U.S. patent application number 14/192353 was filed with the patent office on 2014-06-26 for methods of treating outer eye disorders using high orp acid water and compositions thereof.
This patent application is currently assigned to APR Nanotechnologies s.a.. The applicant listed for this patent is APR Nanotechnologies s.a.. Invention is credited to Yongge Chen, Roberto De Noni.
Application Number | 20140178500 14/192353 |
Document ID | / |
Family ID | 42697495 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178500 |
Kind Code |
A1 |
Chen; Yongge ; et
al. |
June 26, 2014 |
METHODS OF TREATING OUTER EYE DISORDERS USING HIGH ORP ACID WATER
AND COMPOSITIONS THEREOF
Abstract
The present invention relates to a method of treating an outer
eye disorder selected from a cataract, neovascularization,
keratitis, epithelium deficiency, or chronic opacity, by
administering to the eye a composition comprising acidic
electrolytic water. The present invention also relates to a stable
acidic electrolyzed oxidizing water characterized by low
conductivity, the presence of dissolved chlorine gas (Cl.sub.2),
hypochlorous acid (HOCl) and chloride ions (Cl.sup.-), and by the
presence of negligible quantities of hypochlorite ion
(OCl.sup.-).
Inventors: |
Chen; Yongge; (Guangzhou,
CN) ; De Noni; Roberto; (Fregona, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APR Nanotechnologies s.a. |
Balerna |
|
CH |
|
|
Assignee: |
APR Nanotechnologies s.a.
Balerna
CH
|
Family ID: |
42697495 |
Appl. No.: |
14/192353 |
Filed: |
February 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12816731 |
Jun 16, 2010 |
8691289 |
|
|
14192353 |
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Current U.S.
Class: |
424/661 |
Current CPC
Class: |
A61P 27/12 20180101;
A61P 27/02 20180101; A61K 33/20 20130101 |
Class at
Publication: |
424/661 |
International
Class: |
A61K 33/20 20060101
A61K033/20 |
Claims
1) A method of treating a condition selected from cataract, ocular
keratitis, corneal neovascularization, epithelium deficiency, and
chronic opacity in a lens in an animal patient in need thereof,
comprising topically administering to an eye of said patient a
composition comprising a therapeutically effective amount of a high
ORP acidic water.
2) The method of claim 1, wherein said high ORP acidic water has an
ORP of greater than 1100 mV, and a pH of from 0.5 to 5.0.
3) The method of claim 1, wherein said high ORP acidic water has a
NMR half line width using .sup.17O of from 42 to 60 Hz, and a pH of
about 0.5 to about 5.0.
4) The method of claim 1, wherein said administering step comprises
dropping said composition directly into said eye.
5) The method of claim 1, wherein said administering step comprises
applying said composition to said eye with a piece of gauze.
6) The method of claim 1, wherein said administering step comprises
applying said composition to said eye 1 to 20 times per day, for at
least 14 days.
7) An electrolytic acid water comprising free chlorine, wherein: a)
from 95% to 99.9% of said free chlorine is present in the form of
hypochlorous acid; b) said water has a pH of from 1.0 to 4.0; and
c) said water has an ORP of greater than 1100 mV.
8) An electrolytic acid water comprising free chlorine, wherein: a)
from 90% to 99.9% of said free chlorine is present in the form of
hypochlorous acid; b) said water has a pH of from 1.0 to 4.0; and
c) said water has an ORP of greater than 1100 mV.
9) The electrolytic acid water of claim 7, wherein the relative
amount of HOCl and Cl.sub.2 is from 99.9% HOCl and 0.1% Cl.sub.2 to
95% HOCl to 5% Cl.sub.2.
10) The electrolytic acid water of claim 7, wherein the relative
amount of HOCl and Cl.sub.2 is from 99.5% HOCl and 0.5% Cl.sub.2 to
98.5% HOCl to 1.5% Cl.sub.2.
11) The electrolytic acid water of claim 7, having a NMR half line
width using .sup.17O of from 42 to 60 Hz.
12) The electrolytic acid water of claim 7, wherein said water has
a conductivity of from 1200 to 1400 uS/cm.
13) The electrolytic acid water of claim 7, wherein said water
maintains 90% of said free chlorine after a storage period of 3
months at room temperature.
14) The electrolytic acid water of claim 7, wherein said water
maintains 80% of said free chlorine after a storage period of 3
months at room temperature.
Description
RELATION TO PRIOR APPLICATIONS
[0001] This application claims priority to U.S. Provisional App.
Nos. 61/187,900 filed Jun. 17, 2009, and 61/239,912, filed Sep. 4,
2009. The content of these earlier applications is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to pharmacological methods of
treating outer eye disorders using acid water having a high
oxidation reduction potential (ORP) and compositions thereof.
BACKGROUND OF THE INVENTION
Outer Eye Disorders
[0003] The outer eye includes several organs, including the cornea,
the iris and the lens. The iris is a membrane in the eye,
responsible for controlling the amount of light reaching the
retina. The cornea and lens refract light onto the retina. Healthy
eyes produce clear vision as the result of the transparency of the
cornea and lens. Cataract, a clouding of the lens of the eye, can
obstruct the passage of light and result in a gradual loss of
vision. Unfortunately, very few treatments exist for cataract,
other than to replace the cataractous lens with an artificial lens
through a complicated surgical procedure.
[0004] Disorders such as keratitis, neovascularization, and
epithelium deficiency can also reduce vision by interfering with
the transparency of the cornea. These disorders can result from
numerous causes, including viral and bacterial infections, trauma
and surgery. Antibiotics and anti-viral agents are often used to
treat infectious causes, but in many instances the patient has no
choice but to undergo a complicated surgical procedure to remove
damaged tissue before it can scar and reduce eyesight.
[0005] PCT Publications WO 2004/012748 and WO 2001/054704 teach an
isotonic ionized acidic solution for wound care, and tout the water
based upon its antioxidant characteristics and antimicrobial
properties. The publications state that the solution may be used in
the place of saline in ophthalmic applications such as contact lens
cleaning solutions or for irrigation of the eye during ophthalmic
surgery, and that the properties of the solution depend on the
particular concentration ranges of a mixture of salts.
[0006] There remains a need for pharmacological methods of treating
outer eye disorders, especially those that affect the cornea and
lens. There is particularly a need to methods that prevent further
deterioration of vision in the eye, and that potentially improve
the vision of the patient whose vision has worsened.
High ORP Acid Water
[0007] It is known that aqueous solutions of salts, particularly
sodium chloride, as a consequence of an electrolytic treatment, are
split into two liquid products, one having basic and reducing
characteristics (generally known as cathode water or alkaline
water) and another (generally known as anode water or acid water)
having acid and oxidizing characteristics.
[0008] Conventional electrolytic waters suffer the acknowledged
drawback of having very limited preservation. A few days after
preparation, the product in fact generally tends to degrade and
lose its properties. Known electrolytic waters, therefore, must be
prepared and used substantially on the spot. Accordingly, the
commercial utilization of the product in itself is extremely
disadvantageous, since the shelf life of any ready-made packages is
dramatically limited.
[0009] The stability of an electrolyzed oxidizing water is reported
in the article "Effects of Storage Conditions and pH on Chlorine
Loss in Electrolyzed Oxidizing (EO) Water"--Journal of Agricultural
and Food Chemistry--2002, 50, 209-212 by Soo-Voo Len, et al. In
Soo-Voo Len, electrolyzed water with an acidic pH (2.5-2.6), high
OPR (1020-1120 mV), and a free chlorine content of .about.50 ppm
(53-56 ppm) was generated using a current intensity of 14 Ampere
and 7.4 Volt. Unfortunately, in an open condition at 25.degree. C.,
the chlorine in the electrolyzed water was completely lost after 30
hours when agitated, and after 100 hours when not agitated.
Furthermore, in a closed dark condition at 25.degree. C., the free
chlorine in the electrolyzed water decreased by approximately 40%
after 1400 hours (about 2 months).
[0010] The stability of electrolyzed oxidizing water also is
reported in the article "Effects of storage conditions on chemical
and physical properties of electrolyzed oxidizing water"--Journal
of Food Engineering 65 (2004) 465-471 by Shun-Yao Hsu, et al. In
Shun-Yao Hsu, the electrolyzed water of "formulation J" had an
acidic pH (2.61), high OPR (1147 mV), and a free chlorine content
of 56 ppm. The article reports that in a closed condition at
25-30.degree. C., the free chlorine in the electrolyzed water was
43 ppm after 21 days, a 23% loss.
[0011] Thus, there remains a need for acidic electrolytic water
with a greater chemical stability than traditional waters. There is
a particular need for water with a greater stability during long
term storage, so as to allow for the commercial utilization of
acidic electrolytic water products.
SUMMARY OF THE INVENTION
[0012] It has unexpectedly been discovered that acidic water,
having a high oxidation reduction potential, promotes and
facilitates an ordered regeneration of the eye when impacted by
negative environmental stimuli such as infections and trauma, or
metabolic processes such as aging and diabetes. In particular, it
has been discovered that the water fosters a healthy epithelium on
the cornea, reduces uncontrolled corneal neovascularization, and
encourages ordered protein synthesis in the lens. The water can
thus be used to treat various outer eye disorders that affect the
cornea, lens or iris, including cataract, keratitis,
neovascularization and epithelium deficiency.
