U.S. patent application number 11/656087 was filed with the patent office on 2007-08-23 for methods of treating or preventing inflammation and hypersensitivity with oxidative reductive potential water solution.
This patent application is currently assigned to Oculus Innovative Sciences, Inc.. Invention is credited to Hojabr Alimi, Andres Gutierrez.
Application Number | 20070196357 11/656087 |
Document ID | / |
Family ID | 38198117 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196357 |
Kind Code |
A1 |
Alimi; Hojabr ; et
al. |
August 23, 2007 |
Methods of treating or preventing inflammation and hypersensitivity
with oxidative reductive potential water solution
Abstract
Provided is a method for preventing or treating inflammation and
associated states (e.g. infection, hypersensitivity, pain) by
administering a therapeutically effective amount of an oxidative
reduction potential (ORP) water solution that is stable for at
least about twenty-four hours. The ORP water solution administered
in accordance with the invention can be combined with one or more
suitable carriers and can be administered in conjunction with one
or more additional therapeutic agents.
Inventors: |
Alimi; Hojabr; (Santa Rosa,
CA) ; Gutierrez; Andres; (Petaluma, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
Oculus Innovative Sciences,
Inc.
Petaluma
CA
94954
|
Family ID: |
38198117 |
Appl. No.: |
11/656087 |
Filed: |
January 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60760635 |
Jan 20, 2006 |
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60760567 |
Jan 20, 2006 |
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60760645 |
Jan 20, 2006 |
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60760557 |
Jan 20, 2006 |
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Current U.S.
Class: |
424/114 ;
424/600; 424/661 |
Current CPC
Class: |
A61P 11/02 20180101;
A61P 31/16 20180101; A61P 1/02 20180101; Y02A 50/475 20180101; A61P
1/04 20180101; A61P 31/14 20180101; A61P 19/02 20180101; A61P 31/10
20180101; A61K 33/00 20130101; A61P 31/12 20180101; A61P 37/08
20180101; A61P 21/00 20180101; A61K 45/06 20130101; A61P 17/00
20180101; A61P 31/04 20180101; A61P 37/02 20180101; A61P 43/00
20180101; A61P 11/06 20180101; A61K 33/40 20130101; A61P 29/00
20180101; A61P 31/00 20180101; A61P 25/00 20180101; Y02A 50/30
20180101; A61K 33/20 20130101; A61P 31/20 20180101; A61K 33/20
20130101; A61K 2300/00 20130101; A61K 33/40 20130101; A61K 2300/00
20130101; A61K 33/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/114 ;
424/600; 424/661 |
International
Class: |
A61K 33/14 20060101
A61K033/14 |
Claims
1. A method of preventing or treating inflammation in a patient,
the method comprising administering to the patient a
therapeutically effective amount of an oxidative reductive
potential water solution, wherein the solution is stable for at
least about twenty-four hours and the solution has a pH of from
about 6.4 to about 7.8.
2. The method of claim 1, wherein the oxidative reductive potential
water solution is administered topically.
3. The method of claim 1, wherein the oxidative reductive potential
water solution is administered parenterally.
4. The method of claim 1, wherein the oxidative reductive potential
water solution is administered endoscopically.
5. The method of claim 1, wherein the oxidative reductive potential
water solution is administered to a mucosal surface.
6. The method of claim 1, wherein the oxidative reductive potential
water solution is administered to one or more tissues selected from
the group consisting of nasal, sinus, pharyngeal, tracheal,
pulmonary, esophageal, gastric, intestinal, mesothelial,
peritoneal, synovial, urinary bladder, urethral, vaginal, uterine,
fallopian, pancreatic, nervous, oral, cutaneous, and
subcutaneous.
7. The method of claim 1, wherein the oxidative reductive potential
water solution is administered as a liquid, steam, aerosol, mist or
spray.
8. The method of claim 1, wherein the oxidative reductive potential
water solution is administered by aerosolization, nebulization or
atomization.
9. The method of claim 1, wherein the oxidative reductive potential
water solution is administered in the form of droplets having a
diameter in the range of from about 0.1 micron to about 100
microns.
10. The method of claim 1, wherein the inflammation is acute
inflammation.
11. The method of claim 1, wherein the inflammation is chronic
inflammation.
12. The method of claim 1, wherein the inflammation results from a
hypersensitivity reaction.
13. The method of claim 1, wherein the inflammation is associated
with cellular histamine and pro-inflammatory cytokine release.
14. The method of claim 1, wherein the inflammation is
cell-mediated.
15. The method of claim 1, wherein the oxidative reductive
potential water solution inhibits mast cell degranulation.
16. The method of claim 1, wherein the oxidative reductive
potential water solution inhibits mast cell cytokine secretion.
17. The method of claim 1, wherein the method comprises treating
one or more diseases or conditions selected from the group
consisting of allergic rhinosinusitis, atopic dermatitis, food
allergies, asthma, SLE, autoimmune thyroiditis, sarcoidosis,
inflammatory bowel disease, rheumatoid arthritis, multiple
sclerosis, and rheumatic fever.
18. The method of claim 1, wherein the inflammation is caused by an
autoimmune reaction.
19. The method of claim 1, wherein the inflammation is caused by an
infection.
20. The method of claim 19, wherein the infection is by one or more
microorganisms selected from the group consisting of viruses,
bacteria, and fungi.
21. The method of claim 1, wherein the oxidative reductive
potential water solution is stable for at least about one week.
22. The method of claim 1, wherein the oxidative reductive
potential water solution is stable for at least about two
months.
23. The method of claim 1, wherein the oxidative reductive
potential water solution is stable for at least about six
months.
24. The method of claim 1, wherein the oxidative reductive
potential water solution is stable for at least about one year.
25. The method of claim 1, wherein the pH of the oxidative
reductive potential water solution is from about 7.4 to about
7.6.
26. The method of claim 1, wherein the oxidative reductive
potential water solution comprises a mixture of cathode water and
anode water.
27. The method of claim 1, wherein the oxidative reductive
potential water solution comprises cathode water in an amount of
from about 10% to about 50% by volume of the solution.
28. The method of claim 1, wherein the oxidative reductive
potential water solution comprises cathode water in an amount of
from about 20% to about 40% by volume of the solution.
29. The method of claim 1, wherein the oxidative reductive
potential water solution comprises anode water in an amount of from
about 50% to about 90% by volume of the solution.
30. The method of claim 1, wherein the oxidative reductive
potential water solution comprises from about 10% by volume to
about 50% by volume of cathode water and from about 50% by volume
to about 90% by volume of anode water.
31. The method of claim 1, wherein the oxidative reductive
potential water solution comprises at least one free chlorine
species selected from the group consisting of hypochlorous acid,
hypochlorite ions, sodium hypochlorite, chlorite ions, chloride
ions, dissolved chlorine gas, and mixtures thereof and, optionally,
hydrogen peroxide and/or chlorine dioxide.
32. The method of claim 1, wherein the oxidative reductive
potential water solution comprises from about 15 ppm to about 35
ppm hypochlorous acid.
33. The method of claim 1, wherein the oxidative reductive
potential water solution comprises from about 25 ppm to about 50
ppm sodium hypochlorite.
34. The method of claim 1, wherein the oxidative reductive
potential water solution comprises from about 15 ppm to about 35
ppm hypochlorous acid, from about 25 ppm to about 50 ppm sodium
hypochlorite, a pH of from about 6.2 to about 7.8, and the solution
is stable for at least one week.
35. The method of claim 1, wherein the oxidative reductive
potential water solution has a potential between about -400 mV and
about +1300 mV.
36. The method of claim 1, further comprising administering at
least one therapeutic agent selected from the group consisting of
antibiotics, anti-viral agents, and anti-inflammatory agents, and
combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application Nos. 60/760,635, filed Jan. 20,
2006; 60/760,567, filed Jan. 20, 2006; 60/760,645, filed Jan. 20,
2006; and 60/760,557, filed Jan. 20, 2006; all of which are hereby
incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Inflammation is a biological response that can result from a
noxious stimulus and is normally intended to remove that stimulus
or ameliorate its effects. Although normally intended to promote
survival, inflammation can cause damage to the host, especially in
mammals. The stimulus or insult initiating inflammation can be
caused by endogenous factors (e.g., an auto-antigen or irritating
body fluid) or exogenous factors (e.g., a foreign body or
infectious agent).
[0003] Inflammation has been classified as "acute" and "chronic."
Acute inflammation is typically of relatively short duration,
lasting minutes to hours and, in some cases, a few days. Acute
inflammation can be characterized by the exudation of fluid and
plasma proteins and the accumulation of polymorphonuclear
leukocytes (PMNLs) at the site of the insult. Acute inflammation
usually includes an increase in blood flow to the area of the
insult mediated by cellular molecules released in response to the
insult. Increased vascular permeability also results from cellular
mediators and leads to an accumulation of protein-rich fluid.
Important mediators of this increased blood flow and vascular
permeability include histamine from mast cells, serotonin and
bradykinin.
[0004] In acute inflammation, PMNLs are also attracted to the area
of insult and migrate out of the blood stream toward the insult.
The PMNLs release toxic metabolites and proteinases that can cause
tissue damage. These proteinases include proteins in the complement
system, which can damage cell membranes and kallikreins which
generate bradykinin. Acute inflammation can undergo complete
resolution, lead to the formation of an abscess, result in scarring
fibrosis or progress to chronic inflammation.
[0005] Chronic inflammation is of longer duration, lasting weeks to
months, and possibly years, in which tissue destruction and
biological processes that are intended to repair the injury are
simultaneously ongoing. Chronic inflammation more typically
involves lymphocytes and macrophages and may also include a
proliferation of blood vessels, fibrosis and/or necrosis. Chronic
inflammation can result from a number of conditions including
persistent infections, prolonged exposure to toxic agents, and
autoimmune reactions. Chronic inflammation is often maintained by
the production of cytokines by lymphocytes and macrophages at the
site of the persistent insult. Chronic inflammation can result in
permanent tissue damage or complete healing.
[0006] Hypersensitivity generally refers to inflammation that
causes damage to the host, in which the damage outweighs the
benefit to the host. Hypersensitivity can result in significant
pathology including, e.g., anaphylaxis, transplant rejection, and
autoimmune diseases. The most common type of hypersensititvity is
allergy.
[0007] Independently of the inducing factor--and the length of the
exposure--an inflammatory reaction is mediated by a varied number
and type of cells and molecules, the later including cytokines,
growth factors, clotting factors, enzymes, neurotransmitters and
complement proteins, among others. These molecules are primarily
secreted by fibroblasts, endothelial and infiltrating cells (e.g.
macrophages, lymphocytes, mast cells, polymorphonuclear cells,
etc), and local nerves in response to the insulting agent. The
mixture and amount of cytokines therein released will depend on the
type, concentration and exposure time of the inducing agent.
Therefore, these proteins could mediate from an acute local
inflammatory reaction to systemic life-threatening responses (e.g.
acute systemic inflammatory response syndrome, SIRS; multiple organ
failure as in septic shock; anaphylaxis, etc). In chronic
inflammatory processes, the cytokines continuously recruit more and
more infiltrating cells that generate, for example, granulomas,
induration of the tissues, and encapsulated abscesses. In any case,
proteins secreted during an inflammatory process are central
players in the grade and persistence of the final reaction.
[0008] Stimulation of the aforementioned cells by the induction
agent leads to a cascade of intracellular signaling events that
ultimately result in production and secretion of cytokines and
other inflammatory mediators that constitute the pro-inflammatory
response. While the pro-inflammatory response is crucial for
effective clearance of the pathogen or allergen, the inflammatory
mediators produced cause tissue damage and inflammation. Hence, a
balance needs to be maintained between the activation and
down-regulation of this response in order to avoid severe tissue
damage (Cohen, J.: The immunopathogenesis of sepsis. Nature 2002
420, 885-891). Dysregulation of this response could induce local
damage (e.g. lung fibrosis) or could lead to potentially lethal
conditions like septic shock and systemic inflammatory response
syndrome (SIRS) as previously mentioned. Thus, microbes allergens,
endotoxins, and many other molecules induce the production of
pro-inflammatory mediator proteins by different cells in the human
body. The combined effects of all these molecules in living tissues
could mediate changes in the clotting system, wound healing
process, anti-microbial activity, antibody production and the
perception of pain, among many other reactions.
[0009] The systemic inflammatory response syndrome (SIRS), a
syndrome that encompasses the features of systemic inflammation
without end-organ damage or identifiable bacteremia. SIRS is
separate and distinct from sepsis, severe sepsis or septic shock.
The key transition from SIRS to sepsis is the presence of an
identified pathogen in the blood. The pathophysiology of SIRS
includes, but is not limited to, complement activation, cytokine
and arachidonic acid metabolites secretion, stimulated
cell-mediated immunity, activation of the clotting cascades, and
humoral immune mechanisms. Clinically SIRS is characterized by
tachycardia, tachypnea, hypotension, hypoperfusion, oliguria,
leukocytosis or leukopenia, pyrexia or hypothermia, metabolic
acidosis, and the need for volume support. SIRS may affect all
organ systems and may lead to multiple organ dysfunction syndrome
(MODS). Thus, even in early stages (i.e. SIRS), there is
accumulation of pro-inflammatory cytokines at the primary site of
inflammation and in the blood that can contribute to the
establishment of multi-organ failure and death.
[0010] Typically, inflammation is treated with steroidal or
non-steroidal anti-inflammatory drugs. However, conventional
anti-inflammatory therapy suffers from several drawbacks, e.g.,
systemic toxicity, allergic reactions, insulin resistance,
hypertension, cardiac toxicity, renal toxicity, various
coagulopathies and gastric erosions. Accordingly, there is a need
for mild, yet safe and effective methods for treating or preventing
inflammation.
[0011] The present invention provides such methods. These and other
advantages of the invention, as well as additional inventive
features, will be apparent from the description of the invention
provided herein.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention provides a method of preventing or
treating inflammation in a patient by administering to the patient
a therapeutically effective amount of an oxidative reductive
potential (ORP) water solution, wherein the solution is stable for
at least twenty-four hours. The method of the present invention can
be used in the treatment of inflammation resulting from a variety
of causative factors, e.g., allergic reaction, autoimmune reaction,
infection, contact with one or more inflammation-causing
substances, and combinations of such causative factors.
[0013] The method of the present invention can further include
administering the ORP water solution in conjunction with one or
more therapeutic agents, e.g., one or more compounds selected from
the group consisting of antibiotics, anti-viral agents,
anti-inflammatory agents, and combinations thereof. Administering
such therapeutic agents in conjunction with the ORP water solution
includes administering one or more of such agents, e.g., prior to,
during (e.g., contemporaneously, by co-administration or in
combination with), or following administration of the ORP water
solution.
[0014] The ORP water solution can be administered by any suitable
route in accordance with the present invention, e.g., by delivering
the ORP water solution topically or parenterally, so as to contact
a therapeutically effective amount of the ORP water solution with
one or more affected tissues, which may reside inside or outside of
the body. Accordingly, the invention provides a method wherein the
ORP water solution is administered to one or more tissues, e.g.,
nasal, sinus, pharyngeal, tracheal, pulmonary, esophageal, gastric,
intestinal, mesothelial, peritoneal, synovial, urinary bladder,
urtheral, vaginal, uterine, fallopian, pancreatic, nervous, oral,
cutaneous, and subcutaneous. The ORP water solution can be
administered in any suitable form in accordance with the present
invention, e.g., as a liquid, spray, mist, aerosol or steam, and,
if desired, can be combined with one or more suitable carriers,
e.g., vehicles, adjuvants, excipients, diluents, and the like.
[0015] The ORP water solution administered in accordance with the
present invention can be contained within a sealed container and is
stable for at least twenty-four hours. The ORP water solution
administered in accordance with the invention can be produced by
electrolysis, and preferably comprises a mixture of anode water and
cathode water, which contains one or more species, including, e.g.,
reactive species, ionic species, radical species, precursors
thereof and combinations thereof. The ORP water solution
administered in accordance with the invention exhibits potent
anti-inflammatory activity, yet is virtually free of toxicity to
normal tissues and normal eukaryotic cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates a three-chambered electrolysis cell for
producing an exemplary ORP water solution.
[0017] FIG. 2 illustrates a three-chambered electrolysis cell and
depicts ionic species that are believed to be generated during the
production process.
[0018] FIG. 3 is a schematic flow diagram of a process for
producing an exemplary ORP water solution.
[0019] FIG. 4A-4C depict a graphical comparison of cell viability,
apoptosis and necrosis in human diploid fibroblasts (HDFs) treated
with an exemplary ORP water solution (MCN) versus hydrogen peroxide
(HP).
[0020] FIG. 5 is a graphical comparison of the levels of
8-hydroxy-2'-deoxiguanosine (8-OHdG) adducts in HDFs treated with
an exemplary ORP water solution (MCN) versus 500 .mu.M hydrogen
peroxide (HP).
[0021] FIG. 6 illustrates cellular senescence demonstrated by
.beta.-galactosidase expression in HDFs after chronic exposure to
low concentrations of an exemplary ORP water solution (MCN) versus
hydrogen peroxide (HP).
[0022] FIG. 7 illustrates the effect on degranulation of
antigen-activated mast cells treated with various concentrations of
an exemplary ORP water solution (MCN).
[0023] FIG. 8 comparatively illustrates the effect on degranulation
of antigen-activated mast cells treated with cromoglycate.
[0024] FIG. 9 illustrates the effect on degranulation of
antigen-activated and calcium ionophore (A23187)-activated mast
cells treated with various concentrations of an exemplary ORP water
solution (MCN).
[0025] FIG. 10A-10B are RNAse protection assays illustrating
cytokine mRNA levels after antigen challenge in control versus ORP
water solution-treated mast cells.
[0026] FIG. 11 is a graphical comparison of TNF-.alpha. secretion
by antigen-activated mast cells treated with various concentrations
of an exemplary ORP water solution (MCN).
[0027] FIG. 12 is a graphical comparison of MIP1-.alpha. secretion
by antigen-activated mast cells treated with various concentrations
of an exemplary ORP water solution (MCN).
[0028] FIG. 13 is a graphical comparison of IL-6 secretion by
antigen-activated mast cells treated with various concentrations of
an exemplary ORP water solution (MCN).
[0029] FIG. 14 is a graphical comparison of IL-13 secretion by
antigen-activated mast cells treated with various concentrations of
an exemplary ORP water solution (MCN).
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides a method of preventing or
treating inflammation in a patient, which method comprises
administering to the patient a therapeutically effective amount of
an oxidative reductive potential (ORP) water solution (also known
as super-oxidized water (SOW)), wherein the solution is stable for
at least about twenty-four hours. The method of the present
invention can be used for treating or preventing (e.g., inhibiting
the onset of, inhibiting the escalation of, decreasing the
likelihood of) acute inflammation and chronic inflammation,
including hypersensitivity such as, e.g., in allergies. The
inflammation and hypersensitivity treatable or preventable in
accordance with the method of the present invention can include
inflammation that results from, e.g., contact with a noxious
stimulus, injury, infection, autoimmune reaction, hypersensitivity,
and allergic reaction, including allergic reactions associated with
cellular histamine and pro-inflammatory cytokine release.
[0031] Surprisingly, it has been found that the ORP water solution
administered in accordance with the invention is a highly effective
inhibitor of mast cell degranulation, one of the primary
inflammation and allergy-causing biological cascades. The ORP water
solution administered in accordance with the invention inhibits
degranulation of mast cells regardless of whether they are
activated with an antigen or a calcium ionophore. Also
surprisingly, it has been found that the ORP water solution
administered in accordance with the present invention
non-selectively inhibits the secretion of pro-inflammatory
cytokines in mast cells. For example, the ORP water solution of the
present invention can inhibit the secretion of, e.g., TNF-.alpha.,
MIP1-.alpha., IL-6, and IL-13 in mast cells. It is believed that
the ORP water solution administered in accordance with the
invention also can inhibit the secretion of pro-inflammatory
cytokines in other cytokine-secreting cells including, but not
limited to, macrophages, monocytes, lymphocytes, macrophages, PMN,
fibroblasts and endothelial cells. These findings demonstrate that
the ORP water administered in accordance with the present invention
should exhibit broad anti-inflammatory efficacy.
[0032] The ORP water solution administered in accordance with the
invention preferably inhibits mast cell degranulation by more than
about 50% relative to untreated mast cells, more preferably by more
than about 80% relative to untreated mast cells, still more
preferably by more than about 90% relative to untreated mast cells,
and even more preferably by more than about 95% relative to
untreated mast cells, when contacted with the ORP water solution
for up to about 30 minutes, more preferably up to about 15 minutes,
and still more preferably up to about 5 minutes.
[0033] The ORP water solution administered in accordance with the
invention also preferably inhibits the secretion of TNF-.alpha. by
more than about 50%, more preferably by more than about 60%, still
more preferably by more than about 70%, and even more preferably by
more than about 85%. In addition, the ORP water solution
administered in accordance with the invention also preferably
inhibits the secretion of MIP1-.alpha. by more than 25%, more
preferably by more than about 50%, and still more preferably by
more than about 60%. Further, the ORP water solution administered
in accordance with the invention also preferably inhibits the
secretion of IL-6 and/or IL-13 by more than 25%, more preferably by
more than about 50%, and still more preferably by more than about
60%. In accordance with the method of the invention, secretion of
these and that of other cytokines, can be therapeutically inhibited
down to certain % by the administration of the ORP water solution
alone or in combination with a diluent (e.g., water), by increasing
the concentration of the components of the ORP water solution, by
utilizing special delivery systems and/or by increasing the
exposure time. For instance, cytokine secretion can be
therapeutically inhibited by administering compositions in which
the ORP water solution is diluted, e.g., by a ratio of up to about
50% (vol./vol.) ORP water solution/diluent, by a ratio of up to
about 25% (vol./vol.) ORP water solution/diluent, by a ratio of up
to about 10% (vol./vol.) ORP water solution/diluent, by a ratio of
up to about 5% (vol./vol.) ORP water solution/diluent, or even by a
ratio of up to about 1% (vol./vol.) ORP water solution/diluent.
