| United States Patent Application |
20030194444
|
| Kind Code
|
A1
|
|
Burrell, Robert E.
; et al.
|
October 16, 2003
|
Methods of treating skin and integument conditions
Abstract
Methods of treating skin and integument conditions, particularly with
metal-containing compounds, are disclosed. The metal-containing material
can be, for example, an antimicrobial material, an antibacterial
material, an anti-inflammatory material, an anti-fungal material, an
anti-viral material, an anti-cancer material, a pro-apoptosis material,
and/or an MMP modulating material. In certain embodiments, the
metal-containing material is an atomically disordered, silver-containing
material.
| Inventors: |
Burrell, Robert E.; (Alberta, CA)
; Gillis, Scott H.; (Concord, MA)
; Schechter, Paul; (Dover, MA)
; Wright, John B.; (San Antonio, TX)
; Lam, Kan; (San Antonio, TX)
; Yin, Hua Qing; (Alberta, CA)
|
| Correspondence Name and Address:
|
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
| Serial No.:
|
277362 |
| Series Code:
|
10
|
| Filed:
|
October 22, 2002 |
| U.S. Current Class: |
424/618; 424/647; 424/649 |
| U.S. Class at Publication: |
424/618; 424/647; 424/649 |
| Intern'l Class: |
A61K 033/38; A61K 033/26; A61K 033/24 |
Claims
What is claimed is:
1. A method of treating a subject having a condition selected from skin
conditions and integument conditions, comprising: contacting an area of
the subject having the condition with an atomically disordered,
nanocrystalline metal-containing compound.
2. The method of claim 1, wherein the condition is a hyperproliferative
skin condition.
3. The method of claim 2, wherein the hyperproliferative skin condition is
selected from the group consisting of psoriasis, Reiter's syndrome,
pityriasis rubra pilaris, hyperpigmentation, vitiligo and a
hyperproliferative variant of the disorders of keratinization.
4. The method of claim 1, wherein the condition is an inflammatory skin
condition.
5. The method of claim 4, wherein the inflammatory skin condition is
selected from the group consisting of eczema, erythroderma, an insect
bite, mycosis fungoides, pyoderma gangrenosum, eythrema multiforme,
rosacea, onychomyocosis, acne.
6. The method of claim 1, wherein skin condition is selected from the
group consisting of bacterial conditions, microbial conditions,
inflammatory conditions, fungal conditions, viral conditions, autoimmune
conditions, idiopathic conditions, noncancerous growths and cancerous
skin conditions.
7. The method of claim 1, wherein the metal-containing compound is
selected from the group consisting of metals and alloys.
8. The method of claim 1, wherein the metal-containing compound is
selected from the group consisting of metal oxides, metal nitrides, metal
borides, metal halides and metal hydrides.
9. The method of claim 1, wherein the metal-containing compound comprises
a metal selected from the group consisting of silver, gold, platinum and
palladium.
10. The method of claim 1, wherein the metal-containing compound comprises
silver.
11. The method of claim 1, wherein the metal-containing compound comprises
an ionic compound.
12. The method of claim 1, wherein the metal-containing compound comprises
atoms, molecules or clusters.
13. The method of claim 1, wherein the metal-containing compound comprises
an antimicrobial compound.
14. The method of claim 1, wherein, when contacted with the area of skin
having the skin condition, the metal-containing compound is in a
solution.
15. The method of claim 14, wherein the solution contains at least about
0.001 weight percent of the metal-containing compound.
16. The method of claim 15, wherein the solution contains about 10 weight
percent or less of the metal-containing compound.
17. The method of claim 14, wherein the solution is injected.
18. The method of claim 17, wherein the solution is injected via a
needleless injector.
19. The method of claim 17, wherein the solution is injected via a needle.
20. The method of claim 14, wherein the solution further comprises a
solvent.
21. The method of claim 1, wherein, when contacted with the area of skin
having the skin condition, the metal-containing compound is disposed in a
pharmaceutically acceptable carrier.
22. The method of claim 21, wherein the composition contains at least
about 0.01 weight percent of the metal-containing compound.
23. The method of claim 22, wherein the composition contains about 50
weight percent or less of the metal-containing compound.
24. The method of claim 21, wherein the pharmaceutically acceptable
carrier is selected from the group consisting of creams, ointments, gels,
lotions, pastes and foams.
25. The method of claim 1, wherein, when contacted with the area of skin
having the skin condition, the metal-containing compound is in the form
of a free-standing powder.
26. The method of claim 25, wherein the free-standing powder is inhaled.
27. The method of claim 25, wherein the free-standing powder is injected.
28. The method of claim 1, wherein the condition is selected from the
group consisting of a burn, eczema, erythroderma, an insect bite, mycosis
fungoides, pyoderma gangrenosum, eythrema multiforme, rosacea,
onychomyocosis, acne, psoriasis, Reiter's syndrome, pityriasis rubra
pilaris, hyperpigmentation, vitiligo, hypertropic scarring, keloid,
lichen plainus, age-related skin disorders and hyperproliferative
variants of the disorders of keratinization.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part and claims the benefit
of priority under 35 U.S.C. .sctn.120 of: U.S. patent application Ser.
No. 09/628,735, filed Jul. 27, 2000, and entitled "Treatment of
Hyperproliferative Skin Disorders and Diseases;" U.S. patent application
Ser. No. 09/916,757, filed Jul. 27, 2001, and entitled "Treatment of
Hyperproliferative Skin Disorders and Diseases;" U.S. patent application
Ser. No. 09/840,637, filed Apr. 23, 2001, and entitled "Treatment of
Acne;" U.S. Provisional Patent Application Serial No. 60/285,884, filed
Apr. 23, 2001, and entitled "Therapeutic Treatments Using the Direct
Application of Noble Metal Compositions;" U.S. patent application Ser.
No. 10/131,512, filed Apr. 23, 2002, and entitled "Therapeutic Treatments
Using the Direct Application of Noble Metal Compositions;" U.S. patent
application Ser. No. 10/131,509, filed Apr. 23, 2002, and entitled
"Treatment of Mucosal Membranes;" U.S. patent application Ser. No.
10/131,511, filed Apr. 23, 2002, and entitled "Treatment of Inflammatory
Skin Conditions;" U.S. patent application Ser. No. 10/131,568, filed Apr.
23, 2002, and entitled "Method of Induction of Apoptosis and Inhibition
of Matrix Metalloproteinases Using Antimicrobial Metals;" and U.S. patent
application Ser. No. 10/159,587, filed May 30, 2002, and entitled "Method
of Induction of Apoptosis and Inhibition of Matrix Metalloproteinases
Using Antimicrobial Metals." Each of these applications is incorporated
by reference.
TECHNICAL FIELD
[0002] The invention relates to methods of treating skin and integument
conditions, particularly with metal-containing compounds.
BACKGROUND
[0003] It is generally desirable to treat a subject (e.g., a human) that
has an undesirable condition. Many different compositions have been
developed to treat undesirable conditions. For example, certain forms of
silver have been reported to be effective in treating some undesirable
skin conditions.
SUMMARY
[0004] The invention relates to methods of treating skin and integument
conditions, particularly with metal-containing compounds.
[0005] In one aspect, the invention features a method of treating a
subject having a skin or an integument condition. The method includes
contacting an area of the subject having the condition with an atomically
disordered, nanocrystalline metal-containing compound.
[0006] Embodiments of the methods can include one or more of the following
features.
[0007] The condition can be, for example, a hyperproliferative skin
condition or an inflammatory skin condition.
[0008] Examples of hyperproliferative skin conditions include psoriasis,
Reiter's syndrome, pityriasis rubra pilaris, hyperpigmentation, vitiligo
and a hyperproliferative variant of the disorders of keratinization.
[0009] Examples of inflammatory skin conditions include eczema,
erythroderma, an insect bite, mycosis fungoides, pyoderma gangrenosum,
eythrema multiforme, rosacea, and acne.
[0010] An example of an integument condition is onychomyocosis.
[0011] The condition can be a bacterial condition, a microbial condition,
an inflammatory condition, a fungal condition, a viral condition, an
autoimmune condition, an idiopathic condition, a noncancerous growth
and/or a cancerous condition.
[0012] The metal-containing compound can be a metal or an alloy.
[0013] The metal-containing compound can be, for example, a metal oxide, a
metal nitride, a metal boride, a metal halide or a metal hydride.
[0014] The metal-containing compound can contain, for example, silver,
gold, platinum and/or palladium.
[0015] The metal-containing compound can be an ionic compound.
[0016] The metal-containing compound can be in the form of atoms,
molecules and/or clusters.
[0017] The metal-containing compound can be an antimicrobial compound.
[0018] The metal-containing compound can be in a solution when contacted
with the area of skin having the skin condition. The solution can contain
at least about 0.001 weight percent of the metal-containing compound,
and/or up to about 10 weight percent or less of the metal-containing
compound. The solution can be injected. The solution can be injected, for
example, via a needleless injector or a needle. The solution can include
a solvent.
[0019] The metal-containing compound can be disposed in a pharmaceutically
acceptable carrier when contacted with the area of skin having the skin
condition. The composition can contain, for example, at least about 0.01
weight percent of the metal-containing compound, and/or up to about 50
weight percent or less of the metal-containing compound. The
pharmaceutically acceptable carrier can be, for example, a cream, an
ointment, a gel, a lotion, a paste and/or a foam.
[0020] The metal-containing compound can be in the form of a free-standing
powder when contacted with the area of skin having the skin condition.
The free-standing powder can be, for example, inhaled and/or injected.
[0021] The methods can include monitoring a subject after contacting the
subject with the metal-containing material. For example, a subject can be
monitored at relatively regular intervals (e.g., about once an hour,
about once every eight hours, about once a day, about once a week, about
two times a month, about three times a month, about four times a month).
[0022] Other features and advantages of the methods will be apparent from
the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a schematic view of a deposition system;
[0024] FIG. 2 is a graph showing the efficacy of different forms of silver
on erythema;
[0025] FIG. 3 is a graph showing the efficacy of different forms of silver
on edema;
[0026] FIG. 4 is a graph showing MMP activity of incision fluids recovered
from incisions dressed with materials;
[0027] FIG. 5 is a graph showing total protease activity of incision
fluids recovered from different dressings;
[0028] FIG. 6 is a graph showing the concentrations (ng/ml) of active
MMP-9 in fluid samples recovered from ulcers dressed with different
materials;
[0029] FIG. 7 is a graph showing the concentrations (ng/ml) of active
MMP-2 in fluid samples recovered from ulcers dressed with different
materials;
[0030] FIG. 8 is a graph showing the concentrations (pg/ml) of active
TNF-.alpha. in fluid samples recovered from ulcers dressed with different
materials; and
[0031] FIG. 9 is a graph showing the concentrations (pg/ml) of active
IL-1.beta. in fluid samples recovered from ulcers dressed with different
materials.
DETAILED DESCRIPTION
[0032] The inventors have discovered that certain metal-containing
materials (e.g., antimicrobial, atomically disordered, nanocrystalline
silver-containing materials) can be used to treat a subject with a
condition by contacting an area of the subject having the condition with
the metal-containing material. As explained below, the metal-containing
material can be in any of a variety of forms when delivered to a subject,
and the metal-containing material can be delivered to a subject in a
variety of ways. As also explained below, the metal-containing material
can be used to treat various subjects, conditions, and condition
locations.
[0033] Without wishing to be bound by theory, it is believed that the
therapeutic properties of the metal-containing materials may be explained
by one or more potential mechanisms. In one potential mechanism, it is
believed that the metal-containing material (e.g., antimicrobial,
atomically disordered, nanocrystalline silver-containing materials) forms
one or more metastable, relatively high level metal hydroxide species
(e.g., Ag(OH).sub.4.sup.3-, Ag(OH).sub.6.sup.3-) that either directly or
indirectly (e.g., via the formation of one or more biological mediators)
provide the observed therapeutic properties. In another potential
mechanism, it is believed that the metal-containing material is capable
of releasing clusters of the metal (e.g., clusters of Ag.sup.0, clusters
of Ag.sup.+, clusters containing both Ag.sup.+ and Ag.sup.0) that provide
the observed therapeutic properties. It is believed that combinations of
potential mechanisms may result in the observed therapeutic effect of the
metal-containing material.
[0034] In general, clusters refer to relatively small groups of atoms,
ions or the like. For example, a cluster can contain at least two (e.g.,
at least three, at least four, at least five, at least six, at least
seven, at least eight, at least nine, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 20, at least 30, at
least 40, at least 50, at least 60, at least 70, at least 80, at least
90) atoms, ions or the like, and/or at most 1,000 (e.g., at most 900, at
most 800, at most 700, at most 600, at most 500, at most 400, at most
300, at most 200, at most 100) atoms, ions or the like. Clusters are
described, for example, in R. P. Andres et al., "Research Opportunities
on Cluster and Cluster-Assembled Materials", J. Mater. Res. Vol. 4, No 3,
1989, p. 704. In certain embodiments, a cluster (e.g., a cluster
containing silver) can contain less than the 14 atoms and have a normal
face centered cubic crystal lattice.
[0035] Materials
[0036] The metal-containing material can be an ionic material or a
non-ionic material. The metal-containing material can be, for example, an
atom, a molecule, or a cluster.
[0037] In general, the metal-containing material is a metal or an alloy.
Examples of metals that can be contained in metal-containing materials
include Group I A metals, Group II A metals, Group III A metals, Group IV
A metals, Group V A metals, Group VI A metals, Group VII A metals, Group
VIII A metals, Group I B metals, Group II B metals, members of the
lanthanide metal series, and members of the actinide metal series. In
certain embodiments, metal-containing materials contain silver, gold,
platinum, palladium, iridium, zinc, copper, tin, antimony, and/or
bismuth. Examples of silver-containing metals include
colloidal silver,
silver nitrate and silver sulfadiazine.
[0038] In addition to one or more metals, a metal-containing material can
contain oxygen, nitrogen, carbon, boron, sulfur, a halogen (e.g.,
fluorine, chlorine, bromine, iodine) and/or hydrogen. Examples of such
metal-containing materials include metal oxides, metal nitrides, metal
carbides, metal borides, metal sulfides, metal halides (e.g., metal
fluorides, metal chlorides, metal bromides, metal iodides) and metal
hydrides. In certain embodiments, a metal-containing material contains at
least about one atomic percent (e.g., at least about three atomic
percent, at least about five atomic percent) and/or at most about 20
atomic percent (e.g., at most about 15 atomic percent, at most about 12
atomic percent, at most about 10 atomic percent) of nonmetallic elements.
For example, in some embodiments, a silver-containing material can
contain oxygen in an amount from about five atomic percent to about 20
atomic percent (e.g., from about five atomic percent to about 15 atomic
percent, from about eight atomic percent to about 12 atomic percent).
[0039] In certain embodiments, the metal-containing materials are an
antimicrobial material, an atomically disordered crystalline material,
and/or a nanocrystalline material.
[0040] As used herein, an antimicrobial material herein refers to a
material that has sufficient antimicrobial activity to have a beneficial
therapeutic effect. In certain embodiments, an antimicrobial material has
a corrected zone of inhibition ("CZOI") of at least about two millimeters
(e.g., at least about three millimeters, at least about four millimeters,
at lest about five millimeters, at least about six millimeters, at least
about seven millimeters, at least about eight millimeters, at least about
nine millimeters, at least about 10 millimeters). The CZOI of a material
is determined as follows. The material is formed as a coating on a
dressing (see discussion below). Basal medium Eagle (BME) with Earle's
salts and L-glutamine is modified with calf/serum (10%) and 1.5% agar
prior to being dispensed (15 ml) into Petri dishes. The agar containing
Petri dishes are allowed to surface dry prior to being inoculated with a
lawn of Staphylococcus aureus ATCC #25923. The inoculant is prepared from
Bactrol Discs (Difco, M.) which are reconstituted as per the
manufacturer's directions. Immediately after inoculation, the coatings to
be tested are placed on the surface of the agar. The dishes are incubated
for 24 hours at 37.degree. C. After this incubation period, the zone of
inhibition ("ZOI") is measured and the CZOI is calculated as the ZOI
minus the diameter of the test material in contact with the agar. It is
to be noted that, while this test for antimicrobial properties is
performed on materials that are in the form of a coating on a substrate
(e.g., in the form of a dressing), antimicrobial materials are not
limited to materials that are coated on a substrate. Rather, a material
in any form may be antimicrobial, but it is in the form of a coating on a
substrate (e.g., in the form of a dressing) when its antimicrobial
properties are tested according to the procedure described herein.
[0041] As referred to herein, an atomically disordered, crystalline
material (e.g., an atomically disordered, nanocrystalline material) means
a material that has more long range ordered, crystalline structure (a
lesser degree of defects) than the material has in a fully amorphous
state, but that also has less long range, ordered crystalline structure
(a higher degree of defects) than the material has in a bulk crystalline
state, such as in the form of a cast, wrought or plated material.
