U.S. patent application number 10/131568 was filed with the patent office on 2003-01-30 for method of induction of apoptosis and inhibition of matrix metalloproteinases using antimicrobial metals.
Invention is credited to Burrell, Robert Edward, Lam, Kan, Wright, John Barrymore.
Application Number | 20030021854 10/131568 |
Document ID | / |
Family ID | 26963436 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030021854 |
Kind Code |
A1 |
Burrell, Robert Edward ; et
al. |
January 30, 2003 |
Method of induction of apoptosis and inhibition of matrix
metalloproteinases using antimicrobial metals
Abstract
The invention relates to a method to induce apoptosis and to
inhibit matrix metalloproteinases in a disease condition in a human
or animal by contacting hyperplastic tissue, tumor tissue, or a
cancerous lesion with one or more antimicrobial metals, preferably
formed with atomic disorder, and preferably in a nanocrystalline
form. The nanocrystalline antimicrobial metal of choice may be used
in the form of a nanocrystalline coating of one or more
antimicrobial metals, a nanocrystalline powder of one or more
antimicrobial metals, or a solution containing dissolved species
from a nanocrystalline powder or coating of one or more
antimicrobial metals.
Inventors: |
Burrell, Robert Edward;
(Sherwood Park, CA) ; Wright, John Barrymore; (San
Antonio, TX) ; Lam, Kan; (San Antonio, TX) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
5370 MANHATTAN CIRCLE
SUITE 201
BOULDER
CO
80303
US
|
Family ID: |
26963436 |
Appl. No.: |
10/131568 |
Filed: |
April 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10131568 |
Apr 23, 2002 |
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09840637 |
Apr 23, 2001 |
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60285884 |
Apr 23, 2001 |
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Current U.S.
Class: |
424/618 ;
424/649 |
Current CPC
Class: |
A61L 2300/102 20130101;
A61P 17/04 20180101; Y10S 514/902 20130101; Y10S 514/931 20130101;
Y10S 514/826 20130101; A61K 9/0075 20130101; A61L 2300/624
20130101; Y10S 514/83 20130101; Y10S 514/851 20130101; Y10S 514/965
20130101; Y10S 514/954 20130101; A61K 47/34 20130101; A61L 31/082
20130101; A61L 2300/404 20130101; A61L 2300/452 20130101; A61P 9/00
20180101; Y10S 514/912 20130101; Y10S 514/914 20130101; A61P 11/02
20180101; Y10S 514/829 20130101; Y10S 514/825 20130101; A61P 1/02
20180101; A61K 33/243 20190101; A61P 15/00 20180101; A61P 27/16
20180101; A61P 17/06 20180101; A61P 11/04 20180101; A61P 17/10
20180101; Y10S 514/887 20130101; A61K 9/7007 20130101; Y10S 514/953
20130101; Y10S 514/865 20130101; Y10T 428/31855 20150401; Y10S
514/968 20130101; A61L 27/30 20130101; A61P 17/02 20180101; A61P
43/00 20180101; Y10S 514/853 20130101; Y10S 514/90 20130101; Y10S
514/933 20130101; A61P 11/00 20180101; A61P 17/08 20180101; Y10S
514/901 20130101; Y10S 514/969 20130101; A61K 9/0019 20130101; Y10S
514/944 20130101; A61P 13/00 20180101; A61P 19/02 20180101; A61P
31/10 20180101; Y10S 514/861 20130101; A61K 33/40 20130101; A61L
29/10 20130101; A61P 17/00 20180101; A01N 59/16 20130101; A61K
33/38 20130101; A61L 15/46 20130101; A61P 29/00 20180101; Y10S
514/849 20130101; Y10S 514/958 20130101; A61L 2300/104 20130101;
A61P 19/00 20180101; Y10S 514/956 20130101; A61K 47/38 20130101;
Y10S 514/966 20130101; A61K 33/24 20130101; A61P 31/00 20180101;
Y10S 514/88 20130101; Y10S 514/886 20130101; Y10S 514/964 20130101;
A61K 33/242 20190101; A61P 31/04 20180101; Y10S 514/882 20130101;
Y10S 514/932 20130101; A61K 9/0024 20130101; Y10S 424/15 20130101;
Y10S 514/951 20130101; A61L 2300/606 20130101; A61P 13/08 20180101;
Y10S 514/934 20130101; Y10S 514/862 20130101; Y10S 514/925
20130101; Y10S 514/967 20130101; A61P 1/00 20180101; Y10S 514/864
20130101; Y10S 514/881 20130101; A61P 35/00 20180101; A61K 9/0078
20130101; A61K 9/14 20130101; A61L 2300/63 20130101; A61L 17/145
20130101; A61K 33/24 20130101; A61K 2300/00 20130101; A61K 33/38
20130101; A61K 2300/00 20130101; A61K 33/40 20130101; A61K 2300/00
20130101; A01N 59/16 20130101; A01N 25/04 20130101; A01N 25/10
20130101; A01N 25/12 20130101 |
Class at
Publication: |
424/618 ;
424/649 |
International
Class: |
A61K 033/38; A61K
033/24 |
Claims
We claim:
1. A method of inducing apoptosis in a disease condition in a human
or an animal, which comprises: contacting a hyperplastic tissue, a
tumor tissue, or a cancerous lesion with a therapeutically
effective amount of one or more antimicrobial metals in a
crystalline form to provide a localized pro-apoptotic effect,
wherein the one or more antimicrobial metals are characterized by
sufficient atomic disorder, such that the metal, in contact with an
alcohol or water-based electrolyte, releases atoms, ions,
molecules, or clusters of at least one antimicrobial metal at a
concentration sufficient to provide a localized pro-apoptotic
effect.
2. The method as set forth in claim 1, wherein the one or more
antimicrobial metals further inhibit one or more matrix
metalloproteinases or modulate the production of the one or more
matrix metalloproteinases.
3. The method as set forth in claim 2, wherein the antimicrobial
metal is selected from the group consisting of silver, gold,
platinum and palladium.
4. The method as set forth in claim 3, wherein the antimicrobial
metal is nanocrystalline and is formed with sufficient atomic
disorder such that, in contact with an alcohol or water based
electrolyte, the antimicrobial metal releases ions, atoms,
molecules or clusters of the antimicrobial metal on a sustainable
basis.
5. The method as set forth in claim 4, wherein the tumor tissue is
malignant.
6. The method as set forth in claim 4, wherein the tumor tissue or
the cancerous lesion is skin cancer.
7. The method as set forth in claim 6, wherein the skin cancer is
melanoma.
8. The method as set forth in claim 4, wherein the hyperplastic
tissue, the tumor tissue, or the cancerous lesion is in the
lung.
9. The method as set forth in claim 4, wherein the hyperplastic
tissue, the tumor tissue, or the cancerous lesion is in the
liver.
10. The method as set forth in claim 4, wherein the one or more
matrix metalloproteinases are selected from the group consisting of
collagenases, gelatinases, stromelysins, and stromelysin-like
matrix metalloproteinases.
11. The method as set forth in claim 4, wherein the antimicrobial
metal is nanocrystalline silver.
12. The method as set forth in claim 4, wherein the antimicrobial
metal is silver, formed as a composite with oxygen.
13. The method as set forth in claim 4, wherein the antimicrobial
metal is nanocrystalline gold.
14. The method as set forth in claim 4, wherein the antimicrobial
metal is gold, formed as a composite with oxygen.
15. The method as set forth in claim 4, wherein the antimicrobial
metal is nanocrystalline platinum.
16. The method as set forth in claim 4, wherein the antimicrobial
metal is platinum, formed as a composite with oxygen.
17. The method as set forth in claim 4, wherein the one or more
antimicrobial metals are provided as a coating on, or filler in, a
dressing or a hydrated dressing, or in a pharmaceutical composition
with one or more pharmaceutically and dermatogically acceptable
carriers, diluents, or excipients suitable for topical
application.
18. The method as set forth in claim 17, wherein the pharmaceutical
composition includes a nanocrystalline powder of one or more
antimicrobial metals, or a solution containing dissolved species
from a nanocrystalline powder or coating of one or more
antimicrobial metals.
19. The method as set forth in claim 18, wherein the pharmaceutical
composition is a gel, cream, lotion, paste, or ointment containing
the antimicrobial metal powder in an amount of 0.01-10% by weight,
or a liquid formulated as a topical solution, spray, mist, drops,
infusion or instillation containing 0.001-10% by weight of the
antimicrobial metal.
20. The method as set forth in claim 19, wherein the antimicrobial
metal is nanocrystalline silver.
21. The method as set forth in claim 19, wherein the antimicrobial
metal is silver, formed as a composite with oxygen.
22. The method as set forth in claim 19, wherein the antimicrobial
metal is nanocrystalline gold.
23. The method as set forth in claim 19, wherein the antimicrobial
metal is gold, formed as a composite with oxygen.
24. The method as set forth in claim 19, wherein the antimicrobial
metal is nanocrystalline platinum.
25. The method as set forth in claim 19, wherein the antimicrobial
metal is platinum, formed as a composite with oxygen.
26. The method of claim 18, wherein the antimicrobial metal is in a
powder form and is delivered directly to a locus of the
hyperplastic tissue, the tumor tissue, or the cancerous lesion.
27. The method of claim 26, wherein the powder is sized with
particulates no larger than 2 .mu.m.
28. The method of claim 27, wherein the powder is sized with
particulates no larger than 1 .mu.m.
29. The method of claim 28, wherein the antimicrobial metal is
nanocrystalline silver.
30. The method of claim 28, wherein the antimicrobial metal is
nanocrystalline silver, formed as a composite with oxygen.
31. The method as set forth in claim 28, wherein the antimicrobial
metal is nanocrystalline gold.
32. The method as set forth in claim 28, wherein the antimicrobial
metal is gold, formed as a composite with oxygen.
33. The method as set forth in claim 28, wherein the antimicrobial
metal is nanocrystalline platinum.
34. The method as set forth in claim 28, wherein the antimicrobial
metal is platinum, formed as a composite with oxygen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of co-pending
U.S. patent application Ser. No. 09/840,637 filed Apr. 23, 2001.
This application also claims priority from U.S. Provisional Patent
Application No. 60/285,884, filed Apr. 23, 2001. To the extent that
they are consistent herewith, the aforementioned applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention provides a method to induce apoptosis
and to inhibit matrix metalloproteinases in a disease condition in
a human or animal by contacting hyperplastic tissue, tumor tissue,
or a cancerous lesion with antimicrobial metals; methods for the
preparation of the antimicrobial metals; pharmaceutical
compositions comprising same; and uses thereof.
BACKGROUND OF THE INVENTION
[0003] Apoptosis and matrix metalloproteinases (MMPs) have been
implicated in many pathological diseases, such as cancer. Apoptosis
or programmed cell death is a system which removes unnecessary,
aged, or damaged cells. Apoptosis occurs during development to
ensure proper formation of the fingers and toes in the fetus and of
synapses between neurons in the brain; and serves to eradicate
virus-infected cells, unnecessary immune cells, cells with DNA
damage, and cancer cells. Decreased apoptosis has been implicated
in developmental malformation, cancer and autoimmune disease, while
enhanced apoptosis has been associated with degenerative diseases
such as Alzheimer's disease, AIDS dementia, and Huntington's
disease.
[0004] Caspases are a family of proteases, including initiator
(activator) and effector (executioner) protease types, which
regulate proteolysis during apoptosis. Apoptosis is triggered by
signals which are either internal or external to the cell. In a
healthy cell, the protein Bcl-2 is expressed on the surface and is
bound to the protein Apaf-1. Internal damage in the cell causes
Bcl-2 to release Apaf-1 and to no longer keep cytochrome c from
leaking out of the mitochondria. The released cytochrome c and
Apaf-1 bind to molecules of caspase 9. The resulting complex of
cytochrome c, Apaf-1, caspase 9, and ATP aggregates in the cytosol.
In cleaving a protein, caspase 9 activates other caspases, leading
to digestion of structural proteins on the cytoplasm, degradation
of chromosomal DNA, and phagocytosis of the cell. With regard to
external signals (e.g., as in cytotoxic T cells inducing apoptosis
in a virus-infected cell), binding of a death activator (FasL and
TNF) to the Fas and TNF receptor proteins on the surface of a
target cell activates caspase 8, which activates other caspases
leading to phagocytosis of the target cell.
[0005] Cancer cells may have mechanisms to avoid apoptosis. For
example, some B-cell leukemias and lymphomas express high levels of
Bcl-2, thus blocking apoptotic signals they may receive. Melanoma
cells avoid apoptosis by inhibiting the expression of the gene
which encodes Apaf-1. Lung and colon cancer cells secrete a
molecule which binds to FasL, inhibiting its binding to Fas.
Currently, radiation and standard chemotherapeutic drugs are used
to induce apoptosis in some types of cancer cells; however, with
such treatments having undesirable side effects and some cancers
being resistant to such therapies, there exists a need to provide
an effective approach which lacks such side effects, and
demonstrates minimal interference with normal cell function.
[0006] Cancer tissue may also be treated with inhibitors of MMPs.
MMPs have been associated with diseases associated with the
excessive degradation of extracellular matrix, such as tumor
invasion and metastasis, arthritic diseases (rheumatoid arthritis
and osteoarthritis), bone resorptive diseases (such as
osteoporosis), enhanced collagen destruction associated with
diabetes, periodontal disease, corneal ulceration, and ulceration
of the skin. MMPs are a family of at least 20 enzymes (proteases)
which are involved in the degradation of connective tissues, such
as collagen, elastins, fibronectin, laminin, and other components
of the extracellular matrix. Such components are present in the
linings of joints, interstitial connective tissues, basement
membranes, and cartilage. MMPs are present in various cell types
which reside in or are associated with connective tissue, such as
fibroblasts, monocytes, macrophages, endothelial cells, and
invasive or metastatic tumor cells. Expression of MMPs may be
induced by a variety of factors, including growth factors,
cytokines, chemical agents, physical stress, cell-matrix
interactions, cell-cell interactions and oncogenic transformation.