[0013] Therefore, in one embodiment, the invention provides a
method of treating an outer eye disorder selected from cataract,
keratitis, corneal neovascularization and epithelium deficiency in
an animal patient in need thereof, comprising topically
administering to an eye of said patient a composition comprising a
therapeutically effective amount of a high ORP acidic water. The
composition is preferably in the form of an eye drop. In another
embodiment, the invention provides a method for improving opacity
in a lens of an animal patient in need thereof, comprising
administering to an eye of said patient a composition comprising a
therapeutically effective amount of a high ORP acidic water.
[0014] The methods can be performed ad libitum in response to
observable irritation. An effective treatment for these disorders
typically requires a plurality of administrations, extending days,
months or even years of the patient's life. The water can be
defined by several characteristics, including pH and ORP, in
addition to other characteristics including cluster size (as
measured by NMR half line width), and the content of various
chlorine/chloride species.
[0015] It also has unexpectedly been discovered that acidic
nanoclustered water having a particular composition of chlorine
species has a greater chemical stability than traditional waters.
The unique composition can result from particular membrane and
electrodes used in the electrolyzing equipment, which can produce a
high current intensity without causing the electrodes to break up
on their surface and release heavy metals that may adversely affect
stability.
[0016] Additional embodiments and advantages of the invention will
be set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The embodiments and advantages of the invention
will be realized and attained by means of the elements and
combinations particularly pointed out in the appended claims. It is
to be understood that both the foregoing general description and
the following detailed description are exemplary and explanatory
only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0018] FIG. 1 is a schematic view of an electrolytic device
comprising an electrolysis chamber and two electrodes.
[0019] FIG. 2 is side cross-view of a human eye, depicting the
various components of the eye.
[0020] FIG. 3 is a graph illustrating the modulation of the immune
system cytokines by Acidic Nanoclustered Water.
[0021] FIG. 4 is a set of graphs illustrating the concentration of
various chlorine species as a function of pH.
[0022] FIG. 5 is a graph illustrating the loss of free chlorine in
high and low chloride Acidic Nanoclustered Water in an open,
agitated, exposed condition over 24 hours.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention may be understood more readily by
reference to the following definitions and detailed description of
preferred embodiments of the invention and the non-limiting
Examples included therein.
Definitions and Use of Terms
[0024] "Pharmaceutically acceptable" means that which is useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic and neither biologically nor otherwise undesirable and
includes that which is acceptable for veterinary use as well as
human pharmaceutical use.
[0025] As used herein, the term "fluid" is used to reference any
pure fluid, solution or suspension which is capable of producing a
non-spontaneous chemical reaction if subjected to electrolysis. One
highly preferred fluid is water. The term "water" is used to
reference any type of water, such as tap water, filtered water,
deionized water, and distilled water. Once subjected to
electrolysis, the water separates into two liquid fractions, which
for the sake of simplicity are referenced here as acid water or
anode water and as cathode water or alkaline water.
[0026] The term "high ORP water" refers to water having an
oxidation reduction potential greater that +600. The ORP preferably
ranges from +600 to +1350 mV, more preferably from +800, +900, or
+1000 mV to +1300 mV, most preferably from +1100 to +1250 mV.
[0027] The term "acid water" or "acidic water" refers to water
having a pH less than 7.0. The pH of the acid water preferably
ranges from 0.5, 1.0 or 2.0 to 6.5, 6.0, 5.0, 4.0, or 3.0, and most
preferably ranges from 1.0 to 4.0.
[0028] The term "electrolytic water," when used herein, means water
produced by the process of electrolysis, and is preferably
characterized by an oxide reduction potential (ORP) and/or pH that
reflects its acid or alkaline nature.
[0029] The term "nanoclustered water," when used herein, refers to
water having a reduced cluster size, typically induced by
electrolysis. The size of the cluster can be measured by its NMR
half line width, and in preferred embodiments the water has a NMR
half line width using .sup.17O of less than about 60, 56, or 52 Hz,
preferably greater than about 42 or 45 Hz.
[0030] As used herein, "therapeutically effective amount" refers to
an amount sufficient to elicit the desired biological response. The
therapeutically effective amount or dose can depend on the age, sex
and weight of the patient, and the current medical condition of the
patient. The skilled artisan will be able to determine appropriate
dosages depending on these and other factors in addition to the
present disclosure.
[0031] The terms "treating" and "treatment," when used herein,
refer to the medical management of a patient with the intent to
cure, ameliorate, stabilize, or prevent a disease, pathological
condition, or disorder. This term includes active treatment, that
is, treatment directed specifically toward the improvement of a
disease, pathological condition, or disorder, and also includes
causal treatment, that is, treatment directed toward removal of the
cause of the associated disease, pathological condition, or
disorder. In addition, this term includes palliative treatment,
that is, treatment designed for the relief of symptoms rather than
the curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0032] When the context allows, the term "significantly" can be
interpreted to mean a level of statistical significance, in
addition to "substantially." The level of statistical significance
can be, for example, of at least p<0.05, of at least p<0.01,
of at least p<0.005, or of at least p<0.001. When a
measurable result or effect is expressed or identified herein, it
will be understood that the result or effect can be evaluated based
upon its statistical significance relative to a baseline.
[0033] The term "outer eye disorder," as used herein, refers to any
disorder of the cornea, iris or lens, that is associated with
irregular growth or structuring of protein or cellular components.
Examples of outer eye disorders include keratitis,
neovascularization, epithelium deficiency and cataracts. The
keratitis can be superficial, ulcerative (i.e. corneal ulcer),
hypopyon (i.e. hypopyon ulcer), mycotic (caused by fungus), or deep
(perforating through all layers of cornea). Furthermore, the
keratitis may result from bacteria, vitamin A deficiencies,
viruses, trauma (usually following insertion of an object into the
eye), abrasion, surgery, fungi, or parasites. The
neovascularization can be localized, deep, or resulting in buildup
of tissue (pannus). Furthermore, the neovascularization can result
from, or be associated with a lack of oxygen to the eye, trauma,
abrasion, surgery, age related macular degeneration, inflammation
and myopia.
[0034] The term "cataract," as used herein, refers to a variety of
conditions that create a cloudy or calcified lens that obstructs
vision. The cataract can be an infantile, juvenile, or presenile
cataract. Alternatively, the cataract can be an age-related or
senile cataract. Furthermore, the cataract can be either a
traumatic cataract or a congenital cataract. The cataract can be
located in a variety of regions within the lens. For example, the
cataract can be an anterior subcapsular polar cataract (within the
front, center lens surface), a posterior subcapsular polar cataract
(within the rear, center lens surface), a cortical cataract
(radiating from the center to the edge of the lens), a nuclear
cataract (in the center of the lens), or combinations thereof. The
cataract can also be associated with other disorders, such as
diabetic cataract and toxic cataract. Furthermore, the cataract can
be at a variety of stages such as immature cataract (partially
opaque lens), mature cataract (completely opaque lens), or
hypermature cataract (liquefied cortical matter, also known as a
Morgagnian cataract).
[0035] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0036] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" or like terms include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an ingredient" includes mixtures of two or
more ingredients, and the like. The word "or" or like terms as used
herein means any one member of a particular list and also includes
any combination of members of that list.
Discussion
[0037] As discussed above, it has now been discovered that acidic
electrolyzed water can be used to effectively treat patients
suffering from outer eye disorders. This discovery is based on the
results of experimentation described in the "Examples" below, which
revealed that a composition of acidic nanoclustered water, unlike
saline, affects the healing of the eye in a way that can be very
useful in fostering correct protein and cellular structure and
organization, and reducing opacity in the cornea and lens. It also
has been discovered that acidic electrolyzed water of a particular
chemical composition can remain stable for significantly longer
period of time than previous electrolyzed oxidizing waters.
[0038] Therefore, in one embodiment, the invention provides a
method of treating an outer eye disorder selected from cataract,
keratitis, corneal neovascularization and epithelium deficiency in
an animal patient in need thereof, comprising topically
administering to an eye of said patient a composition comprising a
therapeutically effective amount of a high ORP acidic water. The
composition is preferably in the form of an eye drop.
[0039] Importantly, the method can decrease opacity of the lens,
reduce loss of vision, increase visual acuity, increase contrast
sensitivity, and reduce halos. Therefore, in another embodiment,
the invention provides a method for improving opacity, reducing
loss of vision, increasing visual acuity, increasing contrast
sensitivity, or reducing halos in a lens of an animal patient in
need thereof, comprising administering to an eye of said patient a
composition comprising a therapeutically effective amount of a high
ORP acidic water.
[0040] The step of administering the composition can be performed
between 1 and 20 times per day using any method known in the art,
but is preferably undertaken more than once during a 24 hour
period. The administration can be carried out for a period of time
including one week, 10 days, two weeks, three weeks, one month, or
continually as a maintenance therapy. In a particular embodiment,
the administration step comprises dropping the composition directly
into the eye. In another embodiment, the administration step
comprises applying the composition to the eye with gauze. In yet
another embodiment, the administering step comprises applying the
composition to the eye with an eye washer.
[0041] The method can further comprise, before the administering
step, diagnosing said eye as having a cataract or other outer eye
disorder.
[0042] The step of administering the composition can be performed
between 1 and 4 times per day using any method known in the art.