Examples of defects include point defects, vacancies, line defects, grain
boundaries, subgrain boundaries and amorphous regions. Point defects are
defects on a size scale of no more than about four atomic spacings. A
vacancy is the omission of an atom from its regular atomic site in the
crystal lattice. Line defects are defective regions (e.g., edge
dislocations, screw dislocations) that result in lattice distortions
along a line (which may or may not be a straight line), and generally
have a longer scale than point defects. In an edge dislocation, a lattice
displacement is produced by a plane of atoms that forms a terminus of the
lattice. In a screw dislocation, part of the lattice is displaced with
respect to an adjacent part of the lattice. Grain boundaries separate
regions having different crystallographic orientation or misorientation
(e.g., high angle grain boundaries, low angle grain boundaries, including
tilt boundaries and twist boundaries). Subgrain boundaries refer to low
angle grain boundaries. An amorphous region is a region that does not
exhibit long range, ordered crystalline structure. In certain
embodiments, an atomically disordered, crystalline material (e.g., an
atomically disordered, nanocrystalline material) has a degree of atomic
disorder that is about the same as the degree of atomic disorder of the
nanocrystalline silver coating of a member of the Acticoat.RTM. family of
dressings (Smith & Nephew, Hull, UK) (e.g., an Acticoat.RTM. dressing, an
Acticoat7.RTM. dressing, an Acticoat.RTM. moisture coating dressing, an
Acticoat.RTM. absorbent dressings). In some embodiments, an atomically
disordered, crystalline material (e.g., an atomically disordered,
nanocrystalline material) has a degree of atomic disorder that is about
the same as the degree of atomic disorder of the nanocrystalline silver
coatings having a CZOI of at least five millimeters that are disclosed in
the examples of Burrell et al., U.S. Pat. No. 5,958,440. In certain
embodiments, an atomically disordered, crystalline material (e.g., an
atomically disordered, nanocrystalline material), when contacted with an
alcohol or water-based electrolyte, is released into the alcohol or
water-based electrolyte (e.g., as ions, atoms, molecules and/or clusters)
over a time scale of at least about one hour (e.g., at least about two
hours, at least about 10 hours, at least about a day). Examples of
alcohols and/or water-based electrolytes include body fluids (e.g.,
blood, urine, saliva) and body tissue (e.g., skin, muscle, bone).
[0042] As referred to herein, a nanocrystalline material is a single-phase
polycrystal or a multi-phase polycrystal having a maximum dimension of
about 100 nanometers or less (e.g., about 90 nanometers or less, about 80
nanometers or less, about 70 nanometers or less, about 60 nanometers or
less, about 50 nanometers or less, about 40 nanometers or less, about 30
nanometers or less, about 25 nanometers or less) in at least one
dimension.
[0043] Examples of antimicrobial metal-containing materials (which may or
may not also be an atomically disordered crystalline material or a
nanocrystalline material) include antimicrobial silver-containing
materials (e.g., antimicrobial silver, antimicrobial silver alloys,
antimicrobial silver oxides, antimicrobial silver carbides, antimicrobial
silver nitrides, antimicrobial silver borides, antimicrobial silver
sulfides, antimicrobial silver halides, antimicrobial silver hydrides),
antimicrobial gold-containing materials (e.g., antimicrobial gold,
antimicrobial gold alloys, antimicrobial gold oxides, antimicrobial gold
carbides, antimicrobial gold nitrides, antimicrobial gold borides,
antimicrobial gold sulfides, antimicrobial gold halides, antimicrobial
gold hydrides), antimicrobial platinum-containing materials (e.g.,
antimicrobial platinum, antimicrobial platinum alloys, antimicrobial
platinum oxides, antimicrobial platinum carbides, antimicrobial platinum
nitrides, antimicrobial platinum borides, antimicrobial platinum
sulfides, antimicrobial platinum halides, antimicrobial platinum
hydrides), antimicrobial palladium-containing materials (e.g.,
antimicrobial palladium, antimicrobial palladium alloys, antimicrobial
palladium oxides, antimicrobial palladium carbides, antimicrobial
palladium nitrides, antimicrobial palladium borides, antimicrobial
palladium sulfides, antimicrobial palladium halides, antimicrobial
palladium hydrides), antimicrobial iridium-containing materials (e.g.,
antimicrobial iridium, antimicrobial iridium alloys, antimicrobial
iridium oxides, antimicrobial iridium carbides, antimicrobial iridium
nitrides, antimicrobial iridium borides, antimicrobial iridium sulfides,
antimicrobial iridium halides, antimicrobial iridium hydrides),
antimicrobial zinc-containing materials (e.g., antimicrobial zinc,
antimicrobial zinc alloys, antimicrobial zinc oxides, antimicrobial zinc
carbides, antimicrobial zinc nitrides, antimicrobial zinc borides,
antimicrobial zinc sulfides, antimicrobial zinc halides, antimicrobial
zinc hydrides), antimicrobial copper-containing materials (e.g.,
antimicrobial copper, antimicrobial copper alloys, antimicrobial copper
oxides, antimicrobial copper carbides, antimicrobial copper nitrides,
antimicrobial copper borides, antimicrobial copper sulfides,
antimicrobial copper halides, antimicrobial copper hydrides),
antimicrobial tin-containing materials (e.g., antimicrobial tin,
antimicrobial tin alloys, antimicrobial tin oxides, antimicrobial tin
carbides, antimicrobial tin nitrides, antimicrobial tin borides,
antimicrobial tin sulfides, antimicrobial tin halides, antimicrobial tin
hydrides), antimicrobial antimony-containing materials (e.g.,
antimicrobial antimony, antimicrobial antimony alloys, antimicrobial
antimony oxides, antimicrobial antimony carbides, antimicrobial antimony
nitrides, antimicrobial antimony borides, antimicrobial antimony
sulfides, antimicrobial antimony halides, antimicrobial antimony
hydrides), antimicrobial bismuth containing materials (e.g.,
antimicrobial bismuth, antimicrobial bismuth alloys, antimicrobial
bismuth oxides, antimicrobial bismuth carbides, antimicrobial bismuth
nitrides, antimicrobial bismuth borides, antimicrobial bismuth sulfides,
antimicrobial bismuth halides, antimicrobial antimony hydrides).
[0044] Examples of nanocrystalline metal-containing materials (which may
or may not also be an antimicrobial material or an atomically disordered
crystalline material) include nanocrystalline silver-containing materials
(e.g., nanocrystalline silver, nanocrystalline silver alloys,
nanocrystalline silver oxides, nanocrystalline silver carbides,
nanocrystalline silver nitrides, nanocrystalline silver borides,
nanocrystalline silver sulfides, nanocrystalline silver halides,
nanocrystalline silver hydrides), nanocrystalline gold-containing
materials (e.g., nanocrystalline gold, nanocrystalline gold alloys,
nanocrystalline gold oxides, nanocrystalline gold carbides,
nanocrystalline gold nitrides, nanocrystalline gold borides,
nanocrystalline gold sulfides, nanocrystalline gold halides,
nanocrystalline gold hydrides), nanocrystalline platinum-containing
materials (e.g., nanocrystalline platinum, nanocrystalline platinum
alloys, nanocrystalline platinum oxides, nanocrystalline platinum
carbides, nanocrystalline platinum nitrides, nanocrystalline platinum
borides, nanocrystalline platinum sulfides, nanocrystalline platinum
halides, nanocrystalline platinum hydrides), nanocrystalline
palladium-containing materials (e.g., nanocrystalline palladium,
nanocrystalline palladium alloys, nanocrystalline palladium oxides,
nanocrystalline palladium carbides, nanocrystalline palladium nitrides,
nanocrystalline palladium borides, nanocrystalline palladium sulfides,
nanocrystalline palladium halides, nanocrystalline palladium hydrides),
nanocrystalline iridium-containing materials (e.g., nanocrystalline
iridium, nanocrystalline iridium alloys, nanocrystalline iridium oxides,
nanocrystalline iridium carbides, nanocrystalline iridium nitrides,
nanocrystalline iridium borides, nanocrystalline iridium sulfides,
nanocrystalline iridium halides, nanocrystalline iridium hydrides),
nanocrystalline zinc-containing materials (e.g., nanocrystalline zinc,
nanocrystalline zinc alloys, nanocrystalline zinc oxides, nanocrystalline
zinc carbides, nanocrystalline zinc nitrides, nanocrystalline zinc
borides, nanocrystalline zinc sulfides, nanocrystalline zinc halides,
nanocrystalline zinc hydrides), nanocrystalline copper-containing
materials (e.g., nanocrystalline copper, nanocrystalline copper alloys,
nanocrystalline copper oxides, nanocrystalline copper carbides,
nanocrystalline copper nitrides, nanocrystalline copper borides,
nanocrystalline copper sulfides, nanocrystalline copper halides,
nanocrystalline copper hydrides), nanocrystalline tin-containing
materials (e.g., nanocrystalline tin, nanocrystalline tin alloys,
nanocrystalline tin oxides, nanocrystalline tin carbides, nanocrystalline
tin nitrides, nanocrystalline tin borides, nanocrystalline tin sulfides,
nanocrystalline tin halides, nanocrystalline tin hydrides),
nanocrystalline antimony-containing materials (e.g., nanocrystalline
antimony, nanocrystalline antimony alloys, nanocrystalline antimony
oxides, nanocrystalline antimony carbides, nanocrystalline antimony
nitrides, nanocrystalline antimony borides, nanocrystalline antimony
sulfides, nanocrystalline antimony halides, nanocrystalline antimony
hydrides), nanocrystalline bismuth containing materials (e.g.,
nanocrystalline bismuth, nanocrystalline bismuth alloys, nanocrystalline
bismuth oxides, nanocrystalline bismuth carbides, nanocrystalline bismuth
nitrides, nanocrystalline bismuth borides, nanocrystalline bismuth
sulfides, nanocrystalline bismuth halides, nanocrystalline antimony
hydrides).
[0045] Examples of atomically disordered, crystalline metal-containing
material (which may or may not also be an antimicrobial material or a
nanocrystalline material) include atomically disordered, crystalline
silver-containing materials (e.g., atomically disordered, crystalline
silver; atomically disordered, crystalline silver alloys; atomically
disordered, crystalline silver oxides; atomically disordered, crystalline
silver carbides; atomically disordered, crystalline silver nitrides;
atomically disordered, crystalline silver borides; atomically disordered,
crystalline silver sulfides; atomically disordered, crystalline silver
halides; atomically disordered, crystalline silver hydrides), atomically
disordered, crystalline gold-containing materials (atomically disordered,
crystalline gold; atomically disordered, crystalline gold alloys;
atomically disordered, crystalline gold oxides; atomically disordered,
crystalline gold carbides; atomically disordered, crystalline gold
nitrides; atomically disordered, crystalline gold borides; atomically
disordered, crystalline gold sulfides; atomically disordered, crystalline
gold halides; atomically disordered, crystalline gold hydrides),
atomically disordered, crystalline platinum-containing materials (e.g.,
atomically disordered, crystalline platinum; atomically disordered,
crystalline platinum alloys; atomically disordered, crystalline platinum
oxides; atomically disordered, crystalline platinum carbides; atomically
disordered, crystalline platinum nitrides; atomically disordered,
crystalline platinum borides; atomically disordered, crystalline platinum
sulfides; atomically disordered, crystalline platinum halides; atomically
disordered, crystalline platinum hydrides), atomically disordered,
crystalline palladium-containing materials (e.g., atomically disordered,
crystalline palladium; atomically disordered, crystalline palladium
alloys; atomically disordered, crystalline palladium oxides; atomically
disordered, crystalline palladium carbides; atomically disordered,
crystalline palladium nitrides; atomically disordered, crystalline
palladium borides; atomically disordered, crystalline palladium sulfides;
atomically disordered, crystalline palladium halides; atomically
disordered, crystalline palladium hydrides), atomically disordered,
crystalline iridium-containing materials (e.g., atomically disordered,
crystalline iridium; atomically disordered, crystalline iridium alloys;
atomically disordered, crystalline iridium oxides; atomically disordered,
crystalline iridium carbides; atomically disordered, crystalline iridium
nitrides; atomically disordered, crystalline iridium borides; atomically
disordered, crystalline iridium sulfides; atomically disordered,
crystalline iridium halides; atomically disordered, crystalline iridium
hydrides), atomically disordered, crystalline zinc-containing materials
(e.g., atomically disordered, crystalline zinc; atomically disordered,
crystalline zinc alloys; atomically disordered, crystalline zinc oxides;
atomically disordered, crystalline zinc carbides; atomically disordered,
crystalline zinc nitrides; atomically disordered, crystalline zinc
borides; atomically disordered, crystalline zinc sulfides; atomically
disordered, crystalline zinc halides; atomically disordered, crystalline
zinc hydrides), atomically disordered, crystalline copper-containing
materials (e.g., atomically disordered, crystalline copper; atomically
disordered, crystalline copper alloys; atomically disordered, crystalline
copper oxides; atomically disordered, crystalline copper carbides;
atomically disordered, crystalline copper nitrides; atomically
disordered, crystalline copper borides; atomically disordered,
crystalline copper sulfides; atomically disordered, crystalline copper
halides; atomically disordered, crystalline copper hydrides), atomically
disordered, crystalline tin-containing materials (e.g., atomically
disordered, crystalline tin; atomically disordered, crystalline tin
alloys; atomically disordered, crystalline tin oxides; atomically
disordered, crystalline tin carbides; atomically disordered, crystalline
tin nitrides; atomically disordered, crystalline tin borides; atomically
disordered, crystalline tin sulfides; atomically disordered, crystalline
tin halides; atomically disordered, crystalline tin hydrides), atomically
disordered, crystalline antimony-containing materials (e.g., atomically
disordered, crystalline antimony; atomically disordered, crystalline
antimony alloys; atomically disordered, crystalline antimony oxides;
atomically disordered, crystalline antimony carbides; atomically
disordered, crystalline antimony nitrides; atomically disordered,
crystalline antimony borides; atomically disordered, crystalline antimony
sulfides; atomically disordered, crystalline antimony halides; atomically
disordered, crystalline antimony hydrides), atomically disordered,
crystalline bismuth-containing materials (e.g., atomically disordered,
crystalline bismuth; atomically disordered, crystalline bismuth alloys;
atomically disordered, crystalline bismuth oxides; atomically disordered,
crystalline bismuth carbides; atomically disordered, crystalline bismuth
nitrides; atomically disordered, crystalline bismuth borides; atomically
disordered, crystalline bismuth sulfides; atomically disordered,
crystalline bismuth halides; atomically disordered, crystalline bismuth
hydrides).
[0046] Subjects
[0047] The metal-containing material can be used to treat, for example a
human or an animal (e.g., a dog, a cat, a horse, a bird, a reptile, an
amphibian, a fish, a turtle, a guinea pig, a hamster, a rodent, a cow, a
pig, a goat, a primate, a monkey, a chicken, a turkey, a buffalo, an
ostrich, a sheep, a llama).
[0048] Conditions and Condition Locations
[0049] The conditions that can be treated with the metal-containing
material include, for example, bacterial conditions, microbial
conditions, inflammatory conditions, fungal conditions, viral conditions,
autoimmune conditions, idiopathic conditions, noncancerous growths and/or
cancerous conditions (e.g., tumorous conditions, hematologic
malignancies). Such conditions can be associated with, for example, one
or more prions, parasites, fungi, viruses and/or bacteria. In general,
the location of the condition to be treated corresponds to the type of
condition to be treated.
[0050] In some embodiments, the condition can be a skin condition or a
integument condition (e.g., a microbial skin condition, an inflammatory
skin condition, a fungal skin condition, a viral skin condition, an
autoimmune skin condition, an idiopathic skin condition, a cancerous skin
condition, a microbial integument condition, an inflammatory integument
condition, a fungal integument condition, a viral integument condition,
an autoimmune integument condition, an idiopathic integument condition, a
cancerous integument condition). Examples of skin conditions or
integument conditions include bums, eczema (e.g., atopic eczema,
acrodermatitis continua, contact allergic dermatitis, contact irritant
dermatitis, dyshodrotic eczema, pompholyx, lichen simplex chronicus,
nummular eczema, seborrheic dermatitis, stasis eczema), erythroderma,
insect bites, mycosis fungoides, pyoderma gangrenosum, eythrema
multiforme, rosacea, onychomyocosis, acne (e.g., acne vulgaris, neonatal
acne, infantile acne, pomade acne), psoriasis, Reiter's syndrome,
pityriasis rubra pilaris, hyperpigmentation, vitiligo, hypertropic
scarring, keloids, lichen plainus, age-related skin disorders (e.g.,
wrinkles, cellulite) and hyperproliferative variants of the disorders of
keratinization (e.g., actinic keratosis, senile keratosis). Generally,
the treatment of skin or integument conditions involves contacting the
metal-containing material with the area of the skin having the condition.
As an example, a skin or integument condition can be treated by
contacting the area of skin having the condition with a dressing having a
coating of the metal-containing material. As another example, a skin or
integument condition can be treated by contacting the area of skin having
the condition with a solution containing the metal-containing material.
As an additional example, a skin or integument condition can be treated
by contacting the area of skin having the condition with a pharmaceutical
carrier composition containing the metal-containing material. In the case
of onychomycosis, the material may be applied to the nail in an
appropriate form (see below) such that the material penetrates the hard
nail to contact the affected area.
[0051] In certain embodiments, the condition can be a respiratory
condition (e.g., a microbial respiratory condition, an inflammatory
respiratory condition, a fungal respiratory condition, a viral
respiratory condition, an autoimmune respiratory condition, an idiopathic
respiratory condition, a cancerous respiratory condition). Examples of
respiratory conditions include asthma, emphysema, bronchitis, pulmonary
edema, acute respiratory distress syndrome, bronchopulmonary dysplasia,
pulmonary fibrosis, pulmonary atelectasis, tuberculosis, pneumonia,
sinusitis, pharyngitis, mucositis, stomatitis, chronic obstructive
pulmonary disease, bronchiectasis, lupus pneumonitis and cystic fibrosis.
In general, the treatment of respiratory conditions involves contacting
the metal-containing material with the area of the respiratory system
having the condition. Areas of the respiratory system include, for
example, the oral cavity, the nasal cavity, and the lungs. As an example,
certain respiratory conditions can be treated by inhaling a free standing
powder of the metal-containing material (e.g., with a dry powder
inhaler). As another example, certain respiratory conditions can be
treated by inhaling an aerosol containing the metal-containing material
(e.g., with an inhaler).