[0034] The method of the present invention can be used for treating
or preventing cell-mediated inflammation , which results from an
autoimmune reaction, including, but not limited to, SLE, autoimmune
thyroiditis, sarcoidosis, inflammatory bowel disease, rheumatoid
arthritis, rheumatic fever, psoriasis, pemphigus, erythema
multiforme, other bullous diseases of the skin, and atopias. The
method of the invention can be used for treating or preventing
inflammation, which results from infection, allergens, foreign
bodies, and autoimmune processes. The method of the invention can
also be used for treating or preventing inflammation, which results
from infection, e.g., from an infection by one or more
microorganisms selected from the group consisting of viruses,
bacteria, and fungi, including hypersensitivity and
autoimmune-mediated inflammation resulting from infection.
[0035] The method of the present invention can be used for treating
or preventing inflammation associated with an upper respiratory
condition. When the inflammation is associated with an upper
respiratory condition, the ORP water solution is preferably
administered to the upper airway, e.g., as a spray, mist, aerosol
or steam, so as to contact one or more upper airway tissues
affected by the condition. Any suitable method can be employed for
delivering the ORP water solution to the upper airway so as to
treat or prevent one or more upper respiratory conditions in
accordance with the present invention, including one or more routes
of administration described herein.
[0036] The method of the present invention can be used for
preventing or treating inflammation affecting one or more upper
respiratory airway tissues (e.g., nasal tissue, sinus tissue) or
lung tissues. Such conditions can include, for example, sinusitis
(e.g., rhinosinusitis, acute sinusitis, chronic sinusitis, and the
like), pharyngitis, asthma, and the like, which are preventable or
treatable with the ORP solution administered in accordance with the
invention.
[0037] Chronic sinusitis typically refers to inflammation of the
sinuses that continues for at least 3 weeks, but the inflammation
can (and often does) continue for months or even years. Allergies
are frequently associated with chronic sinusitis. In addition,
patients with asthma have a particularly high frequency of chronic
sinusitis. Inhalation of airborne allergens (substances that
provoke an allergic reaction), such as dust, mold, and pollen,
often set off allergic reactions (e.g., allergic rhinitis) that, in
turn, may contribute to sinusitis (particularly rhinosinusitis or
rhinitis). People who are allergic to fungi can develop a condition
called "allergic fungal sinusitis." Damp weather or pollutants in
the air and in buildings also can affect people subject to chronic
sinusitis.
[0038] Like acute sinusitis, chronic sinusitis is more common in
patients with immune deficiency or abnormalities of mucus secretion
or movement (e.g., immune deficiency, HIV infection, cystic
fibrosis, Kartagener's syndrome). In addition, some patients have
severe asthma, nasal polyps, and severe asthmatic responses to
aspirin and aspirin-like medications (so-called non-steroidal
anti-inflammatory drugs, or NSAIDs). These latter patients have a
high frequency of chronic sinusitis.
[0039] A doctor can diagnose sinusitis by medical history, physical
examination, X-rays, and if necessary, MRIs or CT scans (magnetic
resonance imaging and computed tomography). After diagnosing
sinusitis and identifying a possible cause, a doctor can prescribe
a course of treatment that will reduce the inflammation and relieve
the symptoms. Treating acute sinusitis typically requires
re-establishing drainage of the nasal passages, controlling or
eliminating the source of the inflammation, and relieving the pain.
Doctors generally recommend decongestants to reduce the congestion,
antibiotics to control a bacterial infection, if present, and pain
relievers to reduce the pain.
[0040] When treatment with drugs fails, surgery may be the only
alternative for treating chronic sinusitis, e.g., removal of
adenoids, removal of nasal polyps, repair of a deviated septum,
endoscopic sinus surgery, and the like. It is believed that the
administration of ORP water in accordance with the method of the
present invention can be used for treating chronic sinusitis and
inflammation associated therewith as an alternative to potentially
avoid more aggressive therapies, such as antibiotics and
surgery.
[0041] With regard to pharyngitis, it is estimated that worldwide,
1 to 2% of all visits to doctors' offices, clinics and emergency
rooms are because of pharyngitis. In the United States and Mexico,
pharyngitis and tonsillitis is believed to account for about 15 and
12 million consultations per year, respectively. These cases are
typically caused by various bacteria and viruses. Also, pharyngitis
and tonsillitis caused by group A .beta.-hemolytic Streptococcus
can significantly raise the risk of rheumatic fever in poor
populations; however it is believed that only 5 to 15% of
pharyngitis cases are caused by this bacterium, and that the rest
of the acute cases are due to bacteria and viruses of little
epidemiological relevance. The latter cases tend to be
self-limiting in a few days and do not leave sequelae.
[0042] It has been verified that a great number of doctors
worldwide prescribe antibiotics indiscriminately for acute
pharyngitis. This occurs in a daily practice, often because
patients tend to request powerful antibiotics. Unfortunately, it is
difficult to establish an accurate diagnosis of streptococcal
pharyngitis/tonsillitis clinically and the cost/benefit ratio of
treating acute pharyngitis/tonsillitis with antibiotics is
questionable.
[0043] It is believed that the method of the present invention
provides a safe, efficacious and cost-effective adjuvant therapy
for the treatment or prevention of acute pharyngitis and/or
tonsillitis due to bacteria and/or viruses. . The empirical
treatment of acute pharyngitis/tonsillitis may begin with
administering an ORP water solution in accordance with the present
invention, and, depending on evolution or the result of the rapid
test for Streptococcus, antibiotics may be initiated from 48-72
hours thereafter only if needed. The method of the present
invention may thus allow the use of antibiotics to be deferred and,
at the same time, reduce the symptomatology of the patient and
accelerate the patient's recovery if the pharyngitis/tonsillitis is
not from group A Streptococcus. The adjuvant use of an ORP water
solution of the present invention with antibiotics for the
treatment of streptococcal pharyngitis/tonsillitis also may shorten
the period of clinical response and decrease the incidence of
recurrences.
[0044] The method of the present invention also can be used for
treating or preventing inflammation associated with
hypersensitivity. Historically, hypersensitivity reactions have
been classified as one of four types, from which significant
disease can result. The ORP water solution administered in
accordance with the invention can be used to treat and/or prevent
(e.g., inhibit the onset of, inhibit the escalation of or decrease
the likelihood of) one or more of such reactions. Type I
hypersensitivity typically results from the combination of an
antigen with an antibody bound to a mast cell or basophil. Type I
reactions occur within minutes of exposure to the antigen in
individuals who have been previously sensitized to the antigen. In
humans, Type I reactions are mediated by IgE which has high
affinity Fc receptors on mast cells and basophils.
[0045] Mast cells' role in Type I hypersensitivity is especially
important because they reside in tissues under the epithelial
surface near blood vessels and nerves. Multiple clinical symptoms
observed in atopic dermatitis, allergic rhinitis and atopic asthma
are produced by IgE-antigen stimulation of mast cells located in
distinct affected tissues. The currently accepted view of the
pathogenesis of atopic asthma is that allergens initiate the
process by triggering IgE-bearing pulmonary mast cells (MCs) to
release mediators such as histamine, leukotrienes, prostaglandins,
kininis, platelet activating factor (PAF), etc. in the so-called
early phase of the reaction (see Kumar et al., Robbins & Cotran
Pathologic Basis of Disease, 2004, pp. 193-268, which is hereby
incorporated by reference). In turn, these mediators induce
bronchoconstriction and enhance vascular permeability and mucus
production. According to this model, following mast cell activation
in the late phase, those cells secrete various cytokines, including
tumor necrosis factor alpha (TNF-.alpha.), IL-4, IL-5 and IL-6,
which participate in the local recruitment and activation of other
inflammatory cells such as eosinophils, basophils, T lymphocytes,
platelets and mononuclear phagocytes. These recruited cells, in
turn, contribute to the development of an inflammatory response
that may then become autonomous and aggravate the asthmatic
symptoms. This late phase response constitutes a long term
inflammatory process which will induce changes in surrounding
tissues (Kumar et al., pp. 193-268). Clinically, Type I reactions
can have local effects such as allergic rhinitis, or systemic
effects as is found in anaphylaxis which manifests with itching,
hives, respiratory distress, and circulatory collapse.
[0046] Type II hypersensitivity is mediated by antibodies directed
to antigens on the surfaces of cells and in the extracellular
space. These antibodies can direct cell lysis or result in
opsonization of the target molecules (preparation for phagocytosis
by other cells). Alternatively, the antibodies can be directed to
and activate cell surface receptors. Conditions resulting from Type
II reactions include transfusion reactions, Graves disease
(thyrotoxicosis), drug reactions, pernicious anemia, and acute
rheumatic fever. In rheumatic fever the antibodies are formed
against Streptococcal antigens but, cross-react with human tissues
such as heart valves.
[0047] Type III hypersensitivity is caused by immune complexes,
which are combinations of antibodies and other host immune system
proteins, most typically complement proteins. It is the normal
function of antibodies to bind and active complement. However, when
the resulting macromolecular immune complexes are not adequately
processed, they can lead to persistent tissue damage. Macrophages
and PMNLs can be activated by immune complexes and lead to the
release of toxic chemicals by these cells. Immune complex reactions
can be local and may result in conditions such as, e.g., the arthus
reaction or cause systemic disease such as serum sickness or some
of the aspects of systemic lupus erythematous (SLE).
[0048] Type IV hypersensitivity is cell mediated and is sometimes
called delayed-type hypersensitivity. Type IV hypersensitivity is
mediated by T lymphocytes and often results in the formation of a
granulomatous reaction. In a granulomatous reaction, a form of
macrophage called an epitheloid cells attempts to, but fails, to
digest an antigen. The antigen's persistence leads to the release
of cytokines that attract additional lymphocytes resulting in
chronic foci of inflammation. The foci have high concentrations of
cyotoxic T-lymphocytes which release granzymes and perforins which
are toxic to adjacent cells. Type IV hypersensitivity is a
prominent component of autoimmune diseases such as, e.g.,
Sjogrren's Syndrome, Sarcoidosis, and contact dermatitis.
[0049] Pathologic states can combine different types of
hypersensitivity reactions. In autoimmune diseases host antigens
stimulate hypersensitivity with serious consequences for the host.
For example, in SLE host antigens induce Type II reactions against
blood cells while Type III reactions lead to blood vessel and renal
glomerular damage. In addition, hypersensitivity reactions are also
seen in iatragenic conditions such as drug reactions and transplant
rejection. Transplant rejection includes components of Type II and
Type IV hypersensivity. Accordingly, ORP water solution used in
accordance with the invention in transplantable organs or cells
could greatly reduced the possibility of being rejected by the
host.
[0050] It has been found that the ORP water solution administered
in accordance with the invention is virtually free of toxicity to
normal tissues and normal mammalian cells. The ORP water solution
administered in accordance with the invention causes no significant
decrease in the viability of eukaryotic cells, no significant
increase in apoptosis, no significant acceleration of cell aging
and/or no significant oxidative DNA damage in mammalian cells. The
non-toxicity is particularly advantageous, and perhaps even
surprising, given that the disinfecting power of the ORP water
solution administered in accordance with the invention is roughly
equivalent to that of hydrogen peroxide, yet is significantly less
toxic than hydrogen peroxide is to normal tissues and normal
mammalian cells. These findings demonstrate that the ORP water
solution administered in accordance with the present invention is
safe for use, e.g., in mammals, including humans.
[0051] For the ORP water solution administered in accordance with
the invention, the cell viability rate is preferably at least about
65%, more preferably at least about 70%, and still more preferably
at least about 75% after an about 30 minute exposure to the ORP
water solution. In addition, the ORP water solution administered in
accordance with the invention preferably causes only up to about
10% of cells, more preferably only up to about 5% of cells, and
still more preferably only up to about 3% of cells to expose
Annexin-V on their cellular surfaces when contacted with the ORP
water solution for up to about thirty minutes or less (e.g., after
about thirty minutes or after about five minutes of contact with
the ORP water solution).
[0052] Further, the ORP water solution administered in accordance
with the invention preferably causes less than about 15% of cells,
more preferably less than about 10% of cells, and still more
preferably less than about 5% of cells to express the
SA-.beta.-galactosidase enzyme after chronic exposure to the OPR
water solution. The ORP water solution administered in accordance
with the invention preferably causes caused the same fraction of
the oxidative DNA adduct formation caused by saline solution, e.g.,
less than about 20% of the oxidative DNA adduct formation, less
than about 10% of the oxidative DNA adduct formation, or about 5%
or less of the oxidative DNA adduct formation normally caused by
hydrogen peroxide in cells treated under equivalent conditions.
[0053] The ORP water solution administered in accordance with the
invention produces no significant RNA degradation. Accordingly, RNA
extracted from human cell cultures after an about 30 minutes
exposure to the ORP water solution or r at about 3 hours after an
about 30 minute-exposure, , and analyzed by denaturing gel
electrophoresis, will typically show no significant RNA degradation
and will typically exhibit two discreet bands corresponding to the
ribosomal eukaryotic RNAs (i.e. 28S and 18S) indicating that the
ORP water solution administered in accordance with the invention
leaves the RNA substantially intact. Similarly, RNA extracted from
human cell cultures after about 30 minutes of exposure to the ORP
water solution or after about 3 hours of exposure, can be subjected
reverse transcription and amplification (RT-PCR) of the
constitutive human GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
gene and result in a strong GAPDH band on gel electrophoresis of
the RT-PCR products. By contrast, cells treated with HP for a
similar period show significant RNA degradation and little if any
GAPDH RT-PCR product.
[0054] The ORP water solution used in accordance with the present
invention can be administered using any suitable method of
administration known in the art. For instance, the ORP water
solution can be administered parenterally, endoscopically or
directly to the surface of any affected biological tissue, e.g., to
the skin and/or one or more mucosal surfaces. Parenteral
administration can include using, for example, administering the
ORP water solution intramuscularly, subcutaneously, intravenously,
intra-arterially, intrathecally, intravesically or into a synovial
space. Endoscopic administration of the ORP water solution can
include using, e.g., bronchoscopy, colonoscopy, sigmoidoscopy,
hysterscopy, laproscopy, athroscopy, gastroscopy or a transurethral
approach. Administering the ORP water solution to a mucosal surface
can include, e.g., administration to a nasal, oral, tracheal,
bronchial, esophageal, gastric, intestinal, peritoneal, urethral,
vesicular, urethral, vaginal, uterine, fallopian, and synovial
mucosal surface.
[0055] Parenteral administration also can include administering the
ORP water solution used in accordance with the invention
intravenously, subcutaneously, intramuscularly, or
intraperitoneally. The ORP water solution of the present invention
can be administered intravenously as described, e.g., in U.S. Pat.
Nos. 5,334,383 and 5,622,848 (hereby incorporated by reference),
which describe methods of treating viral myocarditis, multiple
sclerosis, and AIDS via intravenous administration of ORP water
solutions. Other applications include the treatment of any
hypersensitivity and infectious processes, as mentioned above.
[0056] The ORP water solution used in accordance with the invention
can be administered topically, e.g., as a liquid, spray, mist,
aerosol or steam by any suitable process, e.g., by aerosolization,
nebulization or atomization. The ORP solution of the present
invention can be administered to the upper airway as a steam or a
spray. When the ORP water solution is administered by
aerosolization, nebulization or atomization, it is preferably
administered in the form of droplets having a diameter in the range
of from about 0.1 micron to about 100 microns, preferably from
about 1 micron to about 10 microns. In one embodiment, the method
of the present invention includes administering the ORP water
solution in the form of droplets having a diameter in the range of
from about 1 micron to about 10 microns to one or more mucosal
tissues, e.g., one or more upper respiratory tissues and/or lung
tissues.
[0057] Methods and devices, which are useful for aerosolization,
nebulization and atomization, are well known in the art. Medical
nebulizers, for example, have been used to deliver a metered dose
of a physiologically active liquid into an inspiration gas stream
for inhalation by a recipient. See, e.g., Pat. No. 6,598,602
(hereby incorporated by reference). Medical nebulizers can operate
to generate liquid droplets, which form an aerosol with the
inspiration gas. In other circumstances medical nebulizers may be
used to inject water droplets into an inspiration gas stream to
provide gas with a suitable moisture content to a recipient, which
is particularly useful where the inspiration gas stream is provided
by a mechanical breathing aid such as a respirator, ventilator or
anaesthetic delivery system.
[0058] An exemplary nebulizer is described, for example, in WO
95/01137, which describes a hand held device that operates to eject
droplets of a medical liquid into a passing air stream (inspiration
gas stream), which is generated by a recipient's inhalation through
a mouthpiece. Another example can be found in U.S. Pat. No.
5,388,571 (hereby incorporated by reference), which describes a
positive-pressure ventilator system which provides control and
augmentation of breathing for a patient with respiratory
insufficiency and which includes a nebulizer for delivering
particles of liquid medication into the airways and alveoli of the
lungs of a patient. U.S. Pat. No. 5,312,281 (hereby incorporated by
reference) describes an ultrasonic wave nebulizer, which atomizes
water or liquid at low temperature and reportedly can adjust the
size of mist. In addition, U.S. Pat. No. 5,287,847 (hereby
incorporated by reference)describes a pneumatic nebulizing
apparatus with scalable flow rates and output volumes for
delivering a medicinal aerosol to neonates, children and adults.
Further, U.S. Pat. No. 5,063,922 (hereby incorporated by reference)
describes an ultrasonic atomizer. The ORP water solution also may
be dispensed in aerosol form as part of an inhaler system for
treatment of infections in the lungs and/or air passages or for the
healing of wounds in such parts of the body.
[0059] For larger scale applications, a suitable device may be used
to disperse the ORP water solution into the air including, but not
limited to, humidifiers, misters, foggers, vaporizers, atomizers,
water sprays, and other spray devices. Such devices permit the
dispensing of the ORP water solution on a continuous basis. An
ejector which directly mixes air and water in a nozzle may be
employed. The ORP water solution may be converted to steam, such as
low pressure steam, and released into the air stream. Various types
of humidifiers may be used such as ultrasonic humidifiers, stream
humidifiers or vaporizers, and evaporative humidifiers. The
particular device used to disperse the ORP water solution may be
incorporated into a ventilation system to provide for widespread
application of the ORP water solution throughout an entire house or
healthcare facility (e.g., hospital, nursing home, etc.).
[0060] In accordance with the invention, the ORP water solution can
be administered alone or in combination with one or more
pharmaceutically acceptable carriers, e.g., vehicles, adjuvants,
excipients, diluents, combinations thereof, and the like, which are
preferably compatible with one or more of the species that exist in
the ORP water solution. One skilled in the art can easily determine
the appropriate formulation and method for administering the ORP
water solution used in accordance with the present invention. Any
necessary adjustments in dose can be readily made by a skilled
practitioner to address the nature and/or severity of the condition
being treated in view of one or more clinically relevant factors,
such as, e.g., side effects, changes in the patient's overall
condition, and the like.
[0061] For example, the ORP water solution can be formulated by
combining or diluting the ORP water solution with up to about 25%
(wt./wt. or vol./vol.) of a suitable carrier, up to about 50%
(wt./wt. or vol./vol.) of a suitable carrier, up to about 75%
(wt./wt. or vol./vol.) of a suitable carrier, up to about 90%
(wt./wt. or vol./vol.) of a suitable carrier, up to about 95%
(wt./wt. or vol./vol.) of a suitable carrier, or even with up to
about 99% (wt./wt. or vol./vol.) or more of a suitable carrier.
Suitable carriers can include, e.g., water (e.g., distilled water,
sterile water, e.g., sterile water for injection, sterile saline
and the like). Suitable carriers also can include one or more
carriers described in U.S. patent application Ser. No. 10/916,278
(hereby incorporated by reference). Exemplary formulations can
include, e.g., solutions in which the ORP water solution is diluted
with sterile water or sterile saline, wherein the ORP water
solution is diluted by up to about 25% (vol./vol.), by up to about
50% (vol./vol.), by up to about 75% (vol./vol.), by up to about 90%
(vol./vol.), by up to about 95% (vol./vol.), or by up to 99%
(vol./vol.) or more of a suitable carrier.
[0062] The ORP water solution administered in accordance with the
invention can further be combined with (or be administered in
conjunction with) one or more additional therapeutic agents, e.g.,
one or more active compounds selected from the group consisting of
antibacterial agents (e.g., antibiotics), anti-viral agents,
anti-inflammatory agents, and combinations thereof.
[0063] The therapeutically effective amount administered to the
patient, e.g., a mammal, particularly a human, in the context of
the present invention should be sufficient to affect a therapeutic
or prophylactic response in the patient over a reasonable time
frame. The dose can be readily determined using methods that are
well known in the art. One skilled in the art will recognize that
the specific dosage level for any particular patient will depend
upon a variety of potentially therapeutically relevant factors. For
example, the dose can be determined based on the strength of the
particular ORP water solution employed, the severity of the
condition, the body weight of the patient, the age of the patient,
the physical and mental condition of the patient, general health,
sex, diet, the frequency of applications, and the like. The size of
the dose also can be determined based on the existence, nature, and
extent of any adverse side effects that might accompany the
administration of a particular ORP water solution. It is desirable,
whenever possible, to keep adverse side effects to a minimum.