The MMP family includes collagenases, gelatinases, stromelysins and
stromelysin-like proteases as follows:
[0007] i) Collagenases include MMP-1 (interstitial), MMP-8
(neutrophil), and MMP-13, which catalyze the initial degradation of
native collagen types I, II, III and VII. Collagen is an essential
component of the extracellular matrix of tissues such as cartilage,
bone, tendon and skin. MMP-13 is associated with osteoarthritis,
ulcers and malignant tumor invasion.
[0008] ii) Gelatinases include MMP-2 (secreted by fibroblasts and a
wide variety of other cell types) and MMP-9 (released by
mononuclear phagocytes, neutrophils, corneal epithelial cells,
tumor cells, cytotrophoblasts and keratinocytes). The gelatinases
degrade gelatins (denatured collagens) and collagen type IV
(basement membrane).
[0009] iii) Stromelysins include MMP-3, MMP-10 and MMP-11. MMP-3
and MMP-10 are expressed by epithelial cells and carcinomas, and
degrade a broad range of extracellular matrix substrates, including
laminin, fibronectin, proteoglycans, and collagen types IV and IX.
MMP-11 is expressed by fibroblasts, and cleaves serine protease
inhibitors.
[0010] iv) Stromelysin-like MMPs include MMP-12 and MMP-7. MMP-12
is expressed by macrophages and stromal cells, and degrades
elastin. MMP-7 or matrilysin is expressed by mononuclear phagocytes
and sporadically in tumors, and degrades a wide range of matrix
substrates including proteoglycans, gelatins, fibronectin, elastin,
and laminin.
[0011] Inhibitors of MMPs may serve as potential therapeutic
agents. However, certain hydroxamic acids and derivatives thereof
which have been suggested as collagenase inhibitors appear to be
potentially toxic due to the hydroxamic moeity. There thus exists a
need for a non-toxic, effective treatment to inhibit MMPs.
[0012] A treatment which provides a two-fold approach, namely
induction of apoptosis of particular cells and inhibition of MMPs
to eradicate cancer cells, and to reduce tissue damage contributing
to tumor invasion and metastasis, may be desirable. Currently,
cis-platin and its variations have been used as pro-apoptotic,
anti-cancer agents, since the platinum complex attacks the DNA of
tumor cells, thus disrupting RNA synthesis. However, cis-platin can
pass through the blood to the kidneys and be immediately excreted;
bind to proteins and be rendered inactive before reaching the tumor
cells; or attack cells which are not tumors. Since acute toxicity
may occur with long term use, cis-platin is restricted to short
term, high doses. Sustained release to maintain treatment efficacy
and non-toxicity in an anti-tumor agent are desirable.
[0013] While the patent literature reports that silver metal or
silver salts such as silver nitrate, silver halides or silver
sulphadiazine are among useful antibacterial agents, they have not,
to the inventors' knowledge, been known or adopted to induce
apoptosis and/or inhibit MMPs. For tumor tissue and cancerous
lesions, there may be benefits associated with enhanced cellular
apoptosis and reduced levels of MMPs; for example, induction of
apoptosis may aid in tumor suppression by eradicating tumor cells
and by reducing the chance of tumor invasion and metastasis through
inhibition of MMPs. In addition to affecting cancer cells, such
treatment may be beneficial in eradicating excessive, normal cells,
as in hyperplastic tissue in which abnormal multiplication or
increase in the number of cells in a normal arrangement in normal
tissue or an organ has occurred.
SUMMARY OF THE INVENTION
[0014] The inventors have established that antimicrobial metals
formed with atomic disorder and preferably in a nanocrystalline
form, induce apoptosis and inhibit MMPs. Treatment of hyperplastic
tissue, tumor tissue, or cancerous lesions with antimicrobial
metals having pro-apoptotic and anti-MMP effects is thereby
indicated. This new treatment will have the dual advantages of
being both pro-apoptotic and an inhibitor of MMPs. Methods and
formulations of this invention have application to both humans and
animals.
[0015] The antimicrobial metals selected from one or more of
silver, gold, platinum and palladium, are formed with atomic
disorder, such that ions, clusters, atoms or molecules of the
metals are released at a concentration sufficient to provide
localized pro-apoptotic and anti-MMP effects. Most preferably, the
antimicrobial metals are in a nanocrystalline form, and include
sufficient atomic disorder to provide such effects on a sustainable
basis.
[0016] Without being bound by the same, it is believed that the
nanocrystalline antimicrobial metals formed with atomic disorder
are capable of releasing highly active clusters of the
antimicrobial metal (example clusters of Ag.sup.0 or
Ag.sup.+/Ag.sup.0), which are responsible for the surprisingly
enhanced antimicrobial activity and the surprising presence of the
anti-inflammatory activity in the treatment of mucosal membranes,
compared with other known antimicrobials such as silver salts (ex.
silver nitrate), silver zeolites which release only Ag.sup.+, or
silver metal and silver oxide which have only minor solubility.
Clusters are known to be small groups of atoms, ions or the like,
as described by R. P. Andres et al., "Research Opportunities on
Cluster and Cluster-Assembled Materials", J. Mater. Res. Vol 4, No
3, 1989, p. 704. For silver, clusters are believed to contain less
than the 14 atoms of a normal face centered cubic crystal lattice
form of silver.
[0017] The crystalline forms of these antimicrobial metals may be
used in, or formulated from, any of the following formats:
[0018] i) coatings of the antimicrobial metals on medical grade
substrates, for example, dressings, packings, meshes, films,
filtering surfaces, filters, infusers, fibres, containers or vials,
from materials composed of for example polyethylene, high density
polyethylene, polyvinylchloride, latex, silicone, cotton, rayon,
polyester, nylon, cellulose, acetate, carboxymethylcellulose,
alginate, chitin, chitosan and hydrofibres;
[0019] ii) powders, preferably prepared as nanocrystalline powders
of the antimicrobial metals, or as nanocrystalline coatings of the
antimicrobial metals on biocompatible substrates in powder form,
preferably on bioabsorbable and/or hygroscopic substrates such
as:
[0020] Synthetic Bioabsorbable Polymers: for example
polyesters/polyactones such as polymers of polyglycolic acid,
glycolide, lactic acid, lactide, dioxanone, trimethylene carbonate
etc., polyanhydrides, polyesteramides, polyortheoesters,
polyphosphazenes, and copolymers of these and related polymers or
monomers, or
[0021] Naturally Derived Polymers:
[0022] Proteins: albumin, fibrin, collagen, elastin;
[0023] Polysaccharides: chitosan, alginates, hyaluronic acid;
and
[0024] Biosynthetic Polyesters: 3-hydroxybutyrate polymers;
[0025] iii) occlusions or hydrated dressings, in which the dressing
is impregnated with a powder or solution of the antimicrobial
metals, or is used with a topical formulation of the antimicrobial
metals, with such dressings for example as hydrocolloids,
hydrogels, polyethylene, polyurethane, polyvinylidine, siloxane or
silicone dressings;
[0026] iv) gels, formulated with nanocrystalline powders or
solutions of the antimicrobial metals with such materials as
carboxymethylcellulose, alginate, chitin, chitosan and hydrofibres,
together with such ingredients as preservatives, pectin and
viscosity enhancers;
[0027] v) creams, lotions, pastes and ointments formulated with
nanocrystalline powders or solutions of the antimicrobial metals,
for example as emulsions or with drying emollients; and
[0028] vi) liquids, formulated as solutions by dissolving
nanocrystalline coatings or powders of the antimicrobial metals,
for example as topical solutions, aerosols, mists, sprays, drops,
infusions and instillation solutions for body cavities such as the
bladder, prostate, lung, and liver.
[0029] Solutions of the antimicrobial metals lose some activity
with aging and are thus either stabilized or generated fresh for
administration. Alternatively, the antimicrobial metals may be
packaged for convenient solution generation, for instance in a
pervious membrane such as a tea bag type infuser. Other two part or
two phase systems may be used in which the nanocrystalline metal is
separated from the water or electrolyte solvent, for example in kit
form, with the antimicrobial metal being provided in dissolving
capsules, as a coating on the inside of vials or containers, on
substrates such as dressing, separated by a membrane which can be
perforated, or in a separate container from the carrier, in a tea
bag-type infuser etc.
[0030] In the above formats, the nanocrystalline antimicrobial
metals are formulated from nanocrystalline coatings or
nanocrystalline powders of the nanocrystalline antimicrobial
metals, or from solutions prepared by dissolving the
nanocrystalline coatings or powders therein. The formulations
include a therapeutically effective amount of the coatings or
powders, and most preferably, the following amounts:
1 For coatings: 150-3000 nm thick coatings for substrates, or
thicker for forming powders (such coatings can be used to generate
0.001 to 10% by weight solutions) For gels, creams 0.01-30% by
weight, more preferably 0.01-10% by etc.: weight and most
preferably 0.1-5% by weight of the antimicrobial or noble metal
powder For liquids: 0.001-10% by weight, more preferably 0.01 to 5%
by weight and most preferably 0.1 to 1% by weight of the
antimicrobial or noble metal (generated from any format, including
coatings, flakes, powders).
[0031] Concentrations of the antimicrobial or noble metal species
in solution will vary according to the application, formulation and
subject, but will generally range from 1-5000 .mu.g/ml, more
preferably 20-3000 .mu.g/ml, more preferably 40-800 .mu.g/ml, and
most preferably 50-500 .mu.g/ml.
[0032] Nanocrystalline coatings of the antimicrobial metals are
most preferably deposited onto substrates such as dressings, for
example one or more layers of medical dressing materials which can
be laminated with uncoated layers of medical dressing materials.
The coatings can be prepared by known techniques for preparing
nanocrystalline coatings, but are most preferably prepared by
physical vapour deposition under conditions which create atomic
disorder. The nanocrystalline coatings may be prepared to create an
interference colour so as to provide an indicator, as described in
prior patent application WO 98/41095, published Sep. 24, 1998, and
naming inventors R. E. Burrell and R. J. Precht.
[0033] Nanocrystalline powders of the antimicrobial metals may be
prepared as nanocrystalline coatings, preferably of the above
thickness, on powdered substrates such as chitin, or may be
prepared as nanocrystalline coatings on a substrate such as a
silicon wafer, and then scraped off as a nanocrystalline powder.
Alternatively, fine grained or nanocrystalline powders of the
antimicrobial metals may be cold worked to impart atomic disorder,
as taught in prior patent applications WO 93/23092, published Nov.
25, 1993, and WO 95/13704, published May 26, 1995, both of which
name Burrell et al., as inventors.
[0034] Thus, the invention broadly provides a method of inducing
apoptosis in a disease condition in a human or an animal, which
comprises:
[0035] contacting a hyperplastic tissue, a tumor tissue, or a
cancerous lesion with a therapeutically effective amount of the
antimicrobial metals in a crystalline form to provide a localized
pro-apoptotic effect, wherein the antimicrobial metals are
characterized by sufficient atomic disorder, such that the metal,
in contact with an alcohol or water-based electrolyte, releases
atoms, ions, molecules, or clusters of at least one antimicrobial
metal at a concentration sufficient to provide a localized
pro-apoptotic effect. The antimicrobial metals further inhibit one
or more matrix metalloproteinases or modulate the production of the
one or more matrix metalloproteinases.
[0036] As used herein and in the claims, the terms and phrases set
out below have the meanings which follow.
[0037] "Apoptosis" means programmed cell death which removes
unnecessary, aged, or damaged cells.
[0038] "Pro-apoptotic effect" means that atoms, ions, molecules or
clusters of the antimicrobial or noble metal are released to
contact a human or animal cell in a concentration sufficient to
induce apoptosis.
[0039] "Anti-MMP effect" means that atoms, ions, molecules or
clusters of the antimicrobial or noble metal are released to
contact a human or animal cell in a concentration sufficient to
inhibit one or more matrix metalloproteinases or modulate the
production of one or more matrix metalloproteinases.
[0040] "Matrix metalloproteinases" is meant to refer to any
protease of the family of MMPs which are involved in the
degradation of connective tissues, such as collagen, elastins,
fibronectin, laminin, and other components of the extracellular
matrix, and associated with conditions in which excessive
degradation of extracellular matrix occurs, such as tumor invasion
and metastasis.
[0041] "Gelatinases" is meant to refer to MMP-2 (secreted by
fibroblasts and a wide variety of other cell types) and MMP-9
(released by mononuclear phagocytes, neutrophils, corneal
epithelial cells, tumor cells, cytotrophoblasts and keratinocytes).
The gelatinases degrade gelatins (denatured collagens) and collagen
type IV (basement membrane).
[0042] "Fibroblast" means a connective tissue cell which is a
flat-elongated cell with cytoplasmic processes at each end having a
flat, oval vesicular nucleus. Fibroblasts which differentiate into
chondroblasts, collagenoblasts, and osteoblasts form the fibrous
tissues in the body, tendons, aponeuroses, supporting and binding
tissues of all sorts.
[0043] "Neutrophil" means a granulocyte which arises from the bone
marrow and is fully mature when it is released into the
circulation. It functions in cellular defense primarily in
phagocytosis.
[0044] "Polymorphonuclear leukocyte" is meant to refer to
neutrophils. The name derives from the multiple lobes of the mature
neutrophil's nucleus.
[0045] "Hyperplasia" means abnormal multiplication or increase in
the number of cells in a normal arrangement in normal tissue or an
organ.
[0046] "Tumor" means a spontaneous growth of tissue in which
multiplication of cells is abnormal, uncontrolled and progressive.
A tumor serves no useful function and grows at the expense of the
healthy organism.
[0047] "Malignant" means a tumor which has the properties of
anaplasia, invasion, and metastasis.
[0048] "Benign" means a tumor which is not malignant, recurrent,
invasive, or progressive. A tumor or growth which is benign is
noncancerous.