[0052] In some embodiments, the condition can be a musculo-skeletal
condition (e.g., a microbial musculo-skeletal condition, an inflammatory
musculo-skeletal condition, a fungal musculo-skeletal condition, a viral
musculo-skeletal condition, an autoimmune musculo-skeletal condition, an
idiopathic musculo-skeletal condition, a cancerous musculo-skeletal
condition). A musculo-skeletal condition can be, for example, a
degenerative musculo-skeletal condition (e.g., arthritis) or a traumatic
musculo-skeletal condition (e.g., a tom or damaged muscle). Examples of
musculo-skeletal conditions include tendonitis, osteomyelitis,
fibromyalgia, bursitis and arthritis. Generally, the treatment of
musculo-skeletal conditions involves contacting the metal-containing
material compound with the area of the musculo-skeletal system having the
condition. Areas of the musculo-skeletal system include, for example, the
joints, the muscles, and the tendons. As an example, certain
musculo-skeletal conditions can be treated by injecting (e.g., via a
small needle injector) a solution containing the metal-containing
material into the subject. As another example, certain musculo-skeletal
conditions can be treated by injecting (e.g., via a needleless injector)
a free standing powder of the metal-containing material into the subject.
As an additional example, certain musculo-skeletal conditions can be
treated by using a pharmaceutical carrier composition of the
metal-containing material, such as a penetrating pharmaceutical carrier
composition of the metal-containing material (e.g., a composition
containing DMSO).
[0053] In certain embodiments, the condition can be a circulatory
condition (e.g., a microbial circulatory condition, an inflammatory
circulatory condition, a fungal circulatory condition, a viral
circulatory condition, an autoimmune circulatory condition, an idiopathic
circulatory condition, a cancerous circulatory condition). As referred to
herein, circulatory conditions include lymphatic conditions. Examples of
circulatory conditions include arteriosclerosis, septicemia, leukemia,
ischemic vascular disease, lymphangitis and atherosclerosis. In general,
the treatment of circulatory conditions involves contacting the
metal-containing material with the area of the circulatory system having
the condition. Areas of the circulatory system include, for example, the
heart, the lymphatic system, blood, blood vessels (e.g., arteries,
veins). As an example, certain circulatory conditions can be treated by
injecting (e.g., via a small needle injector) a solution containing the
metal-containing material into the subject. As another example, certain
circulatory conditions can be treated by injecting (e.g., via a
needleless injector) a free standing powder of the metal-containing
material into the subject.
[0054] In some embodiments, the condition can be a mucosal or serosal
condition (e.g., a microbial mucosal or serosal condition, an
inflammatory mucosal or serosal condition, a fungal mucosal or serosal
condition, a viral mucosal or serosal condition, an autoimmune mucosal or
serosal condition, an idiopathic mucosal or serosal condition, a
cancerous mucosal or serosal condition). Examples of mucosal or serosal
conditions include pericarditis, Bowen's disease, stomatitis,
prostatitis, sinusitis, digestive disorders, esophageal ulcer, gastric
ulcer, duodenal ulcer, espohagitis, gastritis, enteritis, enterogastric
intestinal hemorrhage, toxic epidermal necrolysis syndrome, Stevens
Johnson syndrome, cystic fibrosis, bronchitis, pneumonia (e.g.,
nosocomial pneumonia, ventilator-assisted pneumonia), pharyngitis, common
cold, ear infections, sore throat, sexually transmitted diseases (e.g.,
syphilis, gonorrhea, herpes, genital warts, HIV, chlamydia), inflammatory
bowel disease, colitis, hemorrhoids, thrush, dental conditions, oral
conditions, conjunctivitis, and periodontal conditions. Generally, the
treatment of mucosal or serosal conditions involves contacting the
metal-containing material with the area of a mucosal or serosal region
having the condition. Mucosal or serosal areas include, for example, the
oral cavity, the nasal cavity, the colon, the small intestine, the large
intestine, the stomach, and the esophagus. As an example, certain mucosal
or serosal conditions can be treated by inhaling a free standing powder
of the metal-containing material (e.g., with a dry powder inhaler). As
another example, certain mucosal or serosal conditions can be treated by
inhaling an aerosol containing the metal-containing material (e.g., with
an inhaler). As an additional example, certain mucosal or serosal
conditions can be treated by gargling or spraying a solution of the
metal-containing material.
[0055] In embodiments in which the metal-containing material is used to
treat hyperproliferation of cell growth (e.g., cancerous conditions, such
as malignant tumors, or non-cancerous conditions, such as benign tumors),
the metal-containing material can be used to induce apoptosis (programmed
cell death), modulate matrix metalloproteinases (MMPs) and/or modulates
cytokines by contacting affected tissue (e.g., a hyperplastic tissue, a
tumor tissue or a cancerous lesion) with the metal-containing material.
It has been observed that the metal-containing material (e.g., an
antimicrobial, atomically disordered, silver-containing material) can be
effective in preventing production of a high number of MMPs and/or
cytokines by certain cells without necessarily reducing MMP and/or
cytokine production by the same cells to about zero. It is believed,
however, that in certain embodiments, the metal-containing material can
be used to inhibit MMP and/or cytokine production (e.g., bring MMP and/or
cytokine production to normal levels, desired levels, and/or about zero)
in certain cells.
[0056] MMPs 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.
Examples of MMPs 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). Cytokine refers to a
nonimmunoglobulin polypeptide secreted by monocytes and lymphocytes in
response to interaction with a specific antigen, a nonspecific antigen,
or a nonspecific soluble stimulus (e.g., endotoxin, other cytokines).
Cytokines affect the magnitude of inflammatory or immune responses.
Cytokines can be divided into several groups, which include interferons,
tumor necrosis factor (TNF), interleukins (IL-1 to IL-8), transforming
growth factors, and the hematopoietic colony-stimulating factors. An
example of a cytokine is TNF-.alpha.. A fibroblast is an area 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. Hyperplastic tissue refers to tissue in
which there is an abnormal multiplication or increase in the number of
cells in a normal arrangement in normal tissue or an organ. A tumor
refers to spontaneous growth of tissue in which multiplication of cells
is abnormal, uncontrolled and progressive. A tumor generally serves no
useful function and grows at the expense of the healthy organism. A
cancerous lesion is 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.
[0057] Conditions characterized by undesirable MMP activity include
ulcers, asthma, acute respiratory distress syndrome, skin disorders, skin
aging, keratoconus, restenosis, osteo- and rheumatoid arthritis,
degenerative joint disease, bone disease, wounds, cancer including cell
proliferation, invasiveness, metastasis (carcinoma, fibrosarcoma,
osteosarcoma), hypovolemic shock, periodontal disease, epidermolysis
bullosa, scleritis, atherosclerosis, multiple sclerosis, inflammatory
diseases of the central nervous system, vascular leakage syndrome,
collagenase induced disease, adhesions of the peritoneum, strictures of
the esophagus or bowel, ureteral or urethral strictures, and biliary
strictures. Excessive TNF production has been reported in diseases which
are characterized by excessive MMP activity, such as autoimmune disease,
cancer, cachexia, HIV infection, and cardiovascular conditions.
[0058] Forms of the Material and Methods of Applying the Material
[0059] In general, the metal-containing material can be in any desired
form or formulation. For example, the material can be a coating on a
substrate (e.g., in the form of a dressing), a free standing powder, a
solution, or disposed within a pharmaceutically acceptable carrier.
[0060] In some embodiments, the metal-containing material can act as a
preservative. In such embodiments, a form or formulation containing the
metal-containing material can be prepared without additional
preservatives. Moreover, in embodiments in which the metal-containing
material acts as a preservative, the metal-containing material may be
included in a therapeutic formulation containing other therapeutic agents
(e.g., the metal-containing material may be included primarily in certain
therapeutic compositions to act as a preservative).
[0061] Moreover, the material can be applied to the subject in any of a
variety of ways, generally depending upon the form of the material as
applied and/or the location of the condition to be treated. In general,
the amount of material used is selected so that the desired therapeutic
effect (e.g., reduction in the condition being treated) is achieved while
the material introduces an acceptable level of toxicity (e.g., little or
no toxicity) to the subject. Generally, the amount of the material used
will vary with the conditions being treated, the stage of advancement of
the condition, the age and type of host, and the type, concentration and
form of the material as applied. Appropriate amounts in any given
instance will be readily apparent to those skilled in the art or capable
of determination by routine experimentation. In some embodiments, a
single application of the material may be sufficient. In certain
embodiments, the material may be applied repeatedly over a period of
time, such as several times a day for a period of days or weeks.
[0062] Substrate Coatings
[0063] Examples of commercially available metal-containing materials
include the Acticoat.RTM. family of dressings (Smith & Nephew, Hull, UK),
which are formed of antimicrobial, atomically disordered, nanocrystalline
silver-containing material coated on one or more substrates. Such
dressings include the Acticoat.RTM. dressings, the Acticoat7.RTM.
dressings, the Acticoat.RTM. moisture coating dressings, and the
Acticoat.RTM. absorbent dressings.
[0064] A coating of a metal-containing material (e.g., an antimicrobial,
atomically disordered, nanocrystalline silver-containing material) can be
formed on a substrate using a desired technique. In certain embodiments,
the coating is formed by depositing the material on the substrate surface
using chemical vapor deposition, physical vapor deposition, and/or liquid
phase deposition. Exemplary deposition methods include vacuum evaporation
deposition, arc evaporation deposition, sputter deposition, magnetron
sputter deposition and ion plating.
[0065] In some embodiments, the coating is prepared using physical vapor
deposition. FIG. 1 shows a vapor deposition system 100 that includes a
vacuum chamber 110, an energy source 120 (e.g., an electron beam source,
an ion source, a laser beam, a magnetron source), a target 130 and a
substrate 140. During operation, energy source 120 directs a beam of
energy 122 to target 130, causing material 132 to be removed (e.g., by
evaporation) from target 130 and directed to a surface 142 of substrate
140. At least a portion of the removed material 132 is deposited on
surface 142.
[0066] In general, the values of the system parameters (e.g., the
temperature of surface 142, the pressure of chamber 110, the angle of
incidence of removed material 132 on surface 142, the distance between
target 130 and surface 142) can be selected as desired. The temperature
of surface 142 can be relatively low during the deposition process. For
example, during the deposition process, the ratio of the temperature of
substrate 140 to the melting point of the material forming target 130 (as
determined in using Kelvin) can be about 0.5 or less (e.g., about 0.4 or
less, about 0.35 or less, about 0.3 or less).
[0067] The pressure in chamber 110 can be relatively high. For example,
vacuum evaporation deposition, electron beam deposition or arc
evaporation, the pressure can be about 0.01 milliTorr or greater. For gas
scattering evaporation (pressure plating) or reactive arc evaporation,
the pressure in chamber 110 can be about 20 milliTorr or greater. For
sputter deposition, the pressure in chamber 110 can be about 75 milliTorr
or greater. For magnetron sputter deposition, the pressure in chamber 110
can be about 10 milliTorr or greater. For ion plating, the pressure in
chamber 110 can be 200 milliTorr or greater.
[0068] The angle of incidence of removed material 132 on surface 142
(.theta.) can be relatively low. For example, the angle of incidence of
removed material 132 on surface 142 can be about 75.degree. or less
(e.g., about 60.degree. or less, about 45.degree. or less, about
30.degree. or less).
[0069] The distance between target 130 and surface 142 can be selected
based upon the values of the other system parameters. For example, the
distance between target 130 and surface 142 can be about 250 millimeters
or less (e.g., about 150 millimeters or less, 125 millimeters or less,
about 100 millimeters or less, about 90 millimeters or less, about 80
millimeters or less, about 70 millimeters or less, about 60 millimeters
or less, about 50 millimeters or less, about 40 millimeters or less).
[0070] As noted above, it is believed that, the metal-containing material,
when contacted with an alcohol or water-based electrolyte, can be
released into the alcohol or water-based electrolyte (e.g., as ions,
atoms, molecules and/or clusters). It is also believed that the ability
to release the metal (e.g., as atoms, ions, molecules and/or clusters) on
a sustainable basis from a coating is generally dependent upon 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,
it is believed that the amount of metal species released per unit time
increases. For example, a silver metal film deposited by magnetron
sputtering at a ratio of substrate temperature to the target melting
point of less than about 0.5 and a working gas pressure of about 0.93
Pascals (about seven milliTorr) releases approximately 1/3 of the silver
ions that a film deposited under similar conditions, but at four Pascals
(about 30 milliTorr), will release over 10 days. Coatings formed with an
intermediate structure (e.g., lower pressure, lower angle of incidence
etc.) have been observed to have metal (e.g., silver) release values
intermediate to these values as determined by bioassays. In general, to
obtain relatively slow release of the metal, the coating should have a
relatively low degree of atomic disorder, and, to obtain relatively fast
release of the metal, the coating should have a relatively high degree of
atomic disorder.
[0071] For continuous, uniform coatings, the time for total dissolution is
generally a function of coating thickness and the nature of the
environment to which the coating is exposed. The release of metal is
believed to increase approximately linearly as the thickness of the
coating is increased. For example, it has been observed that a two fold
increase in coating thickness can result in about a two fold increase in
longevity.
[0072] In certain embodiments, it is possible to manipulate the degree of
atomic disorder, and therefore the metal release from a coating, by
forming a thin film coating with a modulated structure. For example, a
coating deposited by magnetron sputtering such that the working gas
pressure was relatively low (e.g., about two Pascals or about 15
milliTorr) for about 50% of the deposition time and relatively high
(e.g., about four Pascals or 30 milliTorr) for the remaining time, can
result in a relatively rapid initial release of metal (e.g., ions,
clusters, atoms, molecules), followed by a longer period of slow release.
This type of coating is can be particularly effective on devices such as
urinary catheters for which an initial rapid release is advantageous to
achieve quick antimicrobial concentrations followed by a lower release
rate to sustain the concentration of metal (e.g., ions, clusters, atoms,
molecules) over a period of weeks.
[0073] It is further believed that the degree of atomic disorder of a
coating can be manipulated by introducing one or more dissimilar
materials into the coating. For example, one or more gases can be present
in chamber 10 during the deposition process. Examples of such gases
include oxygen-containing gases (e.g., oxygen, air, water),
nitrogen-containing gases (e.g., nitrogen), hydrogen-containing gases
(e.g., water, hydrogen), boron-containing gases (e.g., boron),
sulfur-containing gases (e.g., sulfur) and halogen-containing gases
(e.g., fluorine, chlorine, bromine, iodine). The additional gas(es) can
be co-deposited or reactively deposited with material 132. This can
result in the deposition of an oxide, nitride, carbide, boride, sulfide,
hydride and/or halide material (e.g., an oxide of a metal-containing
material, a nitride of a metal-containing material, a carbide of a
metal-containing material, a boride of a metal-containing material, a
sulfide of a metal-containing material, a hydride of a metal-containing
material, a halide of a metal-containing material). Without wishing to be
bound by theory, it is believed that atoms and/or molecules of the
additional gas(es) may become absorbed or trapped in the material,
resulting in enhanced atomic disorder. The additional gas(es) may be
continuously supplied during deposition, or may be pulsed to (e.g., for
sequential deposition). In embodiments, the material formed can be
constituted of a material with a ratio of material 132 to additional
gas(es) of about 0.2 or greater. The presence of dissimilar atoms or
molecules in the coating can enhance the degree of atomic disorder of the
coating due to the difference in atomic radii of the dissimilar
constituents in the coating.
[0074] The presence of dissimilar atoms or molecules in the coating may
also be achieved by co-depositing or sequentially depositing one or more
additional metals (e.g., one or more additional antimicrobial metals).
Such additional metals include, for example, Ta, Ti, Nb, Zn, V, Hf, Mo,
Si, Al, and other transition metals. It is believed that the presence of
dissimilar metals (one or more primary metals and one or more additional
metals) in the coating can reduce atomic diffusion and stabilize the
atomically disordered structure of the coating. A coating containing
dissimilar metals can be formed, for example, using thin film deposition
equipment with multiple targets. In some embodiments, sequentially
deposited layers of the metals are discontinuous (e.g., islands within a
the primary metal). In certain embodiments, the weight ratio of the
additional metal(s) to the primary metal(s) is greater than about 0.2.
[0075] While FIG. 1 shows one embodiment of a deposition system, other
embodiments are possible. For example, the deposition system can be
designed such that during operation the substrate moves along rollers.
Additionally or alternatively, the deposition system may contain multiple
energy sources, multiple targets, and/or multiple substrates. The
multiple energy sources, targets and/or substrates can be, for example,
positioned in a line, can be staggered, or can be in an array.
[0076] In certain embodiments, two layers of the material are deposited on
the substrate to achieve an optical interference effect. Alternatively,
the two layers can be formed of different materials, with the outer (top)
of the two layers being formed of an antimicrobial, atomically
disordered, nanocrystalline silver-containing material, and the inner of
the two layers having appropriate reflective properties so that the two
layers can provide an interference effect (e.g., to monitor the thickness
of the outer (top) of the two layers).
[0077] The substrate can be selected as desired. The substrate may be
formed of one layer or multiple layers, which may be formed of the same
or different materials.