[0064] Factors, which can be taken into account for a specific
dosage can include, for example, bioavailability, metabolic
profile, time of administration, route of administration, rate of
excretion, the pharmacodynamics associated with a particular ORP
water solution in a particular patient, and the like. Other factors
can include, e.g., the potency or effectiveness of the ORP water
solution with respect to the particular condition to be treated,
the severity of the symptoms presented prior to or during the
course of therapy, and the like. In some instances, what
constitutes a therapeutically effective amount also can be
determined, in part, by the use of one or more of the assays, e.g.,
bioassays, which are reasonably clinically predictive of the
efficacy of a particular ORP water solution for the treatment or
prevention of a particular condition.
[0065] The ORP water solution used in accordance with the present
invention can be administered, alone or in combination with one or
more additional therapeutic agents, to a patient, e.g., a human,
e.g., to treat an existing condition. The ORP water solution of the
present invention also can be administered prophylactically, alone
or in combination with one or more additional therapeutic agents,
to a patient, e.g., a human, that has been exposed to one or more
causative agents associated with the condition. For example, the
ORP water solution can be suitably administered prophylactically to
a patient that has been exposed to one or more inflammation-causing
microorganisms (e.g., infections, viruses, bacteria and/or
fungi)--or hypersensitivity epitope or allergen--to inhibit or
decrease the likelihood of inflammation (and even infection)
associated with the microorganism or epitope in a patient, or
decrease the severity of an inflammation (and even infection or
allergy) that develops as a result of such exposure.
[0066] One skilled in the art will appreciate that suitable methods
of administering the ORP water solution used in accordance with the
present invention are available, and, although more than one route
of administration can be used, a particular route can provide a
more immediate and more effective reaction than another route. The
therapeutically effective amount can be the dose necessary to
achieve an "effective level" of the ORP water solution in an
individual patient, independent of the number of applications a
day. The therapeutically effective amount can be defined, for
example, as the amount required to be administered to an individual
patient to achieve a blood level, tissue level, and/or
intracellular level of the ORP water solution (or one or more
active species contained therein) to prevent or treat the condition
in the patient.
[0067] When the effective level is used as a preferred endpoint for
dosing, the actual dose and schedule can vary depending, for
example, upon interindividual differences in pharmacokinetics,
distribution, metabolism, and the like. The effective level also
can vary when the ORP water solution is used in combination with
one or more additional therapeutic agents, e.g., one or more
anti-infective agents, one or more "moderating," "modulating" or
"neutralizing agents," e.g., as described in Pat. Nos. 5,334,383
and 5,622,848 (hereby incorporated by reference), one or more
anti-inflammatory agents, and the like.
[0068] An appropriate indicator can be used for determining and/or
monitoring the effective level. For example, the effective level
can be determined by direct analysis (e.g., analytical chemistry)
or by indirect analysis (e.g., with clinical chemistry indicators)
of appropriate patient samples (e.g., blood and/or tissues). The
effective level also can be determined, for example, by direct or
indirect observations such as, e.g., the concentration of urinary
metabolites, changes in markers associated with the condition
(e.g., viral count in the case of a viral infection),
histopathology and immunochemistry analysis, positive changes in
image analysis (e.g. X ray, CT scan, NMR, PET, etc), nuclear
medicine studies, decrease in the symptoms associated with the
conditions, and the like.
[0069] Conventional ORP water solutions have an extremely limited
shelf-life, usually only a few hours. As a result of this short
lifespan, using conventional ORP water solutions requires the
production to take place in close proximity to the point of use.
From a practical standpoint, this means that the facility, e.g., a
healthcare facility such as a hospital, must purchase, house and
maintain the equipment necessary to produce conventional ORP water
solution. Additionally, conventional manufacturing techniques have
not been able to produce sufficient commercial-scale quantities to
permit widespread use, e.g., as a general disinfecting agent for
healthcare facilities.
[0070] Unlike conventional ORP water solutions, the ORP water
solution administered in accordance with the invention is stable
for at least about twenty-hours after its preparation. In addition,
the ORP water solution administered in accordance with the
invention is generally environmentally safe and, thus, avoids the
need for costly disposal procedures. Preferably, the ORP water
solution administered in accordance with the invention is stable
for at least about one week (e.g., one week, two weeks, three
weeks, four weeks or more.), and more preferably at least about two
months. Still more preferably, the ORP water solution administered
in accordance with the invention is stable for at least about six
months. Even more preferably, the ORP water solution administered
in accordance with the invention is stable for at least about one
year, and most preferably is stable for more than about one year,
e.g., at least about two years or at least about three years.
[0071] Stability can be measured based on the ability of the ORP
water solution to remain suitable for one or more uses, for
example, inhibiting mast cell degranulation, inhibiting cytokine
secretion, decontamination, disinfection, sterilization,
anti-microbial cleansing, and wound cleansing, for a specified
period of time after its preparation under normal storage
conditions (e.g., room temperature). The stability of the ORP water
solution administered in accordance with the invention also can be
measured by storage under accelerated conditions, e.g., from about
30.degree. C. to about 60.degree. C., in which the ORP water
solution preferably is stable for up to about 90 days, and more
preferably for up to about 180 days.
[0072] Stability also can be measured based on the concentration
over time of one or more species (or precursors thereof) present in
solution during the shelf-life of the ORP water solution.
Preferably, the concentrations of one or more species, e.g., free
chlorine, hypocholorous acid and one or more additional
superoxidized water species and are maintained at about 70% or
greater of their initial concentration for at least about two
months after preparation of the ORP water solution. More
preferably, the concentration of one of more of these species is
maintained at about 80% or greater of their initial concentration
for at least about two months after preparation of the ORP water
solution. Still more preferably, the concentration of one or more
of such species is maintained at about 90% or greater, and most
preferably is maintained at about 95% or greater, of their initial
concentration for at least about two months after preparation of
the ORP water solution.
[0073] Stability also can be determined based on the reduction in
the amount of organisms present in a sample following exposure to
the ORP water solution. Measuring the reduction of organism
concentration can be made on the basis of any suitable organism
including, e.g., bacteria, fungi, yeasts, or viruses. Suitable
organisms can include, e.g., Escherichia coli, Staphylococcus
aureus, Candida albicans, and Bacillus athrophaeus (formerly B.
subtilis).
[0074] The ORP water solution administered in accordance with the
invention can function as a low-level disinfectant capable of a
four log (10.sup.4) reduction in the concentration of live
microorganisms, and also can function as a high-level disinfectant
capable of a six log (10.sup.6) reduction in concentration of live
microorganisms. Preferably, the ORP water solution administered in
accordance with the invention is capable of yielding at least about
a four log (10.sup.4) reduction in total organism concentration,
following exposure for one minute when measured at least about two
months after preparation of the solution. More preferably, the ORP
water solution is capable of a 10.sup.4-10.sup.6 reduction of
organism concentration when measured at least about six months
after preparation of the solution. Still more preferably, the ORP
water solution is capable of a 10.sup.4-10.sup.6 reduction of
organism concentration when measured at least about one year after
preparation of the ORP water solution, and most preferably when
measured more than about one year, e.g., at least about two years
or at least about three years, after preparation of the ORP water
solution.
[0075] For instance, the ORP water solution administered in
accordance with the present invention can be capable of at least
about a five log (10.sup.5) reduction in the concentration of a
sample of live microorganisms from the group consisting of
Pseudomonas aeruginosa, Escherichia coli, Enterococcus hirae,
Acinetobacter baumannii, Acinetobacter species, Bacteroides
fragilis, Enterobacter aerogenes, Enterococcus faecalis, Vancomycin
resistant--Enterococcus faecium (VRE, MDR), Haemophilus influenzae,
Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus,
Proteus mirabilis, Serratia marcescens, Staphylococcus aureus,
Staphylococcus epidermidis, Staphylococcus haemolyticus,
Staphylococcus hominis, Staphylococcus saprophyticus, Streptococcus
pneumoniae, Streptococcus pyogenes, Candida albicans within thirty
seconds of exposure, when measured at least two months after
preparation of the ORP water solution (BioSciences Labs, Montana,
US). Preferably, the ORP water solution is capable of achieving a
10.sup.5 reduction of all these organisms when measured at least
about six months after preparation, and more preferably when
measured at least about one year after preparation.
[0076] The invention also provides methods for killing bacteria in
biofilms, e.g., Pseudomonas aeruginosa in biofilms. The invention
further provides methods for killing of Moraexlla catarrhalis and
antibotic resistant bacteria, e.g., penicillin resistant
Streptococcus. The methods disclosed herein can be used in
accordance with the invention for killing bacteria using ORP water
solutions faster than with using Bacitracin.
[0077] In one embodiment, the ORP water solution administered in
accordance with the invention can reduce a sample of live
microorganisms including, but not limited to, Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans,
from an initial concentration of between about 1.times.10.sup.6 and
about 1.times.10.sup.8 organisms/ml to a final concentration of
about zero organisms/ml within about one minute of exposure when
measured at least about two months after preparation of the ORP
water solution. This corresponds to from about a six log (10.sup.6)
to about an eight log (10.sup.8) reduction in organism
concentration. Preferably, the ORP water solution is capable of
achieving a 10.sup.6-10.sup.8 reduction of Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus or Candida albicans
organisms when measured at least about six months after
preparation, and more preferably when measured at least about one
year after preparation.
[0078] Alternatively, the ORP water solution administered in
accordance with the present invention can produce about a six log
(10.sup.6) reduction in the concentration of a spore suspension of
Bacillus athrophaeus spores within about five minutes of exposure
when measured at least about two months after preparation of the
ORP water solution. Preferably, the ORP water solution administered
in accordance with the invention can achieve about a 10.sup.6
reduction in the concentration of Bacillus athrophaeus spores when
measured at least about six months after preparation, and more
preferably when measured at least about one year after
preparation.
[0079] The ORP water solution administered in accordance with the
invention also can produce about a four log (10.sup.4) reduction in
the concentration of a spore suspension of Bacillus athrophaeus
spores within about thirty (30) seconds of exposure when measured
at least about two months after preparation of the ORP water
solution. Preferably, the ORP water solution can achieve this
reduction in the concentration of Bacillus athrophaeus spores when
measured at least about six months after preparation, and more
preferably when measured, at least about one year after
preparation.
[0080] The ORP water solution administered in accordance with the
invention further can produce about a six log (10.sup.6) reduction
in the concentration of fungal spores, such as Aspergillis niger
spores, within about five to about ten minutes of exposure when
measured at least about two months after preparation of the ORP
water solution. Preferably, the ORP water solution can achieve a
10.sup.6 reduction in the concentration of fungal spores when
measured at least about six months after preparation, and more
preferably when measured at least about one year after
preparation.
[0081] The ORP water solution administered in accordance with the
invention can be acidic, neutral or basic, and generally can have a
pH of from about 1 to about 14. Within this pH range, the ORP water
solution can be safely applied in suitable quantities, e.g., to
surfaces without damaging the surfaces or harming objects, such as
human skin, that comes into contact with the ORP water solution.
Preferably, the pH of the ORP water solution administered in
accordance with the invention is from about 3 to about 8. More
preferably, the pH of the ORP water solution is from about 6.4 to
about 7.8, and still more preferably, the pH is from about 7.4 to
about 7.6.
[0082] The ORP water solution administered in accordance with the
invention can have an oxidation-reduction potential of from about
-1000 millivolts (mV) to about +1150 millivolts (mV). This
potential is a measure of the tendency (i.e., the potential) of a
solution to either accept or transfer electrons that are sensed by
a metal electrode and compared with a reference electrode in the
same solution. This potential may be measured by standard
techniques including, for example, measuring the electrical
potential in millivolts of the ORP water solution relative to
standard reference such as, e.g., a silver/silver chloride
electrode.
[0083] The ORP water solution administered in accordance with the
invention preferably has a potential of from about -400 mV to about
+1300 mV. More preferably, the ORP water solution has a potential
of from about 0 mV to about +1250 mV, and still more preferably
from about +500 mV to about +1250 mV. Even more preferably, the ORP
water solution administered in accordance with the present
invention has a potential of from about +800 mV to about +1100 mV,
and most preferably from about +800 mV to about +1000 mV.
[0084] Various ionic and other species may be present in the ORP
water solution administered in accordance with the invention. For
example, the ORP water solution may contain chlorine (e.g., free
chlorine and bound chlorine), and dissolved oxygen and, optionally,
ozone and peroxides (e.g., hydrogen peroxide). The presence of one
or more of these species is believed to contribute to at least the
disinfectant ability of the ORP water solution to kill a variety of
microorganisms, such as bacteria and fungi, as well as viruses.
Although not wishing to be bound by any particular theory, it is
believed that or more of such species also may contribute the
anti-inflammatory efficacy of the ORP water solution.
[0085] Free chlorine typically includes, but is not limited to,
hypochlorous acid (HClO), hypochlorite ions (ClO.sup.-), sodium
hypochlorite (NaOCl), and precursors thereof. The ratio of
hypochlorous acid to hypochlorite ion is dependent upon pH. At a pH
of 7.4, hypochlorous acid levels are typically from about 25 ppm to
about 75 ppm. Temperature also impacts the ratio of the free
chlorine component.
[0086] Bound chlorine typically includes chlorine in chemical
combination with, e.g., ammonia or organic amines (e.g.,
chloramines). Bound chlorine is preferably present in an amount of
up to about 20 ppm.
[0087] One or more chlorine species, one or more additional
superoxidized water species (e.g., one or more additional oxidizing
species such as, e.g., oxygen) can be present in the ORP water
solution administered in accordance with the invention in any
suitable amount. The levels of these components may be measured by
any suitable method, including methods known in the art.
[0088] The total amount of free chlorine species is preferably from
about 10 ppm to about 400 ppm, more preferably from about 50 ppm to
about 200 ppm, and most preferably from about 50 ppm to about 80
ppm. The amount of hypochlorous acid is preferably from about 15
ppm to about 35 ppm. The amount of sodium hypochlorite is
preferably in the range of from about 25 ppm to about 50 ppm.
Optionally, Chlorine dioxide levels are preferably less than about
5 ppm.
[0089] The chlorine content may be measured by methods known in the
art, such as the DPD colorimeter method (Lamotte Company,
Chestertown, Md.) or other known methods such as, e.g., methods
established by the Environmental Protection Agency. In the DPD
calorimeter method, a yellow color is formed by the reaction of
free chlorine with N,N-diethyl-p-phenylenediamine (DPD) and the
intensity is measured with a calibrated calorimeter that provides
the output in parts per million. Further addition of potassium
iodide turns the solution a pink color to provide the total
chlorine value. The amount of bound chlorine present is then
determined by subtracting free chlorine from the total
chlorine.
[0090] The total amount of oxidizing chemical species present in
the ORP water solution is preferably in the range of about 2
millimolar (mM), which includes the aforementioned chlorine
species, oxygen species, and additional species, including those,
which can be difficult to measure such as, e.g., Cl.sup.-,
ClO.sub.3, Cl.sub.2.sup.-, and ClO.sub.x.
[0091] In one embodiment, the ORP water solution administered in
accordance with the invention comprises one or more chlorine
species and one or more additional superoxidized water species
(e.g., one or more additional oxidizing species such as, e.g.,
oxygen). Preferably, the chlorine species present is a free
chlorine species. The free chlorine species can include one or more
species selected from the group consisting of hypochlorous acid
(HOCl), hypochlorite ions (OCl.sup.-), and sodium hypochlorite
(NaOCl), chloride ion (Cl.sup.-), and optionally, chlorine dioxide
(ClO.sub.2), dissolved chlorine gas (Cl.sub.2), precursors thereof
and mixtures thereof.
[0092] In one embodiment, the ORP water solution includes one or
more chlorine species or one or more precursors thereof, and one or
more additional superoxidized water species or one or more
precursors thereof, and, optionally, hydrogen peroxide, and is
stable for at least about 24 hours, preferably for at least about
one week, more preferably for at about least two months, and still
more preferably for at least about six months after its
preparation. Even more preferably, such ORP water solution is
stable for at least about one year, and most preferably for more
than about one year, e.g., at least about two years or at least
about three years.
[0093] It is also preferred that the ORP water solution includes
one or more chlorine species (e.g., hypocholorous acid and sodium
hypochlorite) or one or more precursors thereof and one or one or
more additional superoxidized water species (e.g., one or more
oxygen species, dissolved oxygen) or one or more precursors thereof
and has a pH of from about 6 to about 8. More preferably from about
6.2 to about 7.8, and most preferably from about 7.4 to about 7.6.
An exemplary ORP water solution administered in accordance with the
present invention can comprise, e.g., from about 15 ppm to about 35
ppm hypochlorous acid, from about 25 ppm to about 50 ppm sodium
hypochlorite, from about 1 ppm to about 4 ppm of one or more
additional superoxidized water species and a pH of from about 6.2
to about 7.8, and can be stable for at least about one week, e.g.,
at least about two months, at least about six months, at least
about one year, or more than about one year, e.g., at least about
two years or at least about three years.
[0094] While in no way limiting the present invention, it is
believed that the control of pH and other variables (e.g.,
salinity) can provide stable ORP water solutions, which contain one
or more chlorine species or precursors thereof, such as, e.g.,
hypochlorous acid and hypochlorite ions, and one or more additional
superoxidized water species (e.g., oxygen) or one or more
precursors thereof.
[0095] The ORP water solutions administered in accordance with the
invention preferably comprises one or more oxidized water species
which can yield free radicals (such as, e.g., hydroxyl radicals) on
exposure to iron. The ORP water can optionally include one or more
chemical compounds generated during the production thereof such as,
e.g., sodium hydroxide (NaOH), chlorine dioxide (ClO.sub.2),
peroxides (e.g., hydrogen peroxide (H.sub.2O.sub.2), and ozone
(O.sub.3) although, it has been reported that sodium hydroxide,
chlorine dioxide, hydrogen peroxide, and ozone may react with
hypocholrite resulting in their consumption and the production of
other chemical species.
[0096] The ORP water solution administered in accordance with the
present invention can be produced by an oxidation-reduction
process, e.g., by an electrolytic process or redox reaction, in
which electrical energy is used to produce one or more chemical
changes in an aqueous solution. Exemplary processes for preparing
suitable ORP water solutions are described, e.g., in U.S. Patent
Application Publication Nos. US 2005/0139808 and US 2005/0142157
(hereby incorporated by reference).
[0097] In the electrolytic process, electrical energy is introduced
into and transported through water by the conduction of electrical
charge from one point to another in the form of an electrical
current. In order for the electrical current to arise and subsist
there should be charge carriers in the water, and there should be a
force that makes the carriers move. The charge carriers can be
electrons, as in the case of metal and semiconductors, or they can
be positive and negative ions in the case of solutions. A reduction
reaction occurs at the cathode while an oxidation reaction occurs
at the anode. At least some of the reductive and oxidative
reactions that are believed to occur are described in International
Application WO 03/048421 A1.
[0098] As used herein, water produced at an anode is referred to as
anode water and water produced at a cathode is referred to as
cathode water. Anode water typically contains oxidized species
produced from the electrolytic reaction while cathode water
typically contains reduced species from the reaction. Anode water
generally has a low pH, typically of from about 1 to about 6.8. The
anode water preferably contains chlorine in various forms
including, for example, chlorine gas, chloride ions, hydrochloric
acid and/or hypochlorous acid, or one or more precursors thereof.
Oxygen in various forms is also preferably present including, for
example, oxygen gas, and possibly one or more species formed during
production (e.g., peroxides, and/or ozone), or one or more
precursors thereof. Cathode water generally has a high pH,
typically from about 7.2 to about 11. Cathode water can contain
hydrogen gas, hydroxyl radicals, and/or sodium ions.
[0099] The ORP water solution administered in accordance with the
invention can include a mixture of anode water (e.g., water
produced in the anode chamber of an electrolytic cell) and cathode
water (e.g., water produced in the cathode chamber of an
electrolysis cell). Preferably, the ORP water solution administered
in accordance with the present invention contains cathode water,
e.g., in an amount of from about 1.0% by volume to about 90% by
volume of the solution. More preferably, cathode water is present
in the ORP water solution in an amount of from about 10% by volume
to about 50% by volume, and still more preferably of from about 20%
by volume to about 40% by volume of the solution, e.g., from about
20% by volume to about 30% by volume of the solution. Additionally,
anode water can be present in the ORP water solution, e.g., in an
amount of from about 50% by volume to about 90% by volume of the
solution. Exemplary ORP water solutions can contain from about 10%
by volume to about 50% by volume of cathode water and from about
50% by volume to about 90% by volume of anode water. The anode and
cathode water can be produced using the three-chambered
electrolysis cell shown in FIG. 1.
[0100] The ORP water solution administered in accordance with the
invention is preferably produced using at least one electrolysis
cell comprising an anode chamber, a cathode chamber and a salt
solution chamber located between the anode and cathode chambers,
wherein at least some of the anode and cathode water are combined
such that the ORP water solution comprises anode water and cathode
water. A diagram of an exemplary three chamber electrolysis cell
that can be used in preparing an exemplary ORP water solution is
shown in FIG. 2.
