mixture can be autoclaved in
order to facilitate dissolution. Simultaneously 15 grams of SPSS
(1000K) can be combined with 200 mL of sterile deionized water and
autoclaved (to 105.degree. C.) as the above described in order to
facilitate dissolution. An alternative method of dissolution
utilizes a roller mill (room temperature). Following autoclaving
the above solutions can be combined and filtered. Separately, 1
liter of 0.5 M CaCl.sub.2 solution in deionized win order to
prepare beads. The beads can be allowed to dwell in the calcium
chloride solution in deionized water is prepared and 10 grams of
SPSS (1000K) added in order to ensure that little SPSS is lost
during the cross-linking step. The alginate solution can be added
in a drop wise fashion to the CaCl.sub.2-SPSS solution and the
formed beads are allowed to stir for 5 minutes and subsequently
filtered through polyester fabric. The beads can be packaged and
refrigerated prior to testing. The calcium PSS-containing beads can
be effective at inhibiting elastase (see bar chart) IMS-70-1,
IMS-70-2, IMS-70-alginate, and IMS-1000-aliginate (FIG. 14).
Similarly the products can be effective against cathepsin G, MMP-8,
and MMP-9. With the alginate-SPSS solution from above, a sheet of
Evolon (130 grams) soft (Freudenberg/Evolon NA) can be immersed so
as to become fully wetted and the fabric removed and excess
alginate solution removed. The fabric can be placed into the
CaCl.sub.2-PSS solution and allowed to dwell until the alginate has
become firm. The fabric composite can be cut to size and sterilized
by electron beam irradiation (25 kG) prior to studies.
Example 8
[0108] Application of Material 18 a Dose Response Study of Sodium
Polystyrene Sulfonate (SPSS) Inhibition of Neutrophil Elastase
[0109] Different amounts of SPSS were incubated with 12.5
milliunits of neutrophil elastase in 1.0 mL of buffer for 2 hours
at 25.degree. C. Aliquots (160 microliters) were mixed with an
elastase specific substrate and the liberation of anilide was
monitored spectrophotometrically. Inhibition was rapid and
irreversible with the full inhibitory effects observed as fast as
the measurements could be made (.about.5 minute intervals). The
rate of inhibition is a consequence of the inhibitor and the
proteins being in the solution phase. Dilutions of elastase were
used to generate a standard curve and concentrations down to 1
microgram/mL inhibited elastase efficiently (.about.80%) but not
completely (FIG. 15). A comparison of SPSS against
condroitin-6-sulfate (C6S) for the inhibition of elastase revealed
that C6S was much less efficient an inhibitor than SPSS. C6S could
not achieve the same level of inhibition (only 45%) and it required
about 21 times the amount of C6S as SPSS to achieve an equivalent
level of inhibition.
Example 9
Derivatization of SPSS with Chlorhexidine
[0110] Sodium Polystyrene Sulfonate (SPSS, 70,000 mw) acquired from
Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES
and purified as described in Example 1 can be dissolved into
deionized water to yield a 10-20% solids solution. Separately,
chlorhexidine diacetate (Sigma) is added to warm deionized water
(35.degree. C.) to yield a 25-30 mmol solution. The chlorhexidine
diacetate solution can be added directly to the SPSS solution and a
precipitate forms. The precipitate is filtered from the mixture and
washed with deionized water. The solid is dried under vacuum to
yield an off-white solid.
Example 10
Derivatization of SPSS with Doxycyline
[0111] Sodium Polystyrene Sulfonate (SPSS, 70,000 mw) acquired from
Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178, UNITED STATES
and purified as described in Example 1 can be dissolved into
deionized water to yield a 10-20% solids solution. Separately,
doxycycline hydrochloride (Sigma) is dissolved into deionized water
(20% w/w) and an equivalent molar amount of doxycycline is added to
the SPSS solution. Upon the addition of doxycycline hydrochloride
to the SPSS solution, a precipitate forms. Following the complete
addition, the lightly yellow-colored solid is filtered, washed with
deionized water and dried under vacuum to yield a light yellow
solid. The doxycycline is insoluble in deionized water but slowly
soluble in the phosphate buffered saline (PBS) aqueous media and
the dissolution measured by UV spectroscopy.
Example 11
Application of Material 18--Antimicrobial Effectiveness of 14%
Ag-PSS
[0112] 14% silver incorporation (substitution) of SPSS to yield a
mixed sodium (86%) silver (14%) compound was tested against
Acinetobacter baumanni, Staphylococcus Aureus, and Pseudomonas
aeruginosa. The MIC.sub.50 and MIC.sub.90 were determined against
all three pathogens. Clinical isolates of Pseudomonas aeruginosa,
Stephylococcus aureus, and Acinetobacter baumanni were grown
overnight and then diluted 1:50 in Mueller Hinton broth. Bacteria
were incubated overnight with different concentrations of Ag-PSS in
96-well format (n=4/sample). Light scattering (600 nm) was used to
measure growth. The data are represented in FIG. 16.
Example 12
Application of Material 18 14% PSS Silver Ointment in the Treatment
of Full Thickness Burns in the Rat (Comb Burn Model)
[0113] Full thickness burn injuries (three--2.0.times.1.0 cm, 0.5
cm interspace, i.e. the space between burn wounds) were made in the
back of an anesthetized animal. The burn wounds were dressed
immediately following the burn and redressed each day thereafter. A
three pronged brass template heated to 100.degree. C. was used to
generate three full thickness burns (1.times.2 cm) on each side of
the spine of Wistar rats (n=6). Each of the burns are separated by
two interspaces (0.5.times.2 cm). Escharotomies were created in the
burn sites and the wounds were dressed with an ointment containing
14% Ag-PSS. Contralateral (control) wounds were dressed with
vehicle ointment only. A. Representative wounds at day 5--Left
side: Ag-PSS ointment, right side: control ointment. B:
Quantitation of Preserved Interspace Tissue. The data detailed in
FIG. 17 reveals that the experimental dressing preserved
approximately twice that of the control ointment.