[0078] In certain embodiments, the substrate can include one or more
layers containing a bioabsorbable material. Bioabsorbable materials are
disclosed, for example, in U.S. Pat. No. 5,423,859. In general,
bioabsorbable materials can include natural bioabsorbable polymers,
biosynethetic bioabsorbable polymers and synthetic bioabsorbable
polymers. Examples of synthetic bioabsorbable polymers include polyesters
and polylactones (e.g., polymers of polyglycolic acid, polymers of
glycolide, polymers of lactic acid, polymers of lactide, polymers of
dioxanone, polymers of trimethylene carbonate, polyanhydrides,
polyesteramides, polyortheoesters, polyphosphazenes, and copolymers of
the foregoing). Examples of natural bioabsorbable polymers include
proteins (e.g., albumin, fibrin, collagen, elastin), polysaccharides
(e.g., chitosan, alginates, hyaluronic acid). Examples of biosynthetic
polymers include polyesters (e.g., 3-hydroxybutyrate polymers).
[0079] In some embodiments, the substrate includes multiple layers (e.g.,
two layers, three layers, four layers, five layers, six layers, seven
layers, eight layers, nine layers, 10 layers). The layers can be
laminated together (e.g., by thermal fusing, stitching and/or ultrasonic
welding).
[0080] One or more layers (e.g., an outer layer) of a multi-layer
substrate can be formed of a perforated (and optionally non-adherent)
material (e.g., a woven material or a non-woven material) that can allow
fluid to penetrate or diffuse therethrough. Such materials include, for
example, cotton, gauze, polymeric nets (e.g., polyethylene nets, nylon
nets, polypropylene nets, polyester nets, polyurethane nets,
polybutadiene nets), polymeric meshes (e.g., polyethylene meshes, nylon
meshes, polypropylene meshes, polyester meshes, polyurethane meshes,
polybutadiene meshes) and foams (e.g., an open cell polyurethane foam).
Examples of commercially available materials include DELNET.TM. P530
non-woven polyethylene veil (Applied Extrusion Technologies, Inc.,
Middletown, Del.), Exu-Dry CONFORMANT2.TM. non-woven polyethylene veil
(Frass Survival Systems, Inc., NY, N.Y.), CARELLE.TM. material (Carolina
Formed Fabrics Corp.), NYLON90.TM. material (Carolina Formed Fabrics
Corp.), N-TERFACE.TM. material (Winfield Laboratories, Inc., Richardson,
Tex.), HYPOL.TM. hydrophilic polyurethane foam (W.R. Grace & Co., NY,
N.Y.).
[0081] One or more layers (e.g., an inner layer) of a multi-layer
substrate can be formed of an absorbent material (e.g., a woven material
or a non-woven material) formed of, for example, rayon, polyester, a
rayon/polyester blend, polyester/cotton, cotton and/or cellulosic fibers.
Examples include creped cellulose wadding, air felt, air laid pulp fibers
and gauze. An example of a commercially available material is SONATRA.TM.
8411 70/30 rayon/polyester blend (Dupont Canada, Mississauga, Ontario).
[0082] One or more layers (e.g., an outer layer) of a multi-layer
substrate can be formed of an occlusive or semi-occlusive material, such
as an adhesive tape or polyurethane film (e.g., to secure the device to
the skin and/or to retain moisture).
[0083] In some embodiments, the layers in a multi-layer substrate are
laminated together (e.g., at intermittent spaced locations) by ultrasonic
welds. Typically, heat (e.g., generated ultrasonically) and pressure are
applied to either side of the substrate 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 can be formed as localized
spots (e.g., circular spots). The spots can have a diameter of about 0.5
centimeter or less.
[0084] The shape of the substrate can generally be varied as desired. For
example, the substrate can be in the shape of a film, a fiber or a
powder.
[0085] The substrate/coating article can be used in a variety of articles.
For example, the article can be in the shape of a medical device.
Exemplary medical devices include wound closure devices (e.g., sutures,
staples, adhesives), tissue repair devices (e.g., meshes, such as meshes
for hernia repair), prosthetic devices (e.g., internal bone fixation
devices, physical barriers for guided bone regeneration), tissue
engineering devices (e.g., for use with a blood vessel, skin, a bone,
cartilage, a liver), controlled drug delivery systems (e.g.,
microcapsules, ion-exchange resins) and wound coverings and/or fillers
(e.g., alginate dressings, chitosan powders). In some embodiments, the
article is a transcutaneous medical device (e.g., a catheter, a pin, an
implant), which can include the substrate/coating supported on, for
example, a solid material (e.g., a metal, an alloy, latex, nylon,
silicone, polyester and/or polyurethane). In some embodiments, the
article is in the form of a patch (e.g., a patch having an adhesive layer
for adhering to the skin, such as a transdermal patch).
[0086] Subsequent to deposition, the material can optionally be annealed.
In general, the anneal is conducted under conditions to increase the
stability (e.g., shelf life) of the material while maintaining the
desired therapeutic activity of the material. In certain embodiments, the
material can be annealed at a temperature of about 200.degree. C. or less
(e.g., about room temperature).
[0087] The substrate/coating is typically sterilized prior to use (e.g.,
without applying sufficient thermal energy to anneal out the atomic
disorder). The energy used for sterilization can be, for example, gamma
radiation or electron beam radiation. In some embodiments, ethylene oxide
sterilization techniques are used to sterilize the substrate/coating.
[0088] Free Standing Powders
[0089] A free standing powder can be prepared by, for example, cold
working or compressing to impart atomic disorder to the powder. In
certain embodiments, a free standing powder is prepared by forming a
coating of the material as described above, and then removing the
material from the surface of the substrate. For example, the material can
be scraped from the surface of the substrate by one or more scrapers. In
embodiments in which the substrate moves during deposition of the
material, the scrapers can remove the material as the substrate moves.
The scrapers can be, for example, suspended above the substrate. Such
scrapers can be, for example, weighted and/or spring loaded to apply
pressure sufficient to remove the material as the substrate moves. In
some embodiments (e.g., when a continuous belt is used), the scrapers can
be located above the end rollers to remove the material with a reverse
dragging action as the substrate rounds the end roller.
[0090] A free standing powder can be used to treat a condition in various
ways. As an example, the powder can sprinkled onto the subject's skin. As
another example, the powder can be inhaled using an inhaler, such as a
dry powder inhaler.
[0091] In certain embodiments (e.g., when the free standing powder is
inhaled), the average particle size of the free standing powder is
selected to reduce the likelihood of adverse reaction(s) of the particles
in the tissue (e.g., tissue contacted by the free standing powder during
inhalation). In embodiments, a free standing powder can have an average
particle size of less than about two microns (e.g., less than about one
micron, less than about 0.5 micron).
[0092] Powder Impregnated Materials
[0093] The metal-containing material can be in the form of a powder
impregnated material. Such powder impregnated materials can, for example,
be in the form of a hydrocolloid having the free standing powder blended
therein. A powder impregnated material can be, for example, in the form
of a dressing, such as a hydrocolloid dressing.
[0094] Solutions
[0095] The compound can be in the form of a solution (e.g., a
solvent-based solution). The solution can be formed, for example, by
dissolving a free standing powder of the material in a solvent for the
powder. As an example, a container (e.g., a tea bag-type container) with
the free standing powder within it can be immersed in the water or
solvent. As another example, a substrate (e.g., in the form of a strip or
a bandage) carrying the material can be immersed in the solvent. In
certain embodiments, it can be preferable to form a solution by
dissolving a free standing powder of the compound in a solvent because
this can be a relatively approach to forming a solution.
[0096] In certain embodiments, the solution containing the compound is
contacted with the subject relatively soon after formation of the
solution. For example, the solution containing the compound can be
contacted with the subject within about one minute or less (e.g., within
about 30 seconds or less, within about 10 seconds or less) of forming the
solution containing the compound. In some embodiments, a longer period of
time lapses before the solution containing the compound is contacted with
the subject. For example a period of time of at least about 1.5 minutes
(e.g., at least about five minutes, at least about 10 minutes, at least
about 30 minutes, at least about one hour, at least about 10 hours, at
least about a day, at least about a week) lapses between the time the
solution containing the compound is formed and the solution containing
the compound is contacted with the subject.
[0097] In some embodiments, lowering the pH of the solution (e.g., to less
than about 6.5, such as from about 3.5 to about 6.5) can allow for a
higher concentration of the dissolved material and/or a faster rate of
dissolution. The pH of the solution can be lowered, for example, by
adding acid to the solution (e.g., by adding CO.sub.2 to the solution to
form carbonic acid).
[0098] A solution containing the compound can be contacted with the
subject with or without the use of a device. As an example, a solution
containing the compound can be contacted with the skin, mouth, ears or
eyes as a rinse, a bath, a wash, a gargle, and/or drops. As another
example, the solution can be injected using a small needle injector
and/or a needleless injector. As an additional example, a solution
containing the compound can be formed into an aerosol (e.g., an aerosol
prepared by a mechanical mister, such as a spray bottle or a nebulizer),
and the aerosol can be contacted with the subject using an appropriate
device (e.g., a hand held inhaler, a mechanical mister, a spray bottle, a
nebulizer, an oxygen tent). As a further example, a solution containing
the compound can be contacted with the second location via a catheter.
[0099] In embodiments in which onychomycosis is being treated, the method
can include first hydrating the nail with urea (1-40%) or lactic acid
(10-15%), followed by treatment with the metal-containing material, which
may contain an appropriate solvent (e.g., DMSO) for penetration through
the nail. Alternatively or additionally, onychomycosis can be treated by
injecting (e.g., via a needleless injector and/or a needle) the
metal-containing material to the affected area.
[0100] Typically, the solvent is a relatively hydrophilic solvent.
Examples of solvents include water, DMSO and alcohols. In certain
embodiments, a water-based solution is a buffered solution. In some
embodiments, a water-based solution contains carbonated water. In
embodiments, more than one solvent can be used.
[0101] In some embodiments, the solution can contain about 0.001 weight
percent or more (e.g., about 0.01 weight percent or more, about 0.02
weight percent or more, about 0.05 weight percent or more, about 0.1
weight percent or more, about 0.2 weight percent or more, about 0.5
weight percent or more, about one weight percent or more) of the compound
and/or about 10 weight percent or less (e.g., about five weight percent
or less, about four weight percent or less, about three weight percent or
less, about two weight percent or less, about one weight percent or less)
of the compound.
[0102] Pharmaceutical Carrier Compositions
[0103] The metal-containing material can disposed (e.g., suspended) within
a pharmaceutically acceptable carrier. The formulation can be, for
example, a semi-solid, a water-based hydrocolloid, an oil-in-water
emulsion, a water-in-oil emulsion, a non-dried gel, and/or a dried gel.
Typically, when disposed in a pharmaceutically acceptable carrier, the
metal-containing material is applied to the skin.
[0104] Examples of pharmaceutically acceptable carriers include creams,
ointments, gels, lotions, pastes, foams and liposomes.
[0105] The formulation can contain about 0.01 weight percent or more
(e.g., about 0.1 weight percent or more, about 0.5 weight percent or
more, about 0.75 weight percent or more, about one weight percent or
more, about two weight percent or more, about five weight percent or
more, about 10 weight percent or more) of the metal-containing material
and/or about 50 weight percent or less (e.g., about 40 weight percent or
less, about 30 weight percent or less, about 20 weight percent or less,
about 20 weight percent or less, about 15 weight percent or less, about
10 weight percent or less, about five weight percent or less) of the
metal-containing material.
[0106] In certain embodiments, the metal-containing material can be
effectively used in the oral cavity when in the form of an article (e.g.,
a tape, a pill, a capsule, a tablet or lozenge) that is placed within the
oral cavity (e.g., so that the subject can suck on the tape, pill,
capsule, tablet or lozenge). In some embodiments, the article can be a
sustained release article (e.g., a sustained release capsule) In certain
embodiments, the article can be an enteric article (e.g., an enteric
coated tablet).
[0107] Formulations can optionally include one or more components which
can be biologically active or biologically inactive. Examples of such
optional components include base components (e.g., water and/or an oil,
such as liquid paraffin, vegetable oil, peanut oil, castor oil, cocoa
butter), thickening agents (aluminum stearate, hydrogen lanolin), gelling
agents, stabilizing agents, emulsifying agents, dispersing agents,
suspending agents, thickening agents, coloring agents, perfumes,
excipients (starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc), foaming agents
(e.g., surfactants), surface active agents, preservatives (e.g., methyl
paraben, propyl paraben) and cytoconductive agents (e.g., betaglucan). In
certain embodiments, a pharmaceutical carrier composition can include a
constituent (e.g., DMSO) to assist in the penetration of skin.
[0108] While the foregoing has described embodiments in which a single
condition is treated, in some embodiments multiple conditions can be
treated. The multiple conditions can be the same type of condition (e.g.,
multiple skin or integument conditions) or different types of conditions.
For example, a dressing formed of one or more substrates coated with an
appropriate metal-containing material (e.g., antimicrobial, atomically
disordered, silver-containing material) can be applied to an area of the
skin having multiple skin or integument conditions (e.g., a burn and
psoriasis) so that the metal-containing material treats the multiple skin
or integument conditions.
[0109] Moreover, while the foregoing has described embodiments that
involve one method of contacting a subject with the metal-containing
material, in other embodiments, more than one method of contacting a
subject with the metal-containing material can be used. For example, the
methods can include one or more of ingestion (e.g., oral ingestion),
injection (e.g., using a needle, using a needleless injector), topical
administration, inhalation (e.g., inhalation of a dry powder, inhalation
of an aerosol) and/or application of a dressing.
[0110] Furthermore, while the foregoing has described embodiments in which
one form of the metal-containing material is used, in other embodiments,
more than one form of the metal-containing material can be used. For
example, the methods can include using the metal-containing material in
the form of a coating (e.g., a dressing), a free standing powder, a
solution and/or a pharmaceutical carrier composition.
[0111] Moreover, the metal-containing material can be used in various
industrial applications. For example, the metal-containing material can
be used to reduce and/or prevent microbial growth on industrial surfaces
(e.g., industrial surfaces where microbial growth may occur, such as warm
and/or moist surfaces). Examples of industrial surfaces include heating
pipes and furnace filters. In certain embodiments, the metal-containing
material can be disposed (e.g., coated or sprayed) on the surface of
interest to reduce and/or prevent microbial growth. This can be
advantageous in preventing the spread of microbes via, for example,
heating and/or air circulation systems within buildings.
[0112] The following examples are illustrative and not intended as
limiting.
EXAMPLES
[0113] Treatment of Hyperproliferative Skin conditions
Example 1
Preparation of Nanocrystalline Silver Coatings on Dressings
[0114] 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 anti-microbial
coating having indicator value. The coating layers were formed by
magnetron sputtering under the conditions set out in the following table.
1
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 3.0 .times. 10.sup.-6 Torr
Anode/Cathode Distance 100 mm 100 mm
Sputtering Time 7.5-9 min
1.5 min
Voltage 369-373 V 346 V
[0115] 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.
[0116] 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#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.
[0117] 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 <1 mg/L).
[0118] 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.
[0119] 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.
[0120] 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, and slits formed
therein before packaging. Alternatively, the dressings may be packaged
with instructions for the clinician to cut the dressing to size and form
the desired length of the slit for the medical device.
[0121] 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:
[0122] 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 1100% rayon on a polyurethane film.
[0123] 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 Nanocrystalline Silver Powders
[0124] Nanocrystalline silver powder was prepared by preparing silver
coatings on silicon wafers, under the conditions set forth in the table
above, and then scraping the coating off using a glass blade.
[0125] Nanocrystalline silver powder was also prepared by sputtering
silver coatings on silicon wafers using Westaim Biomedical NGRC unit, and
then scraping the coating off. The sputtering conditions were as follows:
2
Target: 99.99% Ag
Target Size: 15.24 cm
.times. 1216.125 cm
Working Gas: 75:25 wt % Ar/O.sub.2
Working Gas Pressure: 40 mTorr
Total Current: 40 A
Base
Pressure: 5.0 .times. 10.sup.-5 Torr
Sandvik Belt Speed: 340
mm/min
Voltage: 370 V
[0126] The powder has a particle size ranging from 2 .mu.m to 1100 .mu.m,
with crystallite size of 8 to 10 nm, and demonstrated a positive rest
potential.
Example 3
Treatment of Psoriasis
[0127] This patient was a 58 year old female with psoriatic plaques
covering up to sixty percent of her body. For this patient, psoriatic
plaques first occurred ten years ago and have been treated with the
following:
[0128] 1. Adrenal corticosteroids. Injections gave relief from pruritus
and general discomfort. Treatments led to a rebound effect; i.e.
psoriasis would flare up after treatments wore off. Corticosteroids were
discontinued.
[0129] 2. UV Light and Methotrexate treatments. UV light treatments were
given in conjunction with methotrexate. The UV light treatments caused
bums and new lesions. The methotrexate caused severe nausea. Both
treatments were discontinued.
[0130] 3. Ice Cap Spray. This treatment contained a potent corticosteroid,
and gave some relief but it was taken off the market and is no longer
available.
[0131] 4. Soriatone (acetretin 10 mg). This systemic retinoid treatment
was associated with joint aches and was discontinued.
[0132] 5. Diet. The patient was attempting to control the disease through
diet.
[0133] Nanocrystalline silver was tested as follows. Nanocrystalline
silver was deposited on sheets of high-density polyethylene (HDPE) using
a vapour deposition process as set forth in
Example 1
[0134] Two sheets of this coated HDPE were laminated together around a
core of non-woven rayon polyester, as set forth in Example 1. A 50
mm.times.50 mm (2".times.2") piece of this composite material was
saturated with water and placed centrally on a one and a half year old
150 mm.times.100 mm (6".times.4") psoriatic plaque on the patient's
flank. The nanocrystalline silver coated material was covered with a
piece of low moisture vapour transmission thin polymer film. The polymer
sheet extended 50 mm (2") beyond the nanocrystalline silver coated HDPE
to provide control data regarding occlusion of the psoriatic plaque.