[0101] The electrolysis cell 100 has an anode chamber 102, cathode
chamber 104 and salt solution chamber 106. The salt solution
chamber is located between the anode chamber 102 and cathode
chamber 104. The anode chamber 102 has an inlet 108 and outlet 110
to permit the flow of water through the anode chamber 100. The
cathode chamber 104 similarly has an inlet 112 and outlet 114 to
permit the flow of water through the cathode chamber 104. The salt
solution chamber 106 has an inlet 116 and outlet 118. The
electrolysis cell 100 preferably includes a housing to hold all of
the components together.
[0102] The anode chamber 102 is separated from the salt solution
chamber by an anode electrode 120 and an anion ion exchange
membrane 122. The anode electrode 120 may be positioned adjacent to
the anode chamber 102 with the membrane 122 located between the
anode electrode 120 and the salt solution chamber 106.
Alternatively, the membrane 122 may be positioned adjacent to the
anode chamber 102 with the anode electrode 120 located between the
membrane 122 and the salt solution chamber 106.
[0103] The cathode chamber 104 is separated from the salt solution
chamber by a cathode electrode 124 and a cathode ion exchange
membrane 126. The cathode electrode 124 may be positioned adjacent
to the cathode chamber 104 with the membrane 126 located between
the cathode electrode 124 and the salt solution chamber 106.
Alternatively, the membrane 126 may be positioned adjacent to the
cathode chamber 104 with the cathode electrode 124 located between
the membrane 126 and the salt solution chamber 106.
[0104] The electrodes preferably are constructed of metal to permit
a voltage potential to be applied between the anode chamber and
cathode chamber. The metal electrodes are generally planar and have
similar dimensions and cross-sectional surface area to that of the
ion exchange membranes. The electrodes are configured to expose a
substantial portion of the surface of the ion exchange members to
the water in their respective anode chamber and cathode chamber.
This permits the migration of ionic species between the salt
solution chamber, anode chamber and cathode chamber. Preferably,
the electrodes have a plurality of passages or apertures evenly
spaced across the surface of the electrodes.
[0105] A source of electrical potential is connected to the anode
electrode 120 and cathode electrode 124 so as to induce an
oxidation reaction in the anode chamber 102 and a reduction
reaction in the cathode chamber 104.
[0106] The ion exchange membranes 122 and 126 used in the
electrolysis cell 100 may be constructed of any suitable material
to permit the exchange of ions between the salt solution chamber
106 and the anode chamber 102 such as, e.g., chloride ions
(Cl.sup.-) and between the salt solution salt solution chamber 106
and the cathode chamber 104 such as, e.g., sodium ions (Na.sup.+).
The anode ion exchange membrane 122 and cathode ion exchange
membrane 126 may be made of the same or different material of
construction. Preferably, the anode ion exchange membrane comprises
a fluorinated polymer. Suitable fluorinated polymers include, for
example, perfluorosulfonic acid polymers and copolymers such as
perfluorosulfonic acid/PTFE copolymers and perfluorosulfonic
acid/TFE copolymers. The ion exchange membrane may be constructed
of a single layer of material or multiple layers. Suitable ion
exchange membrane polymers can include one or more ion exchange
membrane polymers marketed under the trademark Nafion.RTM..
[0107] The source of the water for the anode chamber 102 and
cathode chamber 104 of the electrolysis cell 100 may be any
suitable water supply. The water may be from a municipal water
supply or alternatively pretreated prior to use in the electrolysis
cell. Preferably, the water is pretreated and is selected from the
group consisting of softened water, purified water, distilled
water, and deionized water. More preferably, the pretreated water
source is ultrapure water obtained using reverse osmosis
purification equipment.
[0108] The salt water solution for use in the salt water chamber
106 can include any aqueous salt solution that contains suitable
ionic species to produce the ORP water solution. Preferably, the
salt water solution is an aqueous sodium chloride (NaCl) salt
solution, also commonly referred to as a saline solution. Other
suitable salt solutions can include other chloride salts such as
potassium chloride, ammonium chloride and magnesium chloride as
well as other halogen salts such as potassium and bromine salts.
The salt solution can contain a mixture of salts.
[0109] The salt solution can have any suitable concentration. For
example, the salt solution can be saturated or concentrated.
Preferably, the salt solution is a saturated sodium chloride
solution.
[0110] FIG. 2 illustrates what are believed to be various ionic
species produced in the three chambered electrolysis cell useful in
connection with the invention. The three chambered electrolysis
cell 200 includes an anode chamber 202, cathode chamber 204, and a
salt solution chamber 206. Upon application of a suitable
electrical current to the anode 208 and cathode 210, the ions
present in the salt solution flowing through the salt solution
chamber 206 migrate through the anode ion exchange membrane 212 and
cathode ion exchange membrane 214 into the water flowing through
the anode chamber 202 and cathode chamber 204, respectively.
[0111] Positive ions migrate from the salt solution 216 flowing
through the salt solution chamber 206 to the cathode water 218
flowing through the cathode chamber 204. Negative ions migrate from
the salt solution 216 flowing through the salt solution chamber 206
to the anode water 220 flowing through the anode chamber 202.
[0112] Preferably, the salt solution 216 is aqueous sodium chloride
(NaCl), which contains both sodium ions (Na.sup.+) and chloride
ions (Cl.sup.-) ions. Positive Na.sup.+ ions migrate from the salt
solution 216 to the cathode water 218. Negative Cl.sup.- ions
migrate from the salt solution 216 to the anode water 220.
[0113] The sodium ions and chloride ions may undergo further
reaction in the anode chamber 202 and cathode chamber 204. For
example, chloride ions can react with various oxygen ions and other
species (e.g., oxygen containing free radicals, O.sub.2, O.sub.3)
present in the anode water 220 to produce ClOn- and ClO.sup.-.
Other reactions may also take place in the anode chamber 202
including the formation of oxygen free radicals, hydrogen ions
(H.sup.+), oxygen (e.g., as O.sub.2), ozone (O.sub.3), and
peroxides. In the cathode chamber 204, hydrogen gas (H.sub.2),
sodium hydroxide (NaOH), hydroxide ions (OH.sup.-), and other
radicals may be formed.
[0114] The apparatus for producing the ORP water solution also can
be constructed to include at least two three chambered electrolysis
cells. Each of the electrolytic cells includes an anode chamber,
cathode chamber, and salt solution chamber separating the anode and
cathode chambers. The apparatus includes a mixing tank for
collecting the anode water produced by the electrolytic cells and a
portion of the cathode water produced by one or more of the
electrolytic cells. Preferably, the apparatus further includes a
salt recirculation system to permit recycling of the salt solution
supplied to the salt solution chambers of the electrolytic cells. A
diagram of an exemplary process for producing an ORP water solution
using two electrolysis cells is shown in FIG. 3.
[0115] The process 300 includes two three-chambered electrolytic
cells, specifically a first electrolytic cell 302 and second
electrolytic cell 304. Water is transferred, pumped or otherwise
dispensed from the water source 305 to anode chamber 306 and
cathode chamber 308 of the first electrolytic cell 302 and to anode
chamber 310 and cathode chamber 312 of the second electrolytic cell
304. Advantageously, this process can produce from about 1
liter/minute to about 50 liters/minute of ORP water solution. The
production capacity may be increased by using additional
electrolytic cells. For example, three, four, five, six, seven,
eight, nine, ten or more three-chambered electrolytic cells may be
used to increase the output of the ORP water solution administered
in accordance with the invention.
[0116] The anode water produced in the anode chamber 306 and anode
chamber 310 are collected in the mixing tank 314. A portion of the
cathode water produced in the cathode chamber 308 and cathode
chamber 312 is collected in mixing tank 314 and combined with the
anode water. The remaining portion of cathode water produced in the
process is discarded. The cathode water may optionally be subjected
to gas separator 316 and/or gas separator 318 prior to addition to
the mixing tank 314. The gas separators remove gases such as
hydrogen gas that are formed in cathode water during the production
process.
[0117] The mixing tank 314 may optionally be connected to a
recirculation pump 315 to permit homogenous mixing of the anode
water and portion of cathode water from electrolysis cells 302 and
304. Further, the mixing tank 314 may optionally include suitable
devices for monitoring the level and pH of the ORP water solution.
The ORP water solution may be transferred from the mixing tank 314
via pump 317 for application in disinfection or sterilization at or
near the location of the mixing tank. Alternatively, the ORP water
solution may be dispensed into one or more suitable containers for
shipment to a remote site (e.g., warehouse, hospital, etc.).
[0118] The process 300 further includes a salt solution
recirculation system to provide the salt solution to salt solution
chamber 322 of the first electrolytic cell 302 and the salt
solution chamber 324 of the second electrolytic cell 304. The salt
solution is prepared in the salt tank 320. The salt is transferred
via pump 321 to the salt solution chambers 322 and 324. Preferably,
the salt solution flows in series through salt solution chamber 322
first followed by salt solution chamber 324. Alternatively, the
salt solution may be pumped to both salt solution chambers
simultaneously.
[0119] Before returning to the salt tank 320, the salt solution may
flow through a heat exchanger 326 in the mixing tank 314 to control
the temperature of the ORP water solution as needed.
[0120] The ions present in the salt solution are depleted over time
in the first electrolytic cell 302 and second electrolytic cell
304. An additional source of ions periodically can be added to the
mixing tank 320 to replace the ions that are transferred to the
anode water and cathode water. The additional source of ions may be
used, e.g., to maintain a constant pH of the salt solution, which
can to drop (i.e., become acidic) over time. The source of
additional ions may be any suitable compound including, for
example, salts such as, e.g., sodium chloride. Preferably, sodium
hydroxide is added to the mixing tank 320 to replace the sodium
ions (Na.sup.+) that are transferred to the anode water and cathode
water.
[0121] Following its preparation, the ORP water solution can be
transferred to one or more suitable containers, e.g., a sealed
container for distribution and sale to end users such as, e.g.,
health care facilities including, e.g., hospitals, nursing homes,
doctor offices, outpatient surgical centers, dental offices, and
the like. Suitable containers can include, e.g., a sealed container
that maintains the sterility and stability of the ORP water
solution held by the container. The container can be constructed of
any material that is compatible with the ORP water solution.
Preferably, the container is generally non-reactive with one or
more ions or other species present in the ORP water solution.
[0122] Preferably, the container is constructed of plastic or
glass. The plastic can be rigid so that the container is capable of
being stored on a shelf. Alternatively, the container can be
flexible, e.g., a container made of flexible plastic such as, e.g.,
a flexible bag.
[0123] Suitable plastics can include, e.g., polypropylene,
polyester terephthalate (PET), polyolefin, cycloolefin,
polycarbonate, ABS resin, polyethylene, polyvinyl chloride, and
mixtures thereof. Preferably, the container comprises one or more
polyethylenes selected from the group consisting of high-density
polyethylene (HDPE), low-density polyethylene (LDPE), and linear
low-density polyethylene (LLDPE). Most preferably, the container is
constructed of high density polyethylene.
[0124] The container preferably has an opening to permit dispensing
of the ORP water solution. The container opening can be sealed in
any suitable manner. For example, the container can be sealed with
a twist-off cap or stopper. Optionally, the opening can be further
sealed with a foil layer.
[0125] The headspace gas of the sealed container can be air or any
other suitable gas, which preferably does not react with one or
more species in the ORP water solution. Suitable headspace gases
can include, e.g., nitrogen, oxygen, and mixtures thereof.
[0126] The ORP water solution administered in accordance with the
invention also can be used for the prevention or treatment of an
infection, e.g., by one or more infectious pathogens such as, for
example, infectious microorganisms. Such microorganisms can
include, for example, viruses, bacteria, and fungi. The viruses can
include, e.g., one or more viruses selected from the group
consisting of adenoviruses, herpes viruses, coxsackie viruses, HIV,
rhinoviruses, comaviruses, and flu viruses. The bacteria can
include, e.g., one or more bacteria selected from the group
consisting of Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, and Mycobaterium tuberculosis. The fungi can
include, e.g., one or more fungi selected from the group consisting
of Candida albicans, Bacillus subtilis and Bacillus
athrophaeus.
[0127] The ORP water solution administered in accordance with the
invention also can be effective against adenovirus. Preferably, the
ORP water solution administered in accordance with the invention
preferably achieves a log-10 reduction in the adenoviral load of
greater than about 2, more preferably greater than about 2.5, and
still more preferably greater than about 3, after exposure to the
ORP water solution for about 20 minutes, more preferably after
exposure for about 15 minutes, and still more preferably after
exposure for about 10 minutes. The ORP water solution administered
in accordance with the invention also can be effective for reducing
the viral load of HIV-1, preferably by a log reduction factor
greater than about 2, more preferably by a log reduction factor of
greater than about 2.5, and still more preferably by a log
reduction factor of greater than about 3 after exposure to the ORP
water solution for about five minutes.
[0128] In accordance with the method of the present invention,
administering the ORP water solution for the prevention or
treatment of infection also can serve to prevent or treat
inflammation associated with the infection (or the affected
tissues) as described herein.
[0129] The ORP water solution administered in accordance with the
invention also can be used for treating impaired or damaged tissue,
e.g., by contacting one or more impaired or damaged tissues with a
therapeutically effective amount of the ORP water solution. Any
suitable method can be used for contacting the impaired or damaged
tissue, so as to treat the impaired or damaged tissue. For example,
the impaired or damaged tissue can be treated by irrigating the
tissue with the ORP water solution, so as to contact the impaired
or damaged tissue with a therapeutically effective amount of the
ORP water solution. The ORP water solution can be administered as a
steam or a spray, or by aerosolization, nebulization or
atomization, as described herein, so as to contact the impaired or
damaged tissue with a therapeutically effective amount of the ORP
water solution.
[0130] The ORP water solution administered in accordance with the
invention can be used for treating tissues, which have been
impaired or damaged, e.g., by surgery. For instance, the ORP water
solution can be used for treating tissues, which have been impaired
or damaged by an incision. In addition, the ORP water solution can
be used for treating tissues, which have been impaired or damaged
by oral surgery, graft surgery, implant surgery, transplant
surgery, cauterization, amputation, radiation, chemotherapy, and
combinations thereof. The oral surgery can include, for example,
dental surgery such as, e.g., root canal surgery, tooth extraction,
gum surgery, and the like.
[0131] The ORP water solution administered in accordance with the
invention can be used for treating tissues, which have been
impaired or damaged by one or more bums, cuts, abrasions, scrapes,
rashes, ulcers, puncture wounds, combinations thereof, and the
like, which are not necessarily caused by surgery. The ORP water
solution administered in accordance with the invention can be used
for treating impaired or damaged tissue, which is infected, or
tissue impaired or damaged due to infection. Such infection can be
caused by one or more infectious pathogens, such as, e.g., one or
more microorganisms selected from the group consisting of viruses,
bacteria, and fungi, as described herein.
[0132] In accordance with the present invention, administering the
ORP water solution for treating impaired or damaged tissue also can
serve to prevent or treat inflammation associated with the
impairment or damage (or with the impaired or damaged tissue).
[0133] The ORP water solution administered in accordance with the
invention also can be used as a disinfectant to eradicate
microorganisms, including bacteria, viruses and spores, in a
variety of settings, e.g., in the healthcare and medical device
fields, to disinfect surfaces and medical equipment, and also can
be applied in wound care, medical device sterilization, food
sterilization, hospitals, consumer households and
anti-bioterrorism. The ORP water solution can be used for
disinfecting a surface, e.g., by contacting the surface with an
anti-infective amount of the ORP water solution. The surface can be
contacted using any suitable method. For example, the surface can
be contacted by irrigating the surface with the ORP water solution,
so as to disinfect the surface. Additionally, the surface can be
contacted by applying the ORP water solution to the surface as a
steam or a spray, or by aerosolization, nebulization or
atomization, as described herein, so as to disinfect the surface.
Further, the ORP water solution can be applied to the surface with
a cleaning wipe, as described herein. By disinfecting a surface,
the surface may be cleansed of infectious microorganisms.
Alternatively (or additionally), the ORP water solution
administered in accordance with the present invention can be
applied to the surface to provide a barrier to infection, to
thereby disinfect the surface.
[0134] The surface(s) can include one or more biological surfaces,
one or more inanimate surfaces, and combinations thereof.
Biological surfaces can include, for example, tissues within one or
more body cavities such as, for example, the oral cavity, the sinus
cavity, the cranial cavity, the abdominal cavity, and the thoracic
cavity. Tissues within the oral cavity include, e.g., mouth tissue,
gum tissue, tongue tissue, and throat tissue. The biological tissue
also can include muscle tissue, bone tissue, organ tissue, mucosal
tissue, vascular tissue, neurological tissue, and combinations
thereof. Biological surfaces also include any other cultured tissue
in vitro, such as primary and established cell lines, stem cells of
any nature, xenotransplants, tissue substitutes (e.g. made of
collagen or any other organic material in addition or not of
cellular elements), any other tissue-engineered substitutes and
combinations thereof.
[0135] Inanimate surfaces include, for example, surgically
implantable devices, prosthetic devices, and medical devices. In
accordance with the method of the present invention, the surfaces
of internal organs, viscera, muscle, and the like, which may be
exposed during surgery, can be disinfected, e.g., to maintain
sterility of the surgical environment. In accordance with the
present invention, administering the ORP water solution for
disinfecting a surface also can serve to treat or prevent
inflammation affecting one or more biological tissues associated
with such surfaces.
[0136] The ORP water solution may also be applied to humans and/or
animals to treat various conditions, including inflammation,
hypersensitivity, and associated systemic effects associated with
one or more of the following: surgical/open wound cleansing agent;
skin pathogen disinfection (e.g., for bacteria, mycoplasmas, virus,
fungi, prions); battle wound disinfection; wound healing promotion;
burn healing promotion; treatment of stomach ulcers; wound
irrigation; skin fungi; psoriasis; athlete's foot; pinkeye and
other eye infections; ear infections (e.g., swimmer's ear);
lung/nasal/sinus infections; and other medical applications on or
in the human or animal body, as well as environmental remediation.
The use of ORP water solutions as a tissue cell growth promoter is
further described in U.S. Patent Application Publication
2002/0160053 (hereby incorporated by reference).
[0137] The ORP water solution may be used as a disinfectant,
sterilization agent, decontaminant, antiseptic and/or cleanser. The
ORP water solution administered in accordance with the invention is
suitable for use in the following representative applications:
medical, dental and/or veterinary equipment and devices; food
industry (e.g., hard surfaces, fruits, vegetables, meats);
hospitals/health care facilities (e.g., hard surfaces); cosmetic
industry (e.g., skin cleaner); households (e.g., floors, counters,
hard surfaces); electronics industry (e.g., cleaning circuitry,
hard drives); and bio-terrorism (e.g., anthrax, infectious
microbes).
[0138] Organisms that can be controlled, reduced, killed or
eradicated by treatment with the ORP water solution include, but
are not limited to, bacteria, fungi, yeasts, and viruses.
Susceptible bacteria include, but are not limited to, Escherichia
coli, Staphylococcus aureus, Bacillus athrophaeus, Streptococcus
pyogenes, Salmonella choleraesuis, Pseudomonas aeruginosa,
Shingella dysenteriae, and other susceptible bacteria. Fungi and
yeasts that may be treated with the ORP water solution include, for
example, Candida; albicans and Trichophyton mentagrophytes. The ORP
water solution may also be applied to viruses including, for
example, adenovirus, human immunodeficiency virus (HIV),
rhinovirus, influenza (e.g., influenza A), hepatitis (e.g.,
hepatitis A), coronavirus (responsible for Severe Acute Respiratory
Syndrome (SARS)), rotavirus, respiratory syncytial virus, herpes
simplex virus, varicella zoster virus, rubella virus, and other
susceptible viruses.
[0139] The ORP water solution may be applied to disinfect and
sterilize in any suitable manner. For example, to disinfect and
sterilize medical or dental equipment, the equipment can be
maintained in contact with the ORP water solution for a sufficient
period of time to reduce the level of organisms present on the
equipment to a desired level. Alternatively, the ORP water solution
can be applied to medical or dental equipment by immersing the
equipment in a container with or without the application of
enhancing physical procedures, e.g. ultrasound, shakers, heaters,
and the like.
[0140] For disinfection and sterilization of hard surfaces, the ORP
water solution can be applied to the hard surface directly from a
container in which the ORP water solution is stored. For example,
the ORP water solution can be poured, sprayed or otherwise directly
applied to the hard surface. The ORP water solution can then be
distributed over the hard surface using a suitable substrate such
as, for example, cloth, fabric or paper towel. In hospital
applications, the substrate is preferably sterile. Alternatively,
the ORP water solution can first be applied to a substrate such as
cloth, fabric or paper towel. The wetted substrate can then be
contacted with the hard surface. Alternatively, the ORP water
solution can be applied to hard surfaces by dispersing the solution
into the air as described herein. The ORP water solution can be
applied in a similar manner to humans and animals.
[0141] The ORP water solution also can be applied with a cleaning
wipe comprising a water insoluble substrate and the ORP water
solution as described herein, wherein the ORP water solution is
dispensed onto the substrate. The ORP water solution can be
impregnated, coated, covered or otherwise applied to the substrate.
Preferably, the substrate is pretreated with the ORP water solution
before distribution of the cleaning wipes to end users.
[0142] The substrate for the cleaning wipe can be any suitable
water-insoluble absorbent or adsorbent material. A wide variety of
materials can be used as the substrate. It should have sufficient
wet strength, abrasivity, loft and porosity. Further, the substrate
should not adversely impact the stability of the ORP water
solution. Examples include non woven substrates, woven substrates,
hydroentangled substrates and sponges.
[0143] The substrate can have one or more layers. Each layer can
have the same or different textures and abrasiveness. Differing
textures can result from the use of different combinations of
materials or from the use of different manufacturing processes or a
combination thereof. The substrate should not dissolve or break
apart in water. The substrate can thereby provide a vehicle for
delivering the ORP water solution to the surface to be treated.