The administration can be carried out for a period of time
including one week, 10 days, two weeks, three weeks, one month, or
continually as a maintenance therapy. In a particular embodiment,
the administration step comprises dropping the composition directly
into the eye. In another embodiment, the administration step
comprises applying the composition to the eye with gauze. In yet
another embodiment, the administering step comprises applying the
composition to the eye with an eye washer.
[0043] The method can further comprise, before the administering
step, performing surgery on said eye.
[0044] The Acid Waters
[0045] Acid water can be obtained with a water electrolysis method
as described below. The electrolytic acid waters can differ from
similar products substantially in their stability, which is at
least partly due to the higher performance of the nano-coated
electrodes and the electrolysis process. In conventional processes,
even when the water is subjected to a filtration step before
electrolysis, the electrodes tend to break up on their surface
during the process, releasing large amounts of heavy metals
(particularly of the metal or metals of which the cathode and anode
are made). However, these acid waters can be free from heavy metals
because said metals, if present, are present in a quantity which is
below the limits that can be detected with ordinary analytical
methods. For example, the water according to the invention can have
a cadmium concentration of less than 5 .mu.g/l, less than 10
.mu.g/l of chromium, less than 5 .mu.g/l of lead, and less than 20
.mu.g/l of nickel. Suitable test methods for these heavy metals are
described in Table 1 below:
TABLE-US-00001 TABLE 1 Heavy Metal Testing Methods TEST TESTING
METHOD Cadmium APAT CNR IRSA 3120/2003 Total Chromium APAT CNR IRSA
3150/2003 Lead APAT CNR IRSA 3230/2003 Nickel APAT CNR IRSA
3220/2003 Fixed Residue at 180.degree. C. APAT CNR IRSA
2090A/2003
[0046] Although one does not intend to be bound to any particular
theory, it is believed that the absence of heavy metals is one of
the main reasons for the unusual and advantageous stability over
time of the electrolytic acid water. The expression "stability over
time" is used to mean that the acid water, if kept sheltered from
the light, air and heat, keeps its chemical and physical
properties, particularly its pH, ORP and/or NMR half line width,
substantially unchanged for greater than 60 or 90 days, preferably
greater than 180 days, even more preferably greater than 365 days,
up to two, three or even five years. By substantially unchanged, it
is meant that the property under evaluation does not vary by more
than 50, 30, 15, 10, 5, or even 3% during the applicable time
frame.
[0047] Although the stability time depends on the steps taken to
preserve the solution, it must be noted that for equal storage
conditions, an acidic water obtained by using an electrolytic
device as defined above has shown a distinctly higher stability
than known similar products, which in the best cases have shown a
shelf life of only 60-90 days. Therefore, these products must be
obtained and used over a short period or even simultaneously with
their production. Therefore, the electrolytic acidic water
according to the invention can be useful also for applications in
locations (Third World countries) and situations (scarcity of water
to provide electrolysis) in which favorable conditions for its
production are not available.
[0048] The ORP of the electrolytic acid water preferably ranges
from +600 to +1350 mV, more preferably from +800, +900, 1000 or
+1100 mV to +1300, 1250 or +1200 mV, most preferably from +1100 to
+1250 mV. The pH of the acid water preferably ranges from 0.5 or
1.0 to 6.5, 6.0, 5.0, 4.0, or 3.0, and most preferably ranges from
1.0 to 4.0.
[0049] Nuclear magnetic resonance .sup.17O NMR measures,
particularly when evaluated at the half way point of the water
peak, are useful to measure the quality of acid waters of the
current invention, because they reflect intrinsic properties of the
water structure such as the median molecular cluster size of
H.sub.2O molecules, and the distribution of molecular cluster
sizes, in addition to contaminants such as ionic species within the
water. The expression "molecular cluster" designates the number of
molecules of water which are coordinated in an ordered
structure
[0050] In most preferred embodiments, the .sup.17O NMR half line
width for the acid water is equal to or greater than 42, 45, 46, or
47, and less than 100, 75, 60, 56, 53, 51, 50 or 49 Hz, wherein the
range can be selected from any of the foregoing endpoints. Thus,
for example, in preferred embodiments, the acid water of the
present invention has an NMR half line width ranging from 45 to
less than 51 Hz, or 45 to less than 50 Hz, or 46 to less than 50
Hz.
[0051] The acid water may also be characterized by the presence and
quantity of chlorine species in the water. One of the following
assays or any combination of the following assays may be used to
characterize the water. According to the free chlorine assay
(spectrophotometric method), or the total chlorine assay
(spectrophotometric method), the water may be defined as containing
less than 85, 70, 60, 55, 52 or even 50 mg/l of chlorine species,
optionally limited by a lower bound of 20, 30 or 40 mg/l. According
to the total chlorine assay (iodometric method), the water may be
defined as containing less than 80, 70, 65, or even 62 mg/l of
chlorine species, optionally limited by a lower bound of 20, 30 or
40 mg/l. According to the UNI 24012 (Mercurimetric method) chloride
assay, the water may contain greater than 50, 100, 130, 150 or even
170 mg/l of chloride, and/or less than 250 or 200 mg/l. Chlorites
(as ClO.sub.2-), when measured by EPA 300.1 (1997) (detection limit
100 ug/l), are preferably non-detectable. Chlorates (ClO.sub.3-),
when measured by EPA 300.1 (1997) (detection limit 0.1 mg/l), are
preferably present in an amount less than 10, 5, 2, or even 1
mg/l.
[0052] Although in certain embodiments the acid water may contain
oxidizing chlorine species in amounts of up to 60 or even 100 mg/l,
in a preferred embodiment the acid water according to the invention
is essentially free of oxidizing chlorine species, or other anionic
residues of salts that are generated during the electrolytic
process, i.e. less than 10 or even 5 mg/l, and preferably
undetectable.
[0053] In a particularly desirable embodiment, the water can be
characterized by conductivity, the presence of dissolved chlorine
gas (Cl.sub.2), hypochlorous acid (HOCl) and chloride ions
(Cl.sup.-), and by the presence of negligible quantities of
hypochlorite ion (OCl.sup.-). In water, the relative amount of
chlorine and hypochlorous acid is strongly affected by the amount
of chlorides. Specifically, an increase in chlorides results in an
increase in the amount of chlorine gas with respect to hypochlorous
acid as according to the following equilibrium:
Cl.sup.-+H.sup.++HOClCl.sub.2+H.sub.2O
[0054] Because of the relationship between the amount of chlorides
and the amounts of chlorine gas and hypochlorous acid, the amount
of chloride in the water is preferably very low (less than 200 ppm)
to ensure that the free chlorine in the water is almost exclusively
in the form of hypochlorous acid.
[0055] The relationship between the four species Cl.sub.2, HOCl,
Cl.sup.-, and OCl.sup.- can be understood using the disassociation
equilibria of gaseous chlorine in water as described below, in
which Cl.sub.2, HOCl, and OCl.sup.- are the three possible forms of
free total chlorine:
Cl.sub.2+H.sub.2O=Cl.sup.-+H.sup.++HOCl
K.sub.a1.apprxeq.3.times.10.sup.-4
HOCl=H.sup.++OCl.sup.-K.sub.a2.apprxeq.2.9.times.10.sup.-8
[0056] As can be seen in the above equations, chlorine generation
occurs in the presence of an excess of Cl.sup.-. Furthermore, the
amount of the three forms of free total chlorine as a function of
pH and Cl.sup.- can be determined algebraically by using the above
described equilibria as follows:
.alpha.Cl.sub.2=[H.sup.+].sup.2[Cl.sup.-]/([H.sup.+].sup.2[Cl.sup.-]+[H.-
sup.+]K.sub.a1+K.sub.a1K.sub.a2)
.alpha.HClO=[H.sup.+]K.sub.a1/([H.sup.+].sup.2[Cl.sup.-]+[H.sup.+]K.sub.-
a1+K.sub.a1K.sub.a2)
.alpha.ClO.sup.-=K.sub.a1K.sub.a2/([H.sup.+].sup.2[Cl.sup.-]+[H.sup.+]K.-
sub.a1+K.sub.a1K.sub.a2)
[0057] These equations can be used to simulate the chlorine
concentration at different pH values. For example, FIG. 3 is a
graphical simulation of the concentration of various chlorine
species as a function of pH. As can be seen in FIG. 3, at a typical
pH for the water of 2.8-3.0 (indicated by the green dash-and-dotted
line), free chlorine is predominantly present as Cl.sub.2 and HClO,
and the relative amount of the two species is strongly affected by
the amount of chlorides, and increase of which results in an
increase of the amount of chlorine gas with respect to HClO.
[0058] It is generally recognized that diluted hypochlorous acid
solutions are unstable due to decomposition. This decomposition can
occur according to a first pathway:
2HOCl.fwdarw.2HCl+O.sub.2
2HCl+2HOCl.fwdarw.2Cl.sub.2+2H.sub.2O
[0059] Or as according to a second pathway, in which chlorous acid
(HClO.sub.2) is an intermediate in the formation of chloric acid
(HClO.sub.3):
2HOCl.fwdarw.[HClO.sub.2]+HCl
2HOCl+[HClO.sub.2].fwdarw.HClO.sub.3+HCl
HClO.sub.3+HCl+HOCl.fwdarw.HClO.sub.3+Cl.sub.2+H.sub.2O
[0060] Kinetic studies have indicated that both decomposition
pathways are pH dependant and increase with concentration,
temperature, and exposure to light. Furthermore, the first process
can be accelerated by catalysts, and the second process can be
accelerated in the presence of other electrolytes, notably chloride
ions. Due to decomposition, hypochlorite solutions can be more
stable than hypochlorous acid solutions. For this reason,
commercial solutions often have neutral or alkaline pH, which
causes the free chlorine to exist as hypochlorite and not
hypochlorous acid.