[0049] "Cancerous lesion" means a tumor of epithelial tissue, or
malignant, new growth made up of epithelial cells tending to
infiltrate surrounding tissues and to give rise to metastases. As
used in reference to the skin, a cancerous lesion means a lesion
which may be a result of a primary cancer, or a metastasis to the
site from a local tumor or from a tumor in a distant site. It may
take the form of a cavity, an open area on the surface of the skin,
skin nodules, or a nodular growth extending from the surface of the
skin.
[0050] "Metastasis" means the movement or spreading of cancer cells
from one organ or tissue to another via the bloodstream, or lymph
system.
[0051] "Metal" or "metals" includes one or more metals whether in
the form of substantially pure metals, alloys or compounds such as
oxides, nitrides, borides, sulphides, halides or hydrides.
[0052] "Antimicrobial metals" are silver, gold, platinum,
palladium, iridium, zinc, copper, tin, antimony, bismuth, or
mixtures of these metals with same or other metals, silver, gold,
platinum and palladium being preferred, and silver being most
preferred.
[0053] "Noble metals" are silver, gold, platinum and palladium, or
mixtures of such metals with same or other metals, with silver
metal being the most preferred.
[0054] "Antimicrobial effect" means that atoms, ions, molecules or
clusters of the antimicrobial or noble metal are released into the
electrolyte which the coating contacts in concentration sufficient
to inhibit microbial growth on and in the vicinity of the coating.
The most common methods of measuring an antimicrobial effect are a
zone of inhibition test (which indicates an inhibitory effect,
whether microbiostatic or microbiocidal) or a logarithmic reduction
test (which indicates a microbiocidal effect). In a zone of
inhibition test (ZOI) the material to be tested is placed on a
bacterial lawn (or a lawn of other microbial species) and
incubated. A relatively small or no ZOI (ex. less than 1 nun)
indicates a non-useful antimicrobial effect, while a larger ZOI
(ex. greater than 5 mm) indicates a highly useful antimicrobial
effect. The ZOI is generally reported as a corrected zone of
inhibition (CZOI), wherein the size of the test sample is
subtracted from the zone. A logarithmic reduction test in viable
bacteria is a quantitative measure of the efficacy of an
antibacterial treatment; for example, a 5 log reduction means a
reduction in the number of microorganisms by 100,000-fold (e.g., if
a product contained 100,000 pertinent microorganisms, a 5 log
reduction would reduce the number of pertinent microorganisms to
1). Generally, a 3 log reduction represents a bactericidal effect.
The logarithmic reduction test involves combining the inoculum with
the test treatment, incubating the inoculum with the test
treatment, recovering the bacteria or other microbial species, and
enumerating the bacteria or other microbial species using serial
dilutions. Examples of these tests are set out in the examples
which follow.
[0055] "Biocompatible" means generating no significant undesirable
host response for the intended utility. Most preferably,
biocompatible materials are non-toxic for the intended utility.
Thus, for human utility, biocompatible is most preferably non-toxic
to humans or human tissues.
[0056] "Sustained release" or "sustainable basis" are used to
define release of atoms, molecules, ions or clusters of an
antimicrobial metal that continues over time measured in hours or
days, and thus distinguishes release of such metal species from the
bulk metal, which release such species at a rate and concentration
which is too low to be therapeutically effective, and from highly
soluble salts of antimicrobial metals such as silver nitrate, which
releases silver ions virtually instantly, but not continuously, in
contact with an alcohol or electrolyte.
[0057] "Atomic disorder" includes high concentrations one or more
of: point defects in a crystal lattice, vacancies, line defects
such as dislocations, interstitial atoms, amorphous regions, grain
and sub grain boundaries and the like relative to its normal
ordered crystalline state. Atomic disorder leads to irregularities
in surface topography and inhomogeneities in the structure on a
nanometer scale.
[0058] "Normal ordered crystalline state" means the crystallinity
normally found in bulk metal materials, alloys or compounds formed
as cast, wrought or plated metal products. Such materials contain
only low concentrations of such atomic defects as vacancies, grain
boundaries and dislocations.
[0059] "Diffusion", when used to describe conditions which limit
diffusion in processes to create and retain atomic disorder, i.e.
which freeze-in atomic disorder, means diffusion of atoms (adatom
diffusion) and/or molecules on the surface or in the matrix of the
material being formed.
[0060] "Alcohol or water-based electrolyte" is meant to include any
alcohol or water-based electrolyte that the antimicrobial materials
of the present invention might contact in order to activate (i.e.
cause the release of species of the antimicrobial metal) into same.
The term is meant to include alcohols (short chain (C.sub.6 or
less) and preferably C.sub.4 or less), water, gels, fluids,
solvents, and tissues containing, secreting, or exuding water or
water-based electrolytes, including body fluids (for example blood,
urine, or saliva), and body tissue (for example skin).
[0061] "Bioabsorbable" as used herein in association includes
substrates which are useful in medical devices, that is which are
biocompatible, and which are capable of bioabsorption in period of
time ranging from hours to years, depending on the particular
application.
[0062] "Bioabsorption" means the disappearance of materials from
their initial application site in the body (human or mammalian)
with or without degradation of the dispersed polymer molecules.
[0063] "Colour change" is meant to include changes of intensity of
light under monochromatic light as well as changes of hue from
white light containing more than one wavelength.
[0064] An "interference colour" is produced when light impinges on
two or more partly reflective surfaces separated by a distance
which bears the right relationship to the wavelength of the light
to be removed by destructive interference.
[0065] "Partly reflective" when used to describe the base or top
layer materials, means that the material has a surface which
reflects a portion of incident light, but which also transmits a
portion of the incident light. Reflection occurs when a ray of
incoming light encounters a boundary or interface characterized by
a change in refractive index between two media. For the top layer
of the antimicrobial materials of this invention, that interface is
with air. For the base layer, the interface is with the top layer.
The reflectance of the base and top layers is balanced so as to
generate an interference colour.
[0066] "Partly light transmissive" when used to describe a thin
film of the top layer material means that the thin film is capable
of transmitting at least a portion of incident visible light
through the thin film.
[0067] "Detectable" when used to describe a colour change means an
observable shift in the dominant wavelength of the reflected light,
whether the change is detected by instrument, such as a
spectrophotometer, or by the human eye. The dominant wavelength is
the wavelength responsible for the colour being observed.
[0068] "Cold working" as used herein indicates that the material
has been mechanically worked such as by milling, grinding,
hammering, mortar and pestle or compressing, at temperatures lower
than the recrystallization temperature of the material. This
ensures that atomic disorder imparted through working is retained
in the material.
[0069] "Pharmaceutically- or therapeutically-acceptable" is used
herein to denote a substance which does not significantly interfere
with the effectiveness or the biological activity of the active
ingredients (pro-apoptotic and anti-MMP properties) and which has
an acceptable toxic profile for the host to which it is
administered.
[0070] "Therapeutically effective amount" is used herein to denote
any amount of a formulation of the antimicrobial or noble metals
which will exhibit either or both of a pro-apoptotic and anti-MMP
effect, when applied to the affected area. A single application of
the formulations of the present invention may be sufficient, or the
formulations may be applied repeatedly over a period of time, such
as several times a day for a period of days or weeks. The amount of
the active ingredient, that is the antimicrobial or noble metal in
the form of a coating, powder or dissolved in liquid solution, will
vary with the conditions being treated, the stage of advancement of
the condition, the age and type of host, and the type and
concentration of the formulation being applied. Appropriate amounts
in any given instance will be readily apparent to those skilled in
the art or capable of determination by routine experimentation.
[0071] "Carrier" means a suitable vehicle including one or more
solid, semisolid or liquid diluents, excipients or encapsulating
substances which are suitable for administration to the area.
[0072] "Nanocrystalline" is used herein to denote single-phase or
multi-phase polycrystals, the grain size of which is less than
about 100, more preferably <50, even more preferably <40,
even more preferably <30, and most preferably <25 nanometers
in at least one dimension. The term, as applied to the crystallite
or grain size in the crystal lattice of coatings, powders or flakes
of the antimicrobial or noble metals, is not meant to restrict the
particle size of the materials when used in a powder form.
[0073] "Powder" is used herein to include particulates of the
antimicrobial or noble metals ranging from nanocrystalline (less
than 100 nm) to submicron sized powders up to flakes.
[0074] Preferably, powders of the antimicrobial or noble metals
used in the present invention are sized at less than 100 .mu.m, and
more preferably less than 40 .mu.m, and most preferably less than
10 .mu.m.
[0075] "Grain size", or "crystallite size" means the size of the
largest dimension of the crystals in the antimicrobial metal
coating or powder.
[0076] "Hydrocolloid" means a synthetically prepared or naturally
occurring polymer capable of forming a thickened gel in the
presence of water and polyols (swelling agent). The swelling agent
must be capable of swelling the hydrocolloid chosen in order to
form the gel phase.
[0077] "Hydrogels" means a hydrocolloid swollen with water or
another hydrophilic liquid which is used for absorbing or retaining
moisture or water.
[0078] "Gel" means a composition that is of suitable viscosity for
such purposes, e.g., a composition that is of a viscosity that
enables it to be applied and remain on the skin.
[0079] When used herein and in the claims, the term
"nanocrystalline antimicrobial metal" and similar terminology, such
as "nanocrystalline coatings or powders" is meant to refer to
antimicrobial metals formed with atomic disorder and having a
nanocrystalline grain size.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] FIG. 1 is a graph showing MMP activity of incision fluids
recovered from incisions dressed with silver-coated dressing (AB),
silver nitrate moistened gauze (SN), silver-coated AgHDPE, and
control dressing on each post-incision day.
[0081] FIG. 2 is a graph showing total protease activity of
incision fluids recovered from the silver-coated dressing (AB),
silver nitrate moistened gauze (SN dressing), silver-coated AgHDPE,
and control dressing over a duration of 5 days.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0082] Crystalline forms of the antimicrobial metals Ag, Au, Pt,
and Pd can be prepared as coatings or powders, or as solutions
prepared by dissolving the coatings or powders. The crystalline
coatings or powders are most preferably formed with atomic disorder
in accordance with the techniques published in the prior patent
applications of Burrell et al, see for example WO 93/23092,
published Nov. 25, 1993, WO 95/13704, published May 26, 1995 and WO
98/41095, published Sep. 24, 1998.
[0083] Pharmaceutical formulations for treatment of hyperplastic
tissue, tumor tissue, or cancerous lesions utilize the
antimicrobial or noble metals in powder, coatings or solution form.
Preparation of the antimicrobial or noble metals as powders or
coatings is set out below in section A, format for formulations are
set forth in section B, sterilization in section C, and
formulating, dosages and treatment are set forth in section D.
[0084] A. Preparation of Crystalline Forms of the Antimicrobial
Metals with Atomic Disorder
[0085] a) Antimicrobial Metal Coatings on Dressings or Other
Substrates
[0086] Dressings or other substrates such as packings, vials,
fabric, or fibres etc. may be coated with antimicrobial coatings
formed with atomic disorder. The description below is directed to
coatings on dressing materials, but the coating techniques are
equally applicable to coating other substrates. Dressings coated
with antimicrobial metals in accordance with the invention include
one or more layers of medical dressing materials. Multiple layers
may be laminated together by known means such as low temperature
thermal fusing, stitching or, most preferably, ultrasonic
welding.
[0087] The dressing may be formed to include an occlusive or
semi-occlusive layer such as an adhesive tape or polyurethane film
in order to secure the dressing in place, and retain moisture for
release of ions, atoms, molecules or clusters of the antimicrobial
metal (hereinafter antimicrobial metal species).
[0088] The preferred and alternate compositions of the dressing
layers, together with the preferred nanocrystalline antimicrobial
metal coatings, are set out in further detail below.
[0089] i) Dressing Materials
[0090] The dressing is formed of a perforated, preferably
non-adherent material which allows for fluids to penetrate or
diffuse there through in either or both directions. The perforated
material may be formed of a woven or non-woven, non-woven being
preferred, fabric such as cotton, gauze, a polymeric net or mesh
such as polyethylene, nylon, polypropylene or polyester, an
elastomer such as polyurethane or polybutadiene elastomers, or a
foam such as open cell polyurethane foam. Exemplary perforated,
non-adherent materials useful for the dressing include non-woven
meshes such as DELNET.TM. P530, which is a non-woven veil formed of
high density polyethylene using extrusion, embossing and
orientation processes, produced by Applied Extrusion Technologies,
Inc. of Middletown, Del., USA. This same product is available as
Exu-Dry CONFORMANT 2.TM. Wound Veil, from Frass Survival Systems,
Inc., Bronx, N.Y., USA as a subset of that company's Wound Dressing
Roll (Non-Adherent) products. Other useful non-woven meshes include
CARELLE.TM. or NYLON 90.TM., available from Carolina Formed Fabrics
Corp., N-TERFACE.TM., available from Winfield Laboratories, Inc.,
of Richardson, Tex., USA. Exemplary woven meshes may be formed from
fibreglass or acetate, or cotton gauze. An exemplary hydrophilic
polyurethane foam is HYPOL.TM., available from W.R. Grace &
Co., New York, N.Y., USA.
[0091] For ease of ultrasonic welding for lamination, at least one
dressing layer is preferably formed from a polymeric material which
is amenable to ultrasonic welding, that is which will melt on the
application of localized heat and then fuse multiple layers
together on cooling.
[0092] If desired, a second, absorbent layer is formed from an
absorbent material for holding sufficient moisture next to the
affected area in order to activate the antimicrobial metal coating,
that is to cause release of ions, molecules, atoms or clusters of
the antimicrobial metal in order to cause pro-apoptotic and
anti-MMP effects. Preferably, the absorbent material is an
absorbent needle punched non-woven rayon/polyester core such as
SONTARA.TM. 8411, a 70/30 rayon/polyester blend commercially
available from Dupont Canada, Mississauga, Ontario, Canada. This
product is sold by National Patent Medical as an American White
Cross sterile gauze pad. However, other suitable absorbent
materials include woven or non-woven materials, non-woven being
preferred made from fibers such as rayon, polyester,
rayon/polyester, polyester/cotton, cotton and cellulosic fibers.
Exemplary are creped cellulose wadding, an air felt of air laid
pulp fibers, cotton, gauze, and other well known absorbent
materials suitable for medical dressings.
[0093] A third layer of the dressing, if used, is preferably formed
of perforated, non-adherent material such as used in the first
layer. This allows moisture penetration as sterile water and the
like are added in order to activate the antimicrobial metal
coating.
[0094] Additional layers may be included between or above the
first, second and third layers as is well known in medical
dressings. The coated dressing layers may be combined with an
adhesive layer, in a well known manner.
[0095] The dressing may be used as a single layer, or may be used
as multiple layers laminated together at intermittent spaced
locations across the dressing by ultrasonic welds. Ultrasonic
welding is a known technique in the quilting art. Briefly, heat
(generated ultrasonically) and pressure are applied to either side
of the dressing at localized spots through an ultrasonic horn so as
to cause flowing of at least one of the plastic materials in the
first and second layers and the subsequent bonding together of the
layers on cooling. The welds appear at localized circular spots and
are preferably less than 0.5 cm in diameter.
[0096] The use of ultrasonic welding of the layers at spaced
locations has the advantage of retaining the absorbent and moisture
penetration properties of the dressing layers, while retaining the
conforming properties of the dressing. Edge seams, stitching and
adhesives have the disadvantage of interfering with one or more of
these desirable properties of the dressings. Furthermore, by
spacing the welds at intermittent locations across the dressing,
the dressing may be cut to smaller sizes, as needed, without
causing delamination. Preferred spacings of about 2.5 cm between
welds allows the dressing to be cut down to about 2.5 cm sizes,
while maintaining at least one weld to hold the laminated layers
together.
[0097] ii) Nanocrystalline Coatings of Antimicrobial Metals
[0098] The coated substrate, for example a dressing, preferably
includes a nanocrystalline coating of one or more of the
antimicrobial metals. The coating is applied to one or more of the
dressing layers, but is most preferably applied at least to the
skin facing layer.
[0099] The nanocrystalline coating is most preferably formed with
atomic disorder in accordance with the procedures set out above and
as described in WO 93/23092, WO 95/13704, and WO98/41095, and as
set out below. Most preferably, the coating is formed as a
multilayer coating of the antimicrobial metals, having a top and a
base layer, as set below, to produce an interference colour. In
this way, the coating provides not only the active ingredient for
treatment of hyperplastic tissue, tumor tissue, or a cancerous
lesion, but also acts as an indicator of activation of the
dressing. As the top layer of the coating is activated with an
alcohol or water-based electrolyte, such as sterile water or
ethanol, even minor dissolution of the antimicrobial metal results
in a detectable colour change, indicating that the coating has been
activated. If there is no colour change, additional moisture might
be provided to the dressing by adding water, until a colour change
is detected. Once activated, the dressing should be maintained in a
moist condition, for example by the addition of sterile water, if
necessary.
[0100] iii) Multilayer Nanocrystalline Coatings of Antimicrobial
Metals With Interference Colour
[0101] The coated substrates, for example dressings may include the
antimicrobial metal coating formed with at least two metal layers,
a base layer and a top layer over the base layer, so as to produce
an interference colour, as set forth in WO 98/41095, the teachings
of which are incorporated herewith by reference. The indicator
colour can function as an indicator when contacted with a water or
alcohol based electrolyte, since the coating will change colour. An
exemplary multilayer nanocrystalline coating of silver with a blue
interference colour is set forth in Example 1.
[0102] iv) Nanocrystalline Coatings of Antimicrobial Metals
Containing Atomic Disorder
[0103] The coatings of the present invention are formed in a
crystalline form from one or more antimicrobial metals with atomic
disorder. The production of atomic disorder through physical vapour
deposition techniques is described in WO 93/23092 and WO 95/13704,
and as outlined below.
[0104] The antimicrobial metal is deposited as a thin metallic film
on one or more surfaces of the dressing by vapour deposition
techniques. Physical vapour techniques, which are well known in the
art, all deposit the metal from the vapour, generally atom by atom,
onto a substrate surface. The techniques include vacuum or arc
evaporation, sputtering, magnetron sputtering and ion plating. The
deposition is conducted in a manner to create atomic disorder in
the coating as defined above. Various conditions responsible for
producing atomic disorder are useful. These conditions are
generally those which one has been taught to avoid in thin film
deposition techniques, since the object of most thin film
depositions is to create a defect free, smooth and dense film (see
for example J. A. Thornton, "Influence of Apparatus Geometry and
Deposition Conditions on the Structure and Topography of Thick
Sputtered Coatings," J. Vac. Sci. Technol., 11(4), 666-670,
1974).
[0105] The preferred conditions which are used to create atomic
disorder during the deposition process include:
[0106] a low substrate temperature, that is maintaining the surface
to be coated at a temperature such that the ratio of the substrate
temperature to the melting point of the metal (in degrees Kelvin)
is less than about 0.5, more preferably less than about 0.35 and
most preferably less than about 0.3; and optionally one or both
of:
[0107] a higher than normal working gas pressure (or ambient
pressure in depositions not using a working gas), i.e. for vacuum
evaporation: e-beam or arc evaporation, greater than 0.001 Pa (0.01
mT), gas scattering evaporation (pressure plating) or reactive arc
evaporation, greater than 2.67 Pa (20 mT); for sputtering: greater
than 10 Pa (75 mT); for magnetron sputtering: greater than about
1.33 Pa (10 mT); and for ion plating: greater than about 26.67 Pa
(200 mT); and
[0108] maintaining the angle of incidence of the coating flux on
the surface to be coated at less than about 750, and preferably
less than about 30.degree..
[0109] For economic reasons, the thin metal film has a thickness no
greater than that needed to provide release of antimicrobial metal
species on a sustainable basis over a suitable period of time, and
to generate the desired interference colour. Within the preferred
ranges of thicknesses set out above, the thickness will vary with
the particular metal in the coating (which varies the solubility
and abrasion resistance), and with the degree of atomic disorder in
(and thus the solubility of) the coating. The thickness will be
thin enough that the coating does not interfere with the
dimensional tolerances or flexibility of the device for its
intended utility.
[0110] The therapeutic effect of the material so produced is
achieved when the coating is brought into contact with an alcohol
or a water based electrolyte, thus releasing metal ions, atoms,
molecules or clusters. The concentration of the metal species which
is needed to produce a therapeutic effect will vary from metal to
metal.
[0111] The ability to achieve release of metal atoms, ions,
molecules or clusters on a sustainable basis from a coating is
dictated by a number of factors, including coating characteristics
such as composition, structure, solubility and thickness, and the
nature of the environment in which the device is used. As the level
of atomic disorder is increased, the amount of metal species
released per unit time increases. For instance, a silver metal film
deposited by magnetron sputtering at T/Tm<0.5 and a working gas
pressure of about 0.93 Pa (7 mT) releases approximately 1/3 of the
silver ions that a film deposited under similar conditions, but at
4 Pa (30 mT), will release over 10 days. Films that are created
with an intermediate structure (ex. lower pressure, lower angle of
incidence etc.) have Ag release values intermediate to these values
as determined by bioassays. This then provides a method for
producing controlled release metallic coatings. Slow release
coatings are prepared such that the degree of disorder is low while
fast release coatings are prepared such that the degree of disorder
is high.
[0112] For continuous, uniform coatings, the time required for
total dissolution will be a function of film thickness and the
nature of the environment to which they are exposed. The
relationship in respect of thickness is approximately linear, i.e.
a two fold increase in film thickness will result in about a two
fold increase in longevity.
[0113] It is also possible to control the metal release from a
coating by forming a thin film coating with a modulated structure.
For instance, a coating deposited by magnetron sputtering such that
the working gas pressure was low (ex. 2 Pa or 15 mT) for 50% of the
deposition time and high (ex. 4 Pa or 30 mTorr) for the remaining
time, has a rapid initial release of metal ions, followed by a
longer period of slow release. This type of coating is extremely
effective on devices such as urinary catheters for which an initial
rapid release is required to achieve immediate antimicrobial
concentrations followed by a lower release rate to sustain the
concentration of metal ions over a period of weeks.
[0114] The substrate temperature used during vapour deposition
should not be so low that annealing or recrystallization of the
coating takes place as the coating warms to ambient temperatures or
the temperatures at which it is to be used (ex. body temperature).
This allowable .DELTA.T, that the temperature differential between
the substrate temperature during deposition and the ultimate
temperature of use, will vary from metal to metal. For the most
preferred metal, Ag, preferred substrate temperatures of -20 to
200.degree. C. , more preferably -10.degree. C. to 100.degree. C.
are used.
[0115] Atomic order may also be achieved, in either or both of the
base and top layers by preparing composite metal materials, that is
materials which contain one or more antimicrobial metals in a metal
matrix which includes atoms or molecules different from the
antimicrobial metals.
[0116] The preferred technique for preparing a composite material
is to co- or sequentially deposit the antimicrobial metal(s) with
one or more other inert, biocompatible metals selected from Ta, Ti,
Nb, Zn, V, Hf, Mo, Si, Al and alloys of these metals or other metal
elements, typically other transition metals. Such inert metals have
a different atomic radii from that of the antimicrobial metals,
which results in atomic disorder during deposition. Alloys of this
kind can also serve to reduce atomic diffusion and thus stabilize
the disordered structure. Thin film deposition equipment with
multiple targets for the placement of each of the antimicrobial and
biocompatible metals is preferably utilized. When layers are
sequentially deposited the layer(s) of the biocompatible metal(s)
should be discontinuous, for example as islands within the
antimicrobial metal matrix. The final weight ratio of the
antimicrobial metal(s) to biocompatible metal(s) should be greater
than about 0.2. The most preferable biocompatible metals are Ti,
Ta, Zn and Nb. It is also possible to form the antimicrobial
coating from oxides, carbides, nitrides, sulphides, borides,
halides or hydrides of one or more of the antimicrobial metals
and/or one or more of the biocompatible metals to achieve the
desired atomic disorder.
[0117] Another composite material may be formed by reactively co-
or sequentially depositing, by physical vapour techniques, a
reacted material into the thin film of the antimicrobial metal(s).
The reacted material is an oxide, nitride, carbide, boride,
sulphide, hydride or halide of the antimicrobial and/or
biocompatible metal, formed in situ by injecting the appropriate
reactants, or gases containing same, (ex. air, oxygen, water,
nitrogen, hydrogen, boron, sulphur, halogens) into the deposition
chamber. Atoms or molecules of these gases may also become absorbed
or trapped in the metal film to create atomic disorder. The
reactant may be continuously supplied during deposition for
codeposition or it may be pulsed to provide for sequential
deposition. The final weight ratio of reaction product to
antimicrobial metal(s) should be greater than about 0.05. Air,
oxygen, nitrogen and hydrogen are particularly preferred reactants,
with oxygen being most preferred.
[0118] The above deposition techniques to prepare composite
coatings may be used with or without the conditions of lower
substrate temperatures, high working gas pressures and low angles
of incidence previously discussed. One or more of these conditions
are preferred to retain and enhance the amount of atomic disorder
created in the coating.
[0119] The most preferred composite coatings are formed by
sputtering silver, under conditions set out above, in an atmosphere
containing oxygen, so as to form a coating of silver as a composite
coating with oxygen.
[0120] Dressings coated with the antimicrobial coatings of this
invention may be sterilized in the manner set out below.
[0121] b) Powders of Atomically Disordered Antimicrobial Metals
[0122] Crystalline powder forms of the antimicrobial or noble
metals (particularly preferred being Ag, Au, Pt, and Pd) can be
prepared as free standing powders, by coating powdered substrates,
or from coatings on substrates which are then collected, for
example by scaping and then sized. The powders may be prepared as
pure metals, metal alloys or compounds such as metal oxides or
metal salts, by vapour deposition, mechanical working, or
compressing to impart the atomic disorder. The crystalline powders
are formed with atomic disorder in accordance with the techniques
set out above and as published in the prior patent applications of
Burrell et al., see for example WO 93/23092, published Nov. 25,
1993, and WO 95/13704, published May 26, 1995. The atomic disorder
will most typically be formed in the metal powders during physical
vapour deposition as set out above for coatings or by mechanically
imparting the disorder, such as by milling, grinding, hammering,
mortar and pestle or compressing, under conditions of low
temperature (i.e., temperatures less than the temperature of
recrystallization of the material) to ensure that annealing or
recyrstallization does not take place.
[0123] Alternatively, the powders may be formed by inert-gas
condensation techniques, which are modified to provide atomic
disorder in the powder produced, as taught in WO 95/13704 to
Burrell et al.
[0124] Powders of the antimicrobial or noble metals are preferably
formed by physical vapour deposition (PVD) onto a substrate such as
a cold finger, a silicon wafer, solid plates, a rotating cylinder,
a continuous belt in a roll coater, or on steel collectors in known
PVD coaters. Preparation of powders of the present invention by
sputtering onto a continuous belt in a roll coater, or other some
other moving or rotating substrate surface is particularly
advantageous, inasmuch as it can quickly and easily yield a
relatively large supply of free-standing powder at a relatively low
cost. A stainless steel belt can be used in the roll coating
process without the need to provide additional cooling of the
substrate. The powders or coatings and then are then scraped off to
form a powder, and may be sized to avoid overly large particulates.
The powders are scraped off the moving surface with scrapers which
contact the moving surface at an angle sufficient to remove the
coating in flake or powder form. The coating may be scraped off
with scrapers angled for forward cutting of the coating from the
moving surface, or with scrapers which remove the coating from the
moving surface by reverse dragging action on the surface. The
scrapers may be suspended above the belt, and either weighted or
spring loaded to apply pressure sufficient to remove the coating
from the moving surface. With a continuous belt, the scrapers can
conveniently be located above the end rollers to remove the coating
with a reverse dragging action as the belt rounds the end
roller.
[0125] Alternatively, the powders of the antimicrobial or noble
metals may be formed on powdered substrates which are
biocompatible, or otherwise compatible for the end use of the
powder. Particularly preferred powdered substrates are
hydrocolloids, particularly those which are bioabsorbable and/or
hygroscopic powders such as chitin. Exemplary bioabsorbable and/or
hygroscopic powders are composed of:
[0126] Synthetic Bioabsorbable Polymers: for example
polyesters/polyactones such as polymers of polyglycolic acid,
glycolide, lactic acid, lactide, dioxanone, trimethylene carbonate
etc., polyanhydrides, polyesteramides, polyortheoesters,
polyphosphazenes, and copolymers of these and related polymers or
monomers.
[0127] Naturally Derived Polymers:
[0128] Proteins: albumin, fibrin, collagen, elastin;
[0129] Polysaccharides: chitosan, alginates, hyaluronic acid;
and
[0130] Biosynthetic Polyesters: 3-hydroxybutyrate polymers.
[0131] The powders may be incorporated into or onto medical
dressings or pharmaceutical formulations, by any methods known in
the art. For example, the powders may be layered onto the
substrates (dressings or powders), mechanically fixed within the
fibres of the dressings, impregnated into dressings by physical
blowing, or added to topical pharmaceutical ingredients.
[0132] Preferably, powders of the present invention are sized at
less than 100 .mu.m, and more preferably less than 40 .mu.m, and
most preferably about 3-5 .mu.m in size. For direct application to
a locus of the hyperplastic tissue, tumor tissue, or a cancerous
lesion, powders are preferably sized less than 2 .mu.m, and more
preferably less than 1 .mu.m.
[0133] B. Formulations for Administration
[0134] 1. Coated substrates coated with antimicrobial metals formed
with atomic disorder are well described above. These techniques can
be used to coat dressings, meshes, films, filtering surfaces,
filters, packing fibres, the insides of vials or containers etc.
The coated substrates in the form of dressings for example, can be
used directly on the affected area, or they can be used to generate
powders, liquid or other formulations as set out below.
[0135] 2. Powders of the antimicrobial metals formed with atomic
disorder are set out above, and may be used in that form directly
on the affected area, or in other formulations such as dressings,
occlusions, creams, liquids etc. Alternatively, powders may be
formulated within liquid pervious membranes such as filters, meshes
and the like, such as a tea bag-type infuser, for generating
liquids containing dissolved species of the antimicrobial
metal.
[0136] 3. Occlusions may include a hydrated dressing, with a
sealing material overlaid on the outside, to the area, e.g. skin
cancer, to be treated. The term hydrated dressing is meant to
include non-hydrated dressings which become hydrated upon contact
with an alcohol or water-based electrolyte. Occlusion prevents loss
of the therapeutic agent from the area, promotes hydration of the
area, and increases the temperature of the area. Examples of
hydrated dressings include hydrocolloids, hydrogels, polyethylene,
polyurethane, polyvinylidine, and siloxane or silicone dressings.
The hydrated dressing can either be impregnated with a solution or
powder of the antimicrobial metals of this invention, or can be
used with a topical formulation of the antimicrobial metals of this
invention.
[0137] An exemplary occlusion is a hydrocolloid dressing
impregnated with silver. Alternatively, one might use a
non-impregnated hydrocolloid dressing to occlude nanocrystalline
silver-containing gel placed on a problematic area. A hydrocolloid
is a synthetically prepared or naturally occurring polymer capable
of forming a thickened gel in the presence of water and polyols
(swelling agent). The swelling agent is a hydrophilic liquid
capable of swelling the hydrocolloid chosen in order to form the
gel phase. The hydrocolloid may be selected from the group
comprising:
[0138] i representative natural or synthetically modified
polysaccharides (e.g., cellulose or its derivatives such as
carboxymethylcellulose, hydroxypropylcellulose, methylcellulose,
hydroxypropylmethylcellulose or hydroxyethylcellulose, starch,
glycogen, gelatin, pectin, chitosan and chitin; and
[0139] ii representative gums from algal extracts, seed extracts,
or plant exudates (e.g., gum arabic, locust bean gum, karaya gum,
gum tragacanth, ghatti gum, agar-agar, carrageenans, alginates,
carob gum, guar gum, xanthan gum); and
[0140] iii synthetic polymers which may be either linear or
crosslinked (e.g. polymers prepared from N-vinyl lactams, e.g.
N-vinyl-2-pyrrolidone, 5-methyl-N-vinyl-2-pyrrolidone).
[0141] The hydrocolloid is present in an amount of from about 0.1%
to 20% of the weight and preferably 1% to 10%. The hydrocolloid can
range for example, from 1 to 10% of the total weight of the
composition. Alternatively, the hydrocolloid may be in the form of
a powder whose average particle size is less than 100 .mu.m
preferably less than 50 .mu.m.
[0142] The swelling agent should be non-volatile, and allow the gel
to remain as a gel during use, hence preserving the swollen
condition of the hydrocolloid. Varieties of non-volatile swelling
agents include room temperature liquid polyols (including
polyhydric alcohols) such as glycerol; room temperature solid
polyols (including polyhydric alcohols) such as sorbitol,
erythritol, threitol, ribotol, arabinitol, xylitol, allitol,
talitol, mannitol, glucitol, glactitol, iditol, pentaerythritol,
heptitol, octitol, nonitol, decitol, and dodecitol, blended with a
room temperature liquid polyol; monoanhydroalditols (such as
styracitol, polyalitol, D-fructose, 1,4 anhydro-D-mannitol and 1,4
anhydro-D-glucitol) blended with a room temperature liquid polyol;
monosaccharides (such as pentoses, hexoses, and heptoses) blended
with a room temperature liquid polyol; and ether alcohols blended
with a room temperature liquid polyol.
[0143] Hydrocolloid dressings often comprise a wafer constructed
from a thin layer of polyurethane film with an adhesive contact
layer containing a hydrocolloid composition and securing the
dressing to the area, and the polyurethane film being impermeable
to water and microorganisms. Hydrocolloid dressings may be prepared
by dispersing a composition in gel form of hydrocolloids with a
swelling agent into a strong pressure sensitive adhesive.
Alternatively, the gel and the adhesive may be mixed in a latex
solution. Alternatively, exemplary products are available
commercially, for example DuoDERM.TM. (ConvaTec Canada, 555, Dr.
Frederik Philips, Suite 110, St-Laurent, Quebec, H4M 2.times.4);
and Tegasorb.TM. (3M Health Care, 300 Tartan Drive, London,
Ontario, Canada, N5V 4M9). The hydrocolloid dressing may be
impregnated with a solution or powder of the antimicrobial metal by
blending the solution or powder of the antimicrobial metal into a
liquid phase during the manufacture of the hydrocolloid dressing,
or by sprinkling and then pressing a powder of the antimicrobial
metal into the surface of the hydrocolloid dressing. Further, the
hydrocolloid dressing can be used with a topical formulation of the
antimicrobial metals of this invention. Upon application, the
dressing surface gels upon continued contact with moisture or
exudate from the area, e.g. skin. With the incorporation of an
antimicrobial metal such as silver (0.01-10%, preferably 0.1-1% by
weight), the dressing is advantageous in being impermeable to water
and microorganisms, and presenting antimicrobial and
anti-inflammatory effects as mediated by the antimicrobial
metal.
[0144] 4. Gels--Nanocrystalline gels may be formed from the
nanocrystalline metal powder in admixture with gelling agents such
as hydrocolloids and hydrogels in powder form. Exemplary gelling
agents include carboxymethyl cellulose (CMC), polyvinyl alcohol
(PVA), collagen, pectin, gelatin, agarose, chitin, chitosan, and
alginate, with the gelling agent comprising between about 0.01-20%
w/v.
[0145] 5. Creams Lotions, Pastes, Ointments, Foams--The
antimicrobial metals may be incorporated into creams, lotions,
pastes, ointments or foams formulated with nanocrystalline powders
or solutions of the antimicrobial metals, for example as emulsions
or with drying emollients. Ointments and creams can be formulated
with an aqueous or oily base with the addition of suitable
thickening and/or gelling agents. Such bases may include water
and/or an oil such as liquid paraffin or a vegetable oil such as
peanut oil or castor oil. An exemplary base is water. Thickening
agents which can be used according to the nature of the base
include aluminum stearate, hydrogenated lanolin, and the like.
Further, lotions can be formulated with an aqueous base and will,
in general, also include one or more of the following: stabilizing
agents, emulsifying agents, dispersing agents, suspending agents,
thickening agents, coloring agents, perfumes, and the like.
Ointments and creams can also contain excipients, such as starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, and talc, or mixtures thereof. Lotions
may be formulated with an aqueous or oily base and will, in
general, also include one or more of the following: stabilizing
agents, emulsifying agents, dispersing agents, suspending agents,
thickening agents, coloring agents, perfumes, and the like. Foams
may be formed with known foaming or surface active agents.
[0146] 6. Liquids--The crystalline forms of the antimicrobial
metals may be incorporated into liquids, formulated as solutions,
dispersion or suspensions by dissolving nanocrystalline coatings or
powders of the antimicrobial metals, for example as topical
solutions, aerosols, mists, sprays, drops, and instillation
solutions for body cavities such as the bladder, prostate, lung, or
liver. Topical administration of the antimicrobial metal to the
skin may be performed by aerosol, which can be generated by a
nebulizer, or by instillation. The antimicrobial metal may be
administered alone, or with a carrier such as saline solution, an
alcohol, water, or DMSO. An effective daily amount of the
antimicrobial metal will vary with the subject, but will be less
than is toxic while still providing a therapeutic effect.
[0147] Solutions and formulations of the antimicrobial metals may
lose some activity with aging and are thus either stabilized or
generated fresh for administration. Alternatively, the
antimicrobial metals may be packaged for convenient solution
generation, for instance as tea bag type infusers. Other two part
or two phase systems may be used in which the nanocrystalline metal
is separated from the water or alcohol-based electrolyte, for
example in a multicomponent kit form, as set out herein.
[0148] Concentrations of the antimicrobial metal species in
solution will vary according to the application, formulation and
subject, but will generally range from 1-5000 .mu.g/ml, more
preferably 20-3000,g/ml, more preferably 40-800 .mu.g/ml, and most
preferably 50-500 .mu.g/ml. Methods of generating liquids with
appropriate concentrations of the antimicrobial metal through pH
control are set out below.
[0149] 7. Transdermal Patch
[0150] Transdermal patches may provide controlled delivery of the
antimicrobial metal to the area. For example, an adhesive patch or
adhesive matrix patch, can be prepared from a backing material and
an adhesive, such as an acrylate adhesive. Powders or solutions of
the antimicrobial metal may be formulated into the adhesive casting
solution and allowed to mix thoroughly. The solution is cast
directly onto the backing material and the casting solvent is
evaporated in an oven, leaving an adhesive film. Alternatively, a
polyurethane matrix patch can be employed to deliver the
antimicrobial metal to the area. The layers of this patch comprise
a backing, a polyurethane drug/enhancer matrix, a membrane, an
adhesive, and a release liner. The polyurethane matrix is prepared
using a room temperature curing polyurethane prepolymer. Addition
of water, alcohol, and drug to the prepolymer results in the
formation of a tacky firm elastomer that can be directly cast onto
the backing material.
[0151] C. Sterilization
[0152] Dressings with nanocrystalline coatings of a antimicrobial
metal formed with atomic disorder are preferably sterilized without
applying excessive thermal energy, which can anneal out the atomic
disorder, thereby reducing or eliminating a useful release of
antimicrobial metal species. Gamma radiation is preferred for
sterilizing such dressings, as discussed in WO 95/13704. Electron
beam and ethylene oxide sterilization techniques can also be
used.
[0153] It should be appreciated that the use of ultrasonic welding
to laminate the layers of dressings with nanocrystalline coatings
formed from antimicrobial metals with atomic disorder is
advantageous since it achieves bonding in localized spots and
avoids applying heat to any significant portion of the dressing,
thereby avoiding any significant reduction in the solubility of the
antimicrobial metals through annealing out of the atomic
disorder.
[0154] The sterilized dressings, coating, powders or formulations
should be sealed in packaging, containers, or kits which limit
moisture and light penetration to avoid additional oxidation or
reduction of the antimicrobial metal. Polyester peelable pouches
are preferred. The shelf life of coatings or powders thus sealed is
over one year.
[0155] D. Formulating Dosages and Treatment
[0156] Typically, the nanocrystalline antimicrobial metals will be
formulated from the active ingredient, namely nanocrystalline
powders or coatings of the antimicrobial metals, or dissolved
species from such powders or coatings, in the one or more of the
formats set out above. Dressing or powders of the nanocrystalline
antimicrobial metals may be applied directly to the hyperplastic
tissue, tumor tissue, or cancerous lesion, or they may be
formulated as set out below. Depending on the particular
application and dosage form, the powder size might be controlled to
less than 2 .mu.m, more preferably to less than 1 .mu.m.
[0157] In the pharmaceutical compositions, the amount of the
nanocrystalline metal powder may range broadly from about 0.001% to
about 30% by weight, but will more preferably fall in the range of
from about 0.01 to 10% by weight, and most preferably in the range
of about 0.1 to 5% by weight. Typical coating thicknesses are in
the range of 150 to 3000 nm thick. Thicker coatings, up to 10,000
nm thick, can be used to generate powders of the antimicrobial
metal. Coatings of the nanocrystalline antimicrobial metals may be
very thin, or thick, depending on the desired duration of
application on the patient. As liquid formulations, the amount of
dissolved antimicrobial metal will typically range between about
0.001 to 10% by weight, more preferably 0.01 to 1% by weight.
[0158] Besides the active ingredient, pharmaceutical compositions
may also include non-toxic, pharmaceutically acceptable carriers,
diluents and excipients, suitable for topical application, as are
well known, see for example Merck Index, Merck & Co., Rahway,
N.J.; and Gilman et al., (eds) (1996) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8.sup.th Ed., Pergamon
Press. For standard dosages of conventional pharmacological agents,
see, e.g., Physicians Desk Reference (1997 Edition); and U.S.
Pharmacopeia National Formulary (1995) United States Pharmacopeial
Convention Inc., Rockville, Md.
[0159] Dosage forms for the topical administration of compositions
of the nanocrystalline antimicrobial metals include various
mixtures and combinations that can be applied topically and will
permit even spreading and absorption into the cutaneous surfaces.
Examples include sprays, mists, aerosols, lotions, creams,
solutions, gels, ointments, pastes, emulsions, foams and
suspensions. The active compound can be mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants which may be required.
Topical preparations can be prepared by combining the antimicrobial
metal powder with conventional pharmaceutically acceptable carriers
commonly used in topical dry, liquid, cream and aerosol
formulations. Ointment and creams can, for example, be formulated
with an aqueous or oily base with the addition of suitable
thickening and/or gelling agents. An exemplary base is water.
Thickening agents can be used according to the nature of the base.
Lotions can be formulated with an aqueous base and will, in
general, also include one or more of the following: stabilizing
agents, emulsifying agents, dispersing agents, suspending agents,
thickening agents, coloring agents, perfumes, and the like. Powders
can be formed with the aid of any suitable powder base, e.g., talc,
lactose starch and the like. Drops can be formulated with an
aqueous base or non-aqueous base, and can also include one or more
dispersing agents, suspending agents, solubilizing agents, surface
active agents and the like.
[0160] Ointments, pastes, creams and gels also can contain
excipients, such as starch, tragacanth, cellulose derivatives,
silicones, bentonites, silicic acid, and talc, or mixtures thereof.
Powders and sprays also can contain excipients such as lactose,
talc, silicic acid, aluminum hydroxide, and calcium silicates, or
mixtures of these substances. Solutions of nanocrystalline
antimicrobial metals can be converted into aerosols or sprays by
any of the known means routinely used for making aerosol
pharmaceuticals. In general, such methods comprise pressurizing or
providing a means for pressurizing a container of the solution,
usually with an inert carrier gas, and passing the pressurized gas
through a small orifice. Sprays can additionally contain customary
propellants, such as inert gases such as nitrogen, carbon dioxide,
argon or neon.
[0161] Multiple inactive ingredients are generally incorporated in
topical formulations to improve cosmetic acceptability, and are
optional ingredients in the formulations. Such ingredients are
included only in therapeutically acceptable forms and amounts.
Examples of ingredients are emulsifiers, emollients, thickening
agents, solvents, hydrating or swelling agents, flavours,
sweetening agents, surface active agents, colouring agents,
anti-foaming agents, preservatives, fragrances, and fillers may
also be added, as is well known in the art; for example,
preservatives such as methyl paraben and propyl paraben,
texturizing agents, thickeners, anticoagulants such as heparin,
.beta.-glucan, hormones, hyaluronic acid, and the like. Polyvinyl
alcohol is a particularly preferred gelling polymer and also acts
as a texturizing agent, methyl or propyl parabens are particularly
preferred preservatives. These other agents may be included in
amounts in the range of 0.1 to 5 wt %.
[0162] Surface active agents or foaming agents may be added to the
formulations and are particularly advantageous for addition to
liquid formulations for aerosol or foam applications, to form foams
for applications such as to treat cancerous lesions on the skin, or
aerosols for application for respiratory disorders. Surface active
agents selected for use should not substantially interfere with the
pro-apoptotic and anti-MMP effects of the nanocrystalline
antimicrobial metals.
[0163] All agents must be non-toxic and physiologically acceptable
for the intended purpose, and must not substantially interfere with
the activity of the nanocrystalline antimicrobial metals so as to
deleteriously affect the pro-apoptotic and anti-MMP effects.
Ingredients are thus only included in therapeutically acceptable
amounts. Ingredients to be generally avoided or limited in the
formulations of the present invention, at least in amounts greater
than 0.01 wt %, are glycerin, glycerols, chloride salts, aldehydes,
ketones, long chain alcohols, and triethanolamine.
[0164] The dosage of the active ingredients depends upon many
factors that are well known to those skilled in the art, for
example, the particular form of the active ingredient, the
condition being treated, the age, weight, and clinical condition of
the recipient patient, and the experience and judgement of the
clinician or practitioner administering the therapy. A
therapeutically effective amount of the nanocrystalline
antimicrobial metal provides either subjective relief of symptoms
or an objectively identifiable improvement as noted by the
clinician or other qualified observer. The dosing range varies with
the metal used, its form, the route of administration and the
potency of the particular compound.
[0165] When the formulation is in the form of a dressing, the
dressing is placed on the afffected area (e.g, cancerous lesion on
the skin) and, depending on the degree of moisture at the membrane,
may be further moistened with drops of sterile water, tap water,
body fluids such as exudate or, for example 70% ethanol, in order
to activate the coating for release of antimicrobial or noble metal
species. The dressing may be then secured in place with an
occlusive or semi-occlusive layer, such as an adhesive tape or
polyurethane film, which keeps the dressing in a moist environment.
In use, the dressings are kept moist, at 100% relative humidity.
Adding sterile water initially to activate the antimicrobial or
noble metal coating is needed, and then as needed to maintain the
dressing in a moist condition. Dressings may be changed as required
for observation and cleaning. Preferably dressings are changed
daily, but could be left longer, such as 3 days, and can provide a
therapeutic effect for a much longer period of time.
[0166] Other forms of formulations, such as occlusions, gels,
pastes, ointments, creams, emulsions, foams, and liquids may be
prepared in stable forms, or more preferably are prepared fresh
from one or more phases, for instance in multicomponent kit form,
so as to avoid aging and to maximize the therapeutic effectiveness
of the antimicrobial metal component. Formulations are best used
within about 30 days after combining the phases. Suitable kits or
containers are well known for maintaining the phases of
formulations separate until the time of use. For instance, the
antimicrobial metal in powder or coated substrate form may be
packages separately from therapeutically acceptable carriers, and
possibly other ingredients for mixing at the time of use. The
separate coated substrate may be in dressing or patch form for
direct application, or may take other suitable forms to generate
liquid formulations and the like, such as a coating on the inside
surface of a vial or container, a mesh, or a film. For example, the
antimicrobial metal may be provided in a "tea bag"-type infuser or
pouch, for generating liquid formulations at the time of use. The
tea bag-type infuser is advantageous in that the pouch may serve as
a filter for small particulates of the powder that may be
detrimental to administration for some applications such as
aerosols for respiratory disorders. A kit containing the dressing,
coated substrate or powder may provide a sterile carrier such as
water (and other ingredients) in a separate container in dosage
specific amounts. As used herein, the term "kit" is meant to refer
to packaged formulations, whether the ingredients are in separate
phases or mixed, and thus for example, may include a gel in a tube
with all ingredients in admixture, or any formulation wherein the
ingredients are separated from each other.
[0167] Formulations for respiratory disorders may be delivered as
dry powders in dry powder inhalers, or they may be formulated in
liquid formulations for delivery in metered dose inhalers,
aerosols, mists or sprays.
[0168] For liquid formulations, in order to increase the amount of
antimicrobial or noble metal solubilized in the solution, the pH of
the solution during dissolution may be lowered to less than pH 6.5,
more preferably to the range of 3.5 to 6.5, with such acidifying
agents as carbon dioxide (which generated carbonic acid in
solution). This pH range will typically generate concentrations of
silver from atomic disordered silver from 85 .mu.g/ml to 370
.mu.g/ml, and can be adjusted for different desired concentrations.
Dissolution of the antimicrobial metal will typically raise the pH
to 6.5 to 7.0.
[0169] Administration as aerosols produces droplets preferably less
than 10 .mu.m in size, more preferably less than 5 .mu.m in size,
most preferably 1-3 .mu.m in size. Control of the droplet size is
important both to control the dosage delivered and to enhance
delivery to the target tissues, e.g., where the aerosol is inhaled
through the mouth, large droplets of about 10 .mu.m tend to stay in
the throat whereas small droplets (e.g., approximately 1-3 .mu.m)
tend to travel to the aveolar region of the lungs. Thus, depending
on the dosage required and the target tissue, it may be important
to regulate the droplet size of the aerosol. In this respect, it
has been found that droplet size can be regulated, to at least some
extent, by the mechanical mister which is used to produce the
aerosol. In addition, the aerosol's droplet size can be adjusted,
to at least some extent, by modifying the surface tension of the
solution. More particularly, the solution of the antimicrobial
metal typically has water as its solvent, and water has a
relatively high surface tension, so it is relatively
straightforward to create an aerosol having relatively small
droplet size. Surface active agents can be added to the solution so
as to reduce the surface tension of the solution, to create an
aerosol having a relatively large droplet size. By way of example,
such surfactants may comprise sodium alkyl sulfates, sodium lauryl
sulfate, sodium lauroyl sarconsinate, phospholipids, e.g.,
lecithin, sphingomyelin, etc.
[0170] Depending on the application, solutions generated from
powders of the antimicrobial metal should avoid inclusion of
particulates larger than 2 .mu.m, and more preferably no larger
than 1 .mu.m (i.e., submicron) to avoid deleterious immune
responses or toxic effects. Larger particulates may be removed by,
for example filtration. Particulates may be formed in the liquid
and can be removed, for example by filtration. For instance, silver
carbonates may be formed on reaction with the carbonic acid used to
acidify the solution. Particulate generation may also be limited by
diluting the carbonic acid in solution.
[0171] The aerosol may be created by passing a liquid solution of
the antimicrobial metal through a mechanical mister (e.g., a
nebulizer) and may be applied directly with a pressurized pack
(e.g., via a hand inhaler with a propellant such as carbon dioxide
or other gas, with a valve metered dosage) or through some other
delivery system (e.g., an oxygen tent, etc.).
[0172] The invention provides methods of treatment, by
administering a therapeutically effective amount of a
nanocrystalline antimicrobial or noble metal powder, or a solution
derived from a nanocrystalline antimicrobial or noble metal, as
either a topical formulation, or as a coating on medical dressing,
applied to the locally affected area, e.g. hyperplastic tissue,
tumor tissue, or cancerous lesion. A therapeutically effective
amount may be determined by testing formulations containing the
nanocrystalline antimicrobial or noble metals by in vitro or in
vivo testing. Formulations may be applied one or more times a day.
Dressings coated with the nanocrystalline antimicrobial or noble
metals may be changed daily, or even less frequently, and should be
kept in a moist condition with the addition of saline, alcohols, or
more preferably sterile water, in order to release ions, atoms,
molecules or clusters of the nanocrystalline metal, on a sustained
basis.
[0173] Hyperplastic tissue, tumor tissue, or cancerous lesions may
thus be treated by administering a therapeutically effective
solution derived from a nanocrystalline antimicrobial or noble
metal to the affected area; for example, as an infusion or
instillation into a body cavity, e.g., the bladder to put the
solution directly in contact with the bladder wall. Dressings or
transdermal patches coated with the nanocrystalline antimicrobial
metals may be applied internally in direct contact with
hyperplastic tissue, tumor tissue, or cancerous lesion, and
externally upon cancerous lesions, e.g., skin cancers such as
melanoma.
EXAMPLES
Example 1
Preparation of Nanocrystalline Silver Coatings on Dressings
[0174] This example shows the preparation of a bilayer
nanocrystalline silver coating on a dressing material. A high
density polyethylene dressing, DELNET.TM. or CONFORMANT 2.TM. was
coated with a silver base layer and a silver/oxide top layer to
generate a coloured antimicrobial coating having indicator value.
The coating layers were formed by magnetron sputtering under the
conditions set out in Table 1.
2TABLE 1 Sputtering conditions Sputtering Conditions Base Layer Top
Layer Target 99.99% Ag 99.99% Ag Target Size 20.3 cm diameter 20.3
cm diameter Working Gas 96/4 wt % Ar/O.sub.2 96/4 wt % Ar/O.sub.2
Working Gas Pressure 5.33 Pa (40 mT) 5.33 Pa (40 mT) Power 0.3 kW
0.15 kW Substrate Temperature 20.degree. C. 20.degree. C. Base
Pressure 3.0 .times. 10.sup.-6 Torr (4 .times. 10.sup.-4 Pa) 3.0
.times. 10.sup.-6 Torr (4 .times. 10.sup.-4 Pa) Anode/Cathode 100
mm 100 mm Distance Sputtering Time 7.5-9 mm 1.5 min Voltage 369-373
V 346 V
[0175] The resulting coating was blue in appearance. A fingertip
touch was sufficient to cause a colour change to yellow. The base
layer was about 900 nm thick, while the top layer was 100 nm
thick.
[0176] To establish that silver species were released from the
coated dressings, a zone of inhibition test was conducted. Mueller
Hinton agar was dispensed into Petri dishes. The agar plates were
allowed to surface dry prior to being inoculated with a lawn of
Staphylococcus aureus ATCC No. 25923. The inoculant was prepared
from Bactrol Discs (Difco, M.), which were reconstituted as per the
manufacturer's directions. Immediately after inoculation, the
coated materials to be tested were placed on the surface of the
agar. The dishes were incubated for 24 hr. at 37.degree. C. After
this incubation period, the zone of inhibition was calculated
(corrected zone of inhibition =zone of inhibition--diameter of the
test material in contact with the agar). The results showed a
corrected ZOI of about 10 mm, demonstrating good release of silver
species.
[0177] The coating was analyzed by nitric acid digestion and atomic
absorption analysis to contain 0.24+/-0.04 mg silver per mg high
density polyethylene. The coating was a binary alloy of silver
(>97%) and oxygen with negligible contaminants, based on
secondary ion mass spectroscopy. The coating, as viewed by SEM, was
highly porous and consisted of equiaxed nanocrystals organized into
coarse columnar structures with an average grain size of 10 nm.
Silver release studies in water demonstrated that silver was
released continuously from the coating until an equilibrium
concentration of about 66 mg/L was reached (determined by atomic
absorption), a level that is 50 to 100 times higher than is
expected from bulk silver metal (solubility .ltoreq.1 mg/L).