[0144] The substrate can be a single nonwoven sheet or multiple
nonwoven sheets. The nonwoven sheet can be made of wood pulp,
synthetic fibers, natural fibers, and blends thereof. Suitable
synthetic fibers for use in the substrate can include, without
limitation, polyester, rayon, nylon, polypropylene, polyethylene,
other cellulose polymers, and mixtures of such fibers. The
nonwovens can include nonwoven fibrous sheet materials which
include meltblown, coform, air-laid, spun bond, wet laid,
bonded-carded web materials, hydroentangled (also known as
spunlaced) materials, and combinations thereof. These materials can
comprise synthetic or natural fibers or combinations thereof. A
binder can optionally be present in the substrate.
[0145] Examples of suitable nonwoven, water insoluble substrates
include 100% cellulose Wadding Grade 1804 from Little Rapids
Corporation, 100% polypropylene needlepunch material NB 701-2.8-W/R
from American Non-wovens Corporation, a blend of cellulosic and
synthetic fibres-Hydraspun 8579 from Ahlstrom Fibre Composites, and
70% Viscose/30% PES Code 9881 from PGI Nonwovens Polymer Corp.
Additional examples of nonwoven substrates suitable for use in the
cleaning wipes are described in U.S. Pat. Nos. 4,781,974,
4,615,937, 4,666,621, and 5,908,707, and International Patent
Application Publications WO 98/03713, WO 97/40814, and WO 96/14835
(all herby incorporated by reference.).
[0146] The substrate also can be made of woven materials, such as
cotton fibers, cotton/nylon blends, or other textiles. Regenerated
cellulose, polyurethane foams, and the like, which are used in
making sponges, also can be suitable for use.
[0147] The liquid loading capacity of the substrate should be at
least about 50%-1000% of the dry weight thereof, most preferably at
least about 200%-800%. This is expressed as loading 1/2 to 10 times
the weight of the substrate. The weight of the substrate varies
without limitation from about 0.01 to about 1,000 grams per square
meter, most preferably 25 to 120 grams/m.sup.2 (referred to as
"basis weight") and typically is produced as a sheet or web which
is cut, die-cut, or otherwise sized into the appropriate shape and
size. The cleaning wipes will preferably have a certain wet tensile
strength which is without limitation about 25 to about 250
Newtons/m, more preferably about 75-170 Newtons/m.
[0148] The ORP water solution can be dispensed, impregnated,
coated, covered or otherwise applied to the substrate by any
suitable method. For example, individual portions of substrate can
be treated with a discrete amount of the ORP water solution.
Preferably, a mass treatment of a continuous web of substrate
material with the ORP water solution is carried out. The entire web
of substrate material can be soaked in the ORP water solution.
Alternatively, as the substrate web is spooled, or even during
creation of a nonwoven substrate, the ORP water solution can be
sprayed or metered onto the web. A stack of individually cut and
sized portions of substrate can be impregnated or coated with the
ORP water solution in its container by the manufacturer.
[0149] The cleaning wipes optionally can contain additional
components to improve the properties of the wipes. For example, the
cleaning wipes can further comprise polymers, surfactants,
polysaccharides, polycarboxylates, polyvinyl alcohols, solvents,
chelating agents, buffers, thickeners, dyes, colorants, fragrances,
and mixtures thereof to improve the properties of the wipes. These
optional components should not adversely impact the stability of
the ORP water solution. Examples of various components that may
optionally be included in the cleaning wipes are described in U.S.
Pat. Nos. 6,340,663, 6,649,584 and 6,624,135 (hereby incorporated
by reference).
[0150] The cleaning wipes can be individually sealed with a
heat-sealable or glueable thermoplastic overwrap (such as
polyethylene, Mylar, and the like). The wipes can also be packaged
as numerous, individual sheets for more economical dispensing. The
cleaning wipes can be prepared by first placing multiple sheets of
the substrate in a dispenser and then contacting the substrate
sheets with the ORP water solution administered in accordance with
the invention. Alternatively, the cleaning wipes can be formed as a
continuous web by applying the ORP water solution to the substrate
during the manufacturing process and then loading the wetted
substrate into a dispenser.
[0151] The dispenser includes, but is not limited to, a canister
with a closure, or a tub with closure. The closure on the dispenser
is to seal the moist wipes from the external environment and to
prevent premature volatilization of the liquid ingredients.
[0152] The dispenser can be made of any suitable material that is
compatible with both the substrate and the ORP water solution. For
example, the dispenser can be made of plastic, such as high density
polyethylene, polypropylene, polycarbonate, polyethylene
terephthalate (PET), polyvinyl chloride (PVC), or other rigid
plastics.
[0153] The continuous web of wipes can be threaded through a thin
opening in the top of the dispenser, most preferably, through the
closure. A means of sizing the desired length or size of the wipe
from the web can then be desirable. A knife blade, serrated edge,
or other means of cutting-the web to desired size can be provided
on the top of the dispenser, for non-limiting example, with the
thin opening actually doubling in duty as a cutting edge.
Alternatively, the continuous web of wipes can be scored, folded,
segmented, perforated or partially cut into uniform or non-uniform
sizes or lengths, which would then obviate the need for a sharp
cutting edge. Further, the wipes can be interleaved, so that the
removal of one wipe advances the next.
[0154] The ORP water solution alternatively can be dispersed into
the environment through a gaseous medium, such as air. The ORP
water solution can be dispersed into the air by any suitable means.
For example, the ORP water solution can be formed into droplets of
any suitable size and dispersed into a room. For small scale
applications, the ORP water solution can be dispensed through a
spray bottle that includes a standpipe and pump. Alternatively, the
ORP water solution can be packaged in aerosol containers. Aerosol
containers can include the product to be dispensed, propellant,
container, and valve. The valve can include both an actuator and
dip tube. The contents of the container can be dispensed by
pressing down on the actuator. The various components of the
aerosol container should be compatible with the ORP water solution.
Suitable propellants can include a liquefied halocarbon,
hydrocarbon, or halocarbon-hydrocarbon blend, or a compressed gas
such as carbon dioxide, nitrogen, or nitrous oxide. Aerosol systems
preferably yield droplets that range in size from about 0.15 .mu.m
to about 5 .mu.m.
[0155] Applications can also be conducted by using various
hydrosurgery equipments for debriding and cleaning (e.g VersaJet
devices sold in the United States by Smith and Nephew, Debritom in
Europe by Medaxis, JetOx in the United States and Europe by DeRoyal
or PulsaVac in Italy), irrigation systems with negative pressure
(e.g., VAC Instill), and the like.
[0156] Optionally, several adjuvant therapies can also be utilized
in accordance with the invention including bioengineered skin
(Apligraf, Organogenesis, Inc., Canton), acellular skin substitutes
(Oasis Wound Matrix, Healthpoint), ultrasonic application of ORP
water solutions, and local oxygen replacement or hyperbaric oxygen
treatment (such as, e.g., hyperbaric boots, the Vent-Ox
System).
[0157] For some applications, the ORP water solution optionally can
contain a bleaching agent. The bleaching agent can include, e.g.,
any suitable compound that lightens or whitens a substrate. The ORP
water solution containing a bleaching agent can be used in home
laundering to disinfect and sterilize bacteria and germs as well as
brighten clothing. Suitable bleaching agents include, but are not
limited to, chlorine-containing bleaching agents and
peroxide-containing bleaching agents. Mixtures of bleaching agents
also can be added to the ORP water solution. Preferably, the
bleaching agent is added in the form of an aqueous solution to the
ORP water solution.
[0158] Suitable chlonine-containing bleaching agents can include,
e.g., chlorine, hypochlorites, N-chloro compounds, and chlorine
dioxide. Preferably, the chlorine-containing bleaching agent added
to the ORP water solution is sodium hypochlorite or hypochlorous
acid. Other suitable chlorine-containing bleaching agents include,
e.g., chlorine, calcium hypochlorite, bleach liquor (e.g., aqueous
solution of calcium hypochlorite and calcium chloride), bleaching
powder (e.g., mixture of calcium hypochlorite, calcium hydroxide,
calcium chloride, and hydrates thereof), dibasic magnesium
hypochlorite, lithium hypochlorite, chlorinated trisodium phosphate
and mixtures thereof.
[0159] The addition of a bleaching agent to the ORP water solution
can be carried out in any suitable manner. Preferably, an aqueous
solution containing the bleaching agent is first prepared. The
aqueous solution containing the bleaching agent can be prepared
using household bleach (e.g., Clorox.RTM. bleach) or other suitable
source of chlorine-containing bleaching agent or other bleaching
agent. The bleaching agent solution can then be combined with the
ORP water solution.
[0160] The bleaching agent can be added to the ORP water solution
in any suitable amount. Preferably, the ORP water solution
containing a bleaching agent is non-irritating to human or animal
skin. Preferably, the total chloride ion content of the ORP water
solution containing a chlorine-containing bleaching agent is from
about 1000 ppm to about 5000 ppm, and preferably from about 1000
ppm to about 3000 ppm. The pH of the ORP water solution containing
a chlorine-containing bleaching agent is preferably from about 8 to
about 10, and the oxidative-reductive potential is preferably from
about +700 mV to about +800 mV.
[0161] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting in its
scope.
EXAMPLES 1-3
[0162] These examples demonstrate the unique features of the ORP
water solution used in accordance with the invention. The samples
of the ORP water solution in Examples 1-3 were analyzed in
accordance with the methods described herein to determine the
physical properties and levels of ionic and other chemical species
present in each sample. The results obtained for chlorine dioxide,
ozone and hydrogen peroxide are based on standard tests used to
measure such species but may be indicative of different species,
which can also generate positive test results. Further, it has been
reported that chlorine dioxide, ozone and hydrogen peroxide react
with hypocholrite resulting in their consumption and the production
of other compounds (e.g., HCl and O.sub.2.) The pH,
oxidative-reductive potential (ORP) and ionic species present are
set forth in Table 1 for each sample of the ORP water solution.
TABLE-US-00001 TABLE 1 Physical characteristics and ion species
present for the ORP water solution samples EXAMPLE 1 EXAMPLE 2
EXAMPLE 3 pH 7.45 7.44 7.45 ORP (mV) +879 +881 +874 Total Cl.sup.-
(ppm) 110 110 120 Bound Cl.sup.- (ppm) 5 6 6
[0163] The ORP water solution has suitable physical characteristics
for use in, e.g., disinfection, sterilization, cleaning, and/or the
prevention and/or treatment of inflammation, sinusitis,
peritonitis, or infection.
EXAMPLES 4-10
[0164] These examples demonstrate the addition of a bleaching agent
to the ORP water solution according to the invention in various
amounts. In particular, these examples demonstrate the
antimicrobial activity and fabric bleaching ability of the
compositions.
[0165] A 10% Clorox.RTM. bleach solution was prepared using
distilled water. The following solutions were then prepared using
the 10% bleach solution: 80% ORP water solution/20% bleach (Example
4); 60% ORP water solution/40% bleach (Example 5); 40% ORP water
solution/60% bleach (Example 6); 20% ORP water solution/80% bleach
(Example 7); and 0% ORP water solution/100% bleach (Example 8). Two
control solutions were also used for comparison including 100% ORP
water solution/0% bleach (Example 9) and an ORP water solution with
0.01% Tween 20 detergent (Example 10). The physical characteristics
of these samples were determined, specifically pH,
oxidative-reductive potential (ORP), total chlorine (Cl.sup.-)
content, hypochlorous acid (HClO.sup.-) content, and were tested
for chlorine dioxide content and peroxide content, the results are
set forth in Table 2. TABLE-US-00002 TABLE 2 Physical
characteristics of ORP water solution/bleach compositions Total
Cl.sup.- HClO.sup.- pH ORP (ppm) (ppm) Ex. 4 8.92 +789 1248 62 Ex.
5 9.20 +782 2610 104 Ex. 6 9.69 +743 4006 80 Ex. 7 9.86 +730 4800
48 Ex. 8 9.80 +737 5000 50 Ex. 9 7.06 +901 64 32 Ex. 10 6.86 +914
51 26
[0166] The large bolus of chlorine ions added as part of the
bleaching agent prevented the accurate measurement of the chlorine
dioxide and peroxide levels as indicated with the n.d.
designations. Also, the results obtained for chlorine dioxide and
peroxide are based on standard tests used to measure such species
but may be indicative of different species, which can also generate
positive test results. Further, it has been reported that chlorine
dioxide, ozone and hydrogen peroxide react with hypocholrite
resulting in their consumption and the production of other
compounds (e.g., HCl and O.sub.2.) As these examples demonstrate,
the hypochlorous acid levels of the ORP water solution with and
without the addition of a bleaching agent are similar.
[0167] The samples of Examples 4-10 were subjected to a high spore
count test using Bacillus subtilis var. niger spores (ATCC #9372
obtained from SPS Medical of Rush, N.Y.). Spore suspensions were
concentrated (by evaporation in a sterile hood) to 4.times.10.sup.6
spores per 100 microliters. A 100 microliter sample of the spore
suspension were mixed with 900 microliters of each of the samples
in Examples 4-10. The samples were incubated at room temperature
for periods of 1 to 5 minutes as set forth in Table 3. At the
indicated times, 100 microliters of the incubated samples were
plated onto individual TSA plates and incubated for 24 hours at
35.degree. C..+-.2.degree. C., after which the number of resulting
colonies on each plate was determined. The control plates
demonstrated that the starting spore concentrations were
>1.times.10.sup.6 spores/100 microliters. The concentration of
Bacillus spores for the various samples at the various incubation
times (as the average of two determinations) is set forth in Table
3. TABLE-US-00003 TABLE 3 Bacillus spore concentrations 1 minute 2
minutes 3 minutes 4 minutes 5 minutes Ex. 4 >>1000 411 1 0 2
Ex. 5 >>1000 1000 1 0 0 Ex. 6 >>1000 >>1000
>1000 22 0 Ex. 7 >>1000 >>1000 >1000 15 0 Ex. 8
>>1000 >>1000 >1000 3 1 Ex. 9 >>1000 74 0 0 0
Ex 10 >>1000 239 3 0 0
[0168] As these results demonstrate, as the concentration of bleach
(as 10% aqueous bleach solution) increases, the amount of Bacillus
spores killed is reduced for the samples incubated for 2-3 minutes.
However, for samples incubated for 5 minutes, the bleach
concentration does not impact Bacillus spore kill. Further, the
results demonstrate that the addition of 0.01% detergent to the ORP
water solution does not reduce spore kill.
[0169] The samples of Examples 4-10 were subjected to a fabric
bleaching test. The fabric upon which the samples were tested was a
100% rayon children's t-shirt with dark blue dye patches. Two inch
square pieces of dyed fabric were placed into 50 mL plastic tubes.
Each fabric piece was covered by a sample of the solution in
Examples 4-10. The elapsed time until complete bleaching was
obtained, as determined by the whitening of the fabric, is set
forth in Table 4. TABLE-US-00004 TABLE 4 Time until complete
bleaching of fabric sample Example Time Ex. 4 39 minutes Ex. 5 23
minutes Ex. 6 18 minutes Ex. 7 19 minutes Ex. 8 10 minutes Ex. 9
>6 hours Ex. 10 >6 hours
[0170] As demonstrated by these examples, as the concentration of
the ORP water solution increases in the composition, the time until
complete bleaching is achieved increases.
EXAMPLE 11
[0171] The purpose of this study was to assess the safety of the
test an exemplary ORP water solution, Microcyn, when administered
as drops into the nasal cavity of rabbits. Thirty-three rabbits
were randomly assigned to two groups, Groups I and II. Group I (18
animals) served as the control group and Group II (15 animals) was
dosed with the test article. On Day -1 or Day 0, body weights were
recorded and blood samples were, collected for analysis of selected
parameters. On Day 0, 500 .mu.L of sterile saline was administered
to the Group I animals and 500 .mu.L of the test article (at a 50%
concentration) was administered to Group n annuals. Both the
control and the test articles were administered twice daily as
drops into the right nostril. The animals were dosed in the same
manner on Days 1-6. Animals were observed daily for signs of
pharmacologic and/or toxicologic effects with special attention
paid to the nose. Body weights were recorded weekly through study
termination. On Day 7, one-third of the animals from each group
were selected for blood collection, sacrifice and necropsy. The
remaining animals continued to be dosed through Day 14, when half
of the animals from each group were selected for blood collection,
sacrifice and necropsy. On Day 21, after a 7-day recovery period),
the remaining animals had blood collected and were sacrificed and
necropsied. Samples of the nasal mucosa from both nostrils were
collected from each animal for histopathological analysis.
[0172] The necropsy consisted of gross observations of the
respiratory tract. The entire nasal passage and associated bone
were taken and fixed in buffered formalin. Samples of any visible
abnormalities in the respiratory tract were also collected for
histopathology. Three biopsy samples (anterior, middle and
posterior nasal cavity) per nostril (treated right and untreated
left) were examined. The microscopic histopathology of the nasal
mucosa included: integrity of epithelium, presence or loss of
epithelial cilia, inflammatory cell infiltration, edema, presence
of goblet cells, hyperplasia of glands, changes in number or
characteristics of blood vessels and any other changes or
observations.
[0173] The results (in-life observations including nasal
observations, body weights, blood analysis, gross necropsy and
histopathology results) from the test group were compared to the
control group. The test group was not significantly different from
animals treated with saline in terms of mild irritation.
EXAMPLE 12
[0174] This example illustrates the lack of toxicity from the use
of an exemplary ORP water solution.
[0175] The characterization of local and systemic toxicity from
topically applied Microcyn 60 to a deep wound was evaluated in
rats. No abnormalities, significant differences in the parameters
of the blood chemistry or hematic cytology were observed, nor
anomalies in the autopsies. The skin irritation gradings and the
histopathology of the wounds and the tissues around the place of
application did not reveal any difference between the wounds
treated with Microcyn 60 and those of the control group treated
with saline solution.
[0176] The systemic toxicity of Microcyn 60 was also evaluated by
means of an intraperitoneal injection in mice. For this, five mice
were injected with a single dose (50 mL/kg) of Microcyn 60 by the
intraperitoneal route. In the same way, five control mice were
injected with a single dose (50 mL/kg) of saline solution (sodium
chloride at 0.9%). In this investigation, neither mortality nor any
evidence of systemic toxicity was observed in any of the animals
that received the single intraperitoneal dose of Microcyn 60,
indicating that the LD.sub.50 is above 50 mL/kg.
[0177] Microcyn 60 was administered by the oral route to rats to
allow its absorption and to characterize any inherent toxic effect
of the product. In this study, a single dose (4.98 mL/kg) was
administered by esophageal tube to three albino rats of the
Sprague-Dawley strain. There was no mortality, nor were there
clinical signs or abnormalities in the autopsies of any of the
animals exposed to the single oral dose of Microcyn 60.
[0178] The potential of topically applied Microcyn 60 for ocular
irritation was also evaluated in rabbits. Ocular irritation was not
observed nor any other clinical sign in any animal exposed to
Microcyn 60 by topical administration through the ocular route.
[0179] Microcyn 60 was applied by the inhalatory route to rats to
determine potential acute toxicity by inhalation. All the animals
showed a very slight or slight reduction in activity and
piloerection after the exposure, but they were all asymptomatic on
the following day. Mortality or abnormalities were not observed at
autopsy of the animals exposed to Microcyn 60 by inhalation.
[0180] Evaluation of the potential for sensitization of the skin
with Microcyn 60 was carried out in guinea pigs using a modified
occlusion patch method (Buehler). Irritation was not observed in
the animals of the control group after a simple treatment
challenge, nor in the animals evaluated (treated by induction)
after challenge with the treatment. These studies demonstrate that
Microcyn 60 does not provoke a sensitizing reaction.
[0181] Thus, when it has been applied to the intact skin, deep open
dermal wounds, in the conjunctival sac, by oral and inhalation
routes or by means of intraperitoneal injection, Microcyn 60 has
not shown adverse effects related to the product. There is also
experience in having treated more than thousands of patients with
wounds of very diverse nature in the skin and mucosae, with
excellent antiseptic and cosmetic results. Accordingly, topically
applied Microcyn 60 should be effective and well-tolerated in this
clinical trial.
[0182] Microcyn 60 is packaged in transparent 240 mL PET sealed
bottles. This product is stored at ambient temperature and remains
stable for up to 2 years in such bottles. From its profile of high
biological safety, Microcyn 60 can be safely disposed of, e.g.,
emptied into the sink without risk of contamination or
corrosion.
EXAMPLE 13
[0183] This example illustrates a clinical study, which can be used
to determine the effectiveness of an exemplary ORP water solution
for treating pharyngitis.
[0184] Multiple microbial trials have been run with Microcyn 60,
both in the United States and in Mexico. Eradication of more than
90% of the bacteria occurs in the first few seconds of exposure.
The antibacterial and antimycotic activity that Microcyn 60
exhibits in accordance with this standard is summarized in Table 5.
TABLE-US-00005 TABLE 5 Kill Times. Time of action (reduction
Bacterium Catalog below 99.999%) Ps. aeruginosa ATCC 25619 1 min
St. aureus ATCC 6538 1 min E. coli ATCC 11229 1 min S. typhi CDC 99
1 min C. albicans ATCC 1 min B. subtilis 9372 Low spore (10.sup.4)
10 min High spore (10.sup.6 ) 15 min
[0185] The sporicidal activity trial was carried out in accordance
with the PAHO [Pan-American Health Organization]/WHO protocol.