[0061] Although one does not intend to be bound to any particular
theory, it is believed that the low chloride ion content is one of
the main reasons for the unusual and advantageous stability over
time of the electrolytic acid water, both to evaporation and self
decomposition. Preferably, the amount of chlorides both at the
beginning and at the end of the electrolytic process is low (200
ppm or lower), so that the water comprises chlorine in the form of
HClO. For example, in a particular embodiment, the water can
comprise .about.50 ppm of free chlorine, and .about.200 ppm of
chloride ions. At pH 2.80, this corresponds to 99.3% HClO, and 0.7%
dissolved gaseous chlorine.
[0062] The conductivity of the water preferably ranges from 900 to
1800 uS/cm, and more preferably ranges from 1000, 1100, 1200, or
1300 to 1400, 1500, 1600, or 1700 uS/cm. The free chlorine content
of the water preferably ranges from 20 to 80 ppm, more preferably
ranges from 30 or 40 to 60 or 70 ppm, and most preferably is about
50 ppm. The chloride ion content of the water preferably ranges
from 150 to 250 ppm, more preferably ranges from 160, 170, 180 or
190 to 210, 220, 230, or 240 ppm, and most preferably is about 200
ppm. The chlorite content of the water preferably ranges from 50 to
150 ppb, more preferably ranges from 60, 70, 80 or 90 to 110, 120,
130, or 140 ppb, and most preferably is about 100 ppb. The chlorate
content of the water preferably ranges from 0.5 to 1.5 ppm, more
preferably ranges from 0.6, 0.7, 0.8, or 0.9 to 1.1, 1.2, 1.3, or
1.4 ppb, and most preferably is about 1 ppm.
[0063] Due to its chemical composition and acidity, the free
chlorine in the water can be present in the form of hypochlorous
acid (HOCl) and chlorine gas (Cl.sub.2). The relative amount of
HOCl and Cl.sub.2 in the water preferably ranges from 99.9% HOCl
and 0.1% Cl.sub.2 to 95% HOCl to 5% Cl.sub.2, more preferably
ranges from 99.5% HOCl and 0.5% Cl.sub.2 to 98.5% HOCl to 1.5%
Cl.sub.2, and most preferably is about 99.3% HOCl and 0.7%
Cl.sub.2.
[0064] Because the free chlorine in the water is present in the
form of HOCl and Cl.sub.2 in the ranges described above, the water
can be highly stable against both evaporation and self
decomposition. In an exposed, non-agitated state at a temperature
of 25.degree. C., the water preferably maintains a level of
chlorine for a time of 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In an
exposed, agitated state at a temperature of 25.degree. C., the
water preferably maintains a level of chlorine for a time of 8, 12,
16, 20, or 24 hours. In a closed state at a temperature of
25.degree. C., the water preferably maintains about 90% of the free
chlorine after a time of 3 months, and about 85% of the free
chlorine after a time of 12 months. In a closed state at a
temperature of 30.degree. C., the water preferably maintains about
90% of the free chlorine after a time of 3 months, and about 80% of
the free chlorine after a time of 12 months. In a closed state at a
temperature of 40.degree. C., the water preferably maintains about
90% of the free chlorine after a time of 3 months, and about 85% of
the free chlorine after a time of 12 months.
[0065] Making Electrolytic Acid Water
[0066] The electrolytic acid water can be prepared, for example, by
using the methods and electrolysis devices described in PCT
Publications WO 2008/131936 and WO 2007/048772. The contents of
said applications are hereby incorporated by reference as if fully
set forth herein.
[0067] Referring now to FIG. 1, the electrolysis device can
comprise an electrolysis chamber 3 divided into two portions by a
membrane 4, and a pair of electrodes 1 and 2 within said
chamber.
[0068] Preferably, both electrodes of the device are nano-coated
electrodes as defined below. However, the advantages in terms of
low cost and efficiency of the electrolysis process, as well as the
advantages in terms of water stability over time, can be obtained
also if only one of the two electrodes is nano-coated as defined
above.
[0069] Preferably, the device according to the invention also
comprises a membrane 4 adapted to divide the at least one chamber
into two half-chambers, wherein the half-chamber that contains the
anode is termed an anode half-chamber, and the half-chamber that
contains the cathode is termed a cathode half-chamber. The membrane
is advantageously an ultrafiltration membrane which can occupy the
chamber partially or totally.
[0070] The membrane 4 can be of the type used in conventional
electrolytic cells, but is preferably based on size exclusion
technology at the nano-scale. Preferably, the membrane is made of
ceramic material with open porosity, coated with metallic
nano-particles, preferably nano-particles of oxides of zirconium,
yttrium, aluminum or mixtures thereof. The metallic nano-particles
used to make the coating are preferably in powder form. As regards
the size distribution within the powder, preferably an amount at
least equal to 70%, 75%, or 80% by weight of the particles that are
present in the powder, more preferably at least equal to 85%, have
a particle diameter ranging from 30 to 100 nm, 40 to 70 nm, or 50
to 60 nm.
[0071] By resorting to nanometer particles to manufacture the
membrane 4, the average pore size of the final membrane has been
found to be extremely constant over time and adaptable according to
the requirements of how the water is to be processed. Preferably,
the average pore size is from about 120 to about 180 nm (mean or
median). Size constancy over time and constancy of the pore
dimensions themselves are two aspects which differentiate the
ceramic membrane described here from the textile membranes
conventionally used in equivalent devices (which are instead
subject to rapid deterioration over time). It is preferred that at
least 50%, 70%, 90%, 95%, 98% or 99% of the pores have a diameter
between 120 and 180 nm. These aspects have shown a positive effect
on the stability of the water obtained after electrolysis, where
this effect combines with, and augments, the stabilizing effect
produced by the use of an electrode as defined above.
[0072] Importantly, the nano-sized dimensional features of the
membrane and electrodes enhance the amount of active surface per
unit of geometric surface, which creates a high apparent current
density (i.e. the current intensity per unit of geometric surface).
As a result, a high current intensity (ampere) and electric
potential (voltage) can be provided to the solution, which can
impart unique chemical and biological characteristics to the water.
Preferably, the water is produced by applying a current intensity
in the range of about 100 to about 39 ampere (24 to 18 volt) to a
diluted sodium chloride solution in deionized water. By applying
the high current intensity, a chemical composition of a low
chloride ion content, low chlorite and chlorate content, and high
hypochlorous acid content can be achieved.
[0073] The amount of current applied to the water preferably ranges
from 30 to 120 ampere, more preferably ranges from 40, 50 or 60
ampere to 90, 100, or 110 ampere, and most preferably is about 80
ampere. The amount of voltage applied to the water preferably
ranges from 15 to 35 volt, more preferably ranges from 16, 17, or
18 volt to 22, 23, or 24 volt, and most preferably is about 20
volt.
[0074] In a preferred electrolysis device, each half-chamber is
connected to the outside of the device through: [0075] openings 7
and 8 arranged in the upper part of the half-chamber from which the
water to be subjected to electrolysis is inserted, and [0076]
additional openings 5 and 6 arranged in the lower part of the
half-chamber which can act as a discharge for the resulting acid
and alkaline fractions (referenced as "acid water" and "alkalescent
water" in FIG. 1). The second opening on the lower part of each
half chamber is provided with closure means (not shown) which is
adapted to prevent the water that has not yet separated from
leaving the half-chamber and are adapted to be opened at the end of
the electrolytic process.
[0077] With specific reference to FIG. 1, the operating mechanism
of a device as described above provided with all the essential and
optional elements that have been listed, therefore entails treating
water by introducing it from above, by means of the water input
ducts, into the two half-chambers of the main chamber. Here, the
water, under the action of the cathode and of the anode previously
connected to the negative and positive poles of an electric voltage
source, is split into positive and negative ions, which, as is
known, are attracted by the respective opposite poles. In passing
from one half-chamber to the other, the nano-porous membrane acts
as a filter for said ions and for any charged particles, allowing
only the particles of sufficiently small size to pass.
[0078] The water input to the unit can be characterized by its
conductivity, preferably measured in .mu.S/cm. Thus, for example,
the water can be described by the consistency of conductivity in
the water input. For example, the conductivity should vary by no
more than 50, 20, 10, 5 or even 2 .mu.S/cm, or 100, 50, 20 or 10%.
The water may also be described by the conductivity of the water
itself. The conductivity can range from 0.5, 1.0 or 1.5 .mu.S/cm to
50, 25, 10, 5 or even 3 .mu.S/cm, based on any selection of
endpoints. The conductivity preferably ranges from 0.5 to 10 or 0.5
to 3 .mu.S/cm, and the most preferred conductivity is about 2
.mu.S/cm. It has been discovered that by controlling the
consistency of the conductivity, and by lowering the conductivity
to the preferred values, one is able to obtain much more consistent
quality electrolyzed water, with a consequent reduction in NMR half
line width. Suitable types of water for input into the unit include
reverse osmosis water, deionized water, and distilled water. A
preferred type of water due to its constant conductivity is osmotic
water prepared by reverse osmosis.