[0178] By varying the coating conditions for the top layer to
lengthen the sputtering time to 2 min, 15 sec., a yellow coating
was produced. The top layer had a thickness of about 140 nm and
went through a colour change to purple with a fingertip touch.
Similarly, a purple coating was produced by shortening the
sputtering time to 1 min, to achieve a top layer thickness of about
65 nm. A fingertip touch caused a colour change to yellow.
[0179] To form a three layer dressing, two layers of this coated
dressing material were placed above and below an absorbent core
material formed from needle punched rayon/polyester (SONTARA.TM.
8411). With the silver coating on both the first and third layers,
the dressing may be used with either the blue coating side or the
silver side in the skin facing position. For indicator value, it
might be preferable to have the blue coating visible. The three
layers were laminated together by ultasonic welding to produce
welds between all three layers spaced at about 2.5 cm intervals
across the dressing. This allowed the dressing to be cut down to
about 2.5 cm size portions for smaller dressing needs while still
providing at least one weld in the dressing portion.
[0180] The coated dressings were sterilized using gamma radiation
and a sterilization dose of 25 kGy. The finished dressing was
packaged individually in sealed polyester peelable pouches, and has
shown a shelf life greater than 1 year in this form. The coated
dressings can be cut in ready to use sizes, such as 5.1.times.10.2
cm strips, before packaging. Alternatively, the dressings may be
packaged with instructions for the patient or clinician to cut the
dressing to size.
[0181] Additional silver coated dressings were prepared in a full
scale roll coater under conditions to provide coatings having the
same properties set out above, as follows:
[0182] i the dressing material included a first layer of silver
coated DELNET, as set out above, laminated to STRATEX, AET,
8.0NP.sub.2-A/QW, which is a layer of 100% rayon on a polyurethane
film.
[0183] ii Silver Foam Dressing--three layers of silver coated high
density polyethylene prepared as above, alternating with two layers
of polyurethane foam, L-00562-6 Medical Foam, available from Rynel
Ltd., Bootbay, Me., USA.
Example 2
Preparation of Atomic Disordered Nanocrystalline Silver Powders
[0184] Nanocrystalline silver coatings were prepared by sputtering
silver in an oxygen-containing atmosphere directly onto an endless
stainless steel belt of a magnetron sputtering roll coater, or onto
silicon wafers on the belt. The belt did not need to be cooled. The
coatings were scraped off with the belt with suspended metal
scrapers as the belt rounded the end rollers. For the coated
silicon wafers, the coatings were scraped off with a knife edge.
The sputtering conditions were as follows:
3TABLE 2 Sputtering Conditions Target 99.99% Ag Target Size 15.24
cm .times. 1216.125 cm (individual, 23 targets) Working Gas 75:25
wt % Ar/O.sub.2 Working Gas 5.33 Pa (40 mT) Pressure Total Current
40 A Base Pressure 5.0 .times. 10.sup.-5 Torr (range: 1 .times.
10.sup.-4 - 9 .times. 10.sup.-7 Torr or 1 .times. 10.sup.-2 - 1.2
.times. 10.sup.-4 Pa) Sandvik Belt Speed 340 mm/mm Voltage 370 V
Note pressure conversions to Pa herein may not be accurate, most
accurate numbers are in torr, mTorr units.
[0185] The powder had a particle size ranging from 2 .mu.m to 100
.mu.m, with grain or crystallite size of 8 to 10 nm (i.e.,
nanocrystalline), and demonstrated a positive rest potential.
[0186] Similar atomic disordered nanocrystalline silver powders
were formed as set forth hereinabove by magnetron sputtering onto
cooled steel collectors, under conditions taught in the prior
Burrell et al. patents to produce atomic disorder.
Example 3
Preparation of Gels
[0187] No. 1
[0188] A commercial carboxymethyl cellulose/pectin gel
(DuoDERM.TM., ConvaTec Canada, 555, Dr. Frederik Philips, Suite
110, St-Laurent, Quebec, H4M 2.times.4) was combined with
nanocrystalline silver powder prepared as set forth in Example 3 to
produce a gel with 0.1% silver. A logarithmic reduction test was
performed as follows in the gel using Pseudomonas aeruginosa. The
inoculum was prepared by placing 1 bacteriologic loopful of the
organism in 5 mL of trypticase soy broth and incubating it for 3-4
h. The inoculum (0.1 mL) was then added to 0.1 mL of gel and
vortexed (triplicate samples). The mixture was incubated for
one-half hour. Then 1.8 mL of sodium thioglycollate-saline (STS)
solution was added to the test tube and vortexed. Serial dilutions
were prepared on 10-1 to 10-7. A 011 mL aliquot of each dilution
was plated in duplicate into Petri plates containing Mueller-Hinton
agar. The plates were incubated for 48 h and then colonies were
counted. Surviving members of organisms were determined and the
logarithmic reduction compared to the initial inoculum was
calculated. The logarithmic reduction for this mixture was 6.2,
indicating a significant bactericidal effect.
[0189] No. 2
[0190] Carboxymethyl cellulose (CMC) fibers were coated directly to
produce an atomic disordered nanocrystalline silver coating, using
magnetron sputtering conditions similar to those set forth in
Example 1. The CMC was then gelled in water by adding 2.9 g to 100
mL volume. This material was tested using the method of No. 1. The
material generated a 5.2 logarithmic reduction of Pseudomonas
aeruginosa, demonstrating that the gel had a significant
bactericidal effect.
[0191] No. 3
[0192] An alginate fibrous substrate was directly coated with an
atomic disordered nanocrystalline silver coating using magnetron
sputtering conditions similar to those set forth in Example 1. The
alginate (5.7 g) was added to 100 mL volume of water to create a
gel. This material was tested using the method of No. 1. The
material generated a 5.2 logarithmic reduction of Pseudomonas
aeruginosa, demonstrating that the gel had a significant
bactericidal effect.
[0193] No. 4
[0194] A commercial gel containing CMC and alginate (Purilin gel,
Coloplast) was mixed with a atomic disordered nanocrystalline
silver powder to give a product with 0.1% silver. This was tested
as above with both Pseudomonas aeruginosa and Staphylococcus
aureus. Zone of inhibition data was also generated for this gel as
follows. An inoculum (Pseudomonas aeruginosa and Staphylococcus
aureus) was prepared as in No. 1 and 0.1 mL of this was spread onto
the surface of Mueller-Hinton agar in a Petri dish. A six mm hole
was then cut into the agar at the center of the Petri dish and
removed. The well was filled with either 0.1 mL of the silver
containing gel, a mupirocin containing cream or a mupirocin
containing ointment. The Petri plates were then incubated for 24 h
and the diameter of the zone of inhibition was measured and
recorded.
[0195] The silver containing gel produced 9 mm zone of inhibition
against both Pseudomonas aeruginosa and Staphylococcus aureus,
while the mupirocin cream and ointment produced 42 and 48 mm zones
against Staphylococcus aureus and 0 mm zones against Pseudomonas
aeruginosa.
[0196] The silver containing gel reduced the Pseudomonas aeruginosa
and Staphylococcus aureus properties by 4.4 and 0.6 log reductions,
respectively, showing good bactericidal activity. The mupirocin
cream and ointment generated 0.4 and 0.8, and 0.8 and 1.6, log
reductions against Staphylococcus aureus and Pseudomonas
aeruginosa, respectively. The silver gel had both a greater
bactericidal effect and spectrum of activity than the mupirocin
containing products.
[0197] Nos. 5-10
[0198] The formula for Nos. 5-10 are summarized in Table 7. Zones
of inhibitions were determined as in No. 4 and log reductions were
determined as in No. 1.
[0199] All formulae provided a broader spectrum of activity and a
greater bactericidal effect than did mupirocin in a cream or
ointment form. The mupirocin cream produced zones of inhibition of
42 and 0, and log reduction of 0.4 and 0.8, against Staphylococcus
aureus and Pseudomonas aeruginosa, respectively.
4TABLE 3 Formulae for Gel Nos. 5-10 and Efficacy Results Silver Log
Log CMC PVA Powder .beta.- Methyl Propyl CZOI CZOI Red'n Red'n #
(%) (%) (%) glucan paraben paraben S. aureus P. aeruginosa S.
aureus P. aeruginosa 5 2 0.1 11 13 1.4 >6 6 2 0.5 0.1 0.1 0.02
14 15 3.3 >6 7 2 0.5 0.1 13 14 2 N/A 8 2 0.5 0.1 0.1 14 14 2 N/A
9 2 0.5 0.1 0.20 14 14 2 N/A 10 2 0.5 0.1 0.5 0.1 0.20 14 14 2
>6
[0200] No. 11
[0201] A commercially available gel (glyceryl polymethacrylate) was
blended with nanocrystalline silver powder of Example 3 to produce
a gel with a silver content of 0.1%. This gel was tested as in Nos.
5-10 and was found to produce zones of 15 mm against both
Staphylococcus aureus and Pseudomonas aeruginosa. Log reductions of
1.7 and >5 were produced against Staphylococcus aureus and
Pseudomonas aeruginosa. This gel product had a greater spectrum of
activity than did mupirocin cream or ointment.
[0202] Testing of the above preparations for antimicrobial effect
was conducted to ensure that the antimicrobial metals, such as the
nanocrystalline silver in these gels, are effectively released.
Example 4
Effects of Antimicrobial Silver on Apoptosis and Matrix
Metalloproteinases in a Porcine Model
[0203] A porcine model was used to examine the effects of an
antimicrobial metal formed with atomic disorder, preferably silver,
on apoptosis and matrix metalloproteinases. Young, commercially
produced, specific pathogen free domestic swine (20-25 kg) were
used in these studies. The animals were conditioned in an animal
facility for one week prior to any experimental manipulation.
Typically, three animals were used in each experiment. The animals
received water and hog ration (Unifeed.TM., Calgary, Alberta)
without antibiotics ad libitum, were housed individually in
suspended stainless steel cages (5'.times.6'), and maintained is a
controlled environment with 12 hours of light per day. The study
was approved by the University of Calgary Animal Care Committee and
was conducted in accordance with guidelines established by the
Canadian Council on Animal Care.
[0204] Antimicrobial silver metal was administered in the form of a
dressing. The dressings included:
[0205] i) AB--nanocrystalline silver-coated dressing (the non-foam,
three-layer dressing as set out in Example 1), comprising two
layers of silver-coated high density polyethylene (HDPE) bonded on
either side of an absorbent rayon/polyester core;
[0206] ii) AgHDPE--nanocrystalline silver coated HDPE layers
aseptically separated from the absorbent core of the AB
dressings;
[0207] iii) Control--identical in construction to the AB dressing
except that the HDPE was not coated with nanocrystalline
silver;
[0208] iv) Gauze--the absorbent rayon/polyester core of the AB
dressings;
[0209] v) cHDPE--the uncoated HDPE aseptically removed from the
absorbent core of the control dressings; and
[0210] vi) SN--sterile piece of the gauze dressing to which 24
.mu.g silver /square inch (from silver nitrate) was added. This
amount of silver is equivalent to the amount of silver released
from a square inch of the AB dressing immersed in serum over a 24
hour period, as determined by atomic absorption analysis.
[0211] Dressings (i)-(iii) were gamma sterilized (25 kGy) prior to
use. All dressings were moistened with sterile water prior to
application to the incision. In some cases, the incisions were
covered with a layer of occlusive polyurethane (Tegaderm.TM., 3M
Corp., Minneapolis, Minn.).
[0212] Three isolates of bacteria were used in the inoculum,
including Pseudomonas aeruginosa, Fusobacterium sp., and
coagulase-negative staphylococci (CNS) (Culture Collection,
University of Calgary, Calgary, Alberta). The bacterial strains
were grown under appropriate conditions overnight prior to the day
of surgery. On the morning of surgery, the organisms were washed
with sterile water and resuspended to a final density of
approximately 10.sup.7 CFU/mL. The bacteria were mixed together in
a ratio of 1:0.5:1 (Pseudomonas:CNS:Fusobacterium) in water.
Sufficient inoculum was prepared to wet the incision created in
each experiment. This procedure resulted in the incisions initially
being evenly contaminated with approximately 8.times.10.sup.4
CFU/cm.sup.2.
[0213] Prior to treatment, animals were sedated by an intramuscular
injection of a mixture of 10 mg/kg ketamine (Ketalean.TM., MTC
Pharmaceuticals, Cambridge, ON) and 0.2 mg/kg acepromazine
(Atravet.TM., Ayerst Laboratories), followed by complete anesthesia
induced by mask inhalation of 1-2% halothane (MTC Pharmaceuticals).
Following induction of anesthesia, the dorsal and lateral thorax
and abdomen of each animal was clipped using a #40 Osler blade and
the skin subsequently scrubbed with a non-antibiotic soap, and
allowed to dry prior to incision.
[0214] Animals typically received 20 full-thickness incisions, 10
on each side of the dorsal thorax. The incisions were created using
a 2 cm diameter trephine. An epinephrine solution was then applied
to the incisions to provide for complete hemostasis prior to
inoculation. The incisions were contaminated by covering them with
gauze sponges soaked with the bacterial inoculum. The wet sponges
were covered with an occlusive barrier and allowed to stand for 15
minutes. In some instances, an incision was then sampled to
determine the initial inoculum. Following any requisite sampling,
the incisions were dressed with the appropriate dressings and
covered with an occlusive layer that was secured with
Elastoplast.TM. tape (Smith & Nephew, Lachine, QC). All animals
received narcotic pain medication (Torbugesic.TM., Ayerst
Laboratories, Montreal, QC, 0.2 mg/kg), as required.