[0186] The virucidal activity of Microcyn 60 has recently been
confirmed in studies carried out in the United States against HIV
and its activity against Listeria monocytogenes, MRSA and
Mycobacterium tuberculosis has also been demonstrated. Thus, it has
been demonstrated that Microcyn 60, when it is administered as
recommended, can eradicate bacteria, fungi, viruses and spores from
one to fifteen minutes of exposure.
[0187] Additionally, the following is a clinical study that can be
used to assess the efficacy of Microcyn 60 for the treatment of
pharyngitis/tonsilitis. In this study, 40 patients with acute
pharyngitis/tonsillitis caused by group A .beta.-hemolytic
Streptococcus and who have not received treatment are recruited.
The inclusion criteria are as follows: age 12 to 40 years and two
or more of the following symptoms: oropharyngeal burning; pain on
swallowing; pharyngeal erythema or of the tonsils (with or without
exudate); cervical lymphadenopathy; and positive immunoassay for
group A Streptococcus antigen (StrepA Test-Abbott Labs). The
exclusion criteria are as follows: fever >38.degree. C.;
bronchospasm (excluded by the clinic); severe cough;
sinusitis-rhinitis (excluded by the clinic); esophageal reflux
(excluded by the clinic); use of antibiotics in the two weeks prior
to the study; patients who have taken part in another clinical
study in the last 8 weeks; rheumatic fever; poststreptococcal
glomerulonephritis; severe chronic cardiopathy; severe renal,
hepatic or pulmonary insufficiencies; and pregnancy or
lactation.
[0188] At the beginning of the study, patients may use such
concomitant medicines as antipyretics and analgesics, including
paracetamol and acetylsalicylics but not anti-inflammatories such
as ibuprofen, Mesulid, COX-2 inhibitors, or steroids. Written
informed consent must be obtained before the patient submits to any
specific procedure of the study.
[0189] The patients are evaluated in three visits. In the first
visit, the patient clinically presents acute
pharyngitis/tonsillitis, and the clinical history is taken, and a
medical examination, rapid immunoassay for Streptococcus, and
taking of a pharyngeal exudate is carried out. After being declared
eligible and after having signed the letter of informed consent,
the patient is prescribed two oropharyngeal cleansings of 30 sec
and 5 mL Microcyn 60 each. These rinsings are done every 3 h for a
total of four times a day for 3 days.
[0190] The second is made 72 h after having been treated with
Microcyn 60. In the second visit, the clinical evolution and side
effects of Microcyn 60 are evaluated. A new pharyngeal exudate is
taken, and it will be decided, in accordance with the clinical
evolution, if the continuing treatment will be with antibiotics or
a palliative. A third visit is done after 10 days to discharge the
patient.
[0191] To be eligible and clinically evaluated in this study, each
patient must present A P-hemolytic Streptococcus
pharyngitis/tonsillitis confirmed by culture. All the patients must
comply with 18 rinsings of 30 sec and 5 mL of Microcyn 60 each, or
a maximum of 24 rinsings in the space of 72 h.
[0192] The primary parameter of efficacy is a reduction by 3 orders
of magnitude in the bacterial load of the initial culture compared
to the culture taken after the administration of Microcyn 60. This
bacteriological evaluation is realized 72 h after treatment with
Microcyn 60. Secondary parameters of efficacy are the improvement
reported clinically, with particular emphasis on the reduction of
pharyngeal pain and dysphagia. Clinical symptoms are reported in
visits 1, 2 and 3.
[0193] Tolerance is evaluated by reports of adverse events. An
adverse event is defined as any symptomatic declaration of the
patient who submits to the treatment with Microcyn 60, related or
not to the antiseptic, that appears in the course of the
treatment.
[0194] The results of bacteriological efficacy (the principal
criterion of efficacy) are issued by a bacteriologist independently
of the clinical symptoms. The tests for the group A Streptococcus
antigen and the initial pharyngeal exudate culture are done in the
first visit (Visit 1), in accordance with the Schedule of
Evaluations and before the administration of Microcyn 60. The
second taking and culture of pharyngeal exudate is carried out 72 h
after the administration of Microcyn 60 (Visit 2). An antibiogram
is done on all the cultures to determnine the bacterial resistance
to penicillin, erythromycin, clarithromycin and lincomycin by means
of the standard diffusion disc test. Bacteriological efficacy is
defined as the reduction by three orders of magnitude of the
bacterial count between the initial culture and the culture taken
72 h after administering Microcyn 60.
[0195] Bacteriological failure is indicated by a reduction of less
than three orders of magnitude of the bacterial count in the
culture at 72 h posttreatment. Indeterminate responses are
documented in those cases in which the transport of the sample has
been delayed for more than 48 h, in those cases in which the swab
has not been immersed in the transport medium, or in those cases in
which the sample has been lost. These cases are outside the
analysis of the study and are replaced by new cases until those of
forty eligible patients have been completed.
[0196] The follow-up and reporting phase begins when the patient
finishes the administration of Microcyn 60, and from the second
visit. In this evaluation, according to the clinical evolution and
the presence of possible adverse effects, the patients are
categorized as follows:
[0197] Therapeutic failures if their initial signs and symptoms
have not been eliminated or if there is worsening of their general
condition with systemic symptoms. In these cases an oral antibiotic
is prescribed, such as procaine penicillin, clarithromycin or
azithromycin at the dose and for the time that the treating doctor
indicates, and they are evaluated in one week.
[0198] Clinically cured if the symptoms and signs that were present
in Visit 1 have been eliminated. In these cases in which the acute
process is resolved, the patient is discharged and reported as
clinically cured. In any case, the patient is asked to return for a
third check-up visit in one week.
[0199] Indeterminate evolution. The evolution of any patient who
could not have been evaluated clinically for any good reason; for
example, a coinfection, or if the evaluation was done very late,
later than 72 h. In these cases, the patients is still able to be
included in the analysis of the study provided it is possible to
document the result of the pharyngeal exudate and culture at 72
h.
[0200] The statistical analysis used in this clinical study takes
into account all the patients who have received at least 18
rinsings of Microcyn 60 of 30 sec each in a period of 72 h. This
same criterion is considered to include any patient in the analysis
of tolerance. The principal criterion for analysis of efficacy is
the reduction of the bacterial count of .beta.-hemolytic
Streptococcus by three orders of magnitude in the culture carried
out at 72 h posttreatment with Microcyn 60. The statistical
analysis is realized by means of a Wilcoxon paired samples test.
Statistical analysis of the clinical variables is realized using
the ANOVA test for quantitative variables. The minimal evaluable
number of patients is 30 patients.
[0201] An adverse event is any contrary medical occurrence in a
patient or subject of clinical investigation to whom a
pharmaceutical product is administered and that does not
necessarily have a causal relationship with that medicine. An
adverse event can, therefore, be any unfavorable and unintended
sign (including an abnormal laboratory finding), symptom or illness
temporarily associated with the use of a medical product, whether
it is considered to be related to this use or not. Preexisting
conditions that deteriorate during a study are reported as adverse
events.
[0202] The treatment is suspended at any time during the 72 h of
duration in case of adverse events that are moderate to severe in
intensity. Subsequent treatment is determined by the treating
doctor. In accordance with this example, the effectiveness of an
ORP water solution of the present invention for treating sinusitis
is thus demonstrated.
EXAMPLE 14
[0203] This example demonstrates the viricidal activity of an
exemplary ORP water solution against Adenovirus-serotype 5. For
this example Adenoviral (Ad) vectors based on human adenovirus type
5 which are E1a-, partially E1-b, and partially E3-deleted were
used. A shuttle plasmid containing the Green Fluorescent Protein
(GFP) reporter gene under the transcriptional control of pCMV was
prepared (pAd-Track ). Homologous recombination of this pShuttle
plasmid with AdEasy 1 plasmid was carried out in electrocompetent
bacteria. Clones that had inserts were tested by restriction
endonuclease digestions. Once confirmed, supercoiled plasmid DMA
was transformed into DH10B cells for large scale amplification.
Subsequently, 293 cells (ATCC 1573) were cultured in serum-free
medium (OptiMEM-GIBCO) and transfected with recombinant plasmid
digested with Pad. Infected cells were monitored for cytopathic
effect, collected and lysed with three cycles of freezing and
thawing. The resultant viruses (AdGFP) were purified with AdenoPure
columns (BD Clontech) according to the manufacturer's instructions.
Viruses were quantitated by OD 260/280. Final yield was
1.52.times.10.sup.11 pfu/mL.
[0204] The efficacy of the ORP water solution for inactivating
adenovirus encoding the green fluorescence protein gene (AdGFP),
was evaluated using a test based on the detection of fluorescence
emission from HeLa cells infected with either, control AdGFP
viruses or ORP water solution-treated AdGFP, using
fluorescence-activated flow cytometry. Infection of HeLa cells was
always carried out with 7.5.times.10.sup.7 pfu/mL (i.e. 150
m.o.i.). In all test conditions, cells appeared normal under light
microscopy. The background fluorescence measured in control HeLa
cells was 0.06%. After infection with control AdGFP, 88.51% of HeLa
cells expressed GFP. Following exposure to the ORP water solution,
adenovirus infectivity decreased inversely proportionally to the
exposure period. Accordingly, ORP water solution-treated virus for
1, 5, and 10 min could only express GFP in 2.8%, 0.13%, and 0.09%
of HeLa cell cultures, respectively. Considering the
autofluorescence and the initial viral load for all tested
conditions (i.e. 7.5.times.10.sup.7 pfu), the infectious titer was
6.6.times.10.sup.7 pfu in the control AdGFP-HeLa group. In the
groups where the virus had been treated with the ORP water
solution, the infectious titers were 2.0.times.10.sup.6,
5.2.times.10.sup.4 and 2.2.times.10.sup.4 at one, five and ten
minutes of virus exposure to the ORP water solution, respectively.
Therefore, the log-10 reduction factor was 1.5, 3.1, and 3.5 at
one, five and ten minutes of viral exposure to the ORP water
solution. Taken together, these results demonstrate that the virus
exposure to the ORP water solution for 5 minutes achieves a log-10
reduction in the viral load of >3.
EXAMPLE 15
[0205] This example demonstrates the viricidal effectiveness of an
exemplary ORP water solution against HIV using the United States
Environmental Protection Agency protocol for disinfection of
inanimate environmental surfaces.
[0206] The SF33 strain of HIV-1 used for this study. Peripheral
blood mononuclear cells from healthy donors were activated with PHA
(3 .mu.g/mL, Sigma) and human IL-2 (20 U/mL, Roche) in HUT media
for three days. Cells were washed and infected with SF33 strain.
Supernatant was collected on days 4 and 6, and tested for the p24
HIV-1 antigen by ELISA (Beckman Coulter). Superantant was
centrifuged to remove cell and debris at 3000 RPM for 20 min at
room temperature. Supematant was removed, aliquoted, and the virus
was stored at -80.degree. C. until the day of use.
[0207] Frozen aliquots were thawed at 37.degree. C. for two minutes
immediately prior to its use. Serial logarithmic dilutions (-1 to
-5) in HUT medium were used. Films of virus were prepared by
spreading 0.2 ml of virus inoculum uniformly over the bottoms of 55
cm.sup.2 sterile polystyrene Petri dishes. The virus films were
air-dried at room temperature (21.degree. C.) in a biological
safety cabinet until they looked visibly dry (20 minutes). (To
assure that the virus strain (SF33) was capable of replicating and
causing cytopathic effects, the procedure was repeated with a viral
suspension that had remained in HUT medium without being
dried.)
[0208] The control film was exposed to 2 ml HUT media for five
minutes. The virus was then scraped and diluted. Separate dried
films were exposed to 2 ml each of the ORP water solution for five
minutes at room temperature. Following the exposure time, the
plates were scraped and their contents were resuspended. The
virus-ORP water solution mixture was immediately diluted (10:1) in
HUT medium. Serial log dilutions of this resulting suspension were
assayed for infectivity. (To control for a possible direct
cytotoxic effect of ORP water solution on MT-2 cells, a 2 ml
aliquot of ORP water solution was diluted serially (10:1 to 10:5)
in medium and inoculated into MT-2 cell cultures.)
[0209] The MT-2 cell line was used as the indicator cell line in
the infectivity assays. This line shows a cytopathic effect
consisting of sincitia formation when infected with HIV-1. Four
microwells were inoculated with 0.2 ml of each dilution of the
reconstituted virus suspension from test (reconstituted in ORP
water) and control (reconsituted with control medium) groups.
Uninfected cell controls were inoculated with test medium only.
Cultures were incubated at 37.degree. C. and 5% CO.sub.2.
[0210] The cultures were scored periodically every two days for the
presence or absence of cytopathic effect as well as presence of
p24-HIV-1 antigen by ELISA. Experimental infection with control
HIV-1 exerted a cytopathic effect and Ag p24 protein release into
the supernatant in infected MT-2 cultures. In contrast, treatment
of HV-1 with the ORP water solution for five minutes, achieved a
log reduction factor >3 in the viral load as measured in MT-2
cultures by both assays. These results thus demonstrate the level
of efficacy that is in conformity with the EPA requirements for
HIV-1 virucidal activity on inanimate surfaces.
EXAMPLE 16
[0211] This example demonstrates the effect of an exemplary ORP
water solution versus hydrogen peroxide (HP) on the viability of
human diploid fibroblasts (HDFs). To study this potential toxicity,
HDFs were exposed in vitro to ORP water solution and hydrogen
peroxide (HP). HP is known to be toxic to eukaryotic cells,
increasing apoptosis and necrosis and reducing cellular viability.
In this example, cell viability, apoptosis and necrosis were
measured in HDFs exposed to pure ORP water solution and 880 mM HP
(a concentration employed for antiseptic uses of HP) for 5 and 30
minutes.
[0212] HDF cultures were obtained from three different foreskins,
which were pooled and cryopreserved together for the purpose of
this study. Only diploid cells were used for all experiments. On
cell cycle analysis, DNA diploidy was defined as the presence of a
single G0-G1 peak with a CV</=7% and a corresponding G2/M peak
collected from at least 20,000 total events. FIG. 4A-4C disclose
the results where exposure times of 5 and 30 minutes are depicted
in white and black bars, respectively. Simultaneous analyses of
these parameters were performed in the same cell populations by
flow cytometry using: A) 7-aminoactinomycin D (7AAD); B) Annexin
V-FITC; and C) Propidium iodide. FIG. 4A-4C disclose percentage
values expressed as mean.+-.SD (n=3).
[0213] Cell viability was 75% and 55% after a 5 minute exposure to
antiseptic concentrations of full strength-ORP water solution and
880 mM HP , respectively (FIG. 4A). The effect of full strength ORP
water solution on cell viability was comparable to a very diluted
HP solution considered sublethal but not disinfectant (i.e. 500
.mu.M). If the exposure was prolonged to 30 min, cell viability
further decreased to 70% and 5%, respectively. Apparently, the ORP
water solution induced cell death through necrosis because 15% of
the cells incorporated propidium iodide in the flow cytometry
analysis at both times (FIG. 4C). Apoptosis does not seem to be the
mechanism by which the ORP water solution induces cell death
because only 3% of ORP water solution-treated cells exposed
Annexin-V in the cellular surface (a marker of apoptosis) (FIG.
4B). This percentage was actually similar to the one measured in
the control group. On the contrary, HP induced necrosis in 20% and
75% of treated cells and apoptosis in 15% and 20% after 5 and 30
min of exposure, respectively. Altogether these results show that
the (undiluted) ORP water solution is far less toxic for HDFs than
an antiseptic concentration of HP.
EXAMPLE 17
[0214] This example demonstrates the effect of an exemplary ORP
water solution relative to hydrogen peroxide (HP) on oxidative DNA
damage and formation of the DNA adduct 8-hydroxy-2'-deoxiguanosine
(8-OHdG) in HDFs. It is known that the production of 8-OHdG adducts
in a cell is a marker of oxidative damage at specific residues of
DNA. In addition, high cellular levels of this adduct correlate
with mutagenesis, carcinogenesis and cellular aging.
[0215] FIG. 5 shows the levels of 8-OHdG adducts present in DNA
samples from HDFs after control treatments, ORP water solution
treatments and HP-treatments for 30 minutes. DNA was extracted
right after the exposure (T0, white bars) or three hours after the
challenge period (T3, black bars). DNA was digested and the 8-OHdG
adducts were measured by ELISA kit as per the manufacturer's
instructions. Values are shown (ng/mL) as mean.+-.SD (n=3). The
exposure to ORP water solution for 30 minutes did not increase the
formation of adducts in the treated cells in comparison to control
cells after incubation for 30 minutes. In contrast, the treatment
with 500 .mu.M HP for 30 minutes increased the number of 8-OHdG
adducts by about 25 fold relative to the control-treated or ORP
water solution-treated cells.
[0216] The ORP water solution-treated cells were able to decrease
the levels of 8-OHdG adducts if left in supplemented DMEM for 3
hours after exposure to the ORP water solution. Despite being
allowed the same 3 hour recovery period, HP-treated cells still
presented about 5 times more adducts than control-treated or ORP
water solution treated cells. Altogether, these results demonstrate
that acute exposure to the ORP water solution does not induce
significant DNA oxidative damage. These results also indicate that
the ORP water solution will not likely induce mutagenesis or
carcinogenesis in vitro or in vivo.
EXAMPLE 18
[0217] This example demonstrates the effects on HDFs of chronic
exposure to low concentrations of an exemplary ORP water solution
versus HP. It is known that chronic oxidative stress induces
premature aging of cells. In order to mimick a prolonged oxidative
stress, primary HDF cultures were chronically exposed to low
concentrations of the ORP water solution (10%) or HP (5 .mu.M)
during 20 population doublings. The expression and activity of the
SA-.beta.-galactosidase enzyme has previously been associated with
the senescence process in vivo and in vitro. In this example the
expression of the SA-.beta.-galactosidase enzyme was analyzed after
one month of continuous exposure of HDF to the ORP water solution
or HP. The results are depicted in FIG. 6. The expression of the
enzyme SA-.beta.-galactosidase was analyzed by counting the number
of blue cells in 20 microscopic fields. FIG. 6 shows that only HP
treatment accelerated the aging of cells as indicated by the number
of cells over-expressing SA-.beta.-galactosidase (n=3). Chronic
treatment with a low dose of HP increased the SA-.beta.-Gal
expression in 86% of cells while the treatment with the ORP water
solution did not induce the overexpression of this protein. It can
be concluded from this example that ORP water solution is not an
inducer of premature cellular aging.
EXAMPLE 19
[0218] This example demonstrates the effect of an exemplary ORP
water solution on the reduction of peritoneal bacterial load and on
the reduction in the length of hospital stay in patients with
peritonitis. All patients admitted to the Hospital Ruben Lenero in
Mexico City from June 2004 to January 2005, and with a diagnosis of
acute generalized, secondary peritonitis, were included in the ORP
water solution-treated group. Secondary peritonitis was defined as
the result of the loss of integrity of the gastrointestinal or
genito-urinary tract leading to contamination of the peritoneal
space. Retrospective analysis of paired-cases presenting similar
peritoneal infections between 2003 and 2004 at the same Institution
was undertaken for the control group. Twenty consecutive patients
were prospectively included in the ORP water solution-treated group
(i.e. study group).
[0219] Upon admission, all patients underwent open surgery and
intra-operative peritoneal lavage ("IOPL") of all quadrants of the
abdomen. Intraoperative peritoneal-culture samples were taken in
both groups. IOPL was performed with 10 L of saline solution in
both groups and followed by 5 L of the ORP water solution in the
study group only. The excess ORP water solution was removed and no
further rinsing was conducted. The abdominal cavity was covered
with a plastic mesh in both groups. However, in the study group, a
dressing soaked in ORP water solution was left on top of the mesh.
The dressing was changed t.i.d. Emperic antimicrobial therapy was
started in all patients with two antibiotics including clindamycin
and cefotaxime or amikacin. Post-operative management in the study
group included daily irrigation of the mesh with 100 mL of the ORP
water solution t.i.d., without further rinsing or lavage. Severe
cases of peritonitis required re-laparotomy and IOPL every 72
hours. Cultures of the peritoneal fluid for aerobic bacteria and
fungi were taken every 72 hours in both groups for up to one week.
The duration of length of stay in the hospital was recorded.
[0220] Twenty control cases were selected from the medical records
of the Institution and paired to the study group by age, sex and
etiology of peritonitis. The control and study populations were
comparable in age, sex and prognostic factors at entry. The
anatomic origin and etiology of peritonitis was also similar for
both groups (Table 6). TABLE-US-00006 TABLE 6 Diagnoses. DIAGNOSIS
CONTROL STUDY TOTAL % Appendicitis 3 6 9 23.0 Post-trauma 1 3 4
10.0 Pancreatitis 6 3 9 23.0 Cholecystitis 1 2 3 7.5 Colon cancer 0
1 1 2.5 Small bowel 4 1 5 12.5 fistula Diverticulitis 1 1 2 5.0
Gastric 4 0 4 10.0 perforation Other Organ 0 2 2 5.0 perforation
Other 0 1 1 2.5 TOTAL 20 20 40 100.0
[0221] Post-operative peritonitis was present in 19 and 17 patients
of the control and study groups, respectively. All cases underwent
surgical treatment followed by IOPL. The types of surgeries
performed in control /study groups, were: appendicectomy (3/6),
gastric resection (4/0), cholecystectomy (1/2), pancreatic
necrosectomy (6/3), small bowel suture/ resection with anastomosis
(4/3), Hartman's operation (1/1), colonic resection (0/1) and
miscellaneous (1/4). The use of antibiotics was very similar in
both groups. For control and study groups, three antibiotics were
administered in 16 and 15 patients and more than 3 antibiotics in 4
and 5 cases, respectively. Patients were kept at the ICU and were
mechanically ventilated post-operatively. Peri-operative
intra-abdominal samples were taken in all 40 patients (Table 7).