[0079] The water preferably contains sodium chloride, or some other
alkali metal salt, to facilitate the electrolysis. The sodium
chloride is preferably pharmaceutical grade. The quantity of sodium
chloride contained in the water is such that the water obtains a
specific level of conductivity. The conductivity of the input
solution preferably ranges from 50 .mu.S/cm to 100 .mu.S/cm, more
preferably ranges from 150 .mu.S/cm to 200 .mu.S/cm, and most
preferably is about 200 .mu.S/cm.
[0080] Also of importance, the filter prevents the transmission of
heavy metals from one chamber to the other. Thus, by introducing
the water into the acidic or alkaline chamber, one is able to
produce alkaline or acid water having practically no contamination
by metallic radicals (or at least beyond the limits of
detection).
[0081] A method of using such a unit for making electrolytic acid
water having a NMR half line width using .sup.17O-NMR of from about
45 to less than 51 Hz comprises:
[0082] (a) providing an electrolysis unit comprising: (i) a cathode
chamber, an anode chamber, and a filter separating said chambers
(preferably characterized by a porosity that allows ionized
fractions of nano-clustered H.sub.2O to pass, such as when the
porosity is predominantly characterized by pores of from about 120
to about 180 nm in diameter (preferably having a mean diameter
between 120 and 180 nm)); and (ii) a cathode situated in said
cathode chamber and an anode situated within said anode chamber,
wherein at least one of said anode and cathode is coated by a
residue of particles in which greater than 70% by weight of said
particles have a diameter of from 40 to 100 nm;
[0083] (b) introducing a solution of water and an alkali metal into
one or both of said chambers; and
[0084] (c) applying an electric potential to said anode and said
cathode, for a time and to an extent sufficient to produce
electrolyzed acidic water having a NMR half line width using
.sup.17O of from about 45 to less than 51 Hz.
[0085] Electrode Construction
[0086] Referring again to FIG. 1, the electrolysis device includes
electrodes 1 and 2 that comprise a surface coating which comprises
nano-particles of one or more metals. Preferably, the electrodes
comprise a core which is made of a metallic material, a nonmetallic
material or combinations thereof.
[0087] If the core is made of metallic material, it can be made for
example of an alloy of titanium and platinum or an alloy of steel
and graphite. If the core is made of a nonmetallic material, it can
be made for example of graphite. The core may also comprise
different layers, such as for example a core made of graphite which
is coated with an outer layer of metal, for example titanium. The
term "metal" references both a metal and chemical compounds which
comprise said metal, such as its oxides. A preferred core is made
of TiO.sub.2.
[0088] The electrode can be characterized with respect to known
electrodes substantially due to the presence of a nanometer
covering (hereinafter also referenced as coating) which is
extremely smooth, i.e., a layer for covering the core which
includes metallic nano-particles.
[0089] The metals of which the nano-particles of the coating are
made are selected preferably among one or more of titanium,
iridium, yttrium, ruthenium, zinc, zirconium platinum, selenium,
tantalum and compounds thereof. Preferred metal compounds are
oxides of the mentioned metals. A preferred coating comprises
ZrO.sub.2, ZnO, Ru.sub.2O.sub.3, IrO.sub.2 and Y.sub.2O.sub.3, or
TiO.sub.2, Pt/ZrO.sub.2, SnO.sub.2, Ta.sub.2O.sub.5, and IrO.sub.2.
Preferably, the various metals are used in powder form.
[0090] The coating can also comprise a nonmetallic carrier
material, for example particles of one or more polymers. The
polymer can be synthetic (such as for example plastics, acrylic
polymers, et cetera) or partly synthetic (such as for example
modified celluloses, modified starches, et cetera). The metallic
nano-particles comprised within the coating are preferably used in
powder form. As regards the size distribution within the powder,
preferably an amount at least equal to 70%, 75%, or 80% by weight
of the particles that are present in the powder, more preferably at
least equal to 85%, has a particle diameter ranging from 40 to 100
nm, 50 to 90 nm, or 60 to 80 nm.
[0091] The electrode coating can be provided by means of
nanotechnology techniques which are known to a person skilled in
the art and are adapted to produce a smooth surface, for example by
sintering the powder or the mixtures of metallic nano-powders.
[0092] The individual metals in powder form can be applied to the
electrode so as to produce the coating: 1) as a preformed mixture,
and/or 2) in the form of discrete layers which are applied
sequentially and mutually superimposed and wherein each layer
consists of a single metal, and/or 3) in the form of discrete
layers which are applied sequentially and mutually superimposed and
in which each layer consists of two or more metals but not
simultaneously of all the metals that are present in the
coating.
[0093] Preferably, the method comprises the step (A) of preparing
the coating of the electrode by sintering powders of nano-particles
of one or more metals as defined above directly on the core of the
electrode. Preferably, step (A) comprises the following steps to be
performed in the order in which they are listed here:
[0094] (A1) preparing one or more powders of metallic
nano-particles as defined above,
[0095] (A2) dissolving the one or more powders of nano-particles in
a suitable solvent and in at least such a quantity as to be able to
dissolve all the powder to be applied, obtaining one or more
solutions, and
[0096] (A3) sintering the one or more solutions obtained in the
preceding step on a metal plate, preferably passivated on its
surface, which will form the core of the electrode.
[0097] Preferably: [0098] the one or more powders of metallic
nano-particles of step (A1) is a combination of powders of
ZrO.sub.2, ZnO, Ru.sub.2O.sub.3, IrO.sub.2 and Y.sub.2O.sub.3, or
TiO.sub.2, Pt/ZrO.sub.2, SnO.sub.2, Ta.sub.2O.sub.5, and IrO.sub.2,
advantageously obtained by hydrothermal chemical processing, at
least 70%, 75%, or 80% and more preferably at least 85% by weight
of the particles in the powder have a diameter ranging from 60 to
80 nm; [0099] the solvent of step (A2) in which each powder is
dissolved is preferably a 30% solution by weight of hydrochloric
acid in water, in at least such an amount as to be able to dissolve
all the powder to be applied, [0100] step (A3) consists in
sintering the aqueous solutions of hydrochloric acid obtained from
step A(2) on both faces of a TiO.sub.2 plate which is passivated on
its surface and has a thickness ranging from 0.15 to 0.35 mm,
wherein sintering may occur according to the steps listed below in
Table 2:
TABLE-US-00002 [0100] TABLE 2 Sintering Steps Sintering Dosage per
unit Sintering temperature Step Solution surface time (min)
(.degree. C.) 1 IrO.sub.2 0.2 g/m.sup.2 45 450 2 Ru.sub.2O.sub.3
0.2 g/m.sup.2 45 450 3 ZnO + Y.sub.2O.sub.3 0.15 g/m 60 550 (Y at 2
mol) 4 IrO.sub.2 0.25 g/m.sup.2 45 450 5 Ru.sub.2O.sub.3 0.25
g/m.sup.2 60 550 6 ZrO.sub.2 + Y.sub.2O.sub.3 0.1 g/m.sup.2 60 550
(Y at 3 mol) 7 Ru.sub.2O.sub.3 0.15 g/m.sup.2 60 550 8 IrO.sub.2
0.15 g/m.sup.2 60 550 9 IrO.sub.2 + Ru.sub.2O.sub.3 0.15 g/m.sup.2
+ 0.15 g/m.sup.2 60 600 10 ZrO.sub.2 + Y.sub.2O.sub.3 0.1 g/m 60
600 (Y at 3 mol) 11 IrO.sub.2 + Ru.sub.2O.sub.3 0.15 g/m.sup.2 +
0.15 g/m.sup.2 60 600
[0101] Resorting to multiple sintering steps has been found to be
particularly useful in order to eliminate any roughness from the
surface of the electrode and obtain an extremely hard and smooth
surface. An electrode as defined above, used as part of a device
for providing the electrolysis of water, produces the following
advantages: [0102] more efficient electrolysis, in that there is a
lower consumption of salts such as NaCl, used conventionally to
accelerate the electrolysis of low-conductivity fluids such as
water; and [0103] if both electrodes are electrodes according to
the invention, the possibility to provide a continuous change of
polarity of the electrodes ("polarity swapping"). The sudden change
of polarity allows the charged particles that are present in the
fluid subjected to electrolysis to circulate in both directions
instead of just in one (forced by the charge of the particles and
by the unchangeable sign of the electrodes), thus avoiding the
forming of deposit-producing masses at the level of the electrodes
and thus keeping their surface clean and their efficiency at the
maximum level. Moreover, if a semipermeable membrane is provided
within the electrolytic cell and divides the two anode and cathode
half-chambers, the change of polarity avoids the clogging of the
pores of said membrane, extending the life of the device; [0104]
the presence of a nanometer coating determines an accumulation of
charge by the upper electrode to more than 100% with respect to
conventional electrodes. This allows to provide a qualitatively and
quantitatively different electrolysis at significantly higher
potentials, with the effect of, for example, reducing the size of
molecular clusters; [0105] the obtainment of a very high
consistency, smoothness and surface density, aspects which avoid
the solubilization of the electrode itself or the forming of
sediments on its surface, which would then occur in the acid and
alkaline water fractions. The same aspects are also the basis for
the substantially nil release of heavy metals and other compounds
which constitute the surface and core of the electrode within the
acid and alkaline water fractions. As will be mentioned also
hereafter, the absence of heavy metals in the water leads to an
amazing stability thereof over time, with preservation of
characteristics such as ORP, pH and molecular cluster size. This
stability is unknown to known equivalent products. The same aspects
are also the basis for the minimal maintenance required by the
electrode, which can be changed with a significantly lower
frequency than known electrodes, reducing costs and increasing ease
of production; [0106] the possibility to obtain quantum effects
(known in the literature also by the term "nano-effects") by means
of the nanometer dimensions of the coating particles. Briefly, when
nanometer dimensions are reached, the optical, magnetic and
electrical properties of matter change radically. By reducing the
dimensions until the typical nanometer dimensions of so-called
clusters are reached, due to the small number of atoms that are
present in said cluster and to its reduced volume, a discretization
of the energy levels (quantization) becomes apparent in the
electron structure and depends on the size of the cluster, this
phenomenon is known as "quantum size effect" and entirely new
characteristics, which contrast with the ones that are typical of
the material at ordinary dimensions, depend from it. In the present
case, the best performance has been obtained with powders which
have a size distribution centered in an interval ranging from 60 to
80 nm as indicated above. As a whole, the effects described above
produce the simultaneous presence of three factors: stability of
the resulting water, ease of its production (for example thanks to
the lower maintenance costs and to the greater durability of the
device as a whole) and an increase in its quality (especially in
terms of purity and constancy of properties over time). In
particular, the increase in the quality of the water can be
measured both in terms of uniformity of the dimensions of the
molecular clusters (higher percentage of micromolecules with
respect to the number of macromolecular clusters) and in terms of
increased stability over time of the properties given to the water
by the electrolysis itself (above all pH, ORP and cluster size).
The stability increase presumably achieves the preservation over
time of the structural surface characteristics of the electrodes
coated with a nano-coating as described here.
EXAMPLES
Example 1
Objective
[0107] Determine the efficacy of acidic nanoclustered water (ANW)
against several strains of bacteria, viruses and fungi.
[0108] Bacterial Activity
[0109] Bacterial activity was assessed with the method of UNI
(Italian Organization for Standardization) EN 1040 (quantitative
suspension test for the evaluation of basic bactericidal activity
of chemical disinfectants and antiseptics). According to this
method, a substance is classified as bactericidal for a specific
microorganism if it reduces the bacterial count by at least
5-log.sub.10 following 5 minutes of contact at 20.degree. C. ANW
solutions at three different concentrations (80%, 50%, and 25%)
were tested against two strains of bacteria known to cause eye
infections, Staphylococcus aureus (ATCC 6538) and Pseudomonas
aeruginosa (ATCC 15442). Table 3 below shows the antibacterial
effect of the three different concentrations of ANW, with viability
reduction values expressed as the log.sub.10 reduction.
TABLE-US-00003 TABLE 3 Antibacterial Effect of ANW Viability
Reduction Species Solution 80% 50% 25% Staphylococcus aureus ANW
>5.41 >5.41 5.35 (ATCC 6538) Pseudomonas aeruginosa ANW
>5.48 <4.10 <4.10 (ATCC 15442)
[0110] As shown in Table 3, ANW can be classified to be a
bactericidal against both strains at a concentration of 80%.
[0111] Bacterial activity was also assessed against the same two
strains of bacteria in the presence of 5% of human blood in the
medium as organic soil interference. Viability reduction was
assessed after 10, 30, 60 and 120 minutes of exposure to pure ANW
at 31.degree. C. Table 4 below shows the antibacterial effect of
the pure ANW at each time point, with viability reduction values
expressed as the log.sub.10 reduction.
TABLE-US-00004 TABLE 4 Antibacterial Effect of ANW in Presence of
5% Human Blood Viability Reduction Species Solution 10 min 30 min
60 min 120 min Staphylococcus aureus ANW >5.6 >5.6 >5.6
>5.6 (ATCC 6538) Pseudomonas aeruginosa ANW >5.6 >5.6
>5.6 >5.6 (ATCC 15442)
[0112] As shown in Table 4, pure ANW was demonstrated to have a
bactericidal effect against both strains at the lowest tested time
point of 10 minutes.
[0113] Bacterial activity was also assessed against
Propionibacterium acnes bacteria in the presence of 1% fetal bovine
serum in the medium as organic soil interference. Viability
reduction was assessed after 1, 5, 15 and 30 minutes of exposure to
pure ANW at 31.degree. C. Table 5 below shows the antibacterial
effect of the pure ANW at each time point, with viability reduction
values expressed as the log.sub.10 reduction.
TABLE-US-00005 TABLE 5 Antibacterial Effect of ANW in Presence of
1% Fetal Bovine Serum Viability Reduction Species Solution 1 min 5
min 15 min 30 min Propionibacterium acnes ANW >6.9 >6.9 5.3
>6.9 (ATCC 11827)
[0114] As shown in Table 5, pure ANW was demonstrated to have a
bactericidal effect at the lowest tested time point of 1
minute.
[0115] Fungal Activity
[0116] Fungal activity was assessed with the method of UNI EN 1275
(quantitative suspension test for the evaluation of basic
fungicidal activity of chemical disinfectants and antiseptics).
According to this method, a substance is classified as fungicidal
for a specific microorganism if it reduces the fungi count by at
least 4-log.sub.10 following 15 minutes of contact at 20.degree. C.
ANW solutions at three different concentrations (80%, 50%, and 25%)
were tested against two strains of fungus known to cause
infections, Candida albicans (ATCC 10231) and Aspergillus niger
(ATCC 16404). Table 6 below shows the antifungal effect of the
three different concentrations of ANW, with viability reduction
values expressed as the log.sub.10 reduction.
TABLE-US-00006 TABLE 6 Antifungal Effect of ANW Viability Reduction
Species Solution 80% 50% 25% Candida albicans (ATCC ANW >4.37
<3.09 <3.09 10231) Aspergillus niger (ATCC ANW <3.12
<3.12 <3.12 16404)
[0117] As shown in Table 6, ANW can be classified to be a
bactericidal against Candida albicans (ATCC 10231) at a
concentration of 80%.
[0118] Viral Activity
[0119] Viral activity was assessed against Human Immunodeficiency
Virus type 1 (HIV-1), Herpes Simplex Virus type 1 (HSV-1), and
Herpes Simplex Virus type 2 (HSV-2) in the presence of 5% fetal
bovine serum in the medium as organic soil interference. For HIV-1,
viability reduction was assessed after 10 minutes of exposure to
pure ANW at 21.5.degree. C. For HSV-1 and HSV-2, viability
reduction was assessed after 5 minutes of exposure to pure ANW at
35.degree. C. Table 7 below shows the antiviral effect of the pure
ANW on each virus, with viability reduction values expressed as the
log.sub.10 reduction.
TABLE-US-00007 TABLE 7 Antiviral Effect of ANW Viability Species
Solution Exposure Reduction HIV-1 (strain HTLV-III.sub.b) ANW 10
min at 21.5.degree. C. >4.5 HSV-1 (ATCC VR733) ANW 5 min at
35.degree. C. <5.5 HSV-2 (ATCC VR734) ANW 5 min at 35.degree. C.
4.25
[0120] As shown in Table 7, ANW can inactivate the tested
viruses.
Example 2
Objective
[0121] Determine the efficacy of acidic nanoclustered water (ANW)
at promoting corneal healing and cataract healing.
[0122] Corneal Ulceration Healing
[0123] Corneal healing activity was assessed in an in vivo rabbit
model. Corneal eye wounds were experimentally provoked in 8
rabbits. The left and right eyes were then treated with ANW and
saline, respectively, by applying 100 .mu.l of the solutions 4
times per day for 14 consecutive days. On the 4.sup.th, 9.sup.th,
and 14.sup.th day after the surgery, images of each wound were
taken under a slit lamp microscope and the area of each wound was
calculated with the software Topcon IMAGENET 2000. The wound area
was then used to calculate the wound healing rate (WHR) using the
following equation:
WHR=100(1-(wound area at timing point)/(initial wound area))
[0124] Table 8 below shows the Wound Healing Rate of ANW and saline
treated eyes at 4, 9, and 14 days.
TABLE-US-00008 TABLE 8 Corneal Ulceration Healing Effect of ANW and
Saline Wound Healing Rate (WHR) Day 4 Day 9 Day 14 ANW 77.78 .+-.
9.06 83.32 .+-. 12.23 87.20 .+-. 13.16 Saline 71.84 .+-. 19.38
63.45 .+-. 23.02 64.23 .+-. 28.28 T-value 1.36 3.73 3.61 P-value
>0.05 <0.05 <0.05
[0125] As shown in Table 8, ANW was significantly more effective
than saline in corneal ulcer healing at the latter two of the three
time points.
[0126] The wounds were also observed daily for the presence of
wound closure. On day 14, it was observed that half of the corneal
ulcers treated with ANW were healed, while some corneas treated
with saline were still presenting a large ulcer. Exemplary
photographs (not shown) were taken of the corneal wounds in 3 of
the rabbits on day 14.
[0127] The wounds were also observed daily for the presence of
infections and inflammation. ANW was observed to reduce
inflammation after injury. Furthermore, two of the eyes treated
with saline were seriously infected with hypopyon, and the
inflammation of these corneas was too intensive to identify the
pupil. Exemplary photographs and histological images (not shown)
were obtained of the inflammation in 2 of the rabbits.
[0128] Histological evaluation also was used to observe
regeneration of the cornea and scarring. ANW was observed to
increase regeneration and reduce scarring as compared to saline.
Furthermore, epithelium deficiency was observed in all of the eyes
treated with saline, and none of the eyes treated with ANW.
Histological images (not shown) depicting scarring of cornea wounds
were taken in 3 of the rabbits.
[0129] The wounds were also observed for the presence of
angiogenesis. Neovascularization was observed in 35% of the corneas
treated with saline, and none of the eyes treated with ANW.
Exemplary photographs (not shown) depicting angiogenesis were taken
of 2 of the rabbits.
[0130] Cataract Healing
[0131] Cataract healing activity was assessed in an in vivo rat
model. Cataract was induced by intraperitoneal injection of
d-galactose in 1 rat at a dose of 10 g/kg per day (twice/day). The
left and right eyes were then treated with ANW and saline,
respectively, by applying 1 drop of the solutions 4 times per day
for 30 consecutive days. On day 30, it was observed that the
cataract treated with ANW was significantly healed as compared to
the cataract treated with saline. Photographs (not shown) depicting
the cataracts were taken on day 30.
Example 3
Objective
[0132] Determine the safety of acidic nanoclustered water (ANW) in
systemic and topical applications.
[0133] In Vitro Studies
[0134] Citotoxicity was assessed with the method of ISO
(International Organization for Standardization) 10993-5. According
to this method, a substance is classified based on its effect on a
cell culture. 100 .mu.l of pure ANW was applied to a cell culture
of murine fibroblasts L-929 and the cells were evaluated after 24
hours of incubation at 37.degree. C. Some malformed cells were
observed after the period of incubation. Based on these results,
ANW was defined as "slightly cytotoxic" (grade 1 of 4).
[0135] Mutagenicity was assessed with the method of OECD 471.
According to this method, a substance is classified based on its
ability to induce point mutations in bacteria. Five mutant strains
of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, and TA
102) were studied both in the presence and in the absence of ANW.
Based on the results of a reverse mutation assay (Ames' test), the
substance ANW was defined as non mutagenic.
[0136] Systemic Toxicity Studies
[0137] Acute toxicity was assessed with the method of ISO 10993-11,
2006, Biological Evaluation of Medical Devices--Part 11: Tests for
Systemic Toxicity. According to this method, a substance is
classified not toxic if animals injected with the substance do not
show a significantly greater biological reaction than animals
treated with a control article. 10 female Swiss albino mice were
injected by intraperitoneal route with either ANW or saline in the
amount of 50 mL/kg. The animals were observed for clinical signs
immediately after injection, and at 4, 24, 48, and 72.+-.2 hours
after injection. ANW did not induce a significantly greater
biological reaction than the control, and was therefore classified
as not toxic.
[0138] Skin Irritation Studies
[0139] Dermal irritation following acute exposures was assessed
with the method of ISO 10993-10. 0.5 mL of pure ANW was applied
with a patch on the shaved skin of three male albino rabbits. The
patch was held on the skin by means of a non-irritating adhesive
plaster for 4 hours. The skin reaction was observed upon removal of
the patch and 24, 48, and 72 hours after removal. No sign of either
erythema or edema was observed. Based on these results, ANW was
determined to be non-irritating for skin, which a Skin Irritation
Index of 0.00.
[0140] Dermal irritation following repeated exposure also was
assessed with the method of ISO 10993-10. Three male New Zealand
rabbits were treated 5 days a week for 4 weeks with two consecutive
daily administrations of 5 mL of pure ANW or saline as a control
applied with a patch for one hour. The skin reaction was before and
after each application throughout the entire 4 week period. No sign
of either erythema or edema was observed. Furthermore, upon
sacrifice, no signs of inflammation were detected in histological
images. Based on these results, ANW was determined to not exhibit
any significant irritancy in the skin.
[0141] Skin Sensitization Studies
[0142] Delayed-type skin hypersensitivity was assessed with the
method of ISO 10993-10: Guinea-Pig Maximization test. The test used
15 albino female Hartley guinea pigs (10 treated and 5 control).
The injection phase (Day 0) was carried out by administering three
0.1 mL intradermal injections to each animal: (a) complete Freund's
adjuvant, (b) either pure ANW (test) or saline (control), (c)
either ANW (test) or saline (control) mixed together with complete
Freund's adjuvant. A skin massage with 1 mL SLS 10% was then
performed on Day 6. The induction phase (Day 7) was carried out by
applying 1 mL of either the test or the control, left in place for
48 hours with an occlusive patch. The challenge was carried out on
Day 21 through the application to each animal (both treated and
control) of dressings with of 1 mL of ANW on the right side and 1
mL of saline on the left side, left in place for 24 hours.
[0143] Assessments were carried out on the 23rd day (24 hours after
patch and removal) and on the 24th day (48 hours after patch and
removal). The intensity of erythema and/or edema was evaluated
according to the Magnusson and Kligman scale from 0 to 3.
[0144] No abnormalities were observed in either the treated or the
control animals. Based on these results, ANW was determined to not
exhibit delayed contact dermatitis potential.
[0145] Ocular Irritation Studies
[0146] Ocular irritation was assessed with the method of ISO
10993-10. In a first experiment, three New Zealand white rabbits
were treated by instilling 0.1 mL of pure ANW into the left eye,
leaving the right eye untreated as a control. The eyes were
examined 1, 24, 48 and 72 hours after instillation through
fluorescein staining and slit-lamp observation. No signs of
irritation were observed in any of the eyes. Based on these
results, ANW was determined to be a non-irritant for the ocular
tissue of New Zealand White rabbits.
[0147] In a second experiment, ocular irritation was again assessed
with the method of ISO 10993-10. Three New Zealand white rabbits
were treated by instilling 0.1 mL of pure ANW into the left eye as
a test, and instilling 0.1 mL of NaCl containing water (saline)
into the right eye as a control. The treatment was repeated for 30
consecutive days. No signs of irritation were observed in any of
the test or control eyes at any of the observation points. Based on
these results, the test article ANW was determined to be a
non-irritant for the ocular tissue of New Zealand White
rabbits.
[0148] Summary of Toxicology Studies
[0149] Table 9 below shows a synopsis of the safety studies
reported above in Example 3.
TABLE-US-00009 TABLE 9 Synopsis of ANW Safety Studies Effect Method
Results Cytotoxicity In vitro mouse fibroblasts Slightly cytotoxic
L-929 (grade 1 of 4) Acute Skin Irritation Acute exposure in
rabbits Non-irritant Repeated Skin Irritation Repeated exposure
Non-irritant in rabbits Delayed hypersensitivity Maximisation test
in Non-sensitising guinea pigs Primary ocular irritation Acute
administration Non-irritant in rabbits Repeated ocular irritation
Repeated administration Non-irritant in rabbits Genotoxicity Ames
test Non-mutagenic Acute Systemic Toxicity i.p. dosing in mice Not
toxic up to 50 mL/kg i.p.
Example 4
Objective
[0150] Determine the efficacy of acidic nanoclustered water (ANW)
for modulating the activity of the immune system
[0151] In Vitro Study of PBMC Proliferation
[0152] The ability of ANW to inhibit the proliferation of
peripheral blood mononuclear cells (PBMC) was assessed in an in
vitro cellular model using 12 batches of PCMC. In the experiment, 4
of the batches were exposed to betamethasone (10 nM), a
glucocorticoid steroid with anti-inflammatory and immunosuppressive
properties, 4 of the batches were exposed to a 1:10 dilution of
ANW, and the remaining 4 batches were exposed to a 1:20 dilution of
ANW. Table 10 below shows the inhibition effect that was measured
for each of the 12 batches.
TABLE-US-00010 TABLE 10 Inhibition of PBMC Proliferation Batch 1
Batch 2 Batch 3 Batch 4 Mean Betamethasone (10 nM) 87.7% 85.7%
79.4% 88.5% 85.3% Acidic Nanoclustered 39.9% 29.0% 19.3% 16.6%
26.2% Water (1:10) Acidic Nanoclustered 17.3% 22.0% 4.0% 7.0% 12.6%
Water (1:20)
[0153] As shown in Table 10, ANW inhibited PBMC proliferation at
both dilutions. These dilutions have previously been shown to not
be significantly toxic on this cell type in vitro.
[0154] In Vitro Study of T-Cell Activation
[0155] The ability of ANW to modulate immunoregulatory cytokines
was assessed in an in vitro cellular model using PBMC from 4 blood
donors stimulated with purified protein derivatives from
Mycobacterium tuberculosis (PPD), a prototypical Th1 antigen. In
the experiment, batches of PBMC cells were stimulated with PPD
alone, PPD plus betamethasone (10 nM), and PPD plus Acidic
Nanoclustered Water (1:10 dilution). T-Cell activation was measured
by testing levels of three cytokines: IL-10 (expressed in T-Cells),
IFN-gamma (expressed in Th-1 cells), and IL-4 (expressed in Th-2
cells). FIG. 3 shows the levels of each cytokine as compared to
non-stimulated PBMC.
[0156] As shown in FIG. 3, ANW up-regulated IL-10 production by
stimulated PMBC in a statistically significant way (T test:
p<0.05). Interleukin IL-10 is an important immunoregulatory
cytokine. Its main biological function is to limit and terminate
inflammatory responses, and to regulate the differentiation and
proliferation of several immune cells. IL-10 deficiency is regarded
as pathophysiologically relevant in inflammatory disorders
characterized by a type 1 cytokine pattern such as psoriasis. Thus,
the immune-modulating properties of ANW and, specifically, the
IL-10 activating properties of ANW, suggest than ANW can be used as
a direct therapeutic agent for several skin diseases.
Example 5
Objective
[0157] Compare the stability of acidic nanoclustered water (ANW)
with the reported stability of other electrolyzed waters of similar
pH, ORP, and composition.
[0158] Comparison of ANW with Electrolyzed Oxidizing (EO) Water of
Soo-Voo Len
[0159] The stability of ANW in open and closed conditions at
25.degree. C. was compared with the stability of EO water as
reported in the article "Effects of Storage Conditions and pH on
Chlorine Loss in Electrolyzed Oxidizing (EO) Water"--Journal of
Agricultural and Food Chemistry--2002, 50, 209-212 by Soo-Voon Len,
et al.
[0160] In Soo-Voo Len, electrolyzed water with an acidic pH
(2.5-2.6), OPR >1000 mV (1020-1120), and a free chlorine content
.about.50 ppm (53-56 ppm) was generated with an ROX-20TA device
manufactured by Hoshizaki Electric Inc. (Aichi, Japan) using a
current intensity of 14 Ampere and 7.4 Volt. The article reports
that in an open condition at 25.degree. C., the chlorine in the
electrolyzed water was completely lost after 30 hours when
agitated, and after 100 hours when not agitated. Furthermore, as
seen in FIG. 1 of the article, the free chlorine was almost
completely lost after 10 hours in open, agitated, diffused light
conditions. The article also reports that in a closed dark
condition at 25.degree. C., the free chlorine in the electrolyzed
water decreased by approximately 40% after 1400 hours (about 2
months).
[0161] In comparison, ANW stored in an open condition at 25.degree.
C. without agitation completely lost chlorine after 240 hours (10
days), more than twice as long as the EO water in Soo-Voo Len (100
hours). Furthermore, ANW stored in an open condition at 25.degree.
C. with agitation and light completely lost chlorine after 24
hours, more than twice as long as the EO water in Soo-Voo Len (10
hours). Finally, ANW stored in a closed condition at 25.degree. C.
lost 8.44% of free chlorine after 3 months, less than a quarter as
much as was lost from the EO water in Soo-Voo Len after about 2
months (40%).
[0162] Comparison of ANW with Electrolyzed Oxidizing (EO) Water of
Shun-Yao Hsu
[0163] The stability of ANW in closed conditions at around
30.degree. C. was compared with the stability of EO water as
reported in the article "Effects of storage conditions on chemical
and physical properties of electrolyzed oxidizing water"--Journal
of Food Engineering 65 (2004) 465-471 by Shun-Yao Hsu, et al.
[0164] In Shun-Yao Hsu, the electrolyzed water of "formulation J"
had an acidic pH (2.61), OPR=1147 mV, and a free chlorine content
of 56 ppm. The article reports that in a closed condition at
25-30.degree. C., the free chlorine in the electrolyzed water was
43 ppm after 21 days, a 23% loss. In comparison, ANW samples stored
in closed conditions at 25.degree. C., 30.degree. C., and
40.degree. C. without agitation lost 8.44%, 8.64%, and 15.43% of
free chlorine after 3 months and 12.14%, 19.13%, and 18.31% of free
chlorine after 1 year, respectively.
Example 6
[0165] The properties and composition of Acidic Nanoclustered Water
were tested and found to have specifications as reported below in
Table 11. The properties and composition Acidic Nanoclustered Water
were also analyzed as reported below in Table 12.
TABLE-US-00011 TABLE 11 Acid Water Specifications Test Item Method
Specification Appearance Naked eye Liquid Odour Smell
Characteristic Colour Naked eye Colourless pH as is @ 25.degree. C.
<3.00 by Mettler Toledo pHmeter SevenMulti- Potentiometric
Determination (Ph Eur. 2.2.3 - Current Ed.) OxidoReductive as is @
25.degree. C. >1100.0 Potential by Mettler Toledo combination
redox electrode (P/N ORP (mV) 51343200) Potentiometric Tritation
(Ph Eur. 2.2.20 - Current Ed.) Conductivity as is @ 25.degree. C.
<1300 (uS cm.sup.-1) Free Chlorine Internal Method M37-07
40.0-70.0 Assay (mg/l or Spectrophotometric Method ppm) source APAT
IRSA CNR HandBook Volume 2 - Ref 4080 Total Chlorine Internal
Method M37-07 40.0-70.0 Assay (mg/l or Spectrophotometric Method
ppm) source APAT IRSA CNR HandBook Volume 2 - Ref 4080 Total
Chlorine Internal Method M37-07 40.0-70.0 Assay (mg/l or Iodometric
Method ppm) source APAT IRSA CNR HandBook Volume 2 - Ref 4080
Chloride Assay Internal Method M05-08 <200.0 ((mg/l or ppm)
Spectrophotometric Method source APAT IRSA CNR HandBook Volume 2 -
Ref 4090 Chlorites EPA 300.1, 1997 (as ClO.sub.2) <100 (.mu.g/l
or ppb)) Chlorates EPA 300.1, 1997 <1 (mg/l or ppm) .sup.17O-NMR
(Hz) .sup.17O-NMR Spectrometer <50 Linewidth @ 50% Heavy metals
ICP Method <10 ppm [Ag, As, Bi, Cd, Cu, Hg, Mo, Pb, Sb, Sn]
Yttrium by EPA 200.8 1994 (0.1 .mu.g/l detection limit) <0.1 ppm
(ICP Method) Zinc by EPA 200.8 1994 (0.1 .mu.g/l detection limit)
<0.1 ppm (ICP Method) Iridium by EPA 200.8 1994 (0.1 .mu.g/l
detection limit) <0.1 ppm (ICP Method) Titanium by EPA 200.8
1994 (0.1 .mu.g/l detection limit) <0.1 ppm (ICP Method)
Zirconium by EPA 200.8 1994 (0.1 .mu.g/l detection limit) <0.1
ppm (ICP Method) Ruthenium by EPA 200.8 1994 (0.1 .mu.g/l detection
limit) <0.1 ppm (ICP Method)
TABLE-US-00012 TABLE 12 Acid Water Test Results ANW ANW ANW LOT LOT
LOT LCOVI/57 LCOX/5 LCOX/1 Appearance colourless Same Same liquid
with light chlorine smell (like swimming pool) Free Chlorine Assay
(mg/l) 53.1 48.6 49.9 Spectrophotometric Method Total Chlorine
Assay (mg/l) 52.1 48.6 49.0 Spectrophotometric Method Total
Chlorine Assay (mg/l) 60.6 54.9 56.7 Iodometric Method Chloride
Assay (mg/l) 138 194.0 183.4 UNI 24012 (Mercurimetric method)
Chlorites .mu.g/l (as ClO.sub.2) by <100 100 <100 EPA 300.1
1997 (detection limit 100 .mu.g/l) Chlorates mg/l by EPA 1.20 1.5
0.9 300.1 1997 (detection limit 0.1 mg/l) pH 2.59 2.71 2.81 (as is
by Mettler Toledo pH meter Met Rohm 744) ORP by Mettler Toledo
1151.8 1121.7 1110.5 PT4805-60-88TE-S7/120 combination redo
electrode .sup.17O NMR (Linewidth @ 45.76 45.33 46.07 50%-Hz) Heavy
Metals <10 ppm <10 ppm <10 ppm (Ag, As, Bi, Cd, Cu, Hg,
Mo, Pb, Sb, Sn)
Example 6
[0166] The stability of Acidic Nanoclustered Water compositions
containing different amounts of chloride ion were tested both in
storage and in the open air. The low chloride composition contained
less than 200 ppm chloride, and the high chloride composition
contained 1100 ppm chloride.
[0167] To test storage stability, the compositions were stored in a
closed condition at 25.degree. C. and 60% relative humidity, and
were not agitated or exposed to diffused light. After 3 and 12
months, the low chloride composition had a loss of free chlorine of
8.44% and 12.14%, respectively. In contrast, the high chloride
composition had a loss of free chlorine of 27.4% after only 3
months.
[0168] To test open air stability, the two compositions were kept
open, agitated, and exposed to light for 24 hrs at a temperature of
30.degree. C. As illustrated in FIG. 5, the high chloride
composition lost free chlorine at a greater rate than the low
chloride composition.
[0169] These results demonstrate that the stability of ANW is
dependent of chlorine remaining HClO, which prevents both
evaporation and decomposition.
[0170] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0171] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0172] All references cited herein, including patents, patent
applications, and published patent applications, are hereby
incorporated by reference in their entireties, whether or not each
is further individually incorporated by reference.
* * * * *