Example 13
Application of Material 18--Preparation of 50:50 Polylactide
Co-Glycolide (50:50 PLGA) SPSS Microspheres for Controlled Release
of SPSS Using a Water/Oil/Water Emulsion Technique
[0114] SPSS as purified in example 1 was dissolved into deionized
water to prepare about an 8% w/w solution. Separately, PLGA (50:50,
Lactel Polymers) dissolved into dichloromethane to yield a solution
of 5% solids. Two solutions are combined (with the total SPSS added
representing 8% of the PLGA mass and shaken to form an emulsion.
Separately, a 1% polyvinyl alcohol (PVA, 87-89% hydrolyzed)
solution is prepared (roughly 30 times the total volume of the
(PLGA) dichloromethane-aqueous (SPSS) emulsion. The emulsion is
added to the rapidly stirring PVA solution and the mixture slowly
heated to 45.degree. C. The mixture was allowed to stir at
45.degree. C. for 60 minutes and the mixture cooled and filtered.
The tan colored material in the filter was washed with D-mannitol
(2% w/w) and the spheres allowed to dry.
[0115] The microspheres were imaged on a hemocytometer grid and
found to be very regular in shape (FIG. 18) and size considering
that the experiment was carried out using standard laboratory
equipment with few controls in place. An evaluation of 25 randomly
chosen microspheres from the sample revealed that a mean spherical
surface area of 0.0259 mm.sup.2 (r=45 .mu.m), a median of 0.0258
mm.sup.2 (r=45 .mu.m), and a std. dev. of 0.0123 mm.sup.2. The
minimum and maximum surface areas in the sample were found to be
0.00859 (r=26 .mu.m) and 0.0492 mm.sup.2 (r=63 .mu.m) respectively.
These surface areas translate to diameters of ca. 90 .mu.m (mean),
52 .mu.m (min) and 126 .mu.m (max). Overall, these data detail a
uniform process.
[0116] The release of SPSS from the microspheres was followed via
UV spectroscopy and total release was shown to require about 5 days
in PBS at 37.degree. C.
Example 14
Preparation of Material 18 Silver Chondroitin-6-Sulfate (Ag-C6S)
Using Silver Acetate
[0117] Sodium chondroitin-6-sulfate (C6S, 1.5 mmole, shark
cartilage source, Seikagaku, Japan) was added to a small beaker as
received and 20 mL of deionized water was added. The mixture was
stirred until dissolved at room temperature and 1.5 mmole of silver
acetate (250.37 mg, Fluka) were added and allowed to stir until
completely dissolved. To the stirring solution an additional 1.5
mmole of sodium chondroitin sulfate was added and allowed to stir
until completely dissolved. The mixture was filtered, placed in a
lyophilization vessel, frozen and lyophilized to yield about 2.2
grams of 50% substituted Ag-C6S as a flocculent grayish solid. The
Ag-C6S was very soluble at 50 mol % and 100 mol % substitution. The
byproduct sodium acetate was not removed but may be dialyzed away
as needed.
[0118] Using a mass balance approach to determining the amount of
silver incorporated, silver was precipitated as silver chloride,
isolated, dried and weighed. Incorporation was determined to be
approximately 50 mol %.
Example 15
Preparation of Material 18 Mixed Sodium/Silver
Chondroitin-6-Sulfate (Na/Ag-C65) Using Silver Ion Modified Dowex
Mac-3 Resin
[0119] The Mac-3 resin (Dow Chemical) was stirred with 1.0 N NaOH
solution in deionized water for 1 hour and the resin filtered,
washed and re-suspended in deionized water. While stirring with a
mechanical stirrer, silver acetate (Fluka) was added to the mixture
and dissolution of the silver acetate followed. The silver-modified
Dowex MAC-3 was triturated with cold deionized water to remove any
insoluble matter and filtered under vacuum. The resin was rinsed
with isopropanol and allowed to dry in the absence of light.
[0120] Sodium chondroitin-6-sulfate (Seikagaku, Japan) was
dissolved into deionized water and mechanically stirred. A
10.times.(w/w) amount of the silver-modified Dowex MAC-3 was added
and the mix heated to 50.degree. C. and stirred for 2 hours. The
resin was filtered and washed with deionized water and the filtrate
transferred to a lyophilizer vessel and frozen. The frozen solution
was placed on a lyophilizer for 72 hours to yield a light gray
fibrous solid. Silver chloride was precipitated by the addition of
saline to a solution of the Ag-C6S. The AgCl was dried onto the
preweighed filter paper and weighed. Silver incorporation was
determined to be 29% by mass balance. More or less silver
incorporation can be achieved by varying the amount of
silver-modified Dowex MAC-3, for a given amount of starting C6S, up
or down respectively.
Example 16
Preparation of Material 18 Arginine Chondroitin-6-Sulfate
(C6S-Arg)
[0121] Sodium chondroitin-6-sulfate (C6S, 1.5 mmole, shark
cartilage source, Seikagaku, Japan) was added to a small beaker as
received and 20 mL of deionized water was added.
[0122] Separately, arginine base (1.5 mmole, Sigma) in deionized
water (10 mg/mL) is added to account for an equimolar quantity of
sodium chondroitin sulfate and the solution stirred for 1 hour at
room temperature. The solution is transferred into a lyophilizer
container and frozen. The frozen mass is placed under vacuum of the
lyophilizer and the solid polystyrene arginine sulfonate is
isolated as a flocculent off-white solid.
* * * * *