[0215] The experimental and control groups are summarized in Table
4:
5TABLE 4 Experimental and Control Groups Animal # Left Side (Silver
Treatment) Right Side (Controls) Pig 1 Silver nitrate (SN) on gauze
gauze moistened with water Pig 2 AgHDPE dHDPE Pig 3 AB control
[0216] A 2 cm diameter circle of the appropriate dressing was
applied to each incision. For Pig 1, incisions on the left side
were dressed with silver nitrate-moistened (SN) gauze, while
control incisions on the right side received water-moistened gauze
dressing. For Pig 2, the incisions on the left side were dressed
with silver-coated HDPE (AgHDPE), while the control incisions on
the right side received non-coated HDPE (cHDPE). For Pig 3, the
incisions on the left side were dressed with AB dressing, while
incisions on the right side received the vehicle control. For these
experiments, each incision was individually covered with an
occlusive film dressing (Tegaderm.TM., 3M Corp., Minneapolis,
Minn.).
[0217] Each day following incision (up to 5 days), the dressing
materials from each of the experimental and control groups were
collected and pooled within each group. These dressing materials
were then placed in conical centrifuge tube containing glass wool.
The tubes and contents were centrifuged to remove all liquid from
the dressings. The glass wool was then placed into a 5-mL syringe
and pressed to recover the incision fluid from each of the six
sample sets. The incisions were rebandaged in an identical manner
to the original dressing format each time. Incision fluid which
collected under the occlusive dressing was also aspirated and
reserved for analysis. Due to the small volumes collected from each
incision, it was necessary to pool the collected fluid from each of
10 incisions dressed with the same type of dressing. All incision
fluids were stored at -80.degree. C. until analysis.
[0218] Prior to enzyme zymography or activity assays, the protein
concentrations of the incision fluid samples were compared to
ensure that the protein levels in each sample were 10, similar. The
samples were diluted 1:100 in water and assayed using the BCA
Protein Assay System.TM. (Pierce Chemical, Rockford, Ill.). A
standard curve was concurrently constructed using dilutions of
bovine serum albumin. Incision fluid was diluted in water and then
mixed with an equal volume of sample buffer (0.06 M Tris-HCl, pH
6.8; 12% SDS; 10% glycerol; 0.005% bromophenol blue). The samples
were then electrophoresed on 10% polyacrylamide (BioRad,
Mississauga, ON) gels containing 0. 1% gelatin. The proteins were
then incubated in renaturing buffer (2.5% Triton.TM. X-100) for 90
minutes at 37.degree. C. Following enzyme renaturation, the gels
were incubated overnight in substrate buffer (50 mM Tri-HCl, pH
7.8; 5 mM CaCl.sub.2; 200 mM NaCl; 0.02% Brij-35) with or without
10 mM 1,10 phenanthroline. The gels were subsequently stained with
a standard Coomassie Blue stain and destained in methanol/acetic
acid. Unless otherwise indicated, all chemicals were obtained from
Sigma-Aldrich (Oakville, ON).
[0219] The incision fluid samples were assayed for the total amount
of protein present. These values were between 30-80 mg/mL. The
samples from individual animals were even more similar, varying by
only 10-20 mg/mL in the pooled incision fluid.
[0220] i) Assay for Activity of Total MMPs
[0221] The total MMP activity of the incision fluid samples was
determined by incubating diluted incision fluid with a quenched
fluorescein-conjugated substrate (EnzChek DQ gelatin.TM., Molecular
Probes, Eugene, Oreg.) for approximately 20 hours. Following
incubation, the samples were read in a fluorometer (excitation
1=480 nm; emission 1=520 nm). Activity was compared to a
collagenase standard as well as experimental versus control
incision fluids.
[0222] FIG. 1 shows the change in total MMP activity from
differently treated incision sites over a five-day period. The
silver-coated HDPE (AgHDPE) results were essentially identical to
those obtained using the silver-coated dressing (AB). Similarly,
the gauze, non-coated HDPE (cHDPE), and control dressings yielded
results essentially identical to each other and to untreated
incisions under occlusion from which incision fluid was collected.
Only the results from the control, silver-coated dressing (AB),
silver-coated HDPE (AgHDPE), and silver nitrate moistened gauze
(SN) are thus shown. The total MMP activity of the incision fluid
sample from the control dressing was low for the first few days,
then rose dramatically and remained high for the duration of the
experiment. Similarly, the silver-nitrate moistened gauze (SN)
demonstrated an almost identical pattern of total MMP activity.
Results from the silver-coated dressing (AB) yielded dramatically
different results. The level of MMP activity remained steady for
the duration of the experiment and did not spike to high levels.
Instead, it remained at a level roughly 60% lower than the highest
level of activity reached in control or silver-nitrate moistened
gauze (SN).
[0223] ii) Assay for Activity of Gelatinases
[0224] Gelatinases include MMP-2 (secreted by fibroblasts and a
wide variety of other cell types) and MMP-9 (released by
mononuclear phagocytes, neutrophils, corneal epithelial cells,
tumor cells, cytotrophoblasts and keratinocytes). The gelatinases
degrade gelatins (denatured collagens) and collagen type IV
(basement membrane). Zymograms were run to examine changes in the
levels and activity of MMP-9 and MMP-2 over the duration of the
experiment.
[0225] Results of the zymograms for the control and silver nitrate
moistened gauze (SN) appeared to be identical. The levels of MMP-9
declined over the period examined, particularly levels of the
active form of MMP-9. The silver-coated dressing (AB) demonstrated
higher levels of active MMP-9 than for the control. On Day 2, the
silver-coated dressing (AB) showed lower levels of active MMP-9
than in the control. On Day 4, the silver-coated dressing (AB)
showed little active MMP-9. In the control, the amount of the
latent enzyme appeared to decrease while the active form of MMP-9
increased, particularly up to Day 4.
[0226] There was not much difference in the levels of MMP-2
activity for the silver-coated dressing (AB) over the duration of
the experiment. However, there was an increase in the level of
active MMP-2 in the control dressing by Day 5. It was also observed
that the levels of some other, unidentified, gelatinolytic enzymes
also decreased in the silver-coated dressing (AB) compared to the
control.
[0227] iii) Assay of Total Protease Activity
[0228] Since MMPs have proteolytic activity, the total protease
activity in incision fluid samples was assessed by incubating the
samples with 3 mg/mL azocasein in 0.05 M Tris-HCl, pH 7.5 for 24
hours at 37.degree. C. The undigested substrate was then
precipitated by the addition of 20% trichloroacetic acid. The
absorbance of the supernatant was then assessed using a
spectrophotometer, 1=400 nm. The absorbance was compared to a
standard curve prepared with bovine pancreatic trypsin.
[0229] Results paralleled those obtained in the total MMP assay
above. The incision fluid samples for the control and silver
nitrate moistened gauze (SN) demonstrated a pronounced increase in
activity after Day 2 (FIG. 2). Incision fluid from the silver
nitrate moistened gauze (SN) also demonstrated a marked increase in
the total protease activity compared to control dressing incision
fluid (FIG. 1). However, the total protease activity in the
incision fluids of the silver coated dressings (AB) remained
constant over the duration of the experiment.
[0230] Antimicrobial silver was thus demonstrated to be effective
in modulating overall MMP activity. However, silver nitrate was not
effective in modulating MMP activity in spite of the Ag.sup.+
concentration being approximately the same levels as were expected
to be released from the silver-coated dressing (AB) over the same
period of time (24 h) between applications. The reason for the
difference in effects may be related to the inherent nature of the
two silver formulations. In the case of silver nitrate, although
the silver was added to provide a similar final concentration of
Ag.sup.+ as was anticipated to be released from the silver-coated
dressing (AB), the Ag.sup.+ ions were added at once. It would thus
be expected that the serum proteins and chlorides within the
incision fluid would quickly inactivate a large portion of the
Ag.sup.+. In the case of the silver-coated dressing (AB), the
silver is continuously released to maintain a steady-state
equilibrium, maintaining an effective level of silver in the
incision for a prolonged period.
[0231] iv) Apoptosis
[0232] Histological assessment of cell apoptosis was carried out in
order to determine whether the silver-coated dressing (AB) affected
apoptosis within the incision.
[0233] Histological Observations of Porcine Tissue
[0234] Samples of tissue from the incisions were collected daily
for the duration of the experiment, except for Day 1, and examined
for evidence of apoptosis. The samples were fixed in 3.7%
formaldehyde in PBS for 24 hours, then embedded in paraffin, and
cut into 5 mm thick sections. The samples were then de-waxed with
Clearing Solvent.TM. (Stephan's Scientific, Riverdale, N.J.) and
rehydrated through an ethanol:water dilution series. The sections
were treated with 20 mg/mL proteinase K (Qiagen, Germantown, MD) in
10 mM Tris-HCl (pH 7.4) for 30 minutes at room temperature.
[0235] Terminal deoxynucleotidyl transferase nick end labeling
(TUNEL staining) was performed using an In Situ Cell Death
Detection POD Kit.TM. (Boehringer Mannheim, Indianapolis, Ind.).
Using this technique, cells which stain brown are those being
eliminated by apoptosis. Endogenous peroxidase was blocked with 3%
hydrogen peroxide in methanol for 10 minutes at room temperature
then cells were permeabilized with 0.1% Triton.TM. X-1 00 (in 0. 1%
sodium citrate) for 2 minutes on ice. After permeabilization, the
samples were treated with the terminal transferase enzyme solution
incubated in a humidified chamber at 37.degree. C. for 60 minutes.
Following labelling, the samples were washed once with 1.0%
Triton.TM. X-100 and twice with PBS. The sections were incubated
with Converter-POD.TM. (Boehringer Mannheim, Indianapolis, Ind.) in
a humidified chamber at 37.degree. C. for 30 minutes, and repeated
washing with 1.0% Triton.TM. X-100 and PBS. Subsequently, the
samples were incubated with DAB substrate (Vector Laboratory Inc.,
Burlingame, Calif.) for 10 minutes at room temperature and washed
with 1.0% Triton.TM. X-100 and PBS. It was also necessary to
counterstain the sections with hematoxylin nuclear counterstain
(Vector Laboratory Inc., Burlingame, Calif.) for 10 seconds.
[0236] The prepared samples were then ready to be observed by light
microscopy for evidence of apoptosis. For a positive control, the
permeabilized sections were treated with 100 mg /mL DNase I in PBS
for 10 minutes at room temperature to induce DNA strand breaks. For
negative controls, the terminal transferase enzyme, POD or DAB were
omitted between each labelling step.
[0237] In all samples examined, there was little difference between
the control and silver nitrate moistened gauze (SN). However,
significant apoptosis of the cell population was observed in
incisions of the silver-coated dressing (AB). In the control
incision, there were significant numbers of polymorphonuclear
leukocytes (PMNs) and few fibroblasts, while in incisions of the
silver-coated dressing (AB), there were significantly more
fibroblasts and few PMNs.
[0238] Histopathological Scoring of Porcine Tissue
[0239] Animals were anesthetized as described above of Days 1, 4,
and 7. A mid-incision biopsy was collected with a sterile 4 mm
biopsy punch. The tissue was fixed in 10% neutral buffered
formalin, embedded in methacrylate and sectioned (2-5 mm thick).
The sections were stained with Lee's methylene blue and basic
fuschin to demonstrate the cellular organization and bacteria. A
pathologist blinded to the treatments scored the sections based on
the presence of fibroblasts, PMNs and bacteria as follows:
0=absent; +=occasional with 1-5 per high power field of view;
++=moderate with 6-20 per high power field of view; +++=-abundant
with 21-50per high power field of view; ++++=very abundant with
more than 50 per high power field of view (Table 5).
6TABLE 5 Histopathological Scoring of Porcine Tissue Collected on
Days 1, 4 and 7 Day Post- incision Dressing Fibroblasts PMNs
Bacteria 1 Silver-coated (AB) ++ ++ + 1 Control 0 +++ ++++ 4
Silver-coated (AB) ++++ ++ 0 4 Control + ++++ ++++ 7 Silver-coated
(AB) ++++ + 0 7 Control +++ +++ +++
[0240] The microscopic observation of the biopsy samples revealed
that the infiltrating cell types were significantly different
between the control and silver-coated dressings (AB). The control
incisions were characterized by a large numbers of PMNs, while the
silver-coated dressings (AB) demonstrated a larger proportion of
fibroblasts and monocytes. The relative abundance of the
fibroblasts in incisions of the silver-coated dressings (AB) became
increasingly pronounced through to Day 7, as compared to the
control incisions that remained populated largely by PMNs and
monocytes. The staining method enabled staining also of bacteria,
which was abundant in the control incision but generally absent in
the incisions of the silver-coated dressings (AB).
[0241] Incisions treated with the nanocrystalline antimicrobial
silver thus demonstrated more extensive apoptosis than did cells
from incisions treated with either control or silver nitrate
dressings. During the first two days following incision, the cell
type which demonstrated the most pronounced increase in apoptosis
were neutrophils. This suggests that part of the reason for the
moderated neutrophil presence and the resultant modulation of MMP
levels was due to neutrophil apoptosis. It has been shown that the
number of apoptotic cells increases as the incision closes and that
this is part of the mechanism involved in the decrease in
cellularity of the maturing scar tissue (Desmouliere, A., Badid,
C., Bochaton-Piallat, M. and Gabbiani, G. (1997) Apoptosis during
wound healing, fibrocontractive diseases and vascular wall injury.
Int. J. Biochem. Cell Biol. 29: 19-30.). The results suggest that
the maturing of the nascent dermal and epidermal tissues may also
be accelerated in the presence of the nanocrystalline antimicrobial
metals. The findings indicated that acceleration in healing induced
by the nanocrystalline antimicrobial metals is associated with a
reduction of local MMP activity, as well as with an increased
incidence of cell apoptosis within the incision.
[0242] All publications mentioned in this specification are
indicative of the level of skill of those skilled in the art to
which this invention pertains. All publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0243] The terms and expressions in this specification are, unless
otherwise specifically defined herein, used as terms of description
and not of limitation. There is no intention, in using such terms
and expressions, of excluding equivalents of the features
illustrated and described, it being recognized that the scope of
the invention is defined and limited only by the claims which
follow.
* * * * *