TABLE-US-00007 TABLE 7 Microorganisms isolated from intraperitoneal
samples and length of hospital stay in patients with peritonitis.
CONTROL GROUP STUDY GROUP Isolated Isolated strains (n) strains (n)
Organism Peri-op Post-op Hospital Days Peri-op Post-op Hospital
Days C. albicans 10 7 19.4 7 0 6.3 E. coli 3 2 17.6 6 1 10.2 S.
aureus 10 9 22.3 8 1 14.1 coagulase 0 0 0 2 0 17.8 neg. Staph. A.
baumanii 0 0 0 1 0 22.4 E. faecalis 3 3 23.7 1 0 28.6 A.
xilosoxidans 0 0 0 1 0 28.6 P. aeruginosa 2 2 24.0 3 0 33.9 E.
coacae 1 1 13.0 1 0 37.0 TOTAL 29 24 31.9 30 2 22.4
[0222] Samples were obtained in the peri-operative period and in
the following week after intra-operative lavage with saline
solution only (control group) or saline solution and ORP water
solution (study group). The average hospital stay was then analyzed
for each microorganism isolated at entry and for the whole
group.
[0223] Peri-operative samples were taken in all 40 patients (Table
7). The average numbers of microorganisms grown from these samples
were 29 in the control and 30 in the study group. The
microorganisms isolated are shown in Table 8. Escherichia coli,
Enterococcus, Staphylococcus aureus, Pseudomonas aeruginosa and
fungi were isolated from these groups in 3/6, 4/2, 10/8, 2/3 and
10/7 occasions, respectively. Positive cultures for A. xilosoxidans
(1), coagulase negative Staphylococci (2) and A. baumanii (1) were
only found in the study group.
[0224] A second intra-abdominal culture was taken during the first
week after surgery (Table 7). At this time, the average number of
organisms isolated in the control group (24) was almost the same as
in the peri-operative sample (29). Importantly, there was a strong
reduction in the number of positive samples in the study group.
From 30 positive cultures in the peri-operative samples, only one
remained positive for S. aureus and another one for E. coli. In the
analysis of hospital days, the control group had a longer stay
(31.9 days) in comparison to the study group (22.4 days). Thus, the
ORP water solution effectively reduced the peritoneal bacterial
load and length of hospital stay in patients with peritonitis.
[0225] The mortality rates were also analyzed. There were six
deaths in the control group and 3 in the study one. All deaths
occurred in the first 30 days after the first surgery and the
calculated relative risk was higher for the control group (i.e. 3.3
versus 0). However, the sample size was too small to achieve
statistical significance. No local side effects were recorded with
the use of ORP water in the IOPL. Surviving patients in the study
group were followed for 6 to 12 months. None of the 20 patients in
the ORP water-treated group presented intestinal occlusion or data
suggesting sclerosing peritonitis in the follow-up period.
EXAMPLE 20
[0226] This example demonstrates the effectiveness of an exemplary
ORP water solution (Mycrocyn) in inhibiting mast cell
degranulation. Mast cells have been recognized as principal players
in type I hypersensitivity disorders. Multiple clinical symptoms
observed in atopic dermatitis, allergic rhinitis, and atopic asthma
are produced by IgE-antigen stimulation of mast cells located in
distinct affected tissues. The currently accepted view of the
pathogenesis of atopic asthma is that allergens initiate the
process by triggering IgE-bearing pulmonary mast cells (MCs) to
release mediators such as histamine, leukotrienes, prostaglandins,
kininis, platelet activating factor (PAF), etc. in the so-called
early phase of the reaction. In turn, these mediators induce
bronchoconstriction and enhance vascular permeability and mucus
production. According to this model, following mast cell
activation, those cells secrete various pro-inflammatory cytokines
in a late phase, including tumor necrosis factor alpha
(TNF-.alpha.), IL-4, IL-5 and IL-6, which participate in the local
recruitment and activation of other inflammatory cells such as
eosinophils, basophils, T lymphocytes, platelets and mononuclear
phagocytes. These recruited cells, in turn, contribute to the
development of an inflammatory response that may then become
autonomous and aggravate the asthmatic symptoms. This late phase
response constitutes a long term inflammation process which can
induce plastic changes in surrounding tissues (see Kumar et al.,
pp. 193-268).
[0227] Antigenic stimulation of mast cells occurs via the
activation of the high affinity receptor for IgE (the Fc.epsilon.RI
receptor), which is a multimeric protein that binds IgE and
subsequently can be aggregated by the interaction of the
receptor-bound IgE with a specific antigen. Its structure comprises
four polypeptides, an IgE binding a chain, a .beta. chain that
serves to amplify its signaling capacity, and two disulfide-linked
.gamma. chains, which are the principal signal transducers via the
encoded immunoreceptor tyrosine-based (ITAM) activation motif.
Signaling pathways activated by the cross-linking of this receptor
have been characterized using bone marrow-derived mast cells
(BMMC), the rat leukemia cell line RBL 2H3, mouse and rat
peritoneal mast cells, and other mast cell lines, such as MC-9, In
all of them, the presence of antigen bound to IgE causes mast cell
degranulation, calcium mobilization, cytoskeletal re-arrangements
and activation of different transcription factors (NFAT,
NF.kappa.B, AP-1, PU.1, SP1, Ets, etc.) which activate cytokine
gene transcription that culminate with cytokine production.
[0228] Mature murine bone-derived mast cells (BMMC) were loaded
with a monoclonal anti-Dinitrophenol IgE (300 ng/million cell)
during 4 hours at 37.degree. C. Culture media was removed and cells
were resuspended in physiological buffer (Tyrode's Buffer/BSA).
Cells were then treated 15 minutes at 37.degree. C. with distinct
concentrations of the ORP water solution (in its Microcyn
embodiment). Buffer was removed and cells resuspended in fresh
Tyrode's/BSA and stimulated with different concentrations of
antigen (Human Albumin coupled to Dinitrophenol) during a 30 minute
incubation at 37.degree. C. Degranulation was measured by
.beta.-hexosaminidase activity determination in supernatants and
pellets of the stimulated cells, using a colorimetric reaction
based on the capacity of this enzyme to hydrolize distinct
carbohydrates. (.beta.-hexosaminidase has been shown to be located
in the same granules that contain histamine in mast cells.) The
results (FIG. 7) demonstrate that degranulation is significantly
reduced with increasing concentrations of the ORP water
solution.
[0229] Surprisingly, the inhibitory effect of the ORP water
solution (Microcyn) on mast cell degranulation at least is similar
to that observed with the clinically effective "mast cell
stabilizer" and established anti-allergic compound sodium
cromoglycate (Intel.TM.). Degranulation was again measured by
.beta.-hexosaminidase enzymatic activity in the pellet and
supernatant of stimulated cells, using a colorimetric reaction
based on the capacity of this enzyme to hydrolize distinct
carbohydrates. Cells loaded with anti-DNP monoclonal IgE were
stimulated with or without a 15 minute pre-incubation with sodium
cromoglycate (Intel.TM.). Cromoglycate was no more effective than
the ORP water solution in reducing degranulations (Compare FIG.7
with FIG. 8; both achieving at least about 50% reduction in
degranulation.)
EXAMPLE 21
[0230] This example demonstrates the inhibitory activity of an
exemplary ORP water solution on mast cell activation by a calcium
ionophore.
[0231] Mast cells can be stimulated via the activation of calcium
fluxes induced by a calcium ionophore. Signaling pathways activated
by calcium ionophores have been characterized using bone
marrow-derived mast cells (BMMC), the rat leukemia cell line RBL
2H3, mouse and rat peritoneal mast cells, and other mast cell
lines, such as MC-9. In all of these systems the calcium
mobilization causes mast cell degranulation (e.g. histamine
release), cytoskeletal re-arrangements, and activation of different
transcription factors (e.g., NFAT, NF.kappa.B, AP-1, PU.1, SP1,
Ets.) which activate cytokine gene transcription that culminate
with cytokine production and secretion.
[0232] Mature murine BMMC were loaded with a monoclonal
anti-Dinitrophenol IgE (300 ng/million cell) during 4 hours at
37.degree. C. Culture media was removed and cells were resuspended
in physiological buffer (Tyrode's Buffer/BSA). Cells were then
treated for 15 minutes at 37.degree. C. with distinct
concentrations of the ORP water solution (Microcyn). Buffer was
removed and cells were resuspended in fresh Tyrode's/BSA and
stimulated with calcium ionophore (100 mM A23187) during a 30
minute incubation at 37.degree. C.. Degranulation was measured by
.beta.-hexosaminidase activity determination in supernatants and
pellets of the stimulated cells, using a colorimetric reaction
based on the capacity of this enzyme to hydrolyze distinct
carbohydrates. (.beta.-hexosaminidase has been shown to be located
in the same granules that contain histamine in mast cells.) The
results (FIG. 8) demonstrate that degranulation is significantly
reduced with increasing concentrations of the ORP water
solution.
[0233] These results suggest that ORP water solution is a
non-specific inhibitor of histamine release. Thus, ORP water
solution--even at different concentrations--will inhibit the
degranulation of mast cells independently of the stimulus (e.g.
antigen or ionophore). While not desiring to be bound by any
theory, ORP water solution probably modifies the secretory pathway
system at the level of the plasma membrane and/or cytoskeleton.
Because the mechanism of action of ORP water solution is believed
to be non-specific, it is believed that ORP water solution can have
broad potential clinical applications.
EXAMPLE 22
[0234] This example demonstrates the effect of an exemplary ORP
water solution on the activation of mast cell cytokine gene
transcription.
[0235] FIGS 10A and 10B are RNAase protection assays from mast
cells treated with ORP water solution at different concentrations
for 15 minutes and further stimulated by antigen as described in
Example 20. After stimulation, mRNA was extracted using affinity
chromatography columns (RNAeasy kit, Qiagene) and the RNAse
Protection Assay was performed using standard kit conditions
(Clontech, Becton & Dickinson) in order to detect mRNA
production of distinct cytokines after antigen challenge. The
cytokines included TNF-.alpha., LIF, IL13, M-CSF, IL6, MIF and
L32.
[0236] FIGS. 10A and 10B show that the ORP solution water
(Microcyn) did not modify cytokine mRNA levels after antigen
challenge in mast cells irrespective of the concentrations of ORP
water solution or antigen used for the experiment.
[0237] In this study, the level of transcripts (i.e., the RNA
content of stimulated mast cells) of proinflammatory genes was not
changed in ORP water solution-treated mast cells after being
stimulated with various concentrations of antigen. Thus, the ORP
water solution inhibited the secretory pathway of these cytokines
without affecting their transcription.
EXAMPLE 23
[0238] This example demonstrates the inhibitory activity of an
exemplary ORP water solution on mast cell secretion of
TNF-.alpha..
[0239] Mast cells were treated with different concentrations of ORP
water solution for 15 minutes and further stimulated by antigen as
described in Example 20, Thereafter, the tissue culture medium was
replaced and samples of the fresh medium were collected at various
periods of time (2-8 hours) for measuring TNF-.alpha. levels.
Samples were frozen and further analyzed with a commercial ELISA
kit (Biosource) according to the manufacturer's instructions.
[0240] FIG. 11 shows that the level of secreted TNF-.alpha. to the
medium from ORP water solution-treated cells after antigen
stimulation is significantly decreased in comparison to the
untreated cells.
[0241] Since the release of TNF-.alpha. and that of various other
pro-inflammatory molecules depends on a separate secretory pathway
than that of histamine, it is possible that the ORP solution can
stop the secretion of those other cytokines leading the late
inflammatory phase.
[0242] Thus, the ORP water solution inhibited TNF-.alpha. secretion
of antigen-stimulated mast cells. These results are in agreement
with clinical observations that the use of ORP water solutions can
decrease the inflammatory reaction in various wounds after surgical
procedures.
EXAMPLE 24
[0243] This example demonstrates the inhibitory activity of an
exemplary ORP water solution on mast cell secretion of MIP
1-.alpha..
[0244] Mast cells were treated with different concentrations of an
exemplary ORP water solution (Microcyn) for 15 minutes and further
stimulated by antigen as described in Example 20. Thereafter, the
tissue culture medium was replaced and samples of the fresh medium
were collected at various periods of time (2-8 hours) for measuring
MIP 1-.alpha. levels. Samples were frozen and further analyzed with
a commercial ELISA kit (Biosource) according to the manufacturer's
instructions.
[0245] FIG. 12 shows that the level of secreted MIP 1-.alpha. to
the medium from ORP water solution-treated cells after antigen
stimulation was significantly decreased in comparison to the
untreated cells.
[0246] Thus, the ORP water solution inhibited MIP 1-.alpha.
secretion of antigen-stimulated mast cells. These results are in
agreement with clinical observations that the use of ORP water
solutions can decrease the inflammatory reaction in various wounds
after surgical procedures.
[0247] Since the release of MIP 1-.alpha. and that of various other
pro-inflammatory molecules depends on a separate secretory pathway
than that of histamine, it is possible that the ORP solution can
stop the secretion of those other cytokines leading the late
inflammatory phase.
[0248] The results of analogous studies measuring IL-6 and IL-13
secretion are depicted in FIGS. 13 and 14.
[0249] Examples 20-23 and this example further demonstrate that the
ORP water solution is able to inhibit early and late phase allergic
responses initiated by IgE receptor crosslinking.
EXAMPLE 25
[0250] This example demonstrates the results of a toxicity study
using an exemplary ORP water solution.
[0251] An acute systemic toxicity study was performed in mice to
determine the potential systemic toxicity of Microcyn 60, an
exemplary ORP water solution. A single dose (50 mL/kg) of Microcyn
60 was injected intraperitoneally in five mice. Five control mice
were injected with a single dose (50 mL/kg) of saline (0.9% sodium
chloride). All animals were observed for mortality and adverse
reactions immediately following the injection, at 4 hours after
injection, and then once daily for 7 days. All animals were also
weighed prior to the injection and again on Day 7. There was no
mortality during the study. All animals appeared clinically normal
throughout the study. All animals gained weight. The estimated
Microcyn 60 acute intraperitoneal LD50 from this study is greater
than 50 mL/kg. This example demonstrates that Microcyn 60 lacks
significant toxicity and should be safe for therapeutic use
accordance with the invention.
EXAMPLE 26
[0252] This example illustrates a study conducted to determine the
potential cytogenetic toxicity of an exemplary ORP water
solution.
[0253] A micronucleus test was performed using an exemplary ORP
water solution (10% Microcyn.TM.) to evaluate the mutagenic
potential of intraperitoneal injection of an ORP water solution
into mice. The mammalian in vivo micronucleus test is used for the
identification of substances which cause damage to chromosomes or
the mitotic apparatus of murine polychromatic erythrocytes. This
damage results in the formation of "micronuclei," intracellular
structures containing lagging chromosome fragments or isolated
whole chromosomes.The ORP water solution study included 3 groups of
10 mice each (5 males/5 females): a test group, dosed with the ORP
water solution; a negative control group, dosed with a 0.9% NaCl
solution; and a positive control group, dosed with a mutagenic
cyclophosphamide solution. The test and the negative control groups
received an intraperitoneal injection (12.5 ml/kg) of the ORP water
solution or 0.9% NaCl solution, respectively, for two consecutive
days (days 1 and 2). The positive control mice received a single
intraperitoneal injection of cyclophosphamide (8 mg/mL, 12.5 ml/kg)
on day 2. All mice were observed immediately after injection for
any adverse reactions. All animals appeared clinically normal
throughout the study and no sign of toxicity was noted in any
group. On day 3, all mice were weighed and terminated.
[0254] The femurs were excised from the terminated mice, the bone
marrow was extracted, and duplicate smear preparations were
performed for each mouse. The bone marrow slides for each animal
were read at 40.times.magnification. The ratio of polychromatic
erythrocytes (PCE) to normochromatic erythrocytes (NCE), an index
of bone marrow toxicity, was determined for each mouse by counting
a total of at least 200 erythrocytes. Then a minimum of 2000
scoreable PCE per mouse were evaluated for the incidence of
micronucleated polychromatic erythrocytes. Statistical analysis of
the data were done using the Mann and Whitney test (at 5% risk
threshold) from a statistical software package (Statview 5.0.RTM.,
SAS Institute Inc., USA).
[0255] The positive control mice had statistically significant
lower PCEINCE ratios when compared to their respective negative
controls (males : 0.77 vs. 0.90 and females 0.73 vs. 1.02), showing
the toxicity of the cyclophosphamide on treated bone marrow.
However, there was no statistically significant difference between
the PCE/NCE ratios for the ORP water solution-treated mice and
negative controls. Similarly, positive control mice had a
statistically significant higher number of polychromatic
erythrocytes bearing micronuclei as compared to both the ORP water
solution-treated mice (males: 11.0 vs. 1.4/females: 12.6 vs. 0.8)
and the negative controls (males: 11.0 vs. 0.6 /females: 12.6 vs.
1.0). There was no statistically significant difference between the
number of polychromatic erythrocytes bearing micronculei in ORP
water solution-treated and negative control mice.
[0256] This example demonstrates that Microcyn.TM. 10% did not
induce toxicity or mutagenic effects after intraperitoneal
injections into mice.
EXAMPLE 27
[0257] This study demonstrates the lack of toxicity of an exemplary
ORP water solution, Dermacyn.
[0258] This study was done in accordance with ISO 10993-5:1999
standard to determine the potential of an exemplary ORP water
solution, Dermacyn, to cause cytotoxicity. A filter disc with 0.1
mL of Dermacyn was placed onto an agarose surface, directly
overlaying a monolayer of mouse fibroblast cells (L-929). The
prepared samples were observed for cytotoxic damage after 24 hours
of incubation at 37.degree. C. in the presence of 5% CO.sub.2.
Observations were compared to positive and negative control
samples. The Dermacyn containing samples did not reveal any
evidence of cell lysis or toxicity, while positive and negative
control performed as anticipated.
[0259] Based on this study Dermacyn was concluded not to generate
cytotoxic effects on murine fibroblasts.
EXAMPLE 28
[0260] This study was conducted with 16 rats to evaluate the local
tolerability of an exemplary ORP water solution, Dermacyn, and its
effects on the histopathology of wound beds in a model of
full-thickness dermal wound healing. Wounds were made on both sides
of the subject rat. During the healing process skin sections were
taken on either the left or the right sides (e.g., Dermacyn-treated
and saline-treated, respectively).
[0261] Masson's trichrome-stained sections and Collagen Type II
stained sections of the Dermacyn and saline-treated surgical wound
sites were evaluated by a board-certified veterinary pathologist.
The sections were assessed for the amount of Collagen Type 2
expression as a manifestiation of connective tissue proliferation,
fibroblast morphology and collagen formation, presence of
neoepidermis in cross section, inflammation and extent of dermal
ulceration.
[0262] The findings indicate that Dermacyn was well tolerated in
rats. There were no treatment-related histopathologic lesions in
the skin sections from either sides' wounds (Dermacyn-treated and
saline-treated, respectively). There were no relevant
histopathologic differences between the saline-treated and the
Dermacyn-treated wound sites, indicating that the
Dermacyn-treatement was well tolerated. There were no significant
differences between Collagen Type 2 expression between the
saline-treated and the Dermacyn.TM.-treated wound sites indicating
that the Dermacyn does not have an adverse effect on fibroblasts or
on collagen elaboration during wound healing.
EXAMPLE 29
[0263] This example demonstrates the use of an exemplary oxidative
reductive potential water, Microcyn, in accordance with the
invention as an effective antimicrobial solution.
[0264] An In-Vitro Time-Kill evaluation was performed using
Microcyn oxidative reductive potential water. Microcyn was
evaluated versus challenge suspensions of fifty different
microorganism strains--twenty-five American Type Culture Collection
(ATCC) strains and twenty-five Clinical Isolates of those same
species--as described in the Tentative Final Monograph, Federal
Register, 17 Jun. 1994, vol. 59:116, pg. 31444. The percent
reductions and the Log.sub.10 reductions from the initial
population of each challenge strain were determined following
exposures to Microcyn for thirty (30) seconds, one (1) minute,
three (3) minutes, five (5) minutes, seven (7) minutes, nine (9)
minutes, eleven (11) minutes, thirteen (13) minutes, fifteen (15)
minutes, and twenty (20) minutes. All agar-plating was performed in
duplicate and Microcyn was evaluated at a 99% (v/v) concentration.
All testing was performed in accordance with Good Laboratory
Practices, as specified in 21 C.F.R. Part 58.