[0135] The dressing was removed after three days. There was no discernible
change in the plaque at this time. However two days later the area that
was covered with the nanocrystalline silver had the appearance of normal
skin while the rest of the plaque was still rough and unchanged,
including the untreated but occluded area.
[0136] The nanocrystalline silver therapy caused the treated psoriatic
plaque to resolve.
Example 4
Treatment of Psoriasis
[0137] The subject was a 58 year old female with psoriatic plaques over up
to sixty percent of her body. Psoriatic plaques had first occurred 10
years ago and had been treated with the following:
[0138] 1. Adrenal corticosteroids. Injections gave relief from pruritus
and general discomfort. Treatments led to a rebound effect i.e. psoriasis
would flare up after treatments wore off. Corticosteroids were
discontinued.
[0139] 2. UV Light and Methotrexate treatments. UV light treatments were
given in conjunction with methotrexate. The UV light treatments caused
burns and new lesions. The methotrexate caused severe nausea. Both
treatments were discontinued.
[0140] 3. Ice Cap Spray. This treatment contained a potent corticosteroid,
and gave some relief but it was taken off the market and is no longer
available.
[0141] 4. Soriatone (acetretin 10 mg). This systemic retinoid treatment
was associated with joint aches and was discontinued.
[0142] 5. Diet. The patient was attempting to control the disease through
diet.
[0143] Nanocrystalline silver was tested as follows. Nanocrystalline
silver was deposited on sheets of high-density polyethylene (HDPE) using
a vapour deposition process as set forth in Example 1 (top layer). Two
sheets of this coated HDPE were laminated together around a core of
non-woven rayon polyester, as set forth in Example 1. A 50 mm.times.50 mm
(2".times.2") piece of this composite material was saturated with water
and placed centrally on a 125 mm.times.100 mm (5".times.4") psoriatic
plaque on the patient's upper left thigh. The nanocrystalline silver
coated material was covered with a piece of low moisture vapour
transmission thin polymer film. The polymer sheet extended 50 mm (2")
beyond the nanocrystalline silver coated HDPE to provide control data
regarding occlusion of the psoriatic plaque.
[0144] The dressing was removed and the plaque examined after two days.
The area that was covered with the nanocrystalline silver was free of
scaling and only slightly erythematous while the rest of the plaque was
still erythenatous and scaly, including the untreated but occluded area.
The plaque was redressed with a similar 50 mm.times.50 mm (2".times.2")
piece of nanocrystalline silver coated dressing, which was left in place
for a further period of 2 days. The area that was covered with the
nanocrystalline silver remained free of scale and only slightly
erythenatous, while the rest of the plaque was still erythenatous and
scaly, including the area under the occlusive film.
[0145] The nanocrystalline silver therapy caused the treated psoriatic
plaque to resolve.
Example 5
Preparation of Nanocrystalline Gels
[0146] A commercial carboxymethyl cellulose/pectin (Duoderm Convatec.TM.)
was combined with nanocrystalline silver powder prepared as in Example 2
to produce a gel with 0.1% w/v. silver. Carboxymethyl cellulose (CMC)
fibers were coated by magnetron sputtering, under conditions similar to
those set out in Example 1 for the top layer to produce a defective
nanocrystalline silver coating. The CMC was then gelled in water by
adding 2.9 g to 100 mL volume. An alginate fibrous substrate was directly
coated with a defective nanocrystalline silver coating by magnetron
sputtering under coating conditions similar to those set forth in Example
1 for the top layer. The alginate (5.7 g) was added to 100 mL volume of
water to create a gel. A commercial gel containing CMC and alginate
(Purilon gel Coloplast.TM.) was mixed with an atomic disordered
nanocrystalline silver powder prepared as in Example 2 to give a gel
product with 0.1% w/v silver. A commercially available gel
(Lubriderm.TM.--glyceryl polymethacrylate) was blended with atomic
disordered nanocrystalline silver powder prepared as in Example 2, to
prepare a gel with a silver content of 0.1% w/v. A further gel was
formulated with, on w/v basis, 0.1% methyl paraben, 0.02% propyl paraben,
0.5% polyvinyl alcohol (Airvol.TM. PVA 540), 2% CMC, 0.1% nanocrystalline
silver powder prepared as in Example 2, and was brought up to 1000 g with
water.
[0147] Treatment of Inflammatory Skin conditions
Example 1
Preparation of Nanocrystalline Silver Coatings on Dressings
[0148] 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 as described in Example 1 of the Treatment
of Hyperproliferative Skin conditions examples. The coating layers were
formed by magnetron sputtering under the conditions set out in the
following table.
Example 2
Preparation of Nanocrystalline Silver Coating on HDPE Mesh
[0149] The silver coated mesh was produced, as set forth in Example 1, by
sputtering silver onto Delnet, a HDPE mesh (Applied Extrusion
Technologies, Inc., Middletown, Del., USA) using Westaim Biomedical TMRC
unit under the following conditions:
3
Target: 99.99% Ag
Target Size: 15.24 cm
.times. 152.4 cm
Working Gas: 99.375:0.625 wt % Ar/O.sub.2
Working Gas Pressure: 5.33 Pascals (40 mTorr)
Total Current: 22
A
Base Pressure: 5.0 .times. 10.sup.-5 Torr
Sandvik Belt
Speed: 577 mm/min
Voltage: 367 V
[0150] The coating was tested and found to have a weight ratio of reaction
product to silver of between 0.05 and 0.1. The dressing was non-staining
to human skin.
Example 3
Preparation of Atomic Disordered Nanocrystalline Silver Powders
[0151] 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:
4
Target: 99.99% Ag
Target Size: 15.24 cm .times.
1216.125 cm
Working Gas: 75:25 wt % Ar/O.sub.2
Working Gas
Pressure: 5.33 Pascals (40 milliTorr)
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/min
Voltage: 370 V
Note
pressure conversions to Pa herein may not be
accurate, most accurate numbers are in torr, mTorr units.
[0152] 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.
[0153] 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 4
In vitro Activity of Silver Solution against Propionibacterium acne
[0154] An in vitro test was conducted to determine if silver solutions
according to the present invention effectively control Propionibacterium
acne. The silver solution was obtained by static elution of Acticoat.TM.
Burn Wound Dressing (lot #: 00403A-05, Westaim Biomedical Corp., Fort
Saskatchewan, Canada) with nanopure water in a ratio of one square inch
of dressing in five milliliters of water for 24 hours at room
temperature. The silver concentration of the silver solution was
determined by an atomic absorption method. The silver elute was diluted
with nanopure water to 20 .mu.g/ml. The Propionibacterium acne (ATCC No.
0919) was provided by Biofilm Research Group, University of Calgary.
[0155] The inoculum was prepared by inoculating freshly autoclaved and
cooled tubes of Tryptic soy broth (TSB) with P. acne and incubating them
for 2 days at 37.degree. C. in an anaerobic jar. At this time, the
optical density of the suspensions was .about.0.3 at a wavelength of 625
nm.
[0156] The bacterial suspension (100 .mu.L) was mixed with 100 .mu.L of
the silver solution being tested. The final concentration of silver in
these mixtures was 10 .mu.g/ml. The mixtures were incubated in an
anaerobic jar at 37.degree. C. for two hours. The silver was neutralized
by addition of 0.4% STS (0.85% NaCl, 0.4% Sodium thioglycolate, 1%
Tween.TM. 20) and the solution was serially 10-fold diluted with
phosphate-buffered saline. 20 .mu.L aliquots of the original solution and
subsequent dilutions were plated onto TSA drop plates. The drops were
allowed to dry and the plates were incubated in an anaerobic jar at
37.degree. C. for 72 hours at which time the colonies were counted. The
control consisted of 100 .mu.L of bacterial suspension mixed with 100
.mu.L of nanopure water and treated as above.
[0157] The results showed that the silver solution according to the
present invention, at a final concentration of 10 .mu.g/ml, gave 4.3
logarithm reduction in viable P. acne counts in two hours.
Example 5
Treatment of Acne
[0158] A sixteen year old female was diagnosed with acne vulgaris. She had
numerous red papules and pustules on her forehead. Various skin cleansing
regimes and antibiotic (erythromycin and clindomycin) treatments had been
tried and had failed to control the acne. Prior to bedtime, the papules
and pustules on one side of her forehead were moistened and covered with
a nanocrystalline silver coated high density polyethylene mesh, prepared
as in Example 1 (single layer, blue coating). The mesh was then occluded
with a thin film dressing which remained in place for 10 hours. Upon
removal, the papules and pustules were no longer red and were only
slightly raised. Some brown staining of the skin was observed.
Example 6
Treatment of Acne
[0159] A sixteen year old male was diagnosed with acne vulgaris. He had
numerous raised, red papules and pustules on his forehead. Various skin
cleansing regimes and antibiotic treatments had been tried and had failed
to control the acne. The patient was placed on isotretinoin treatment
which controlled his acne well. He did develop a single large pustule on
his forehead which was embarrassing for him. Prior to bedtime, the
pustule was moistened and covered with a nanocrystalline silver coated
high density polyethylene mesh prepared as in Example 2. The mesh was
then occluded with a thin film dressing which remained in place for 10
hours. Upon removal the pustule was no longer red and was only slightly
raised. A second treatment resulted in the disappearance of the pustule.
Example 7
Treatment of Acne
[0160] A sixteen year old female was diagnosed with acne vulgaris. She had
numerous red papules and pustules on her forehead. Various skin cleansing
regimes and antibiotic (erythromycin and clindomycin) treatments had been
tried and had failed to control the acne. Prior to bedtime, the papules
and pustules on one side of her forehead were moistened and covered with
a nanocrystalline silver coated high density polyethylene mesh, prepared
as in Example 2. The mesh was then occluded with a thin film dressing
which remained in place for 10 hours. Upon removal the papules and
pustules were no longer red and were only slightly raised. A second
treatment resulted in the disappearance of the papules and virtual
elimination of the pustules. The silver coated mesh, when prepared as set
forth in Example 2, did not result in any staining of the skin.
Example 8
Treatment of Adult Acne with Silver-Impregnated Hydrocolloid Dressing
[0161] A 49 year old white male experienced occasional acne vulgaris. He
had painful, raised, red papules and pustules on his shoulders. The
patient was treated with a thin hydrocolloid dressing (Craig Medical
Products Ltd., Clay Gate House 46 Albert Rd. North Reigate, Surrey,
United Kingdom) which was impregnated with 1% nanocrystalline silver
powder formed with atomic disorder as in Example 3. Following cleansing,
the pustule was covered with a small disc of the dressing, which remained
in place for 24 hours. Upon removal, the pustule was no longer painful,
red, or raised.
Example 9
Treatment of Eczema
[0162] A twenty-nine year old white female presented with acrodermatitis.
The erythematous area was located on the dorsal surface of the first web
space of the left hand. It was bounded by the metacarpal bones of the
thumb and index finger. The patient also complained of pruritus
associated with the dermatitis. A gel consisting of 0.1% nanocrystalline
silver powder (formed with atomic disorder as in Example 3) and 2%
carboxymethylcellulose was applied to the inflamed area before bedtime.
There was an immediate antipruritic effect that provided the patient with
relief in the short term. The next morning all evidence of acrodermatitis
(i.e. redness disappeared) was gone. The condition had not returned after
two weeks.
Example 10
Allergic Contact Dermatitis
[0163] Skin allergic contact hypersensitivity is caused by excessive
infiltration of eosinophils. An animal model may be used for in vivo
evaluation of eosinophil infiltration in the contact sensitivity reaction
and to determine whether it is associated with allergic skin conditions
such as contact dermatitis. On a gross histology level, this can be
measured by the degree of erythema and edema at the dermatitis site.
Current drugs used for treatment of this and other related eczema
conditions include high potency steroids (Ultravate.TM.), medium potency
steroids (Elocon.TM.) and non steroidal anti-inflammatory compounds
(Protopic.TM. or tacrolimus). These compounds do not always work and may
have undesirable side effects. Several commercially available
anti-inflammatory products were compared to a nanocrystalline silver
powder for the treatment of allergic contact dermatitis as follows.
[0164] Four healthy domestic pigs (approximate weight 20 kg) were used in
the study. All pigs had normal skin prior to induction of eczema with 10%
2,4-dinitrochlorobenzene (DNCB) in acetone. The animals were housed in
appropriate animal facilities with 12 hour light-dark cycles. The pigs
were fed antibiotic-free feed and water ad libitum. The pigs were housed
and cared for in accordance with Canadian Council of Animal Care
guidelines. On day 0, the hair on both left and right back and side were
clipped. The DNCB solution was painted over this area. This was repeated
on day 7 and 11. On day 11, the solution was painted approximately 4
hours before treatment was initiated.
[0165] Treatment groups are shown in the following table. Protopic.TM.
(tacrolimus), Elocon.TM. and Ultravate.TM. were purchased as creams from
the local pharmacy. The nanocrystalline silver powder (1 g/L) was mixed
into a 2% sodium carboxymethyl cellulose (CMC) and water solution at
30.degree. C. using a magnetic stirrer at a high speed (Vista
Scientific). Petrolatum, commercially known as Vaseline.TM., was used as
a control for Elocon.TM. and Ultravate.TM..
5
Day of
Pig # Treatment (Left Side) Control
(Right Side) Treatment
1 Protopic .TM.
(tacrolimus) Protopic .TM. Control Day 0
2 Medium Potency Steroid
Petrolatum Day 0
(Elocon .TM.)
3 2% CMC + 1% 2% CMC Day 0
nanocrystalline silver
(Vista Scientific)
4 High
Potency Steroid Petrolatum Day 0
(Ultravate .TM.)
[0166] Pigs were placed under general anesthetic with ketamine
(Ketalean.TM., MTC Pharmaceuticals, Cambridge, ON; 4-500 mg) and
halothane (MTC Pharmaceuticals). The skin was wiped with a moist gauze
and allowed to dry. Bandages (n=8) containing each treatment were applied
to the left side of the thoracolumbar area of the pig, while control
bandages (n=8) were applied to the right side of the thoracolumbar area
of the pig. Following placement of bandages, they were covered with
Tegaderm.TM. (3M Corp., Minneapolis, Minn.) which was secured with an
Elastoplast.TM. (Smith and Nephew, Lachine, QC) wrap. Bandages with
active agents were changed daily. The skin associated with each bandage
site was scored for severity of erythema (0=normal, 1=slight, 2=moderate,
3=severe, 4=very severe) and swelling (0=normal, 1=slight, 2=moderate,
3=severe, 4=very severe). This was performed on days 0, 1, 2 and 3.
[0167] All pigs remained healthy during the study. Results are shown in
the following tables, and indicated in FIGS. 3 and 4. FIGS. 3 and 4 show
the efficacy of the nanocrystalline silver powder compared to
Protopic.TM., Elocon.TM. and Ultravate.TM. in the treatment of contact
dermatitis in the pig model.
6
Day 0 Day 1 Day 2 Day 3
Treatment (Erythema)
Nanoerystalline silver 3 2 1 0
Protopic .TM. 3 3 1.9 0.4
Elocon .TM. 3 2.4 2.6 2.6
Ultravate .TM. 3 3 3 3
Treatment (Edema)
Nanocrystalline
silver 2 1 0 0
Protopic .TM. 2 2 2 0
Elocon .TM. 2 2 2 0
Ultravate .TM. 2 2 1 0
[0168] The pigs treated with the high (Ultravate.TM.) and medium
(Elocon.TM.) strength steroids showed little to no improvement in the
degree of erythema associated with contact dermatitis. They did, however,
improve in terms of edema in that at Day 3, no swelling was apparent.
Protopic.TM. showed a marked improvement when compared to the steroids in
both the degree of erythema and edema. The largest improvement occurred
with the nanocrystalline silver powder suspended in a 2% carboxymethyl
cellulose gel. Both erythema and edema scores were lower after a single
treatment and were normal after Day 2 (edema) and Day 3 (erythema) of
treatment. Clearly the nanocrystalline silver product was more
efficacious in treating contact dermatitis than the commercially
available products.
Example 11
Preparation of Gels
[0169] No. 1
[0170] A commercial carboxymethyl cellulose/pectin gel (DuoDERM.TM.,
ConvaTec Canada, 555, Dr. Frederik Philips, Suite 110, St-Laurent,
Quebec, H4M 2X4) 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.sup.-1 to 10.sup.-7. A 0.1 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.
[0171] No. 2
[0172] 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.
[0173] No. 3
[0174] 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.
[0175] No. 4
[0176] 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.
[0177] 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.
[0178] 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.
[0179] Nos. 5-10
[0180] The formula for Nos. 5-10 are summarized in the following table.
Zones of inhibitions were determined as in No. 4 and log reductions were
determined as in No. 1.
[0181] 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.
7
Log Log
Ag CZOI CZOI red'n red'n
CMC PVA Powder Beta- Methyl Propyl S. P. S. P.
# (%) (%) (%)
glucan paraben paraben aureus aeruginosa aureus 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
[0182] No. 11
[0183] A commercially available gel (glyceryl polymethacrylate) was
blended with nanocrystalline silver powder 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.
Example 12
Treatment of Adult Acne with Nanocrystalline Silver Gel Occluded by a
Hydrocolloid Dressing
[0184] A 49 year old white male experienced occasional acne vulgaris. He
had painful, raised, red papules and pustules on his shoulders. The
patient was treated with gel formulation No. 5 as set forth in Example
11. Gel formulation No. 5 was applied to the problem area of the
patient's shoulders and then occluded by a thin hydrocolloid dressing
(Craig Medical Products Ltd., Clay Gate House 46 Albert Rd. North
Reigate, Surrey, United Kingdom). The dressing remained in place for 24
hours. Upon removal the pustule was no longer painful, red, or raised.
[0185] Treatment of Mucosal or Serosal Conditions
Example 1
Preparation of Nanocrystalline Silver Coatings on Dressings
[0186] 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 as described in Example 1 of the Treatment
of Hyperproliferative Skin conditions examples. The coating layers were
formed by magnetron sputtering under the conditions set out in the
following table.
Example 2
Preparation of Atomic Disordered Nanocrystalline Silver Powders
[0187] Atomically disordered, nanocrystalline silver powder was prepared
as described in Example 3 in the Treatment of Inflammatory Skin
conditions examples above.
Example 3
[0188] Silver solutions were prepared by immersing AgHDPE mesh from
dressings prepared as in Example 1 in reverse osmosis water that had been
pretreated with CO.sub.2 in order to reduce the pH. Two different
concentrations of silver solutions were prepared by this method, the
concentrations being 85 .mu.g/ml, and 318 .mu.g/ml. Solutions of silver
nitrate were also prepared to use as comparisons in the experiments. The
concentrations of the silver nitrate were 103 ppm of silver and 295 ppm
of silver as determined by Atomic Absorption Spectroscopy.
[0189] The solutions were in turn placed in an ultrasonic nebulizer that
created small droplets containing dissolved and suspended parts of the
silver solution. The output from the nebulizer was directed into a
chamber made from a stainless steel frame and base. Petri dishes
containing Mueller Hinton agar streaked with 4 h old cultures of
Pseudomonas aeruginosa and Staphylococcus aureus, were exposed to the
silver solution aerosols and the silver nitrate aerosols.
[0190] The results of the tests show that silver aerosols of this
invention transmit the antimicrobial activity of the dressings to remote
sites, and such aerosols are more effective than comparable
concentrations of silver nitrate.
[0191] In many instances the delivery of antimicrobial materials may most
expeditiously be accomplished by using aerosols (e.g. treatment of
pneumonia). The drawback of aerosols is the requirement for a high
concentration of the active ingredient to ensure that a sufficient amount
is delivered to achieve the biological effect desired without causing
problems with the carrier solvent (e.g. water). It is preferably that the
equipment for producing an aerosol that contains the dissolved and
suspended components of nanocrystalline silver form droplets of aerosol
direct from the liquid form, and the aerosol droplets must be small
enough to reach the lungs. This means the droplets should be less than
approximately 10 .mu.m. To meet these requirements the aerosol is not
created by first evaporating the liquid then condensing it to form
droplets. Rather, aerosols are generated by 1) mechanical disruption of
the liquid, or 2) air under pressure passing through some form of orifice
that combines the air and the liquid in a way that creates droplets
instead of evaporating the liquid.
[0192] Several experiments were carried out with silver solutions of this
invention and silver nitrate solutions to determine if the antimicrobial
activity of the dressing could be transferred through a direct droplet
aerosol to a Petri dish.
[0193] a) Methods
[0194] i) Equipment
[0195] The method used for the current tests was the mechanical method in
the form of an ultrasonic nebulizer. For convenience an ultrasonic
humidifier was used. The liquid containing the dissolved and suspended
silver from the dressing of Example 1 was placed in the water reservoir
of the humidifier. When power was applied to the humidifier aerosol
droplets of dissolved and suspended silver were generated and flowed from
the output nozzle.
[0196] A test chamber was constructed using a stainless steel frame with a
transparent plastic covering. The frame was placed on a stainless steel
base plate. The output nozzle from the humidifier was modified so that
the aerosol could be directed into the chamber at a height of
approximately 30 cm from the base. The plates and other test samples were
placed on the stainless steel plate and exposed to the aerosol for a
prescribed length of time.
[0197] ii) Solutions
[0198] Solution 1--A silver containing solution was prepared by immersing
518 sq. inches of the dressing from Example 1 in 1 L of reverse osmosis
water, which was treated with CO.sub.2 to maintain a pH of 6.5. After 20
minutes the concentration of silver in the water was 85 .mu.g/ml.
[0199] Solution 2--A solution containing 370 .mu.g/ml of silver from a
dressing from Example 1 was prepared as follows: 1 L of reverse osmosis
water was purged with commercial grade carbon dioxide until the pH was
4.3.
[0200] Sufficient dressing was added to bring the pH up to 6.5. At that
time, the silver concentration was 370 .mu.g/ml.
[0201] Solution 3--Ag as AgNO.sub.3 was prepared by dissolving 0.157 g of
AgNO.sub.3 into 1 L of reverse osmosis water and mixed until dissolved.
The solution was analyzed by Atomic Absorption Spectroscopy and found to
be 102.9 ppm of silver.
[0202] Solution 4--Ag as AgNO.sub.3 was prepared by dissolving 0.427 g of
AgNO.sub.3 into 1 L of reverse osmosis water and mixed until dissolved.
The solution was analyzed by Atomic Absorption Spectroscopy and found to
be 295 ppm of silver.
[0203] iii) Aerosolization Petri dishes, containing Mueller Hinton agar,
were streaked with 4 h old cultures of Pseudomonas aeruginosa or
Staphylococcus aureus. The plates were then weighed and their exposed
outer surfaces were coated with Parafilm to prevent condensation from
occurring on these surfaces. These plates were placed in the aerosol
chamber uncovered. The ultrasonic nebulizer was then started and run for
53 minutes. The plates were then removed from the chamber, the plastic
was removed and the dishes re-weighed so that the amount of moisture
loss/gain could be determined.
[0204] The plates were then placed in a 35.degree. C. incubator for 16 h.
After incubation the pattern and amount of growth was assessed on the
plates for both organisms.
[0205] iv) Viability Assessment
[0206] Three of the six plates made for each organism were tested to
determine if the antimicrobial effect was cidal or static in nature. This
was accomplished by rinsing or placing a piece of the clear section of
agar in the Petri dish plates into Tryptic soy broth in a test tube and
incubating for 4 h or 16 h. If the medium turned turbid in 4 h it would
indicate that the antimicrobial affect was bacteriostatic in nature. If
the organisms took more than 16 h to grow, as indicated by turbidity, it
was considered an indication that both static and cidal effects occurred.
If no growth occurred the effect was bactericidal.
[0207] v) Results--The results are summarized in the following table.
8
Silver from Dressing AgNO.sub.3
P. S. P. S.
Organism aeruginosa aureus aeruginosa aureus
Solutions
1 and 3
Ag concentration 85 85 99 99
(.mu.g/ml)
pH
of test solution 6.5 6.5 About 6.5 about 6.5
Exposure Time 53 53
53 53
(minutes)
Exposed are (sq. in.) 9.8 9.8 9.8 9.8
Exp 0.8 0.8 1.05 1.05
Weight Gain (g)
Growth at 16 h 0 0 0
0
(0-++++) at 48 h 0 ++ 0 ++++
Viable 4 h No No No No
16 h No No No N/A
Solutions 2 and 4
Ag concentration 370
370 300 300
(.mu.g/ml)
pH of test solution 6.5 6.5 About
6.3 about 6.3
Exposure Time 53 53 53 53
(minutes)
Exposed are (sq. in.) 9.8 9.8 9.8 9.8
Exp 1.14 1.14 1.12 1.12
Weight Gain (g)
Growth at 16 h 0 0 0 0
(0-++++) at 48 h 0
0 0 +++
Viable 4 h No No No No
16 h No No No N/A
[0208] vi) Discussion
[0209] At the low concentration of silver in solution, the dressing
generated silver was effective in controlling the growth of both
organisms while the silver nitrate only prevented the growth of P.
aeruginosa. Viability tests showed that at the low concentration, neither
form of silver was completely bacteriocidal although the poor growth on
the dressing aerosol treated plates compared to the silver nitrate
treated plates suggests that a significant log reduction occurred in the
dressing aerosol treated plates.
[0210] At a higher concentration of silver, both dressing generated silver
(370 .mu.g/ml) and AgNO.sub.3 (300 .mu.g/ml) were effective at
controlling P. aeruginosa. Since no re-growth occurred, it is assumed
that the agent at this concentration were bactericidal. Silver generated
from the dressing was more effective than AgNO.sub.3 at controlling S.
aureus. No re-growth occurred on any plates or in the broth indicating a
total kill of the organism while in the AgNO.sub.3 treatment, a large
number of organisms grew at 16 h.
[0211] Based on weight gain during aerosol treatments a dose per unit area
can be calculated. In each case for solution 1 the dose was 8.5 .mu.g/sq.
inch while for solution 2 the dose was 38 .mu.g/sq. inch. These doses, on
a per lung basis, would be less than 10 mg of silver per hour of
treatment. Each hour of treatment with dressing generated silver aerosols
appears to provide at least 48 h of protection. Therefore the dose per
day, from the high concentration treatment, would be about 5 mg or a
little less than the silver released by 2 sq. inches of SSD per day.
[0212] A most significant advantage of using dressing generated silver may
be the lack of a toxic cation such as NO.sub.3 or sulfadiazine.
[0213] Overall, the example demonstrated that the dressing generated
aerosols are operative to transmit the antimicrobial activity of the
dressings to remote sites. Furthermore, the dressing generated aerosols
were more effective than comparable concentrations of silver nitrate.
Example 4
Aerosolized Silver Solutions in Rats
[0214] a) Materials And Methods
[0215] i) Solutions From Atomically Disordered Silver Dressings
[0216] A solution was prepared by sparging CO.sub.2 through 400 ml of
reverse osmosis water for 30 minutes at a flow rate of 1 L/min. The
beaker of water was covered with a piece of parafilm to assist in
maintaining a saturated CO.sub.2 environment. This process resulted in
the pH of the water dropping to about 4. At this point, approximately 600
square inches of silver-coated net (AgHDPE) prepared as in Example 1 was
added to the water and stirred for approximately 40 minutes resulting in
an elevation of the pH to approximately 6.5. The solution was then
transferred to a medical nebulizer and connected to an oxygen cylinder
with a flow rate of 10 L/min. The outflow of the nebulizer was connected
to a sealed animal chamber housing the rats to be dosed. Only the "test"
rats (15 randomly assigned animals) received the dosing. The rats
received two, one-hour aerosol administrations of the solution on the day
of infection. Thereafter, the test rats were dosed three times per day
for an additional three days.
[0217] ii) Animals
[0218] Thirty male Sprague-Dawley rats were obtained from the University
of Calgary, Alberta, Canada breeding colony. These animals were
specific-pathogen free and weighed approximately 300 g. The animals were
housed in groups of 5 in plastic cages with wire mesh tops. The rats had
access to fresh water and rat chow ad libitum. All animals were
maintained in an appropriate facility with 12-hour light/dark cycles and
constant temperature and humidity, according to facility standard
operating procedures. The protocol 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.
[0219] iii) Bacteria
[0220] The bacteria used for infection of these animals were Pseudomonas
aeruginosa strain 579. The dose was previously titrated to ascertain that
a dose of up to 10.sup.10 CFU was not lethal for the animals. The
bacteria were grown overnight in Tryptic soy broth, washed once in
sterile PBS, and re-suspended in a 1/10 volume of sterile PBS.
[0221] iv) Infection
[0222] The rats were anesthetized by inhalation of 2% halothane. A 50
microliter volume of bacterial suspension was intratracheally
administered into the bronchi of each rat. This was performed
non-surgically on intubated animals. The infection process resulted in
the instillation of approximately 2.times.10.sup.9 CFU into the lungs of
each animal.
[0223] v) Sampling
[0224] On each day, a number of animals were sacrificed. The lungs of the
animals were aseptically removed, homogenized, and plated to determine
bacterial levels. A few animals were also subjected to bronchoalveolar
lavage prior to removal of the lungs. In several cases, lung homogenates
and/or lavage fluids were reserved for silver analysis.
[0225] After the first batch of the silver solution was prepared, total
silver analysis indicated that there was about 225 ppm of total silver in
the solution. The solution was reserved for several hours until after
next dosing of the animals. A second silver analysis indicated that the
total silver in solution had dropped to about 166 ppm. The reason for the
drop was immediately apparent as the silver had visibly precipitated out
of solution and had deposited on the surface of the nebulizer. One other
batch of freshly prepared solution had a total silver concentration of
337 ppm. Regardless of the actual numbers, the process of generating the
silver solution results in a significant quantity of silver in the
solution that is aerosolized into the dosing chamber.
[0226] The dosing chamber is not perfect. Although significant amounts of
mist are generated into the chamber, the rates tend to lie on top of one
another and are probably exposed to vastly different levels of the silver
mist.
9
Day Log CFU/Test Lung Log CFU/Control Lung
1 6.2 7.3
2 4.1 7.8
3 0 6.2
4 3.5 4.8
[0227] The bacteriological results gathered from the lungs of the treated
and control animals demonstrated a sharper decline in the numbers of
bacteria present in the lungs following treatment with silver mist as
compared to controls. The results indicated that, in spite of the small
sample sizes and inconsistent exposures, a difference could still be
noted. There was considerable variation in the numbers of bacteria
recovered from individual animals within each treatment group and,
therefore, there was no significant difference in the results. Gross
examination of excised lungs suggested that there may have been less
apparent damage to the lungs in the animals treated with the silver mist
as compared to the untreated, infected animal. This was very encouraging
given the potential anti-inflammatory effects of the nanocrystalline
silver technology.
10
Sacrifice Date Rat Description Total Silver Level
Average
36999 Silver mist 1 0.50 ppm
36999
Silver mist 2 1.13 ppm 0.74 ppm
36999 Silver mist 3 0.58 ppm
37000 Silver mist 4 0.73 ppm
37000 Silver mist 5 0.70 ppm 0.72
ppm
37000 Control 1 0.08 ppm
37000 Control 2 0.10 ppm 0.09
ppm
[0228] The results of the silver analysis appear to indicate that the
amount of silver in the lung either plateaus or each dose of silver mist
deposits a certain amount of silver within the lung and this level is
significantly diminished prior to the next dosing of the animals.
[0229] The results of this experiment indicated that the method employed
to prepare the silver mist solution was reasonably reproducible and
yielded relatively high concentrations of silver in solution. However,
the silver was prone to precipitation and should be freshly prepared
prior to each dosing period. A lengthy period between preparation and
dosing, although resulting in a decrease in the amount of silver in
solution, did not result in a complete elimination of the silver from the
solution or even result in the silver concentration dropping to very low
levels.
[0230] The method employed for exposing the rats to the mist is also prone
to significant variation due to the piling up of the rats and the
resultant inconsistent exposure to the silver-containing mist. However,
the silver analyses suggested that a reasonably uniform dose of silver
was achieved when only a few animals were present within the dosing
chamber.
[0231] Regardless of the difficulties associated with the experiment, the
results were indicative of a therapeutic modality for pulmonary
infections. The results showed that the presence of silver mist was
effective in more rapidly clearing the bacterial load of the infected
lungs than is the host immune system alone. The apparently less severe
pathology associated with the rat lungs treated with the silver mist
showed that the treatment was effective for more than simply assisting in
the killing of invading organisms.
Example 5
Pulmonary Anti-inflammatory Activity
[0232] A solution form nanocrystalline silver coated dressings (AgHDPE)
from Example 1 was prepared by sparging CO.sub.2 through 1000 ml of
reverse osmosis water using commercial CO.sub.2 Soda Syphon Charger. This
process resulted in the pH of the water dropping to about 4. At this
point, approximately 333 ml of the carbonated water was decanted into a
plastic bottle and 333 square inches of nanocrystalline silver-coated net
was added to the water. The nanocrystalline silver-coated net and water
were placed in 37.degree. C. shaker incubator and shaken at 180 RPM for
30 minutes to elevate the pH to approximately 5.8. The solution was then
transferred to a beaker and stirred vigorously for 2 minutes to raise the
pH to approximately at 7.3. The dissolution solution was transferred to a
commercial nebulizer which was connected to a medical air cylinder with a
flow rate of approx. 20 L/min. The outflow of the nebulizer was connected
to a animal chamber housing the rats to be dosed. Only the "test" rats
(12 randomly assigned animals) received the dosing. The rats received
two--1/2 hour aerosol administrations of the test solution on the day of
infection. Thereafter, the test rats were dosed 3 times per day for an
additional one and a half days.
[0233] Thirty male Spragu-Dawley rats were obtained. These animals are
specific-pathogen free and weighed approximately 400 g. The animals are
housed in groups of four in plastic cages with wire mesh tops. The rats
had access to fresh water and rat chow ad libitum. All animals were
maintained in an appropriate facility standard operating procedures.
[0234] The bacteria used for infection of these animals were Pseudomonas
aeruginosa strain 5588. The dose was previously titrated to ascertain
that a dose of up to 10.sup.9 CFU was not lethal for the animals. The
bacteria were grown overnight in Tryptic soy broth, washed once in
sterile PBS and resuspend in sterile PBS. The final concentration of the
inoculum was 4.times.10.sup.9 CFU/ml.
[0235] The rats were anesthetized by inhalation of 2% halothane. A 400
microliter volume of bacterial suspension was intratracheally
administered in the bronchi of each rat. This was performed
non-surgically on intubated animals. The infection process resulted in
the instillation of approximately 10.sup.9 CFU into the lungs of each
animal.
[0236] The three treatment groups of rats and treatment schedules were as
follows:
11
Group 1 Infected, not treated (12 Rats)
Group 2
Infected, animal will be treated by intramuscularly injection of
Tobramycin at 30 mg/kg (12 mg/rat) once daily (12 Rats)
Group 3
Infected and treated, using nanocrystalline silver solution and
nebulizer (Nebulized Ag), three times a day (12 Rats)
Day One
10:00 AM Infection
4:00 PM First treatment (For Group 2,
Nebulized Ag for Group 3)
8:00 PM Nebulized Ag treatment for
Group 3
Day Two
9:00 AM Injection treatment for Group 2,
Nebulized Ag for Group 3
1:00 PM Sacrifice and sample six Rats in
each group
3:00 PM Nebulized Ag treatment for Group 3
8:00 PM Nebulized Ag treatment for Group 3
Day Three
9:00
AM Injection treatment for Group 2, Nebulized Ag for Group 3
1:00
PM Sacrifice and sample six Rats in each group
[0237] On each day, six rats of each group of animals were sacrificed. The
lungs of the animals were aseptically removed, homogenized and plated to
determine bacterial levels. Lung samples were collected for histological
examination. Three lung homogenates were reserved for silver analysis.
Lungs were grossly scored (absent=0, mild=1, moderate=2, and severe=3)
based on the degree and involvement of consolidation, hemorrhage, edema
and necrosis based upon gross observation.
[0238] Histopathology was scored (0-4) based upon the degree of
consolidation and inflammation (neutrophil infiltration). The entire
right middle lobes of all rats were collected for histopathology. As
whole lobes were selected there was no bias toward any sample. All
samples were fixed in neutral buffered formalin at the time the lung was
removed from the thorax. It was fixed overnight, dehydrated and embedded
in was. Sections were obtained which were hydrated and stained with
haematoxylin and eosin.
[0239] All sections were examined by a veterinary pathologist who was
blinded to the treatment groups, until after the samples were scored and
comments were provided. The Scores and comments are provided in Table 5.
(0=normal, 1=slight, 2 moderate, 3 severe, 4 very severe).
[0240] Tissue Colony Counts:
[0241] At 24 hours, there was not a significant reduction in the number of
colony forming units (cfu) in the nebulized Ag group compared to the
control but at 48 hours there was a significant reduction in the
bacterial numbers in the nebulized Ag animals. The Tobramycin treated
animals had a similar cfu counts to the controls at time 24 hours and 48
hours.
12
Control Tobramycin Nebulization
24 h animal
1 0 2 1
2 0 3 1
3 3 1 0
4 3 3 0
5 2 2 3
6 3 2 1
48 h animal
7 2 1
1
8 1 2 1
9 1 1 0
10 1 1 0
11 3 1 1
12 Dead Dead Dead
[0242] Histopathology of Lung Samples:
[0243] Both the control and the Tobramycin treated rats had similar
pathology. These are outlined in Table 6. At 24 and 48 hours severe
infiltration of polymorphonuclear leukocytes (PMN's) into the
interstitial spaces of the lung was observed. These cellular elements
could also be identified in alveolar and bronchiolar spaces but to a
lesser extent.
[0244] The pulmonary vessels were dilated and the alveolar spaces were
filled with proteinaceous material. The silver-nebulized rats had
occasional infiltration of PMN's and no evidence of accumulation of
fluids in alveolar or bronchiolar spaces.
13
Histopathology of Lung Samples Removed from Rats
Inflam Consol
Treatment Time Score Score Comments
Control (1) 24 3 3 Severe infiltration of PMN into interstitial spaces.
Proteinaceous secretion in alveolar spaces. Occasional
PMN in alveolar and bronchiolar space. Consolidation
in
affected areas. Involvement of 70% of sample.
Interstitial
Pneumonia.
Control (2) 24 3 3 Severe infiltration of PMNs into
interstitial spaces
Proteinaceous secretion in alveolar
spaces. Occasional
PMN in alveolar and bronchiolar space.
Consolidation
in affected areas. Involvement of 80% of sample.
Interstitial Pneumonia
Tobramycin (1) 24 3 3 Severe
infiltration of PMNs into interstitial spaces.
Proteinaceous
secretion in alveolar spaces. Occasional
PMN in alveolar and
bronchiolar space. Consolidation
in affected areas.
Involvement of 90% of sample.
Interstitial Pneumonia.
Tobramycin (2) 24 3 3 Severe infiltration of PMNs into interstitial
spaces.
Proteinaceous secretion in alveolar spaces. Occasional
PMN in alveolar and bronchiolar space. Consolidation
in affected areas. Involvement of 80% of sample.
Interstitial
Pneumonia.
Nebulized Ag (1) 24 0 1 No PMNs in area. Slight
consolidation. Normal Lung
Nebulized Ag (2) 24 1 1 Slight
infiltration of PMNs around vessels and
brocheoli.
Control (1) 48 3 3 Severe infiltration of PMNs into interstitial spaces.
Proteinaceous secretion in alveolar spaces. Occasional
PMN in alveolar and bronchiolar space. Consolidation
in
affected areas. Involvement of 80% of sample.
Interstitial
Pneumonia.
Control (2) 48 2 2 Severe infiltration of PMNs into
interstitial spaces.
Proteinaceous secretion in alveolar
spaces. Occasional
PM7N in alveolar and bronchiolar space.
Consolidation
in affected areas. Involvement of 60% of sample.
Interstitial Pneumonia.
Tobramycin (1) 48 3 3 Severe
infiltration of PMNs into interstitial spaces.
Proteinaceous
secretion in alveolar spaces. Occasional
PMN in alveolar and
bronchiolar space. Consolidation
in affected areas.
Involvement of 70% of sample.
Interstitial Pneumonia.
Tobramycin (2) 48 3 3 Severe infiltration of PMNs into interstitial
spaces.
Proteinaceous secretion in alveolar spaces. Occasional
PMN in alveolar and bronchiolar space. Consolidation
in affected areas. Involvement of 70% of sample.
Interstitial
Pneumonia. Slight infiltration of PMNs
around vessels and
brocheoli.
Nebulized Ag (1) 48 1 0 Slight infiltration of PMNs
around vessels and
brocheoli.
Nebulized Ag (2)
Normal lung.
[0245] The nebulized nanocrystalline silver reduced bacterial colonization
in Pseudomonas infected lungs reduced injury as determined by gross
pathology (consolidation, hemorrhage, edema) in Pseudomonas infected
lungs. Further, the nanocrystalline silver delivered by aerosol reduced
pulmonary inflammation (primarily PMN infiltration) in Pseudomonas
infected lungs compared to Tobramycin (IM).
Example 6
Pulmonary Anti-inflammatory Activity
[0246] A solution was prepared by sparging CO.sub.2 through 1000 ml of
reverse osmosis water using commercial CO.sub.2 Soda Syphon Charger. This
process results in the pH of the water dropping to about 4. At this
point, approximately 333 ml of the carbonated water was decanted into a
plastic bottle and 333 square inches of nanocrystalline silver-coated net
was added to the water. The nanocrystalline silver-coated net and water
were placed in 37.degree. C. shaker incubator and shaken at 180 RPM for
30 minutes to elevate the pH to approximately 5.8. The solution was then
transferred to a beaker and stirred vigorously for 2 minutes to raise the
pH to approximately at 7.3. The solution had a final silver concentration
of approximately 400 ppm.
[0247] Test solutions of silver nitrate (400 ppm) and silver acetate (400
ppm) were prepared by dissolving the silver salts in deionized water. A
colloidal silver solution (20 ppm) in was obtained from a commercial
source.
[0248] The dissolution solutions were transferred to commercial nebulizers
which were connected to a Medical air cylinder with a flow rate of
approx. 20 L/min. The outflows of the nebulizers were connected to an
animal chamber housing the rats to be dosed. All rats (40 randomly
assigned animals) received the dosing. The rats received two--1/2 hour
aerosol administrations of the test solutions on the day of infection.
Thereafter, the test rats were dosed 3 times per day for an additional
one and a half days.
[0249] Forty male Sprague-Dawley rats were obtained. These animals are
specific-pathogen free and weighed approximately 400 g. The animals were
housed in groups of four in plastic cages with wire mesh tops. The rats
had access to fresh water and rat chow ad libitum. All animals were
maintained in an appropriate facility with 12-hour light/dark cycles and
constant temperature and humidity, according to facility standard
operating procedures.
[0250] The bacteria used for infection of 20 these animals were
Pseudomonas aeruginosa strain 5588. The dose was previously titrated to
ascertain that a dose of up to 10.sup.9 CFU was not lethal for the
animals. The bacteria were grown overnight in Tryptic soy broth, washed
once in sterile PBS and resuspend in sterile PBS. The final concentration
of the inoculum was 4.times.10.sup.9 CFU/ml.
[0251] The rats were anesthetized by inhalation of 2% halothane. A 400
microliter volume of bacterial suspension was intratracheally
administered into the bronchi of each rat. This was performed
non-surgically on intubated animals. The infection process resulted in
the instillation of approximately 10.sup.9 CFU into the lungs of each
animal.
[0252] Group 1 & 2: Not infected and infected, treated with nebulized
silver nitrate. (10 Rats)
[0253] Group 3 & 4: Not infected and infected, treated with nebulized
colloidal silver. (10 Rats)
[0254] Group 5 & 6: Not infected and infected, treated with nebulized
nanocrystalline silver. (10 Rats)
[0255] Group 7 & 8: Not infected and infected, treated with nebulized
silver acetate. (10 Rats)
[0256] The treatment schedule was as follows:
14
Day One Day Two
10:00 AM
Infection 9:00 AM Third Treatment
4:00 PM First Treatment 1:00 PM
Sacrifice, sample 5 rats/Gp
8:00 PM Second Treatment
[0257] All rats of each group of animals were sacrificed after 24 h. The
lungs of the animals were aseptically removed, homogenized and plated to
determine bacterial levels. Lung samples were collected for histological
examination. Three lung homogenates were reserved for silver analysis.
Lungs were grossly scored (absent=0, mild=1, moderate=2, and severe=3)
based on the degree of involvement of consolidation, hemorrhage, edema
and necrosis based upon gross observation.
[0258] Histopathology was scored (0-4) based upon the degree of
consolidation and inflammation (neutrophil infiltration). The entire
right middle lobes of all rats were collected for histopahtology. As
whole lobes were selected there was no bias toward any sample. All
samples were fixed in neutral buffered formalin at the time the lung was
removed from the thorax. It was fixed overnight, dehydrated and embedded
in wax. Sections were obtained which were hydrated and stained with
haematoxylin and eosin.
[0259] All sections were examined by a veterinary pathologist who was
blinded to the treatment groups, until after the samples were scored and
comments were provided, with scoring being (0=normal, 1=slight,
2=moderate, 3=severe, 4=very severe).
[0260] All rats in the silver nitrate, silver acetate and
colloidal silver
groups had lung that were grossly scored as moderately to severely
inflamed while the lungs of the nanocrystalline group were grossly scored
as normal to slightly inflamed. This was confirmed by the
histopathological analyses.
[0261] The nanocrystalline derived silver solution had pulmonary
anti-inflammatory properties while the other forms of silver did not.
Example 7
Treatment of an Infected Throat with a Nanocrystalline Silver Derived
Solution
[0262] A forty-nine year old male was suffering from an infected throat.
The condition was accompanied by fever and a severe pain that made
swallowing very difficult and limited the patients ability to sleep. A
solution of nanocrystalline derived silver was prepared using a method
similar to Example 1. This solution was gargled for one minute and
repeated 3 times over the next ten minutes. Within an hour the pain was
reduced to the point where the patient could sleep. The treatment was
repeated every four hours for 16 h and then once 8 h later. The throat
infection was cleared as determined by the elimination of fever and pain.
No further symptoms occurred.
Example 8
Preparation of Gels
[0263] Gels were prepared as described above in Example 11 in the
Treatment of Inflammatory Skin Conditions examples above.
[0264] Apoptosis Induction/MMP Modulation
Example 1
Preparation of Nanocrystalline Silver Coatings on Dressings
[0265] This example shows the preparation of a bilayer nanocrystalline
silver coating on a dressing material. A high density polyethylene
dressing, DELNETTM or CONFORMANT 2TM was coated with a silver base layer
and a silver/oxide top layer to generate a coloured antimicrobial coating
having indicator value as described in Example 1 of the Treatment of
Hyperproliferative Skin conditions examples. The coating layers were
formed by magnetron sputtering under the conditions set out in the
following table.
Example 2
Preparation of Atomic Disordered Nanocrystalline Silver Powders
[0266] Atomically disordered, nanocrystalline silver powders were prepared
as described in Example 3 in the Treatment of Inflammatory Skin
Conditions examples above.
Example 3
Preparation of Gels
[0267] Gels were prepared as described above in Example 11 in the
Treatment of Inflammatory Skin Conditions examples above.
Example 4
Effects of Antimicrobial Silver on Apoptosis and Matrix Metalloproteinases
in a Porcine Model
[0268] 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 in 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.
[0269] Antimicrobial silver metal was administered in the form of a
dressing. The dressings included:
[0270] 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;
[0271] ii) AgHDPE--nanocrystalline silver coated HDPE layers aseptically
separated from the absorbent core of the AB dressings;
[0272] iii) Control--identical in construction to the AB dressing except
that the HDPE was not coated with nanocrystalline silver;
[0273] iv) Gauze--the absorbent rayon/polyester core of the AB dressings;
[0274] v) cHDPE--the uncoated HDPE aseptically removed from the absorbent
core of the control dressings; and
[0275] 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.
[0276] 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.).
[0277] 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.
[0278] 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.
[0279] 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.
[0280] The experimental and control groups are summarized in the following
table:
15
Animal # Left Side (Silver Treatment) Right Side
(Controls)
Pig 1 silver nitrate (SN) on gauze gauze
moistened with water
Pig 2 AgHDPE cHDPE
Pig 3 AB control
[0281] 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.).
[0282] 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.
[0283] 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 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).
[0284] 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.
[0285] i) Assay for Activity of Total MMPs
[0286] 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.
[0287] FIG. 4 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).
[0288] ii) Assay for Activity of Gelatinases
[0289] 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.
[0290] 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.
[0291] 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.
[0292] iii) Assay of Total Protease Activity
[0293] 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.
[0294] 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. 5).
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. 4). However, the total protease
activity in the incision fluids of the silver coated dressings (AB)
remained constant over the duration of the experiment.
[0295] 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.
[0296] iv) Apoptosis
[0297] Histological assessment of cell apoptosis was carried out in order
to determine whether the silver-coated dressing (AB) affected apoptosis
within the incision.
[0298] Histological Observations of Porcine Tissue
[0299] 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.
[0300] 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-100 (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.
[0301] 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.
[0302] 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.
[0303] Histopathological Scoring of Porcine Tissue
[0304] 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
21-50 per high power field of view; ++++=very abundant with more than 50
per high power field of view (see the following table).
16
Day Post-
Incision Dressing Fibroblasts PMNs
Bacteria
1 Silver coated ++ ++ +
(AB)
1 Control 0 +++ ++++
4 Silver coated ++++ ++ 0
(AB)
4 Control + ++++ ++++
7 Silver Coated ++++ + 0
(AB)
7 Control +++ +++ +++
[0305] 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).
[0306] 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.
Example 5
Clinical Study on the Effect of Silver-Coated Dressings on MMPs and
Cytokines
[0307] This study was conducted to assess the effect of the silver-coated
dressing on the concentrations of MMPs and cytokines in non-healing
wounds over time during treatment. The modulation of the levels of active
MMPs and cytokines may alleviate the inflammatory response in a wound,
allowing the wound to advance through the subsequent stages of wound
healing culminating in a healed wound.
[0308] A total of 10 patients with non-healing venous stasis ulcers were
randomly assigned to treatment with a silver-coated dressing (5 patients)
or a control dressing (5 patients). The silver-coated dressing was
prepared as in Example 1. The control dressing was identical in
construction to the silver-coated dressing of Example 1, except that the
HDPE was not coated with silver. The ulcers were dressed in appropriate
pressure dressings to correct the underlying medical problem. Samples of
the ulcer fluid were collected before treatment (day 0) and at weekly
intervals (days 1, 7, 14 and 21) by removing the silver-coated dressing
or control dressing, and replacing the dressing with Tegaderm.TM.
occlusive dressing (3M Corp., Minneapolis, Minn.) for one hour to allow
wound fluids to collect. The fluid samples were aspirated from below the
dressing in a syringe, and were frozen at -80.degree. C. until assayed.
[0309] Assays were conducted for active MMP-9, active MMP-2, Tumor
necrosis factor-.alpha. (TNF-.alpha.) and Interleukin-1.beta.
(IL-1.beta.). High levels of MMP-9 and MMP-2 are predominant in
non-healing wounds, with levels decreasing over time in normal healing
wounds. Released by activated macrophages, TNF-.alpha. and IL-1.beta. are
indicators of wound inflammation. Levels of TNF-.alpha. and IL-1.beta.
are elevated in non-healing wounds and increase release of pro-MMPs, for
example, MMP-9 and MMP-2.
[0310] To measure the levels of active MMP-9 and MMP-2, enzyme capture
assays (BioTrak, NJ) were conducted. In this method, active enzyme is
detected through activation of a modified pro-detection enzyme and the
cleavage of its chromogenic peptide substrate. The resultant color is
read by spectrophotometer, and the concentration of MMP is determined by
interpolation of a standard curve, expressed in ng/ml (see results in
FIGS. 6 and 7).
[0311] To assay the levels of cytokines, IL-1.beta. levels were measured
using a sandwich immunoassay (BioTrak, NJ), while TNF-.alpha. levels were
measured by a high sensitivity sandwich antibody assay (BioTrak, NJ). In
both methods, endogenous cytokine is bound to an immobilized antibody and
then detected by an addition of a biotinylated antibody, followed by a
colorimetric substrate. The color is measured by a spectrophotometer, and
the concentrations of TNF-.alpha. and IL-1.beta. are determined by
interpolation of a standard curve and expressed as pg/ml (see results in
FIGS. 8 and 9).
[0312] Total protein levels were measured for each sample to standardize
the measures of the MMPs and cytokines. Total protein levels were
measured using BCA Protein Assay System.TM. (Pierce Chemical, Rockford,
Ill.). No protein level of any sample was significantly different from
the total mean.
[0313] FIG. 6 is a graph showing the concentrations (ng/ml) of active
MMP-9 in fluid samples recovered from ulcers dressed with silver-coated
dressing (Silver) and control dressing (Control) at days 0, 1, 7, 14 and
21. The levels of active MMP-9 decreased to a normal level, and were
suppressed over time with the silver-coated dressing compared to the
control dressing, demonstrating a modulating effect of the silver-coated
dressing.
[0314] FIG. 7 is a graph showing the concentrations (ng/ml) of active
MMP-2 in fluid samples recovered from ulcers dressed with silver-coated
dressing (Silver) and control dressing (Control) at days 0, 1, 7, 14 and
21. The levels of active MMP-2 were not significantly different with the
silver-coated dressing and the control dressing.
[0315] FIG. 8 is a graph showing the concentrations (pg/ml) of TNF-.alpha.
in fluid samples recovered from ulcers dressed with silver-coated
dressing (Silver) and control dressing (Control) at days 0, 1, 7, 14 and
21. The levels of TNF-.alpha. were suppressed over the treatment period,
and did not increase significantly over the treatment period with the
silver-coated dressing, while the levels in the control dressing
increased, demonstrating a modulating effect of the silver-coated
dressing.
[0316] FIG. 9 is a graph showing the concentrations (pg/ml) of IL-1.beta.
in fluid samples recovered from ulcers dressed with silver-coated
dressing (Silver) and control dressing (Control) at days 0, 1, 7, 14 and
21. The levels of IL-1.beta. were not significantly different with the
silver-coated dressing and the control dressing.
[0317] The study suggests that the modulation of the MMP-9 and TNF-.alpha.
levels is responsible for improved wound healing and reduced inflammation
with silver-coated dressings. In comparison, the levels of MMPs and
cytokines did not decrease over time with the control dressings.
[0318] This example and Example 4 above, taken together with the evidence
that the silver materials herein disclosed are capable of reducing
inflammation (see co-pending U.S. patent application Ser. Nos.
10/131,568; 10/131,511; 10/131,509; 10/131,513; and 10/128,208 filed Apr.
23, 2002; and co-pending U.S. patent application Ser. No. 09/840,637
filed Apr. 23, 2001, and U.S. Provisional Patent Application No.
60/285,884 filed Apr. 23, 2001) demonstrates a method of reducing
inflammation in a patient in need thereof, by contacting an area of
inflammation or an inflammatory cell with a therapeutically effective
amount of the antimicrobial metals in a crystalline form. 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 modulate the release
of one or both of MMP-9 and TNF-.alpha.. Excessive TNF production has
been reported in diseases, such as cancer and autoimmune diseases, which
are characterized by elevated MMP activity. In this regard, use of the
nanocrystalline silver of the present invention, when in therapeutically
effective amounts, provides the dual modulation of MMP-9 and TNF-.alpha.
to alleviate the particular condition.
ADDITIONAL EXAMPLES
Example 1
[0319] 6 milligrams of antimicrobial metals with atomic disorder, in
free-standing powder form, are sprinkled lightly onto 6.5 cm2 of burned
tissue, and thereafter wet with a light spray of water or wound exudate
or TDWL (Trans Dermal Water Loss) or other bodily fluids, so as to
provide an antimicrobial treatment to the burned tissue. The treatment is
repeated every 24 hours until the therapeutic effects are no longer
needed.
Example 2
[0320] 0.5 milligrams of antimicrobial metals with atomic disorder, in
free-standing powder form, are injected, using a small-needle drug
delivery system or a needle-less drug delivery system, into gum tissue so
as to treat gingivitis. The treatment is repeated every 3 days until the
therapeutic effects are no longer needed.
Example 3
[0321] A solution of antimicrobial metals with atomic disorder is prepared
by dissolving 6 milligrams of antimicrobial metals with atomic disorder
in 1 gram of water. The solution of antimicrobial metals with atomic
disorder is applied as a rinse or bath or wash to a wound site so as to
provide an antimicrobial treatment to the wound site. The treatment is
repeated every 24 hours until the therapeutic effects are no longer
needed.
Example 4
[0322] A solution of antimicrobial metals with atomic disorder is prepared
by dissolving 6 milligrams of antimicrobial metals with atomic disorder
in 1 gram of water. The solution of antimicrobial metals with atomic
disorder is applied to the interior of the bladder via a catheter so as
to provide antimicrobial treatment to the bladder. The treatment is
repeated every 8 hours until the therapeutic effects are no longer
needed.
Example 5
[0323] A solution of antimicrobial metals with atomic disorder is prepared
by dissolving 6 milligrams of antimicrobial metals with atomic disorder
in 1 gram of water. The solution of antimicrobial metals with atomic
disorder is injected (using a small-needle or needle-less injection
system) under the toenails or into the nail bed and/or the surrounding
tissue of a person suffering from onychomycosis so as to provide an
antimicrobial treatment to the tissue. The treatment is repeated 2 times
a day until the therapeutic effects are no longer needed.
Example 6
[0324] Summary
[0325] Solutions of nanocrystalline noble metals were prepared by
immersing Acticoat.RTM. burn dressings (distributed by Smith & Nephew) in
reverse osmosis water that had been pretreated with CO2 in order to
reduce the pH. Two different concentrations of antimicrobial metals with
atomic disorder solutions were prepared by this method, the
concentrations being 85 mg/mL and 318 mg/mL. Solutions of silver nitrate
were also prepared to use as comparisons in the experiments. The
concentrations of the silver nitrate were 103 ppm of silver and 295 ppm
of silver as determined by Atomic Absorption Spectroscopy.
[0326] The solutions were in turn placed in an ultrasonic nebulizer that
created small droplets containing dissolved and suspended parts of the
solution of nanocrystalline noble metals. The output from the nebulizer
was directed into a chamber made from a stainless steel frame and base.
Petri dishes containing Mueller Hinton agar streaked with 4 h old
cultures of Pseudomonas aerugiona and Staphylococcus aureus were exposed
to nanocrystalline noble metal aerosols and the silver nitrate aerosols.
[0327] The results of the tests show that nanocrystalline noble metal
aerosols transmit the antimicrobial activity of the dressings to remote
sites, and nanocrystalline noble metal aerosols are more effective than
comparable concentrations of silver nitrate.
[0328] Introduction
[0329] In many instances the delivery of antimicrobial materials may most
expeditiously be accomplished by using aerosols (e.g., in the treatment
of pneumonia). The drawback of aerosols is the requirement for a high
concentration of the active ingredient to ensure that a sufficient amount
is delivered to achieve the biological effect desired without causing
problems with the carrier solvent (e.g., water). The essential
requirement of the equipment for producing an aerosol that contains
dissolved and suspended components of antimicrobial metals with atomic
disorder is that it must form droplets of aerosol directly from the
liquid form, and the aerosol droplets must be small enough to reach the
lungs. This means that the droplets should be preferably less than
approximately 10 mm. To meet these requirements, the aerosol cannot be
created by first evaporating the liquid and then condensing it to form
droplets, since this would remove the desired antimicrobial metals with
atomic disorder from the aerosol. There are two methods that can be used
to relatively easily form the droplets directly: (1) mechanical
disruption of the liquid; and (2) air, under pressure, passing through
some form of orifice that combines the air and the liquid in a way that
creates droplets instead of evaporating the liquid.
[0330] Several experiments were carried out with antimicrobial metals with
atomic disorder and silver nitrate solutions to determine if the
antimicrobial activity of the dressing could be transferred through a
direct droplet aerosol to a Petri dish.
[0331] Equipment
[0332] The method used to create an aerosol for these tests was the
mechanical method in the form of an ultrasonic nebulizer. For
convenience, an ultrasonic humidifier was used. The liquid containing the
dissolved and suspended antimicrobial metals with atomic disorder was
placed in the water reservoir of the humidifier. When power was applied
to the humidifier, aerosol droplets of dissolved and suspended
antimicrobial metals with atomic disorder were generated and flowed from
the output nozzle.
[0333] A test chamber was constructed using a stainless steel frame with a
transparent plastic covering. The frame was placed on a stainless steel
plate. The output nozzle from the humidifier was modified so that the
aerosol could be directed into the chamber at a height of approximately
30 cm from the base. The plates and other test samples were placed on the
stainless steel plate and exposed to the aerosol for a prescribed length
of time.
[0334] Solution 1
[0335] A solution of antimicrobial metals with atomic disorder was
prepared by immersing 518 sq. inches of Acticoat.RTM. burn dressing in 1L
of reverse osmosis water, which was treated with CO2 to maintain a pH of
6.5. After 20 minutes the concentration of silver in the water was 85
mg/mL.
[0336] Solution 2
[0337] A solution containing 370 mg/mL of silver from a Acticoat.RTM.
dressing was prepared as follows: 1 L of reverse osmosis water was purged
with commercial grade carbon dioxide until the pH was 4.3. Sufficient
Acticoat.RTM. dressing was added to bring the pH up to 6.5. At that time,
the silver concentration was 370 mg/mL.
[0338] Solution 3
[0339] Ag as AgNO.sub.3 was prepared by dissolving 0.157 g of AgNO.sub.3
into 1 L of reverse osmosis water and mixed until dissolved. The solution
was analyzed by Atomic Absorption Spectroscopy and found to be 102.9 ppm
of silver.
[0340] Solution 4
[0341] Ag as AgNO.sub.3 was prepared by dissolving 0.427 of AgNO.sub.3
into 1 L of reverse osmosis water and mixed until dissolved. The solution
was analyzed by Atomic Absorption Spectroscopy and found to be 295 ppm of
silver.
[0342] Aerosolization
[0343] Petri dishes, containing Mueller Hinton agar, were streaked with 4
h old cultures of Pseudomonas aeruginosa or Staphylococcus aureus. The
plates were then weighed and their exposed outer surfaces were coated
with Parafilm to prevent condensation from occurring on these surfaces.
These plates were placed in the aerosol chamber uncovered. The ultrasonic
nebulizer was then started and run for 53 minutes. The plates were then
removed from the chamber, the plastic was removed and the dishes
re-weighed so that the amount of moisture loss/gain could be determined.
[0344] The plates were then placed in a 35.degree. C. incubator for 16 h.
After incubation the pattern and amount of growth was assessed on the
plates for both organisms.
[0345] Viability Assessment
[0346] Three of the six plates made for each organism were tested to
determine if the antimicrobial effect was cidal or static in nature. This
was accomplished by rinsing or placing a piece of the clear section of
agar in the Petri dish plates into Tryptic soy broth in a test tube and
incubating for 4 h or 16 h. If the medium turned turbid in 4 h it would
indicate that the antimicrobial affect was bacteriostatic in nature. If
the organism took more than 16 h to grow, as indicated by turbidity, it
was considered an indication that both static and cidal effects occurred.
If no growth occurred, the effect was bactericidal.
[0347] Results
[0348] The results for Solutions 1 and 3 are summarized in the following
two table.
17
Antimicrobial Metals
With Atomic Disorder
AgNo.sub.3
Ps. S. Ps. S.
Organism Aeruginasa aureus
Aeruginosa aureus
Ag concentration 85 85 99 99
(.mu.g/mL)
pH of test solution 6.5 6.5 Approx 6.5 Approx 6.5
Exposure time 53 53 53 53
(minutes)
Exposed area (sq in)
9.8 9.8 9.8 9.8
Weight gain (g) 0.8 0.8 1.05 1.05
Growth at
16 h 0 0 0 ++++
(0-++++) at 48 h 0 ++ 0 ++++
Viable 4 h No
Yes No Yes
16 h Yes Yes Yes Yes
[0349] The results for Solutions 2 and 4 are summarized in the following
two table.
18
Antimicrobial Metals
With Atomic Disorder
AgNo.sub.3
Ps. S. Ps. S.
Organism aeruginosa aureus
aeruginosa aureus
Ag concentration 370 370 300 300
(.mu.g/mL)
pH of test solution 6.5 6.5 Approx. 6.3 Approx 6.3
Exposure time 53 53 53 53
(minutes)
Exposed area (sq in)
9.8 9.8 9.8 9.8
Weight gain (g) 1.14 1.14 1.12 1.12
Growth
at 16 h 0 0 0 0
(0-++++) at 48 h 0 0 0 +++
Viable 4 h No No
No No
16 h No No No N/A
[0350] Discussion
[0351] At the low concentration of silver in solution, the Acticoat.RTM.
dressing generated silver was effective at controlling the growth of both
organisms while the silver nitrate only prevented the growth of Ps.
aeruginosa. Viability tests showed that at the low concentration, neither
form of silver was completely bactericidal although the poor growth on
the plates treated with antimicrobial metals with atomic disorder
compared to the silver nitrate treated plates suggests that a significant
log reduction occurred in the plates treated with the aerosol of
antimicrobial metals with atomic disorder.
[0352] At a higher concentration of silver, both antimicrobial metals with
atomic disorder (370 mg/mL) and Ag NO.sub.3 (300 mg/mL) were effective at
controlling P. aeruginosa. Since no re-growth occurred, it is assumed
that the agent at this concentration was bactericidal. Antimicrobial
silver with atomic disorder was more effective than Ag NO.sub.3 at
controlling S. aureus. No re-growth occurred on any plates or in the
broth indicating a total kill of the organism while, in the Ag NO.sub.3
treatment, a large number of organisms grew at 16 h.
[0353] Based on weight gain during aerosol treatments, a dose per unit
area can be calculated. In each case for Solution 1, the dose was 8.5
mg/sq. inch, while for Solution 2, the dose was 38 mg/sq. inch. These
doses, on a per lung basis, would be less than 10 mg of silver per hour
of treatment. Each hour of treatment with antimicrobial silver with
atomic disorder aerosols appears to provide at least 48 h of protection.
Therefore, the dose per day, from the high concentration treatment, would
be about 5 mg or a little less than the silver released by 2 sq. inches
of SSD per day.
[0354] The most significant advantage of using antimicrobial silver with
atomic disorder may be the lack of a toxic action such as NO.sub.3 or
sulfadiazine.
[0355] Conclusions
[0356] (1) Aerosols of antimicrobial metals with atomic disorder transmit
the antimicrobial activity of the dressings to remote sites.
[0357] (2) Aerosols of antimicrobial metals with atomic disorder are more
effective than comparable concentrations of silver nitrate.
[0358] (3) The dose delivered is acceptable and would not appear to be
excessive.
[0359] (4) No toxic cations (NO.sub.3 or sulfadiazine) are introduced to
the patient.
Example 7
Gels of Antimicrobial Metals With Atomic Disorder
[0360] Gel products of antimicrobial metals with atomic disorder encompass
both wet and dry materials.
[0361] A wet gel product of antimicrobial metals with atomic disorder is a
product that provides moisture to a dry skin condition (psoriasis,
eczema, acne, wound, etc.) and facilitates autolytic debridement of
necrotic tissue. It also delivers the antimicrobial and anti-inflammatory
properties of the suspended antimicrobial metals with atomic disorder
powders.
[0362] In many instances it is also beneficial to supply biologically
active molecules to elicit a specific response such as cell migration,
etc. Since these biologically active molecules are susceptible to
microbial degradation if not protected, it is beneficial to include them
in gels of antimicrobial metals with atomic disorder that will provide
the necessary protection.
[0363] Dry gel products of antimicrobial metals with atomic disorder are
physically stabilized (dry or cross-linked) materials that provide
lubricious, antimicrobial, antithrombogenic, and anti-inflammatory
properties to a variety of implantable, trancutaneous or topically
applied devices. The coatings may also provide other benefits such as
accelerating or otherwise facilitating tissue integration by creating a
favorable environment for cell proliferation. This favorable environment
may be created by including cyto-conductive agents or anti-adhesion
agents such as bone morphogenetic proteins, B-glucan hyaluronic acids in
the gel. The gel may be stabilized by cross-linking the gel components
(collagen, gelatin, etc.) or by drying the coated materials.
[0364] Examples of the primary gelling agents are listed in the following
table. Biologically active ingredients that may be used, in any
combination with the primary gelling agents, are given in the subsequent
table. Materials that should not be used with gels of antimicrobial
silver with atomic disorder are given in the final table.
19
Percentage Composition
Material
Carboxymethyl cellulose (CMC) 0.1-10
Polyvinyl
alcohol (PVA) 0.1-10
Collagen 0.1-10
Pectin 0.1-10
Uclatin 0.1-10
Chitin 0.1-10
Chitosan 0.1-10
Alginate 0.1-10
Poly (.alpha.-amino acids)
Polyester
Poly-1-caprolactone
PEG
Cocoa butter
Sepigel
Biologically Active Ingredients
Methyl paraben <3
Propyl parabcn <3
B-gltican <5
Hyaluronic acid
<5
Epidermal growth factor <1
Platelet derived
growth factor <1
Transforming growth factor <1
Vascular endothelial growth factor <1
Interleukins <1
Heparin <1
Bone morphogenctic proteins <5
Non-Compatible Materials
Chloride salts >0.01
Aldehydes >0.01
Ketones >0.01
Long chain alcohols
>0.01
Glycerol >0.01
Triethanolamine >0.01
Example 8
Examples of Gels with Antimicrobial Metals With Atomic Disorder
[0365] Gels were prepared as described above in Example 11 in the
Treatment of Inflammatory Skin Conditions examples above.
[0366] Other embodiments are in the claims.