[0265] The following table summarizes the results of the
abovementioned In-Vitro Time-Kill evaluation at the thirty second
exposure mark for all populations tested which were reduced by more
than 5.0 Log.sub.10: TABLE-US-00008 TABLE 8 30-Second In-Vitro
Kill. Initial Post-Exposure Population Population Log.sub.10
Percent No. Microorganism Species (CFU/mL) (CFU/mL) Reduction
Reduction 1 Acinetobacter baumannii 2.340 .times. 10.sup.9 <1.00
.times. 10.sup.3 6.3692 99.9999 (ATCC #19003) 2 Acinetobacter
baumannii 1.8150 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.2589
99.9999 Clinical Isolate BSLI #061901Ab3 3 Bacteroides fragilis
4.40 .times. 10.sup.10 <1.00 .times. 10.sup.3 7.6435 99.9999
(ATCC #43858) 4 Bacteroides fragilis 2.70 .times. 10.sup.10
<1.00 .times. 10.sup.3 7.4314 99.9999 Clinical Isolate BSLI
#061901Bf6 5 Candida albicans 2.70 .times. 10.sup.10 <1.00
.times. 10.sup.3 6.3345 99.9999 (ATCC #10231) 6 Candida albicans
5.650 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.7520 99.9999
Clinical Isolate BSLI #042905Ca 7 Enterobacter aerogenes 1.2250
.times. 10.sup.9 <1.00 .times. 10.sup.3 6.0881 99.9999 (ATCC
#29007) 8 Enterobacter aerogenes 1.0150 .times. 10.sup.9 <1.00
.times. 10.sup.3 6.0065 99.9999 Clinical Isolate BSLI #042905Ea 9
Enterococcus faecalis 2.610 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.4166 99.9999 (ATCC #29212) 10 Enterococcus faecalis
1.2850 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.1089 99.9999
Clinical Isolate BSLI #061901Efs2 11 Enterococcus faecium 3.250
.times. 10.sup.9 <1.00 .times. 10.sup.3 6.5119 99.9999 VRE, MDR
(ATCC #51559) 12 Enterococcus faecium 1.130 .times. 10.sup.9
<1.00 .times. 10.sup.3 6.0531 99.9999 Clinical Isolate BSLI
#061901Efm1 13 Escherichia coli 5.00 .times. 10.sup.8 <1.00
.times. 10.sup.3 5.6990 99.9998 (ATCC #11229) 14 Escherichia coli
3.950 .times. 10.sup.8 <1.00 .times. 10.sup.3 5.5966 99.9997
Clinical Isolate BSLI #042905Ec1 15 Escherichia coli 6.650 .times.
10.sup.8 <1.00 .times. 10.sup.3 5.8228 99.9998 (ATCC #25922) 16
Escherichia coli 7.40 .times. 10.sup.8 <1.00 .times. 10.sup.3
5.8692 99.9998 Clinical Isolate BSLI #042905Ec2 17 Haemophilus
influenzae 1.5050 .times. 10.sup.9 <1.00 .times. 10.sup.4 5.1775
99.9993 (ATCC #8149) 18 Haemophilus influenzae 1.90 .times.
10.sup.9 <1.00 .times. 10.sup.4 5.2788 99.9995 Clinical Isolate
BSLI #072605Hi 19 Klebsiella oxytoca 1.120 .times. 10.sup.9
<1.00 .times. 10.sup.3 6.0492 99.9999 MDR (ATCC #15764) 20
Klebsiella oxytoca 1.810 .times. 10.sup.9 <1.00 .times. 10.sup.3
6.2577 99.9999 Clinical Isolate BSLI #061901Ko1 21 Klebsiella
pneumoniae 1.390 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.1430
99.9999 subsp. ozaenae (ATCC #29019) 22 Klebsiella pneumoniae 9.950
.times. 10.sup.8 <1.00 .times. 10.sup.3 5.9978 99.9999 Clinical
Isolate BSLI #061901Kpn2 23 Micrococcus luteus 6.950 .times.
10.sup.8 <1.00 .times. 10.sup.3 5.8420 99.9999 (ATCC #7468) 24
Micrococcus luteus 1.5150 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.1804 99.9999 Clinical Isolate BSLI #061901Ml2 25 Proteus
mirabilis 1.5950 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.2028
99.9999 (ATCC #7002) 26 Proteus mirabilis 2.0950 .times. 10.sup.9
<1.00 .times. 10.sup.3 6.3212 99.9999 Clinical Isolate BSLI
#061901Pm2 27 Pseudomonas aeruginosa 6.450 .times. 10.sup.8
<1.00 .times. 10.sup.3 5.8096 99.9999 (ATCC #15442) 28
Pseudomonas aeruginosa 1.3850 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.1414 99.9999 Clinical Isolate BSLI #072605Pa 29
Pseudomonas aeruginosa 5.550 .times. 10.sup.8 <1.00 .times.
10.sup.3 5.7443 99.9999 (ATCC #27853) 30 Pseudomonas aeruginosa
1.1650 .times. 10.sup.9 <1.00 .times. 10.sup.3 6.0663 99.9999
Clinical Isolate BSLI #061901Pa2 31 Serratia marcescens 9.950
.times. 10.sup.8 <1.00 .times. 10.sup.3 5.9978 99.9999 (ATCC
#14756) 32 Serratia marcescens 3.6650 .times. 10.sup.9 <1.00
.times. 10.sup.3 6.5641 99.9999 Clinical Isolate BSLI #042905Sm 33
Staphylococcus aureus 1.5050 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.1775 99.9999 (ATCC #6538) 34 Staphylococcus aureus 1.250
.times. 10.sup.9 <1.00 .times. 10.sup.3 6.0969 99.9999 Clinical
Isolate BSLI #061901Sa1 35 Staphylococcus aureus 1.740 .times.
10.sup.9 <1.00 .times. 10.sup.3 6.2405 99.9999 (ATCC #29213) 36
Staphylococcus aureus 1.1050 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.0434 99.9999 Clinical Isolate BSLI #061901Sa2 37
Staphylococcus epidermidis 1.0550 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.0233 99.9999 (ATCC #12228) 38 Staphylococcus epidermidis
4.350 .times. 10.sup.8 <1.00 .times. 10.sup.3 5.6385 99.9998
Clinical Isolate BSLI #072605Se 39 Staphylococcus haemolyticus
8.150 .times. 10.sup.8 <1.00 .times. 10.sup.3 5.9112 99.9999
(ATCC #29970) 40 Staphylococcus haemolyticus 8.350 .times. 10.sup.8
<1.00 .times. 10.sup.3 5.9217 99.9999 Clinical Isolate BSLI
#042905Sha 41 Staphylococcus hominis 2.790 .times. 10.sup.8
<1.00 .times. 10.sup.3 5.4456 99.9996 (ATCC #27844) 42
Staphylococcus hominis 5.20 .times. 10.sup.8 <1.00 .times.
10.sup.3 5.7160 99.9998 Clinical Isolate BSLI #042905Sho 43
Staphylococcus saprophyticus 9.10 .times. 10.sup.8 <1.00 .times.
10.sup.3 5.9590 99.9999 (ATCC #35552) 44 Staphylococcus
saprophyticus 1.4150 .times. 10.sup.9 <1.00 .times. 10.sup.3
6.1508 99.9999 Clinical Isolate BSLI #042905Ss 45 Streptococcus
pneumoniae 2.1450 .times. 10.sup.9 <1.00 .times. 10.sup.4 5.3314
99.9995 (ATCC #33400) 46 Streptococcus pyogenes 5.20 .times.
10.sup.9 <1.00 .times. 10.sup.3 6.7160 99.9999 (ATCC #19615) 47
Streptococcus pyogenes 2.5920 .times. 10.sup.9 <1.00 .times.
10.sup.3 6.4141 99.9999 Clinical Isolate BSLI #061901Spy7
[0266] While their microbial reductions were measured at less than
5.0 Log.sub.10, Microcyn also demonstrated antimicrobial activity
against the remaining three species not included in Table 8. More
specifically, a thirty second exposure to Microcyn reduced the
population of Streptococcus pneumoniae (Clinical Isolate; BSLI
#072605Spn1) by more than 4.5 Log.sub.10, which was the limit of
detection versus this species. Further, when challenged with
Candida tropicalis (ATCC #750), Microcyn demonstrated a microbial
reduction in excess of 3.0 Log.sub.10 following a thirty second
exposure. Additionally, when challenged with Candida tropicalis
(BSLI #042905Ct), Microcyn demonstrated a microbial reduction in
excess of 3.0 Log.sub.10 following a twenty minute exposure.
[0267] The exemplary results of this In-Vitro Time-Kill evaluation
demonstrate that Microcyn oxidative reductive potential water
exhibits rapid (i.e., less than 30 seconds in most cases)
antimicrobial activity versus a broad spectrum of challenging
microorganisms. Microbial populations of forty-seven out of the
fifty Gram-positive, Gram-negative, and yeast species evaluated
were reduced by more than 5.0 Log.sub.10 within thirty seconds of
exposure to the product.
EXAMPLE 30
[0268] This example demonstrates a comparison of the antimicrobial
activity of an exemplary oxidative reductive potential water,
Microcyn, used in accordance with the invention versus
HIBICLENS.RTM. chlorhexidine gluconate solution 4.0% (w/v) and 0.9%
sodium chloride irrigation (USP).
[0269] An In-Vitro Time-Kill evaluation was performed as described
in Example 29 using HIBICLENS.RTM. chlorhexidine gluconate solution
4.0% (w/v) and a sterile 0.9% sodium chloride irrigation solution
(USP) as reference products. Each reference product was evaluated
versus suspensions of the ten American Type Culture Collection
(ATCC) strains specifically denoted in the Tentative Final
Monograph. The data collected was then analyzed against the
Microcyn microbial reduction activity recorded in Example 29.
[0270] Microcyn oxidative reductive potential water reduced
microbial populations of five of the challenge strains to a level
comparable to that observed for the HIBICLENS.RTM. chlorhexidine
gluconate solution. Both Microcyn and HIBICLENS.RTM. provided a
microbial reduction of more than 5.0 Log.sub.10 following a thirty
second exposure to the following species: Escherichia coli (ATCC
#11229 and ATCC #25922), Pseudomonas aeruginosa (ATCC #15442 and
ATCC #27853), and Serratia marcescens (ATCC #14756). Further, as
shown above in Table 9, Microcyn demonstrated excellent
antimicrobial activity against Micrococcus luteus (ATCC #7468) by
providing a 5.8420 Log.sub.10 reduction after a thirty second
exposure. However, a direct Micrococcus luteus (ATCC #7468)
activity comparison to HIBICLENS.RTM. was not possible because
after a thirty second exposure, HIBICLENS.RTM. reduced the
population by the detection limit of the test (in this specific
case, by more than 4.8 Log.sub.10). It is noted that the sterile
0.9% sodium chloride irrigation solution reduced microbial
populations of each of the six challenge strains discussed above by
less than 0.3 Log.sub.10 following a full twenty minute
exposure.
[0271] Microcyn oxidative reductive potential water provided
greater antimicrobial activity than both HIBICLENS.RTM. and the
sodium chloride irrigation for four of the challenge strains
tested: Enterococcus faecalis (ATCC #29212), Staphylococcus aureus
(ATCC #6538 and ATCC #29213), and Staphylococcus epidermidis (ATCC
#12228). The following table summarizes the microbial reduction
results of the In-Vitro Time-Kill evaluation for these four
species: TABLE-US-00009 TABLE 9 Comparative Results Log.sub.10
Reduction Microorganism Exposure NaCl Species Time Microcyn
HIBICLENS .RTM. Irrigation Enterococcus 30 seconds 6.4166 1.6004
0.3180 faecalis 30 seconds 6.4166 1.6004 0.3180 (ATCC #29212) 1
minute 6.4166 2.4648 0.2478 3 minutes 6.4166 5.2405 0.2376 5
minutes 6.4166 5.4166 0.2305 7 minutes 6.4166 5.4166 0.2736 9
minutes 6.4166 5.4166 0.2895 11 minutes 6.4166 5.4166 0.2221 13
minutes 6.4166 5.4166 0.2783 15 minutes 6.4166 5.4166 0.2098 20
minutes 6.4166 5.4166 0.2847 Staphylococcus 30 seconds 6.1775
1.1130 0.0000 aureus 1 minute 6.1775 1.7650 0.0191 (ATCC #6538) 3
minutes 6.1775 4.3024 0.0000 5 minutes 6.1775 5.1775 0.0000 7
minutes 6.1775 5.1775 0.0000 9 minutes 6.1775 5.1775 0.0000 11
minutes 6.1775 5.1775 0.0267 13 minutes 6.1775 5.1775 0.0000 15
minutes 6.1775 5.1775 0.0191 20 minutes 6.1775 5.1775 0.0000
Staphylococcus 30 seconds 6.2405 0.9309 0.0000 aureus 1 minute
6.2405 1.6173 0.0000 (ATCC #29213) 3 minutes 6.2405 3.8091 0.0460 5
minutes 6.2405 5.2405 0.0139 7 minutes 6.2405 5.2405 0.0000 9
minutes 6.2405 5.2405 0.0113 11 minutes 6.2405 5.2405 0.0283 13
minutes 6.2405 5.2405 0.0000 15 minutes 6.2405 5.2405 0.0000 20
minutes 6.2405 5.2405 0.0615 Staphylococcus 30 seconds 5.6385
5.0233 0.0456 epidermidis 1 minute 5.6385 5.0233 0.0410 (ATCC
#12228) 3 minutes 5.6385 5.0233 0.0715 5 minutes 5.6385 5.0233
0.0888 7 minutes 5.6385 5.0233 0.0063 9 minutes 5.6385 5.0233
0.0643 11 minutes 5.6385 5.0233 0.0211 13 minutes 5.6385 5.0233
0.1121 15 minutes 5.6385 5.0233 0.0321 20 minutes 5.6385 5.0233
0.1042
[0272] The results of this comparative In-Vitro Time-Kill
evaluation demonstrate that Microcyn oxidative reductive potential
water not only exhibits comparable antimicrobial activity to
HIBICLENS.RTM. against Escherichia coli (ATCC #11229 and ATCC
#25922), Pseudomonas aeruginosa (ATCC #15442 and ATCC #27853),
Serratia marcescens (ATCC #14756), and Micrococcus luteus (ATCC
#7468), but provides more effective treatment against Enterococcus
faecalis (ATCC #29212), Staphylococcus aureus (ATCC #6538 and ATCC
#29213), and Staphylococcus epidermidis (ATCC #12228). As shown in
Table 9, Microcyn exemplifies a more rapid antimicrobial response
(i.e., less than 30 seconds) in some species. Moreover, exposure to
Microcyn results in a greater overall microbial reduction in all
species listed in Table 9.
EXAMPLE 31
[0273] This example demonstrates the effectiveness of an ORP water
solution against Penicillin Resistant Streptococcus pneumoniae
(ATCC 51915).
[0274] A culture of Streptococcus pneumoniae was prepared by using
a frozen culture to inoculate multiple BAP plates and incubating
for 2-3 days at 35-37.degree. C. with CO2. Following incubation 3-7
mL of sterile diluent/medium was transferred to each agar plate and
swabbed to suspend the organism. The suspensions from all plates
were collected and transferred to a sterile tube and compared to a
4.0 McFarland Standard. The suspension was filtered through sterile
gauze and vortex mixed prior to use in the testing procedure.
[0275] An inoculum of 0.1 ml of the organism suspension was added
to 49.9 ml of the Microcyn or control substance. At each exposure
period, the test mixture was mixed by swirling. The test mixture
was exposed for 15 seconds, 30 seconds, 60 seconds, 120 seconds, 5
minutes, and 15 minutes at 25.0.degree. C.
[0276] A 1.0 ml sample was removed from the test mixture and added
to 9.0 ml of neutralizer representing a 100 dilution of the
neutralized inoculated test mixture. A 5 ml aliquot of the 100
neutralized inoculated test mixture was transferred to a 0.45
microliter filter apparatus pre-wetted with 10 ml of Butterfield's
Buffer. The filter was rinsed with approximately 50 mL of
Butterfield's Buffer, aseptically removed from the apparatus, and
transferred to a BAP plate. Additional 1:10 serial dilutions were
prepared and one (1.0) ml aliquots of the 10-3-10-4 dilutions of
neutralized inoculated test mixture were plated in duplicate on
BAP.
[0277] The bacterial subculture plates were incubated for 48.+-.4
hours at 35-37.degree. C. in CO2. Subculture plates were
refrigerated for two days at 2-8.degree. C. prior to examination.
Following incubation and storage, the agar plates were observed
visually for the presence of growth. The colony forming units were
enumerated and the number of survivors at each exposure time was
determined. Representative subcultures demonstrating growth were
appropriately examined for confirmation of the test organisms.
[0278] The exemplary ORP water solution, Microcyn, demonstrated a
>99.93197279% reduction of Penicillin Resistant Streptococcus
pneumoniae (ATCC 51915) after 15 second, 30 second, 60 second, 120
second, 5 minute, and 15 minute contact times at 25.0.degree.
C.
EXAMPLE 32
[0279] The objective of this Example is to determine the microbial
activity of an exemplary ORP water solution (Dermacyn) versus
Bacitracin using a bacterial suspension assay.
[0280] Dermacyn is a ready to use product, therefore performing
dilutions during testing was not required. Bacitracin is a
concentrated re-hydrated solution requiring a dilution to 33
Units/ml.
[0281] A purchased spore suspension of B. atropheus at
2.5.times.107 /ml was used for testing. In addition fresh
suspensions of Pseudomonas aeruginosa, and Staphylococcus aureus
were prepared and measured using a spectrophotometer to ensure the
titer was acceptable
[0282] Nine microliters of test substance was added to 100 ul of
microbe suspension. The test mixture was held at 20.degree. C. for
the contact times of 20 seconds, 5 minutes, and 20 minutes. 1.0 ml
of the test mixture (entire mixture) was added to 9,0 ml of
neutralizer for 20 minutes (this is the original neutralization
tube or ONT) 1.0 ml of the neutralized test mixture was plated on
Tryptic Soy Agar in duplicate for the 5 minute and 20 minute
contact times. Additional dilutions and spread plates were used for
the 20 second time point, to achieve countable plates.
[0283] All plates were incubated at 30.degree. C.-35.degree. C. for
a total of 3 days and were evaluated after each day of incubation.
To determine the number of microbes exposed to Dermacyn and
Bacitracin during testing the suspensions Four 10-fold dilutions
were performed and 1.0 ml of the final 2 dilutions was plated in
duplicate, where applicable.
[0284] Dermacyn when challenged with the test organisms showed
total eradication (>4 log reduction) of the vegetative bacteria
at all time points and for spores at the 5, and 20 minute time
points. Bacitracin only produced approximately 1 log reduction.
Microcyn at the 20 second time point showed some reduction in
spores. Bacitracin showed no evidence of lowering the bacterial or
spore populations over the time periods tested.
EXAMPLE 33
[0285] This example demonstrates the effectiveness of two exemplary
ORP water solutions (M1 and M2) against bacteria in biofilms.
[0286] The parental strain for all studies is P. aeruginosa PAO1.
All planktonic strains were grown aerobically in minimal medium
(2.56 g Na.sub.2HPO.sub.4, 2.08 g KH.sub.2PO.sub.4, 1.0 g
NH.sub.4Cl, 0.04 g CaCl.sub.20.2 H.sub.2O, 0.5 g
MgSO.sub.40.7H.sub.2O, 0.1 mg CuSO.sub.40.5H.sub.2O, 0.1 mg
ZnSO.sub.4. H.sub.2O, 0.1 mg FeSO.sub.4.7H.sub.2O, and 0.004 mg
MnCl.sub.2.4H.sub.2O per liter, pH 7.2) at 22.degree. C. in shake
flasks at 220 rpm. Biofilms were grown as described below at
22.degree. C. in minimal medium. Glutamate (130 mg/liter) was used
as the sole carbon source.
[0287] Biofilms were grown as described previously (Sauer et.al.,
J. Bacteriol. 184:1140-1154 (2002),which is hereby incorporated by
reference). Briefly, the interior surfaces of silicone tubing of a
once-through continuous flow tube reactor system were used to
cultivate biofilms at 22.degree. C. Biofilms were harvested after 3
days (maturation-1 stage), 6 days (maturation-2 stage), and 9 days
(dispersion stage) of growth under flowing conditions. Biofilm
cells were harvested from the interior surface by pinching the tube
along its entire length, resulting in extrusion of the cell
material from the lumen. The resulting cell paste was collected on
ice. Prior to sampling, the bulk liquid was purged from the tubing
to prevent interference from detached, planktonic cells.
[0288] The population size of planktonic and biofilm cells was
determined by the number of CFU by using serial dilution plate
counts. To do so, biofilms were harvested from the interior surface
after various periods of time of exposure to SOSs. Images of
biofilms grown in once-through flow cells were viewed by
transmitted light with an Olympus BX60 microscope (Olympus,
Melville, N.Y.) and a .sub.--100 magnification A100PL objective
lens. Images were captured using a Magnafire cooled three-chip
charge-coupled device camera (Optronics Inc., Galena, Calif.) and a
30-ms exposure. In addition, confocal scanning laser microscopy was
performed with an LSM 510 Meta inverted microscope (Zeiss,
Heidelberg, Germany). Images were obtained with a LD-Apochrome
40.sub.--/0.6 lens and with the LSM 510 Meta software (Zeiss).
[0289] A 2-log reduction was observed for M1-treated biofilms
within 60 min of treatment. The finding indicates that every 10.8
min (+/-2.8 min), treatment with M1 results in a 50% reduction in
biofilm viability. TABLE-US-00010 TABLE 10 M1 Killing. Time (min)
Viability (%) 0 100 10 50 20 25 34 12.5 47 6.25 54 3.125
[0290] However, overall M2 was somewhat more effective in killing
biofilms than M1 because the results indicated that every 4.0 min
(+/-1.2 min), treatment with M2 results in a 50% reduction in
biofilm viability. TABLE-US-00011 TABLE 11 M2 Killing. Viability
Time (min) (%) 0 100 2.5 50 7 25 12 12.5 15 6.25 20 3.125
[0291] Thus, ORP water is effective against bacteria in
bioflims.
[0292] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0293] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0294] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *