U.S. patent application number 14/336307 was filed with the patent office on 2015-01-22 for methods of therapeutic treatment of eyes.
The applicant listed for this patent is Revalesio Corporation. Invention is credited to Gregory J. Archambeau, Richard L. Watson, Anthony B. Wood.
Application Number | 20150024010 14/336307 |
Document ID | / |
Family ID | 43124695 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024010 |
Kind Code |
A1 |
Archambeau; Gregory J. ; et
al. |
January 22, 2015 |
METHODS OF THERAPEUTIC TREATMENT OF EYES
Abstract
Provided are electrokinetically-altered aqueous fluids (e.g.,
gas-enriched electrokinetic fluids) comprising an ionic aqueous
solution of charge-stabilized oxygen-containing nanostructures in
an amount sufficient to provide modulation of at least one of
cellular membrane potential and cellular membrane conductivity, and
therapeutic compositions and methods for use in treating an
irritation, infection or inflammatory eye condition, comprising
administering to, by contacting the eye of a subject in need
thereof a therapeutically effective amount of an
electrokinetically-altered aqueous fluid. The
electrokinetically-altered fluids or therapeutic compositions and
methods include electrokinetically-altered ioinic aqueous fluids
optionally in combination with other therapeutic agents. Other
embodiments include particular routes of administration or
formulations for the electrokinetically-altered fluids (e.g.,
electrokinetically-altered gas-enriched fluids) and therapeutic
compositions for use in treating eye conditions. Certain
embodiments relate to cosmetic and/or therapeutic fluids and/or
methods of treatment utilizing the fluids to treat a cosmetic
and/or therapeutic symptom related to eye conditions and/or
diseases.
Inventors: |
Archambeau; Gregory J.;
(Puyallup, WA) ; Wood; Anthony B.; (Tacoma,
WA) ; Watson; Richard L.; (Ruston, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Revalesio Corporation |
Tacoma |
WA |
US |
|
|
Family ID: |
43124695 |
Appl. No.: |
14/336307 |
Filed: |
July 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12769136 |
Apr 28, 2010 |
8784897 |
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14336307 |
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12431577 |
Apr 28, 2009 |
8609148 |
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12769136 |
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11924601 |
Oct 25, 2007 |
8591957 |
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12431577 |
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60862953 |
Oct 25, 2006 |
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60862959 |
Oct 25, 2006 |
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60862955 |
Oct 25, 2006 |
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60862904 |
Oct 25, 2006 |
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60982387 |
Oct 24, 2007 |
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61048357 |
Apr 28, 2008 |
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60862953 |
Oct 25, 2006 |
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60862959 |
Oct 25, 2006 |
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60862955 |
Oct 25, 2006 |
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60862904 |
Oct 25, 2006 |
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60982387 |
Oct 24, 2007 |
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61175409 |
May 4, 2009 |
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Current U.S.
Class: |
424/400 ;
424/613; 435/1.2 |
Current CPC
Class: |
A61K 9/5115 20130101;
A61K 33/00 20130101; A61K 9/10 20130101; B01F 3/0807 20130101; A61K
45/06 20130101; B01F 3/04531 20130101; A61P 27/02 20180101; A61K
2300/00 20130101; A61K 31/573 20130101; B01F 3/0853 20130101; A01N
1/0226 20130101; A61K 31/573 20130101; A61K 9/0048 20130101; A61K
33/00 20130101; B01F 7/00816 20130101; A61K 2300/00 20130101; A61K
33/40 20130101 |
Class at
Publication: |
424/400 ;
424/613; 435/1.2 |
International
Class: |
A61K 33/40 20060101
A61K033/40; A01N 1/02 20060101 A01N001/02; A61K 45/06 20060101
A61K045/06 |
Claims
1.-69. (canceled)
70. A method of harvesting, and/or storing and/or transporting
organs, organ samples, living tissues or cells, comprising
contacting organ, organ sample, living tissue or cells, during
harvesting, storing and/or transporting thereof, with an effective
amount of an ionic aqueous solution of charge-stabilized
oxygen-containing nanobubbles having an average diameter of less
than 100 nanometers persisting in the ionic aqueous fluid
sufficient for preserving, storing and/or transplanting, with less
cell damage, the organ, organ sample, living tissue or cells,
during the harvesting, storing, transporting and/or transplanting
thereof.
71. The method of claim 70, wherein contacting the organ, organ
sample, living tissue or cells, during harvesting, storing,
transporting and/or transplanting thereof with the ionic aqueous
solution comprises perfusing the organ, organ sample, living tissue
or cells with the ionic aqueous solution.
72. The method of claim 70, wherein the organ, organ sample, living
tissue or cells comprises at least one of liver, kidney, heart,
eye, hand, foot, brain and stem cells.
73. The method of claim 70, wherein oxygen is present in the ionic
aqueous solution in an amount selected from the group consisting of
at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm,
at least 50 ppm, or at least 60 ppm oxygen at atmospheric
pressure.
74. The method of claim 70, wherein the amount of charge-stabilized
oxygen-containing nanobubbles in the ionic aqueous solution is at
least 15 ppm at atmospheric pressure.
75. The method of claim 73, wherein at least 90% of oxygen present
in the ionic aqueous solution is in the charge-stabilized
oxygen-containing nanobubbles.
76. The method of claim 70, wherein the ionic aqueous solution
comprises at least one of solvated electrons stabilized by
molecular oxygen, and electrokinetically modified or charged oxygen
species.
77. The method of claim 70, wherein the ionic aqueous solution
comprises saline or oxygen-enriched saline.
78. The method of claim 70, wherein the ionic aqueous solution
further comprises at least one therapeutic agent selected from the
group consisting of: anti-microbial agent, anti-inflammatory agent,
pain reliever, anesthetic, vitamin, cytokine, adjuvant,
preservative, salt, and combinations thereof.
79. A method of providing artificial blood to an organ, organ
sample, living tissue or cells, comprising contacting an organ,
organ sample, living tissue or cells with an effective amount of an
ionic aqueous solution of charge-stabilized oxygen-containing
nanobubbles having an average diameter of less than 100 nanometers
persisting in the ionic aqueous fluid sufficient for preserving,
with less cell damage, the organ, organ sample, living tissue or
cells.
80. The method of claim 79, wherein contacting the organ, organ
sample, living tissue or cells with the ionic aqueous solution
comprises perfusing the organ, organ sample, living tissue or cells
with the ionic aqueous solution.
81. The method of claim 79, wherein the organ, organ sample, living
tissue or cells comprises at least one of liver, kidney, heart,
eye, hand, foot, brain and stem cells.
82. The method of claim 79, wherein oxygen is present in the ionic
aqueous solution in an amount selected from the group consisting of
at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm,
at least 50 ppm, or at least 60 ppm oxygen at atmospheric
pressure.
83. The method of claim 79, wherein the amount of charge-stabilized
oxygen-containing nanobubbles in the ionic aqueous solution is at
least 15 ppm at atmospheric pressure.
84. The method of claim 82, wherein at least 90% of oxygen present
in the ionic aqueous solution is in the charge-stabilized
oxygen-containing nanobubbles.
85. The method of claim 79, wherein the ionic aqueous solution
comprises at least one of solvated electrons stabilized by
molecular oxygen, and electrokinetically modified or charged oxygen
species.
86. The method of claim 79, wherein the ionic aqueous solution
comprises saline or oxygen-enriched saline.
87. The method of claim 79, wherein the ionic aqueous solution
further comprises at least one therapeutic agent selected from the
group consisting of: anti-microbial agent, anti-inflammatory agent,
pain reliever, anesthetic, vitamin, cytokine, adjuvant,
preservative, salt, and combinations thereof.
88. The method of claim 79, wherein contacting the organ, organ
sample, living tissue or cells with the effective amount of the
ionic aqueous solution is during a surgical procedure.
89. The method of claim 88, wherein the surgical procedure
comprises at least one selected from the group consisting of a
coronary surgery, a shock-related procedure, a trauma-related
procedure, and an eye-related surgery.
90. An artificial blood, comprising a physiological saline solution
of charge-stabilized oxygen-containing nanobubbles having an
average diameter of less than 100 nanometers persisting in the
solution sufficient for preserving, with less cell damage, an
organ, organ sample, living tissue or cells.
91. The method of claim 90, wherein oxygen is present in the
physiological saline solution in an amount selected from the group
consisting of at least 15 ppm, at least 25 ppm, at least 30 ppm, at
least 40 ppm, at least 50 ppm, or at least 60 ppm oxygen at
atmospheric pressure.
92. The method of claim 90, wherein the amount of charge-stabilized
oxygen-containing nanobubbles in the physiological saline solution
is at least 15 ppm at atmospheric pressure.
93. The method of claim 90, wherein the physiological saline
solution further comprises at least one therapeutic agent selected
from the group consisting of: anti-microbial agent,
anti-inflammatory agent, pain reliever, anesthetic, vitamin,
cytokine, adjuvant, preservative, salt, and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/769,136 filed Apr. 28, 2010 and entitled
METHODS OF THERAPEUTIC TREATMENT OF EYES (which will issue on Jul.
22, 2014 as U.S. Pat. No. 8,784,897), which is a
continuation-in-part of U.S. patent application Ser. No. 12/431,577
filed Apr. 28, 2009 and entitled METHODS OF THERAPEUTIC TREATMENT
OF EYES (now U.S. Pat. No. 8,609,148 issued on Dec. 17, 2013),
which is a continuation-in-part of U.S. patent application Ser. No.
11/924,601 filed Oct. 25, 2007 and entitled METHODS OF THERAPEUTIC
TREATMENT OF EYES AND OTHER HUMAN TISSUES USING AN OXYGEN-ENRICHED
SOLUTION (now U.S. Pat. No. 8,591,957 issued on Nov. 26, 2013),
wherein both Ser. No. 12/431,577 and Ser. No. 11/924,601 claim
priority to U.S. Provisional Patent Application Serial Nos.
60/862,953 filed Oct. 25, 2006 and entitled METHOD OF THERAPEUTIC
TREATMENTS OF EYES AND OTHER HUMAN TISSUES USING AN OXYGENATED
SOLUTION; 60/862,959 filed Oct. 25, 2006 and entitled WOUND
TREATMENT WITH INFUSION MATERIALS; 60/862,955 filed Oct. 25, 2006
and entitled OXYGENATED SALINE SOLUTION; 60/862,904 filed Oct. 25,
2006 and entitled DIFFUSER/EMULSIFIER; and 60/982,387 filed Oct.
24, 2007 and entitled MIXING DEVICE; wherein Ser. No. 12/431,577
additionally claims priority to U.S. Provisional Patent Application
Ser. No. 61/048,357 filed Apr. 28, 2008 and entitled METHOD OF
THERAPEUTIC TREATMENT OF EYES; and wherein Ser. No. 12/769,136
additionally claims priority to U.S. Provisional Patent Application
Ser. No. 61/175,409 filed May 4, 2009 and entitled
ELECTROKINETICALLY-ALTERED FLUIDS COMPRISING CHARGE-STABILIZED
GAS-CONTAINING NANOSTRUCTURES, all of which are incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] Certain embodiments disclosed herein relate generally to the
field of eye care and treatment, and more particularly to
compositions and methods for treating irritation, infection or
inflammatory eye condition or disease of a subject comprising
administering a therapeutic composition comprising at least one
electrokinetically generated fluid (including gas-enriched (e.g.,
oxygen enriched) electrokinetically generated fluids) as disclosed
herein for modulation of at least one of cellular membrane
potential and cellular membrane conductivity, modulating
intracellular signal transduction by modulation of at least one of
cellular membranes, membrane potential, and membrane proteins such
as membrane receptors, including but not limited to G protein
coupled receptors. Particular aspects relate to treating at least
one disease or condition or symptom thereof associated with eye
care and treatment.
BACKGROUND OF THE INVENTION
[0003] The eye of an animal, particularly a human, has an outer
covering called a cornea, which refracts light rays as a first step
in the process of the animal visualizing an object. The surface of
the animal eye, particularly the human eye, is normally bathed by a
tear film that is secreted by tiny glands around the eye. The tear
film is primarily composed of three layers: mucous, water, and oil.
The mucous layer is closest to the eye organ and serves as an
anchor for the tear film to adhere to the eye. The middle layer is
an aqueous layer, and the outer oil layer seals the tear film and
prevents evaporation. The tear film also contains various nutritive
and protective proteins and peptides. The tear film serves several
purposes: it keeps the eye moist, creates a smooth surface for
light to pass through the eye, nourishes the front of the eye, and
provides protection from injury and infection.
[0004] The eye, particularly the cornea, can become dry, irritated,
or damaged by trauma, disease, natural aging, environmental factors
(such as pollutants and allergens), or by wearing contact lenses.
In some cases, individuals may experience burning, a feeling of
dryness, scratchiness, itchiness, or persistent irritation or
inflammation. This irritation can lead to infection and/or visual
impairment if not treated properly.
[0005] Red, irritated, and scratchy eyes are a common
ophthalmological occurrence affecting millions of Americans each
year. In some cases, individuals may experience burning, a feeling
of dryness, scratchiness, itchiness, or persistent irritation or
inflammation such as is often caused by particles that are lodged
between the eye lid and the eye surface. This irritation can lead
to infection and/or visual impairment if not treated properly.
[0006] Dry Eye.
[0007] Keratoconjunctivitis sicca (KCS), more commonly known as dry
eye, affects more than 40 million Americans and countless more
worldwide. KCS is an under-diagnosed disease which can produce
symptoms of ocular dryness, discomfort, and in severe cases, visual
impairment. KCS is the result of changes in tear film dynamics
leading to one of two conditions: a decrease in aqueous tear
production or an increase in tear evaporation. Both conditions
create a state of hyperosmolar stress, which leads to an
upregulation of several pro-inflammatory mediators and subsequent
ocular surface damage. The mainstay for treatment of dry eye has
been the use of over-the-counter artificial tear products. More
recent advances include the additional use of topical
anti-inflammatory agents to reduce inflammation and increase basal
tear production.
[0008] Allergy Eye.
[0009] Allergic conjunctivitis, or allergy eye, is another fairly
common ocular condition. Allergic conjunctivitis may be seasonal or
perennial, and may be triggered by any number of agents. Several
common treatments for allergy eye presently include antihistamines,
mast cell stabilizers, nonsteroidal anti-inflammatory drugs, or
corticosteroids.
[0010] Allergic conjunctivitis has an immune-mediated inflammatory
componsent. In susceptible individuals, initial exposure to the
causative allergen stimulates production of antigen-specific
antibodies (such as IgE), which bind to the surface of the mast
cells and basophils in the conjunctiva. Typical clinical
manifestations of allergic conjunctivitis include erythema, edema,
and itching. Presently, eye drops approved for the treatment of
allergic conjunctivitis are aimed at preventing or treating
allergic signs and symptoms. Allergy eye occurs in phases or
stages, such that several hours following the initial mast cell
mediated event, the clinical symptoms may worsen during the
so-called late phase reaction. The late phase reaction is
characterized by an influx of inflammatory cells, including
neutrophils, lymphocytes, and eosinophils. This results in the
release of a second wave of inflammatory mediators such as
leukotrienes, prostaglandins, and platelet activating factor
(PAF).
[0011] Prior Approaches.
[0012] The most common approach to treatment of irritated,
allergic, and/or dry eyes has been to supplement the natural ocular
tear film using so-called artificial tears instilled throughout the
day. Examples of artificial tears include buffered, isotonic saline
solutions, and/or aqueous solutions containing water soluble
polymers that render the solutions more viscous and thus less
easily shed by the eye. Tear reconstitution is also attempted by
providing one or more components of the tear film such as
phospholipids and oils. Phospholipid compositions have been shown
to be useful in treating dry eye; see, e.g., McCulley and Shine,
Tear film structure and dry eye, Contactologia, volume 20(4), pages
145-49 (1998); and Shine and McCulley, Keratoconjunctivitis sicca
associated with meibomian secretion polar lipid abnormality,
Archives of Ophth., volume 116(7), pages 849-52 (1998). Examples of
phospholipid compositions for the treatment of dry eye are
disclosed in U.S. Pat. No. 4,131,651 (Shah et al.), U.S. Pat. No.
4,370,325 (Packman), U.S. Pat. No. 4,409,205 (Shively), U.S. Pat.
Nos. 4,744,980 and 4,883,658 (Holly), U.S. Pat. No. 4,914,088
(Glonek), U.S. Pat. No. 5,075,104 (Gressel et al.), U.S. Pat. No.
5,278,151 (Korb et al.), U.S. Pat. No. 5,294,607 (Glonek et al.),
U.S. Pat. No. 5,371,108 (Korb et al.) and U.S. Pat. No. 5,578,586
(Glonek et al.).
[0013] Another approach involves the provision of lubricating
substances in lieu of artificial tears. For example, U.S. Pat. No.
4,818,537 (Guo) discloses the use of a lubricating, liposome-based
composition, and U.S. Pat. No. 5,800,807 (Hu et al.) discloses
compositions containing glycerin and propylene glycol for treating
eye irritation.
[0014] Other approaches include the use of ocular inserts that
provide a tear substitute or stimulation of endogenous tear
production. Such approaches have been met with limited success, and
provide merely temporary and minimal symptomatic relief.
[0015] There is, however, a profound need in the art for novel safe
and effective eye care treatments that allow for alleviation of eye
irritation and inflammation, and/or increases wetting of the
eye.
SUMMARY OF THE INVENTION
[0016] Particular aspects provide a method for treating an eye
irritation, eye infection or inflammatory eye condition or disease,
comprising administering to the eye of a subject in need thereof a
therapeutically effective amount of an electrokinetically-altered
aqueous fluid comprising an ionic aqueous solution of
charge-stabilized oxygen-containing nanostructures substantially
having an average diameter of less than about 100 nanometers and
stably configured in the ionic aqueous fluid in an amount
sufficient for treating an eye irritation, eye infection or
inflammatory eye condition or disease, or at least one symptom
thereof. In certain embodiments, the charge-stabilized
oxygen-containing nanostructures are the major charge-stabilized
gas-containing nanostructure species in the fluid. In particular
embodiments, the percentage of dissolved oxygen molecules present
in the fluid as the charge-stabilized oxygen-containing
nanostructures is a percentage selected from the group consisting
of greater than: 0.01%; 0.1%; 1%; 5%; 10%; 15%; 20%; 25%; 30%; 35%;
40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; and 95%. In
particular aspects, the total dissolved oxygen is substantially
present in the charge-stabilized oxygen-containing nanostructures.
In particular embodiments, the charge-stabilized oxygen-containing
nanostructures substantially have an average diameter of less than
a size selected from the group consisting of: 90 nm; 80 nm; 70 nm;
60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; and less than 5 nm.
[0017] In preferred aspects, the ionic aqueous solution comprises a
saline solution.
[0018] In particular aspects, the fluid is superoxygenated.
[0019] In certain embodiments, the fluid comprises a form of
solvated electrons.
[0020] In certain method aspects, alteration of the
electrokinetically altered aqueous fluid comprises exposure of the
fluid to hydrodynamically-induced, localized electrokinetic
effects. In particular aspects, exposure to the localized
electrokinetic effects comprises exposure to at least one of
voltage pulses and current pulses. In certain embodiments, the
exposure of the fluid to hydrodynamically-induced, localized
electrokinetic effects, comprises exposure of the fluid to
electrokinetic effect-inducing structural features of a device used
to generate the fluid.
[0021] In particular method aspects, the eye irritation, eye
infection or inflammatory eye condition or disease comprises at
least one selected from the group consisting of dry eye, allergy
irritation, corneal irritation, bacterial infection, and cellular
damage (e.g., at least one of dry eye, allergy irritation, and
corneal irritation, preferably, dry eye or allergy irritation). In
certain aspects, the at least one symptom of the eye irritation,
eye infection or inflammatory eye condition or disease is related
to at least one condition selected from the group consisting of:
dry eye-related irritation, dry eye-related cellular damage, and
allergic inflammation.
[0022] In particular method aspects, the electrokinetically altered
aqueous fluid modulates localized or cellular levels of nitric
oxide.
[0023] In certain embodiments, the electrokinetically altered
aqueous fluid promotes a localized decrease at the site of
administration of at least one cytokine selected from the group
consisting of: IL-1-beta, IL-8, TNF-alpha, and TNF-beta.
[0024] Particular method aspects, further comprise a synergistic or
non-synergistic inhibition or reduction in inflammation by
simultaneously or adjunctively treating the subject with another
anti-inflammatory or eye treatment agent (e.g., a steroid or
glucocorticoid steroid such as Budesonide or an active derivative
thereof).
[0025] Particular method aspects, further comprise combination
therapy, wherein at least one additional therapeutic agent is
administered to the patient. In particular aspects, the at least
one additional therapeutic agent is selected from the group
consisting of: anti-microbial agent, anti-inflammatory agent, pain
reliever, anesthetic, vitamin, cytokine, adjuvant, preservative,
salt, inhibitors of MMPs including inhibitor of MMP-9 and MMP-2,
short-acting .beta..sub.2-agonists, long-acting
.beta..sub.2-agonists, anticholinergics, corticosteroids, systemic
corticosteroids, mast cell stabilizers, leukotriene modifiers,
methylxanthines, .beta..sub.2-agonists, albuterol, levalbuterol,
pirbuterol, artformoterol, formoterol, salmeterol, anticholinergics
including ipratropium and tiotropium; corticosteroids including
beclomethasone, budesonide, flunisolide, fluticasone, mometasone,
triamcinolone, methyprednisolone, prednisolone, prednisone;
leukotriene modifiers including montelukast, zafirlukast, and
zileuton; mast cell stablizers including cromolyn and nedocromil;
methylxanthines including theophylline; combination drugs including
ipratropium and albuterol, fluticasone and salmeterol, budesonide
and formoterol; antihistamines including hydroxyzine,
diphenhydramine, loratadine, cetirizine, and hydrocortisone; immune
system modulating drugs including tacrolimus and pimecrolimus:
cyclosporine; azathioprine; mycophenolatemofetil; and combinations
thereof.
[0026] In particular aspects, the at least one additional
therapeutic agent is a TSLP and/or TSLPR antagonist (e.g., at least
one selected from the group consisting of neutralizing antibodies
specific for TSLP and the TSLP receptor, soluble TSLP receptor
molecules, and TSLP receptor fusion proteins, including
TSLPR-immunoglobulin Fc molecules or polypeptides that encode
components of more than one receptor chain).
[0027] In certain aspects, the charge-stabilized oxygen-containing
nanostructures are stably configured in the ionic aqueous fluid in
an amount sufficient to provide, upon contact of a living cell by
the fluid, modulation of at least one of cellular membrane
potential and cellular membrane conductivity. In particular
embodiments, modulation of at least one of cellular membrane
potential and cellular membrane conductivity comprises altering of
a conformation, ligand binding activity, or a catalytic activity of
a membrane associated protein. In certain aspects, the membrane
associated protein comprises at least one selected from the group
consisting of receptors, transmembrane receptors, ion channel
proteins, intracellular attachment proteins, cellular adhesion
proteins, and integrins. In certain aspects, the transmembrane
receptor comprises a G-Protein Coupled Receptor (GPCR). In
particular embodiments, the G-Protein Coupled Receptor (GPCR)
interacts with a G protein a subunit (e.g., comprising at least one
selected from the group consisting of G.alpha..sub.s,
G.alpha..sub.i, G.alpha..sub.q, and G.alpha..sub.12, preferably
G.alpha..sub.q).
[0028] In certain aspects, modulating cellular membrane
conductivity, comprises modulating whole-cell conductance (e.g.,
comprising modulating at least one linear or non-linear
voltage-dependent contribution of the whole-cell conductance). In
certain aspects, modulation of at least one of cellular membrane
potential and cellular membrane conductivity comprises at least one
of: modulation of a calcium-dependant cellular messaging pathway or
system; modulation of phospholipase C activity; modulation of
adenylate cyclase (AC) activity; and modulation of intracellular
signal transduction associated with at least one condition or
symptom selected from the group consisting of: dry eye, allergy
irritation, corneal irritation, bacterial infection, and cellular
damage.
[0029] Particular method embodiments comprise administering the
electrokinetically-altered aqueous fluid to a cell network or
layer, and further comprise modulation of an intercellular junction
therein. In certain aspects, the intracellular junction comprises
at least one selected from the group consisting of tight junctions,
gap junctions, zona adherins and desmasomes. In particular aspects,
the cell network or layers comprise at least one selected from the
group consisting of corneal epithelial layer and corneal
`endothelium`.
[0030] In particular aspects, the electrokinetically-altered
aqueous fluid is oxygenated, and the oxygen in the fluid is present
in an amount of at least 8 ppm, at least 15, ppm, at least 25 ppm,
at least 30 ppm, at least 40 ppm, at least 50 ppm, or at least 60
ppm oxygen at atmospheric pressure.
[0031] In certain aspects, the electrokinetically altered aqueous
fluid comprises at least one of a form of solvated electrons and
electrokinetically modified or charged oxygen species. In certain
embodiments, the form of solvated electrons or electrokinetically
modified or charged oxygen species are present in an amount of at
least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm,
at least 3 ppm, at least 5 ppm, at least 7 ppm, at least 10 ppm, at
least 15 ppm, or at least 20 ppm. In particular aspects, the
electrokinetically-altered aqueous fluid comprises a form of
solvated electrons stabilized, at least in part, by molecular
oxygen.
[0032] In particular aspects, the ability to alter at least one of
cellular membrane potential and cellular membrane conductivity
persists for at least two, at least three, at least four, at least
five, at least 6, at least 12 months, or longer periods, in a
closed gas-tight container.
[0033] In particular aspects, the electrokinetically-altered
aqueous fluid comprises a contact lens solution or multi-use
contact lens solution (e.g., comprising at least one of a storage
solution, a wetting solution, or a cleaning solution.
[0034] In certain embodiments, the electrokinetically-altered
aqueous fluid inhibits the growth of Pseudomonas bacteria.
[0035] In particular aspects, the eye irritation, eye infection or
inflammatory eye condition or disease, or the at least one symptom
thereof, is the result of a wound (e.g, at least one selected from
the group consisting of: lacerations, abrasions, rupture, puncture
wounds, chemical, thermal, or radiation-induced burns, cuts,
scrapes, incisions, blisters, ulcers, and surgical wounds). In
particular embodiments, the surgical wound is a result of at least
one surgery selected from the group consisting of: laser
keratotomy, cataract removal, lens implantation or removal, corneal
alterations, laser-assisted in situ keratomileusis (LASIK),
intraLASIK, extracapsular surgery, phacoemulsification,
vitreoretinal surgery, glaucoma procedures, neuro-ophthalmic
surgery, and strabismus surgery.
[0036] In particular aspects, the eye irritation, eye infection or
inflammatory eye condition or disease, or the at least one symptom
thereof, comprises dry eye, and wherein at least one sign or
symptom selected from the group consisting of: decrease in aqueous
tear production, increase in tear evaporation, and increase in
cellular inflammation is ameliorated. In certain aspects, said
ameliorating is assessed by at least one measurement selected from
the group consisting of corneal staining, conjunctival staining,
tear volume, bulbar conjunctival erythema, tear film height, ocular
surface healing, level of infection, level of inflammation, level
of ocular surface damage, and level of inferior tear meniscus.
[0037] In certain aspects, treating comprises administration by at
least one of topical, inhalation, intranasal, oral and intravenous.
Preferably, administration comprises topically or systemically
contacting said eye of the subject. In certain aspects, said
administering comprises orally administering said medication to the
subject.
[0038] In particular method aspects, said treatment comprises an
adjunct therapy and said subject further undergoes a primary
treatment for an eye condition (e.g., wherein said primary
treatment comprises administering to said subject at least one
medication selected from the group consisting of artificial tears,
anti-inflammatory medications, steroids, antihistamines,
antibiotics, and vasoconstrictor medications). In certain aspects,
said treatment results in a reduction in corneal or conjuctival
staining, and/or results in an increase in tear volume or tear film
height.
[0039] In certain aspects, the amount of oxygen present in
charge-stabilized oxygen-containing nanostructures of the
electrokinetically-alterd fluid is at least 8 ppm, at least 15,
ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 40
ppm, at least 50 ppm, or at least 60 ppm oxygen at atmospheric
pressure.
[0040] In particular aspects, the membrane associated protein
comprises CCR3.
[0041] In certain embodiments, treating the eye irritation, eye
infection or inflammatory eye condition or disease, or at least one
symptom thereof, comprises modulation of intracellular NF-.kappa.B
expression and/or activity.
[0042] Additional aspects provide a method of formulating a
therapeutic agent suitable for use in treating an eye irritation,
eye infection or inflammatory eye condition or disease, comprising:
obtaining a therapeutic agent suitable for use in treating an eye
irritation, eye infection or inflammatory eye condition or disease,
of a subject; and combining the therapeutic agent with an amount of
an electrokinetically altered aqueous fluid comprising an ionic
aqueous solution of charge-stabilized oxygen-containing
nanostructures substantially having an average diameter of less
than about 100 nanometers and stably configured in the ionic
aqueous fluid in an amount sufficient for treating inflammation or
at least one symptom thereof, wherein formulating a therapeutic
agent suitable for use in treating an eye irritation, eye infection
or inflammatory eye condition or disease is afforded. Preferably,
the charge-stabilized oxygen-containing nanostructures are stably
configured in the ionic aqueous fluid in an amount sufficient to
provide, upon contact of a living cell by the fluid, modulation of
at least one of cellular membrane potential and cellular membrane
conductivity.
[0043] Further aspects provide a pharmaceutical composition,
comprising: a therapeutic agent suitable for use treating an eye
irritation, eye infection or inflammatory eye condition or disease,
of a subject; and an amount of an electrokinetically altered
aqueous fluid comprising an ionic aqueous solution of
charge-stabilized oxygen-containing nanostructures substantially
having an average diameter of less than about 100 nanometers and
stably configured in the ionic aqueous fluid in an amount
sufficient for treating inflammation or at least one symptom
thereof.
[0044] Yet further aspects provide pharmaceutical compositions,
prepared by the disclosed methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a partial cross-section, partial block diagram of
a prior art mixing device.
[0046] FIG. 2 is block diagram of an exemplary embodiment of a
mixing device.
[0047] FIG. 3 is an illustration of an exemplary system for
delivering a first material to the mixing device of FIG. 2.
[0048] FIG. 4 is a fragmentary partial cross-sectional view of a
top portion of the mixing device of FIG. 2.
[0049] FIG. 5 is a fragmentary cross-sectional view of a first side
portion of the mixing device of FIG. 2.
[0050] FIG. 6 is a fragmentary cross-sectional view of a second
side portion of the mixing device of FIG. 2.
[0051] FIG. 7 is a fragmentary cross-sectional view of a side
portion of the mixing device of FIG. 2 located between the first
side portion of FIG. 5 and the second side portion of FIG. 6.
[0052] FIG. 8 is a perspective view of a rotor and a stator of the
mixing device of FIG. 2.
[0053] FIG. 9 is a perspective view of an inside of a first chamber
of the mixing device of FIG. 2.
[0054] FIG. 10 is a fragmentary cross-sectional view of the inside
of a first chamber of the mixing device of FIG. 2 including an
alternate embodiment of the pump 410.
[0055] FIG. 11 is a perspective view of an inside of a second
chamber of the mixing device of FIG. 2.
[0056] FIG. 12 is a fragmentary cross-sectional view of a side
portion of an alternate embodiment of the mixing device.
[0057] FIG. 13 is a perspective view of an alternate embodiment of
a central section of the housing for use with an alternate
embodiment of the mixing device.
[0058] FIG. 14 is a fragmentary cross-sectional view of an
alternate embodiment of a bearing housing for use with an alternate
embodiment of the mixing device.
[0059] FIG. 15 is a cross-sectional view of the mixing chamber of
the mixing device of FIG. 2 taken through a plane orthogonal to the
axis of rotation depicting a rotary flow pattern caused by
cavitation bubbles when a through-hole of the rotor approaches (but
is not aligned with) an aperture of the stator.
[0060] FIG. 16 is a cross-sectional view of the mixing chamber of
the mixing device of FIG. 2 taken through a plane orthogonal to the
axis of rotation depicting a rotary flow pattern caused by
cavitation bubbles when the through-hole of the rotor is aligned
with the aperture of the stator.
[0061] FIG. 17 is a cross-sectional view of the mixing chamber of
the mixing device of FIG. 2 taken through a plane orthogonal to the
axis of rotation depicting a rotary flow pattern caused by
cavitation bubbles when a through-hole of the rotor that was
previously aligned with the aperture of the stator is no longer
aligned therewith.
[0062] FIG. 18 is a side view of an alternate embodiment of a
rotor.
[0063] FIG. 19 is an enlarged fragmentary cross-sectional view
taken through a plane orthogonal to an axis of rotation of the
rotor depicting an alternate configuration of through-holes formed
in the rotor and through-holes formed in the stator.
[0064] FIG. 20 is an enlarged fragmentary cross-sectional view
taken through a plane passing through and extending along the axis
of rotation of the rotor depicting a configuration of through-holes
formed in the rotor and through-holes formed in the stator.
[0065] FIG. 21 is an enlarged fragmentary cross-sectional view
taken through a plane passing through and extending along the axis
of rotation of the rotor depicting an alternate offset
configuration of through-holes formed in the rotor and
through-holes formed in the stator.
[0066] FIG. 22 is an illustration of a shape that may be used to
construct the through-holes of the rotor and/or the apertures of
the stator.
[0067] FIG. 23 is an illustration of a shape that may be used to
construct the through-holes of the rotor and/or the apertures of
the stator.
[0068] FIG. 24 is an illustration of a shape that may be used to
construct the through-holes of the rotor and/or the apertures of
the stator.
[0069] FIG. 25 is an illustration of a shape that may be used to
construct the through-holes of the rotor and/or the apertures of
the stator.
[0070] FIG. 26 is an illustration of an electrical double layer
("EDL") formed near a surface.
[0071] FIG. 27 is a perspective view of a model of the inside of
the mixing chamber.
[0072] FIG. 28 is a cross-sectional view of the model of FIG.
27.
[0073] FIG. 29 is an illustration of an experimental setup.
[0074] FIG. 30 illustrates dissolved oxygen levels in water
processed with oxygen in the mixing device of FIG. 2 and stored a
500 ml thin walled plastic bottle and a 1,000 ml glass bottle each
capped at 65.degree. Fahrenheit.
[0075] FIG. 31 illustrates dissolved oxygen levels in water
processed with oxygen in the mixing device of FIG. 2 and stored in
a 500 ml plastic thin walled bottle and a 1,000 ml glass bottle
both refrigerated at 39.degree. Fahrenheit.
[0076] FIG. 32 illustrates the dissolved oxygen retention of a 500
ml beverage fluid processed with oxygen in the mixing device of
FIG. 2.
[0077] FIG. 33 illustrates the dissolved oxygen retention of a 500
ml braun balanced salt solution processed with oxygen in the mixing
device of FIG. 2.
[0078] FIG. 34 illustrates a further experiment wherein the mixing
device of FIG. 2 is used to sparge oxygen from water by processing
the water with nitrogen in the mixing device of FIG. 2.
[0079] FIG. 35 illustrates the sparging of oxygen from water by the
mixing device of FIG. 2 at standard temperature and pressure.
[0080] FIG. 36 is an illustration of an exemplary nanocage.
[0081] FIGS. 37A and B illustrate Rayleigh scattering effects of an
oxygen-enriched fluid;
[0082] FIG. 38 illustrates the cytokine profile of a mitogenic
assay in the presence of a gas-enriched fluid and deionized control
fluid; and
[0083] FIG. 39 illustrates the difference in the growth rates of
Pseudomonas bacteria at various dissolved oxygen saturation
ratios.
[0084] FIGS. 40A and 40B illustrate in vitro healing of wounds
using an oxygen-enriched cell culture media and a non-gas-enriched
media.
[0085] FIGS. 41A through 41F show histological cross-sections of
dermal and epidermal in vivo wound healing.
[0086] FIG. 42 illustrates the expression of Hale's stain in
treated and control healing wounds, used to detect acid
mucopolysaccharides, such as hyaluronic acid;
[0087] FIG. 43 illustrates the expression of von Willebrand's
Factor stain used to detect angiogenesis in treated and control
healing wounds;
[0088] FIG. 44 illustrates the detection of Luna's stain used to
detect elastin in treated and control healing wounds;
[0089] FIG. 45 illustrates the number of mast cells per visual
field for treated and control healing wounds;
[0090] FIG. 46 illustrates the percentage of dead cells at separate
time points in a corneal fibroblast assay using inventive
gas-enriched culture media and control culture media,
[0091] FIG. 47 illustrates the shelf life of the inventive
gas-enriched fluid in a polymer pouch;
[0092] FIG. 48 illustrates the results of contacting splenocytes
with MOG in the presence of pressurized pot oxygenated fluid (1),
inventive gas-enriched fluid (2), or control deionized fluid
(3).
[0093] FIGS. 49-58 show the results of whole blood sample
evaluations of cytokines.
[0094] FIGS. 59-68 show the corresponding cytokine results of
bronchoalveolar lavage fluid (BAL) sample evaluations.
[0095] FIGS. 69-75 shows studies where the Bradykinin B2 membrane
receptor was immobilized onto aminopropylsilane (APS) biosensor.
The Sample plate set up was as designated in FIG. 69 and the
binding of Bradykinin to the immobilized receptor was assessed
according to the sample set up as designated in FIG. 71. Results of
Bradykinin binding are shown in FIG. 72. Bradykinin binding to the
receptor was further titrated according to the set-up as designated
in FIG. 73. As indicated in FIG. 74, Bradykinin binding to the B2
receptor was concentration dependent, and binding affinity was
increased in the proprietary gas-enriched saline fluid of the
instant disclosure compared to normal saline. Stabilization of
Bradykinin binding to the B2 receptor is shown in FIG. 75.
[0096] FIGS. 76-83 show data showing the ability of particular
embodiments disclosed herein to affect regulatory T cells. The
study involved irradiating antigen presenting cells, and
introducing antigen and T cells.
[0097] FIG. 84 shows that the inventive electrokinetically
generated fluids decreased serum uptake of salmon calcitonin and an
animal model. The results are consistent with enhancement of tight
junctions.
[0098] FIGS. 85-89 show the expression levels of of tight
junction-related proteins in lung tissue from the animal model used
to generate the data of FIG. 84.
[0099] FIGS. 90-94 show data obtained from human foreskin
keratinocytes exposed to RDC1676-01 (sterile saline processed
through the instant proprietary device with additional oxygen
added; gas-enriched electrokinetically generated fluid (Rev) of the
instant disclosure) showing up-regulation of NOS1 and 3, and
Nostrin, NOS3.
[0100] FIGS. 95 and 96 show data supporting localized
electrokinetic effects (voltage/current) occurring in a mixing
device comprising insulated rotor and stator features to allow for
detection of voltage/current effects during electrokinetic fluid
generation.
[0101] FIGS. 97A-C show results of nuclear magnetic resonance (NMR)
studies conducted to further characterize the fundamental nature of
the inventive electrokinetically generated fluids. The
electrokinetically generated fluids increased the .sup.13C-NMR
line-widths of the reporter Trehalose solute.
[0102] FIGS. 98 and 99 show results of voltametric studies (i.e.,
square wave voltametry (FIG. 98) and stripping polarography (FIG.
99)) conducted to further characterize the fundamental nature of
the inventive electrokinetically generated fluids. Square wave
voltametry peak differences (with respect to control) unique to the
electrokinetically generated fluids were observed at -0.14V,
-0.47V, -1.02V and -1.36V. Pronounced polaragraphic peaks were seen
at -0.9 volts for the electrokinetically generated Revera and Solas
fluids, and the spectra of the non-electrokinetically generated
blank and saline control fluids show characteristic peaks at -0.19
and -0.3 volts that are absent in the spectra for the
electrokinetically generated fluids.
[0103] FIGS. 100-106 show results of patch clamping techniques that
assessed the effects of the electrokinetically generated fluid test
on epithelial cell membrane polarity and ion channel activity. The
results indicate that the inventive electrokinetically generated
fluids affect a voltage-dependent contribution of the whole-cell
conductance.
[0104] FIGS. 107A-D and 108A-D show data indicating that the
inventive electrokinetically generated fluids (e.g., RDC1676-00,
RDC1676-01, RDC1676-02 and RDC1676-03) protected against
methacholine-induced bronchoconstriction when administered alone or
as diluents for albuterol sulfate in male guinea pigs.
[0105] FIGS. 109-114 show results of budesonide experiments
performed to assess the airway anti-inflammatory properties of the
inventive electrokinetically generated fluids in a Brown Norway rat
ovalbumin sensitization model. The inventive electrokinetically
generated fluids decreased eosinophil count, showed strong synergy
with Budesonide in decreasing eosinophil count, decreased Penh
values, increased Tidal Volume, decreased blood levels of Eotaxin,
significantly enhanced the Blood levels of two major key
anti-inflammatory cytokines, IL10 and Interferron gamma at 6 hours
after challenge as a result of treatment with he inventive
electrokinetically generated fluid (e.g., Rev 60) alone or in
combination with Budesonide, and decreased systemic levels of
Rantes. The data show that there is a substantial synergistic
effect of Budesonide 750 ug/kg and the inventive electrokinetically
generated fluids (e.g., Rev 60).
[0106] FIG. 115 shows that the inventive electrokinetically
generated fluid (e.g., Revera 60 and Solas) reduced DEP-induced
TSLP receptor expression in bronchial epithelial cells (BEC) by
approximately 90% and 50%, respectively, whereas whereas normal
saline (NS) had only a a marginal effect.
[0107] FIG. 116 shows the inventive electrokinetically generated
fluid (e.g., Revera 60 and Solas) inhibited the DEP-induced cell
surface bound MMP9 levels in bronchial epithelial cells by
approximately 80%, and 70%, respectively, whereas normal saline
(NS) had only a marginal effect.
[0108] FIGS. 117 A-C demonstrate the results of a series of patch
clamping experiments that assessed the effects of the
electrokinetically generated fluid (e.g., RNS-60 and Solas) on
epithelial cell membrane polarity and ion channel activity at two
time-points (15 min (left panels) and 2 hours (right panels)) and
at different voltage protocols.
[0109] FIGS. 118 A-C show, in relation to the experiments relating
to FIGS. 117 A-C, the graphs resulting from the subtraction of the
Solas current data from the RNS-60 current data at three voltage
protocols (A. stepping from zero mV; B. stepping from -60 mV; C.
stepping from -120 mV) and the two time-points (15 mins (open
circles) and 2 hours (closed circles)).
[0110] FIGS. 119 A-D demonstrate the results of a series of patch
clamping experiments that assessed the effects of the
electrokinetically generated fluid (e.g., Solas (panels A. and B.)
and RNS-60 (panels C. and D.)) on epithelial cell membrane polarity
and ion channel activity using different external salt solutions
and at different voltage protocols (panels A. and C. show stepping
from zero mV; panels B. and D. show stepping from -120 mV).
[0111] FIGS. 120 A-D show, in relation to the experiments relating
to FIGS. 119 A-D, the graphs resulting from the subtraction of the
CsCl current data (shown in FIG. 119) from the 20 mM CaCl.sub.2
(diamonds) and 40 mM CaCl.sub.2 (filled squares) current data at
two voltage protocols (panels A. and C. stepping from zero mV; B.
and D. stepping from -120 mV) for Solas (panels A. and B.) and
Revera 60 (panels C. and D.).
[0112] FIG. 121 A shows 1 mm2 AFM scan for RNS60-1 (rns60-1 1 um
3D.jpg). The small peaks ("1") represent hydrophobic nanobubbles
which are .about.20 nm wide and .about.1.5 nm tall or smaller.
[0113] FIG. 121 B shows 1 mm2 scan for PNS60-1 (pp60-1 1 um
3d.jpg). This scan reveals peaks ("2") (hydrophobic nanobubbles)
that are substantially larger (.about.60 nm wide and .about.5 nm
tall) than those visible with RNS60-1.
[0114] FIG. 122 A shows quadrants for scaled grading of
conjunctival staining by lissamine green.
[0115] FIG. 122 B shows quadrants for scaled grading of
erythema.
DETAILED DESCRIPTION
[0116] The surface of the animal eye, particularly the human eye,
is normally bathed by a tear film that is secreted by tiny glands
around the eye. The tear film is primarily composed of three
layers: mucous, water, and oil. The mucous layer is closest to the
eye organ and serves as an anchor for the tear film to adhere to
the eye. The middle layer is an aqueous layer, and the outer oil
layer seals the tear film and prevents evaporation. The tear film
also contains various nutritive and protective proteins and
peptides. The tear film serves several purposes: it keeps the eye
moist, creates a smooth surface for light to pass through the eye,
nourishes the front of the eye, and provides protection from injury
and infection.
[0117] The eye, particularly the cornea, can become dry, irritated,
or damaged by trauma, disease, natural aging, environmental factors
(such as pollutants and allergens), or by wearing contact lenses.
In some cases, individuals may experience burning, a feeling of
dryness, scratchiness, itchiness, or persistent irritation or
inflammation. This irritation can lead to infection and/or visual
impairment if not treated properly.
[0118] As summarized above, the most common approach to treatment
of irritated and/or dry eyes has been to supplement the natural
ocular tear film using so-called artificial tears instilled
throughout the day. Other approaches include the use of lubricants,
and ocular inserts that provide a tear substitute or stimulation of
endogenous tear production. Novel, safe and effective approaches
are needed, and are provided herein.
[0119] Certain embodiments disclosed herein relate to providing
compositions and methods of treatment of at least one symptom or
sign of an eye (e.g. corneal) condition or disorder by contacting
the site or administering to a subject, a therapeutic and/or
cosmetic composition comprising a gas-enriched fluid. In certain
specific embodiments, the gas-enriched fluid comprises
oxygen-enriched water.
[0120] Certain embodiments disclosed herein provide compositions
and methods for eye care and treatment comprising administering a
therapeutic composition comprising at least one electrokinetically
generated fluid (including gas-enriched (e.g., oxygen enriched)
electrokinetically generated fluids) as disclosed herein for
regulating or modulating intracellular signal transduction by
modulation of at least one of cellular membranes, membrane
potential, and membrane proteins such as membrane receptors,
including but not limited to G protein coupled receptors.
Particular aspects relate to modulating (e.g., treating or
preventing) at least one disease or condition or symptom thereof
associated with eye care and treatment.
[0121] According to particular aspects, the introduction of
oxygen-rich saline in therapeutic applications has beneficial
effects on human tissues in vivo. Applicants' microarray analyses
have confirmed the anti-inflammatory nature of the inventive fluids
through an increase in the level of anti-inflammatory cytokines,
and such fluids would therefore be advantageous in dry eye as well
as other inflammatory-mediated conditions. According to additional
aspects, the application of oxygen-rich saline to cultured cells
shows no significant levels of toxicity that would preclude in vivo
use.
[0122] Certain embodiments disclosed herein relate to providing
compositions and methods of treatment of at least one symptom or
sign of an eye (e.g. corneal) condition or disorder by contacting
the site or administering to a subject, a therapeutic and/or
cosmetic composition comprising an electrokinetically generated
fluid (e.g., gas-enriched fluid) as described herein. In certain
specific embodiments, a gas-enriched fluid comprises
oxygen-enriched water.
[0123] Particular embodiments disclosed herein relate to
compositions and methods for treating non-responding dry eye
patients with pre-existing ocular surface damage when added to
their current treatment regime. Certain other embodiments disclosed
herein relate to topically applied compositions and methods for
treating dry eyes as a safe and effective adjunct therapy in
conjunction with concomitant therapies. Still other embodiments
relate to compositions and methods for beneficially treating severe
dry eye disease that is unresponsive to standard therapies by
reducing at least one sign or symptom of severe dry eye
disease.
[0124] Other particular embodiments relate to compositions and
methods for treating allergy eye patients. In certain embodiments,
the compositions described herein comprise a preservative free,
buffered saline eye rewetting drop. In particular embodiments, the
rewetting drop is further gas-enriched. In specific embodiments the
gas-enriched fluid is oxygen-enriched water or oxygen-enriched
saline.
Electrokinetically-Generated Fluids:
[0125] "Electrokinetically generated fluid," as used herein, refers
to Applicants' inventive electrokinetically-generated fluids
generated, for purposes of the working Examples herein, by the
exemplary Mixing Device described in detail herein (see also
US200802190088 and WO2008/052143, both incorporated herein by
reference in their entirety). The electrokinetic fluids, as
demonstrated by the data disclosed and presented herein, represent
novel and fundamentally distinct fluids relative to prior art
non-electrokinetic fluids, including relative to prior art
oxygenated non-electrokinetic fluids (e.g., pressure pot oxygenated
fluids and the like). As disclosed in various aspects herein, the
electrokinetically-generated fluids have unique and novel physical
and biological properties including, but not limited to the
following:
[0126] In particular aspects, the electrokinetically altered
aqueous fluid comprise an ionic aqueous solution of
charge-stabilized oxygen-containing nanostructures substantially
having an average diameter of less than about 100 nanometers and
stably configured in the ionic aqueous fluid in an amount
sufficient to provide, upon contact of a living cell by the fluid,
modulation of at least one of cellular membrane potential and
cellular membrane conductivity.
[0127] In particular aspects, electrokinetically-generated fluids
refers to fluids generated in the presence of
hydrodynamically-induced, localized (e.g., non-uniform with respect
to the overall fluid volume) electrokinetic effects (e.g.,
voltage/current pulses), such as device feature-localized effects
as described herein. In particular aspects said
hydrodynamically-induced, localized electrokinetic effects are in
combination with surface-related double layer and/or streaming
current effects as disclosed and discussed herein.
[0128] In particular aspects the administered inventive
electrokinetically-altered fluids comprise charge-stabilized
oxygen-containing nanostructures in an amount sufficient to provide
modulation of at least one of cellular membrane potential and
cellular membrane conductivity. In certain embodiments, the
electrokinetically-altered fluids are superoxygenated (e.g.,
RNS-20, RNS-40 and RNS-60, comprising 20 ppm, 40 ppm and 60 ppm
dissolved oxygen, respectively, in standard saline). In particular
embodiments, the electrokinetically-altered fluids are
not-superoxygenated (e.g., RNS-10 or Solas, comprising 10 ppm
(e.g., approx. ambient levels of dissolved oxygen in standard
saline). In certain aspects, the salinity, sterility, pH, etc., of
the inventive electrokinetically-altered fluids is established at
the time of electrokinetic production of the fluid, and the sterile
fluids are administered by an appropriate route. Alternatively, at
least one of the salinity, sterility, pH, etc., of the fluids is
appropriately adjusted (e.g., using sterile saline or appropriate
diluents) to be physiologically compatible with the route of
administration prior to administration of the fluid. Preferably,
and diluents and/or saline solutions and/or buffer compositions
used to adjust at least one of the salinity, sterility, pH, etc.,
of the fluids are also electrokinetic fluids, or are otherwise
compatible.
[0129] In particular aspects, the inventive
electrokinetically-altered fluids comprise saline (e.g., one or
more dissolved salt(s); e.g., alkali metal based salts (Li+, Na+,
K+, Rb+, Cs+, etc.), alkaline earth based salts (e.g., Mg++, Ca++),
etc., or transition metal-based positive ions (e.g., Cr, Fe, Co,
Ni, Cu, Zn, etc.,), in each case along with any suitable anion
components, including, but not limited to F-, Cl-, Br-, I-, PO4-,
SO4-, and nitrogen-based anions. Particular aspects comprise mixed
salt based electrokinetic fluids (e.g., Na+, K+, Ca++, Mg++,
transition metal ion(s), etc.) in various combinations and
concentrations, and optionally with mixtures of couterions. In
particular aspects, the inventive electrokinetically-altered fluids
comprise standard saline (e.g., approx. 0.9% NaCl, or about 0.15 M
NaCl). In particular aspects, the inventive
electrokinetically-altered fluids comprise saline at a
concentration of at least 0.0002 M, at least 0.0003 M, at least
0.001 M, at least 0.005 M, at least 0.01 M, at least 0.015 M, at
least 0.1 M, at least 0.15 M, or at least 0.2 M. In particular
aspects, the conductivity of the inventive
electrokinetically-altered fluids is at least 10 .mu.S/cm, at least
40 .mu.S/cm, at least 80 .mu.S/cm, at least 100 .mu.S/cm, at least
150 .mu.S/cm, at least 200 .mu.S/cm, at least 300 .mu.S/cm, or at
least 500 .mu.S/cm, at least 1 mS/cm, at least 5, mS/cm, 10 mS/cm,
at least 40 mS/cm, at least 80 mS/cm, at least 100 mS/cm, at least
150 mS/cm, at least 200 mS/cm, at least 300 mS/cm, or at least 500
mS/cm. In particular aspects, any salt may be used in preparing the
inventive electrokinetically-altered fluids, provided that they
allow for formation of biologically active salt-stabilized
nanostructures (e.g., salt-stabilized oxygen-containing
nanostructures) as disclosed herein.
[0130] According to particular aspects, the biological effects of
the inventive fluid compositions comprising charge-stabilized
gas-containing nanostructures can be modulated (e.g., increased,
decreased, tuned, etc.) by altering the ionic components of the
fluids, and/or by altering the gas component of the fluid.
[0131] According to particular aspects, the biological effects of
the inventive fluid compositions comprising charge-stabilized
gas-containing nanostructures can be modulated (e.g., increased,
decreased, tuned, etc.) by altering the gas component of the fluid.
In preferred aspects, oxygen is used in preparing the inventive
electrokinetic fluids. In additional aspects mixtures of oxygen
along with at least one other gas selected from Nitrogen, Oxygen,
Argon, Carbon dioxide, Neon, Helium, krypton, hydrogen and Xenon.
As described above, the ions may also be varied, including along
with varying the gas constitutent(s).
[0132] Given the teachings and assay systems disclosed herein
(e.g., cell-based cytokine assays, patch-clamp assays, etc.) one of
skill in the art will readily be able to select appropriate salts
and concentrations thereof to achieve the biological activities
disclosed herein.
TABLE-US-00001 TABLE 1 Exemplary cations and anions. Name Formula
Other name(s) Common Cations: Aluminum Al.sup.+3 Ammonium
NH.sub.4.sup.+ Barium Ba.sup.+2 Calcium Ca.sup.+2 Chromium(II)
Cr.sup.+2 Chromous Chromium(III) Cr.sup.+3 Chromic Copper(I)
Cu.sup.+ Cuprous Copper(II) Cu.sup.+2 Cupric Iron(II) Fe.sup.+2
Ferrous Iron(III) Fe.sup.+3 Ferric Hydrogen H.sup.+ Hydronium
H.sub.3O.sup.+ Lead(II) Pb.sup.+2 Lithium Li.sup.+ Magnesium
Mg.sup.+2 Manganese(II) Mn.sup.+2 Manganous Manganese(III)
Mn.sup.+3 Manganic Mercury(I) Hg.sub.2.sup.+2 Mercurous Mercury(II)
Hg.sup.+2 Mercuric Nitronium NO.sub.2.sup.+ Potassium K.sup.+
Silver Ag.sup.+ Sodium Na.sup.+ Strontium Sr.sup.+2 Tin(II)
Sn.sup.+2 Stannous Tin(IV) Sn.sup.+4 Stannic Zinc Zn.sup.+2 Common
Anions: Simple ions: Hydride H.sup.- Oxide O.sup.2- Fluoride
F.sup.- Sulfide S.sup.2- Chloride Cl.sup.- Nitride N.sup.3- Bromide
Br.sup.- Iodide I.sup.- Oxoanions: Arsenate AsO.sub.4.sup.3-
Phosphate PO.sub.4.sup.3- Arsenite AsO.sub.3.sup.3- Hydrogen
phosphate HPO.sub.4.sup.2- Dihydrogen phosphate
H.sub.2PO.sub.4.sup.- Sulfate SO.sub.4.sup.2- Nitrate
NO.sub.3.sup.- Hydrogen sulfate HSO.sub.4.sup.- Nitrite
NO.sub.2.sup.- Thiosulfate S.sub.2O.sub.3.sup.2- Sulfite
SO.sub.3.sup.2- Perchlorate ClO.sub.4.sup.- Iodate IO.sub.3.sup.-
Chlorate ClO.sub.3.sup.- Bromate BrO.sub.3.sup.- Chlorite
ClO.sub.2.sup.- Hypochlorite OCl.sup.- Hypobromite OBr.sup.-
Carbonate CO.sub.3.sup.2- Chromate CrO.sub.4.sup.2- Hydrogen
carbonate HCO.sub.3.sup.- Dichromate Cr.sub.2O.sub.7.sup.2- or
Bicarbonate Anions from Organic Acids: Acetate CH.sub.3COO.sup.-
formate HCOO.sup.- Others: Cyanide CN.sup.- Amide NH.sub.2.sup.-
Cyanate OCN.sup.- Peroxide O.sub.2.sup.2- Thiocyanate SCN.sup.-
Oxalate C.sub.2O.sub.4.sup.2- Hydroxide OH.sup.- Permanganate
MnO.sub.4.sup.-
TABLE-US-00002 TABLE 2 Exemplary cations and anions. Formula Charge
Name Mono atomic Cations H.sup.+ 1+ hydrogen ion Li.sup.+ 1+
lithium ion Na.sup.+ 1+ sodium ion K.sup.+ 1+ potassium ion
Cs.sup.+ 1+ cesium ion Ag.sup.+ 1+ silver ion Mg.sup.2+ 2+
magnesium ion Ca.sup.2+ 2+ calcium ion Sr.sup.2+ 2+ strontium ion
Ba.sup.2+ 2+ barium ion Zn.sup.2+ 2+ zinc ion Cd.sup.2+ 2+ cadmium
ion Al.sup.3+ 3 + aluminum ion Polyatomic Cations NH.sub.4.sup.+ 1+
ammonium ion H.sub.3O.sup.+ 1+ hydronium ion Multivalent Cations
Cr.sup.2+ 2 chromium(II) or chromous ion Cr.sup.3+ 3
chromium(III)or chromic ion Mn.sup.2+ 2 manganese(II) or manganous
ion Mn.sup.4+ 4 manganese(IV) ion Fe.sup.2+ 2 iron(II) or ferrous
ion Fe.sup.3+ 3 iron(III) or ferric ion Co.sup.2+ 2 cobalt(II) or
cobaltous ion Co.sup.3+ 3 cobalt(II) or cobaltic ion Ni.sup.2+ 2
nickel(II) or nickelous ion Ni.sup.3+ 3 nickel(III) or nickelic ion
Cu.sup.+ 1 copper(I) or cuprous ion Cu.sup.2+ 2 copper(II) or
cupric ion Sn.sup.2+ 2 tin(II) or atannous ion Sn.sup.4+ 4 tin(IV)
or atannic ion Pb.sup.2+ 2 lead(II) or plumbous ion Pb.sup.4+ 4
lead(IV) or plumbic ion Monoatomic Anions H.sup.- 1- hydride ion
F.sup.- 1- fluoride ion Cl.sup.- 1- chloride ion Br.sup.- 1-
bromide ion I.sup.- 1- iodide ion O.sup.2- 2- oxide ion S.sup.2- 2-
sulfide ion N.sup.3- 3- nitride ion Polyatomic Anions OH.sup.- 1-
hydroxide ion CN.sup.- 1- cyanide ion SCN.sup.- 1- thiocyanate ion
C.sub.2H.sub.3O.sub.2.sup.- 1- acetate ion ClO.sup.- 1-
hypochlorite ion ClO.sub.2.sup.- 1- chlorite ion ClO.sub.3.sup.- 1-
chlorate ion ClO.sub.4.sup.- 1- perchlorate ion NO.sub.2.sup.- 1-
nitrite ion NO.sub.3.sup.- 1- nitrate ion MnO.sub.4.sup.2- 2-
permanganate ion CO.sub.3.sup.2- 2- carbonate ion
C.sub.2O.sub.4.sup.2- 2- oxalate ion CrO.sub.4.sup.2- 2- chromate
ion Cr.sub.2O.sub.7.sup.2- 2- dichromate ion SO.sub.3.sup.2- 2-
sulfite ion SO.sub.4.sup.2- 2- sulfate ion PO.sub.3.sup.3- 3-
phosphite ion PO.sub.4.sup.3- 3- phosphate ion
[0133] The present disclosure sets forth novel gas-enriched fluids,
including, but not limited to gas-enriched ionic aqueous solutions,
aqueous saline solutions (e.g., standard aqueous saline solutions,
and other saline solutions as discussed herein and as would be
recognized in the art, including any physiological compatible
saline solutions), cell culture media (e.g., minimal medium, and
other culture media) useful in the treatment of eye conditions. A
medium, or media, is termed "minimal" if it only contains the
nutrients essential for growth. For prokaryotic host cells, a
minimal media typically includes a source of carbon, nitrogen,
phosphorus, magnesium, and trace amounts of iron and calcium.
(Gunsalus and Stanter, The Bacteria, V. 1, Ch. 1 Acad. Press Inc.,
N.Y. (1960)). Most minimal media use glucose as a carbon source,
ammonia as a nitrogen source, and orthophosphate (e.g., PO.sub.4)
as the phosphorus source. The media components can be varied or
supplemented according to the specific prokaryotic or eukaryotic
organism(s) grown, in order to encourage optimal growth without
inhibiting target protein production. (Thompson et al., Biotech.
and Bioeng. 27: 818-824 (1985)).
[0134] In particular aspects, the electrokinetically altered
aqueous fluids are suitable to modulate .sup.13C-NMR line-widths of
reporter solutes (e.g., Trehelose) dissolved therein. NMR
line-width effects are in indirect method of measuring, for
example, solute `tumbling` in a test fluid as described herein in
particular working Examples.
[0135] In particular aspects, the electrokinetically altered
aqueous fluids are characterized by at least one of: distinctive
square wave voltametry peak differences at any one of -0.14V,
-0.47V, -1.02V and -1.36V; polarographic peaks at -0.9 volts; and
an absence of polarographic peaks at -0.19 and -0.3 volts, which
are unique to the electrokinetically generated fluids as disclosed
herein in particular working Examples.
[0136] In particular aspects, the electrokinetically altered
aqueous fluids are suitable to alter cellular membrane conductivity
(e.g., a voltage-dependent contribution of the whole-cell
conductance as measure in patch clamp studies disclosed
herein).
[0137] In particular aspects, the electrokinetically altered
aqueous fluids are oxygenated, wherein the oxygen in the fluid is
present in an amount of at least 15, ppm, at least 25 ppm, at least
30 ppm, at least 40 ppm, at least 50 ppm, or at least 60 ppm
dissolved oxygen at atmospheric pressure. In particular aspects,
the electrokinetically altered aqueous fluids have less than 15
ppm, less that 10 ppm of dissolved oxygen at atmospheric pressure,
or approximately ambient oxygen levels.
[0138] In particular aspects, the electrokinetically altered
aqueous fluids are oxygenated, wherein the oxygen in the fluid is
present in an amount between approximately 8 ppm and approximately
15 ppm, and in this case is sometimes referred to herein as
"Solas."
[0139] In particular aspects, the electrokinetically altered
aqueous fluid comprises at least one of solvated electrons (e.g.,
stabilized by molecular oxygen), and electrokinetically modified
and/or charged oxygen species, and wherein in certain embodiments
the solvated electrons and/or electrokinetically modified or
charged oxygen species are present in an amount of at least 0.01
ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3
ppm, at least 5 ppm, at least 7 ppm, at least 10 ppm, at least 15
ppm, or at least 20 ppm.
[0140] In particular aspects, the electrokinetically altered
aqueous fluids are suitable to alter cellular membrane structure or
function (e.g., altering of a conformation, ligand binding
activity, or a catalytic activity of a membrane associated protein)
sufficient to provide for modulation of intracellular signal
transduction, wherein in particular aspects, the membrane
associated protein comprises at least one selected from the group
consisting of receptors, transmembrane receptors (e.g., G-Protein
Coupled Receptor (GPCR), TSLP receptor, beta 2 adrenergic receptor,
bradykinin receptor, etc.), ion channel proteins, intracellular
attachment proteins, cellular adhesion proteins, and integrins. In
certain aspects, the effected G-Protein Coupled Receptor (GPCR)
interacts with a G protein a subunit (e.g., G.alpha..sub.s,
G.alpha..sub.i, G.alpha..sub.q, and G.alpha..sub.12).
[0141] In particular aspects, the electrokinetically altered
aqueous fluids are suitable to modulate intracellular signal
transduction, comprising modulation of a calcium dependant cellular
messaging pathway or system (e.g., modulation of phospholipase C
activity, or modulation of adenylate cyclase (AC) activity).
[0142] In particular aspects, the electrokinetically altered
aqueous fluids are characterized by various biological activities
(e.g., regulation of cytokines, receptors, enzymes and other
proteins and intracellular signaling pathways) described in the
working Examples and elsewhere herein.
[0143] In particular aspects, the electrokinetically altered
aqueous fluids display synergy with metformin as shown in working
Examples herein
[0144] In particular aspects, the electrokinetically altered
aqueous fluids reduce DEP-induced TSLP receptor expression in
bronchial epithelial cells (BEC) as shown in working Examples
herein.
[0145] In particular aspects, the electrokinetically altered
aqueous fluids inhibit the DEP-induced cell surface-bound MMP9
levels in bronchial epithelial cells (BEC) as shown in working
Examples herein.
[0146] In particular aspects, the biological effects of the
electrokinetically altered aqueous fluids are inhibited by
diphtheria toxin, indicating that beta blockade, GPCR blockade and
Ca channel blockade affects the activity of the electrokinetically
altered aqueous fluids (e.g., on regulatory T cell function) as
shown in working Examples herein.
[0147] In particular aspects, the physical and biological effects
(e.g., the ability to alter cellular membrane structure or function
sufficient to provide for modulation of intracellular signal
transduction) of the electrokinetically altered aqueous fluids
persists for at least two, at least three, at least four, at least
five, at least 6 months, or longer periods, in a closed container
(e.g., closed gas-tight container).
Anti-Inflammatory Activity of the Electrokinetically-Generated
Gas-Enriched Fluids and Solutions:
[0148] According to certain aspects of the present invention, the
gas-enriched fluids and/or solutions disclosed herein have
anti-inflammatory properties and effects, and can be used as
anti-inflammatory agents for the treatment of subjects afflicted by
diseases or disorders relating to inflammation. FIG. 38 shows the
experimental results of cytokine profiles in stimulated lymphocytes
from a healthy blood donor. As can be seen in FIG. 38, the
inventive oxygen-enriched fluid (water) affected a down regulation
of particular cytokines, especially IL-6, IL-8, and IL-1R.
[0149] Increased production of pro-inflammatory cytokines has been
implicated in the pathogenesis of numerous inflammatory and
autoimmune diseases. Secretion of TNF.alpha. is a primary event in
the initiation of the inflammatory cascade (Brennan F. M., et. al.
Lancet, 1989, 2:244-7; Haworth C, et. al. Eur. J. Immunol. 1991,
21:2575-2579) and directly contributes to the initiation and
maintenance of inflammatory and autoimmune diseases. Other
pro-inflammatory cytokines also play a role, including interleukin
1.beta. (IL-1.beta.), IL-6, IL-8, IL-12 nitric oxide, IFN-.gamma.
and GM-CSF, while anti-inflammatory cytokines such as IL-10 may
reduce disease. Cells of the immune system, macrophages in
particular, secrete many of these cytokines in response to
activating stimuli.
[0150] A variety of cell types are involved in the inflammatory
process. Overproduction of TNF.alpha. by monocytes, macrophages and
other immune cells is a key element in the pathogenesis of a
multitude of diseases. Macrophages and T-cells in particular play a
central role in the initiation and maintenance of the immune
response. Once activated by pathological or immunogenic stimuli,
macrophages respond by releasing a host of cytokines, including
TNF-.alpha., IL-1.beta., IL-8, IL-12, nitric oxide (NO), IL-6,
GM-CSF, G-CSF, M-CSF and others. T-cells release IL-2, IL-4,
INF-.gamma., and other inflammatory cytokines. These cytokines
activate other immune cells and some can also act as independent
cytotoxic agents. Excessive release of macrophage and T-cell
derived inflammatory mediators can particularly lead to damage of
normal cells and surrounding tissues.
[0151] Pro-inflammatory cytokines have been implicated to
contribute to many diseases and disease symptoms including,
diabetes. The induction of NO from smooth muscle cells mediates
decreased mean arterial pressure and systemic vascular resistance
during septic shock, suggesting a fundamental role for NO. Thus,
therapies that target downregulatory effects on IL-8, IL-1.beta.,
and NO could be beneficial in the treatment of inflammatory
diseases or disorders, including sepsis, septic shock, endotoxic
shock and diabetes.
[0152] Overproduction of TNF.alpha. contributes to the clinical
features of numerous autoimmune diseases such as diabetes and
rheumatoid arthritis. Systemic lupus erythematosus (SLE) is also
precipitated by increased IL-1.beta. and TNF.alpha. levels. Within
lupus patients, serum C-reactive protein, IL-1.beta. and TNF.alpha.
levels were higher than in controls, suggesting that an increased
inflammatory response plays a role in the disease (Liou L. B. Clin.
Exp. Rheumatol. 2001, 19:515-523). A study of patients with one
form of SLE, neuropsychiatric lupus erythematosus (NPLE), showed
that the number of peripheral blood mononuclear cells expressing
mRNA for TNF.alpha. as well as the cerebrospinal fluid level of NO
metabolites correlated with NPLE disease severity (Svenungsson E.,
et al. Ann. Rheum. Dis. 2001, 60:372-9).
[0153] IL-1 and TNF.alpha. play a central role in various acute as
well as chronic responses in animal models. Additionally, IL-11,
IFN.alpha. and IFN.beta. may also up-regulate inflammatory
reactions. Conversely, several cytokines may be involved in
down-regulation of inflammatory responses (i.e. IL-4, IL-10, IL-13,
among others). As set forth in Example 1, cells contacted with the
inventive gas-enriched fluid showed an increase in IFN-.gamma.
levels with T3 antigen than in the control culture media with T3
antigen, while IL-8 was lower in the inventive gas-enriched culture
media with T3 antigen than in the control culture media with T3
antigen. Additionally, IL-6, IL-8, and TNF-.alpha. levels were
lower in the inventive gas-enriched media with PHA, than in the
control media with PHA, while IL-1.beta. levels were lower in the
inventive gas-enriched fluid with PHA when compared with control
media with PHA. In the inventive gas-enriched media alone,
IFN-.gamma. levels were higher than in control media. These results
are consistent with an anti-inflammatory microenvironment.
[0154] NO is recognized as a mediator and regulator of inflammatory
responses. It possesses cytotoxic properties toward pathogens, but
can also have deleterious effects on the subject's own tissues.
(Korhonen et al., Curr Drug Targets Inflamm Allergy 4(4): 471-9,
2005). NO reacts with soluble guanylate cyclase to form cyclic
guanosine monophosphate (cGMP), which mediates many of the effects
of NO. NO can also interact with molecular oxygen and superoxide
anion to produce reactive oxygen species that can modify various
cellular functions. These indirect effects of NO have a significant
role in inflammation, where NO is produce in high amounts by
inducible NO synthase (iNOS) and reactive oxygen species are
synthesized by activated inflammatory cells. In fact,
overproduction of NO contributes to impairment of both muscle cell
insulin action and .beta. cell function in obesity, which can lead
to insulin resistance and diabetes.
[0155] NO can be produced by keratinocytes, fibroblasts,
endothelial cells, and possibly others. Some of the vascular
actions of NO include vasodilation, inhibiting platelet adhesion to
the vascular endothelium, inhibiting leukocyte adhesion to the
vascular endothelium, and scavenging superoxides. (Shah et al.,
Env. Health Persp. v. 106 (5): 1139-1143.)
[0156] Furthermore, inhibition of NO synthesis has been shown to
delay wound contraction, alter collagen organization, and alter
neoepidermis thickness. (Amadeu and Costa, J. Cutan. Pathol. 33:
465-473, 2006.) Mast cell migration and angiogenesis in wounds is
also affected by inhibition of NO. (Id.) Without being bound to any
particular theory of mechanism, in certain embodiments the
inventive gas-enriched fluids may be modulating localized and/or
cellular NO production, or degradation, consistent with the
spectrum of wound healing effects illustrated in the Examples
section disclosed herein. Due to variable pathways of regulation,
in certain embodiments, the inventive gas-enriched fluid may
increase NO production and/or retard NO degradation, whereas in
other certain embodiments, the inventive gas-enriched fluid may
decrease NO production and/or hasten NO degradation.
[0157] Specifically, wounds treated with oxygen-enriched saline
solution showed an increase in wound healing at days 4 through 11,
and between days 3 and 11, the new epidermis in wounds treated with
the oxygen-enriched saline solution migrated at two to four times
as fast as the epidermis of the wounds treated with the normal
saline solution, as set forth in Example 9 herein. The study also
showed that between 15 and 22 days, wounds treated by the
oxygen-enriched saline solution differentiated at a more rapid rate
as evidenced by the earlier formation of more mature epidermal
layers. At all stages, the thickening that occurs in the epidermis
associated with normal healing did not occur within the wounds
treated by the oxygen-enriched saline solution.
[0158] Thus, in accordance with this spectrum of wound healing
effects, but without wishing to be bound by any particular theory,
it is believed that the oxygen-enriched saline solution may
modulate the localized and/or cellular level of NO within the
wounds. NO modulates growth factors, collagen deposition,
inflammation, mast cell migration, epidermal thickening, and
neovascularization in wound healing. Furthermore, nitric oxide is
produced by an inducible enzyme that is regulated by oxygen.
[0159] In the case of mast cell migration, differences also
occurred in early and late migration for the oxygen-enriched
solution. This is consistent with what is known in the art
regarding inhibition of NO synthesis (Amadeu and Costa, J. Cutan
Pathol 33: 465-473, 2006).
[0160] Referring now to FIG. 41A through 41F, various illustrations
compare the wound healing results of the porcine epidermal tissues
with or without oxygen-enriched saline solution. As can be seen,
the healing of the control wound and of the wound using the
oxygen-enriched saline solution was followed for days 1, 4 and
16.
[0161] FIG. 41A illustrates the wound healing for the control wound
on day 1. As can be seen, the wound shows epidermal/dermal
thickening and a loss of contour. FIG. 41B illustrates the wound
healing on day 1 for the wound treated using the oxygen-enriched
saline solution. The wound shows normal epidermal/dermal thickness
and normal contouring is typical on a new wound.
[0162] Referring now to FIGS. 41C and 41D, there are illustrated
the wound healing for the control wound on day 4 and the wound
healing for the wound treated with the oxygen-enriched saline
solution on day 4. For the control wound illustrated in FIG. 41C,
the wound shows a 600 micron epidermal spur. In the wound treated
with the oxygen-enriched saline solution in FIG. 41D, there is
illustrated a 1200 micron epidermal spur. Thus, in the first 4 days
of the experiment, the epidermal spur created in the wound treated
using the oxygen-enriched saline solution shows an epidermal growth
rate of twice of that of the wound that was not treated with the
oxygen-enriched saline solution.
[0163] Referring now to FIG. 41E, there is illustrated the control
wound at day 16. The wound shows less differentiated epidermis with
loss of epidermal/dermal contour than that illustrated by the wound
treated with the oxygen-enriched saline solution illustrated in
FIG. 41F. FIG. 41F shows more differentiated epidermis and more
normal epidermal/dermal contouring in the wound.
[0164] In the first two phases of the inflammatory process, the
foreign body is either destroyed, for example, if the foreign body
is an organism, or the tissue around it is loosened, for example,
if it is a splinter. In the healing phase, the inflammation begins
to subside; individual blood vessels and vascular patterns become
normal once again; and repair of the wound commences. The three
main events in the repair process are (1) formation of new
connective tissue by proliferating fibroblasts; (2) regeneration of
epithelium; and (3) outgrowth of new capillaries.
[0165] Even before the inflammation subsides, fibroblasts begin
moving into the injured area from the surrounding normal tissue,
where they usually exist in a dormant state. They migrate by an
amoeboid movement along strands of fibrin and distribute themselves
throughout the healing area. Once fixed into position in the
injured tissue, they begin to synthesize collagen and secrete this
protein, which arranges itself into fibers. The fibers orient
themselves with their longitudinal axes in the direction of the
greatest stress. As the collagen bundles grow in firmness, the
fibroblasts gradually degenerate and attach closely to the bundles,
and the injured area transforms into scar tissue.
[0166] Simultaneously with scar tissue formation, the intact
epidermal cells on the edge of the wound begin to proliferate and
move, as one sheet, toward the center of the injured area. As the
inflammation subsides, a need for a direct supply of blood arises,
and angiogenesis occurs at the wound site.
[0167] Inflammation is a complex process that involves multiple
cell types. For example, mast cells release mediators that trigger
an early phase of vasodilation, accompanied by the separation of
endothelial cells and exposure of collagen fibers in the
subendothelial layer. Fibers in the intercellular gaps that form in
blood vessels trap platelets and trigger the release of mediators
from these cells.
[0168] In addition to platelets, the exposed collagen fibers also
interact with proteins of the plasma that filter through the pores
of the dilated vessel wall, including the triggering factor of the
blood-clotting cascade, increased vasodilation, increased blood
vessel permeability, and chemotaxis.
[0169] Additionally, the complement cascade can be activated by
several stimuli: the injured blood vessels, the proteolytic enzymes
released by the damaged cells, the membrane components of any
participating bacteria, and antigen-antibody complexes. Some of the
activated complement components act as chemotactic factors,
responsible for the influx of leukocytes into the inflamed area,
while others facilitate phagocytosis and participate in cell
lysis.
[0170] In particular aspects, inventive gas-enriched fluids or
solutions also regulate at least one cytokine involved in at least
one aspect of inflammation, the cytokine(s) including, but not
limited to MAF (macrophage activating factor), MMIF (macrophage
migration inhibition factor), MCF (macrophage chemotactic factor),
LMIF (leukocyte migration inhibition factor), HRFs (histamine
releasing factors), TF (transfer factors), interleukins (IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, etc.), TNF-.alpha., TNF-.beta.,
interferons (IFN-.alpha., IFN-.beta., IFN-.gamma., IFN-.zeta.,
IFN-.delta., etc.), G-CSF (granulocyte colony stimulating factor),
GM-CSF (granulocyte-macrophage CSF), M-CSF (macrophage CSF),
multi-CSF (IL-3), fibroblast growth factor (aFGF, bFGF), EGF
(epidermal growth factor), NGF (nerve growth factor), PDGF
(platelet-derived growth factor), VEGF (vascular endothelial growth
factor), transforming growth factors (TGF-.alpha., TGF-.beta.,
etc.), NAP-2 (neutrophil-activating protein 2), PF-4 (platelet
factor 4), thromboglobulin, MCP-1 (monocyte chemoattractant protein
1), MCP-3, MIP-1a, MIP-1.beta.-+(macrophage inflammatory proteins),
RANTES (regulated upon activation normal T expressed and presumably
secreted chemokine), HSPS (heat shock proteins), GRPs
(glucose-regulated proteins), ubiquitin, and others.
[0171] Thus, in certain embodiments, the gas-enriched fluids and/or
therapeutic compositions increase production and/or secretion of
anti-inflammatory molecules or cytokines or decrease the
degradation of anti-inflammatory molecules or cytokines, thereby
alleviating or preventing at least one symptom of inflammation. In
other embodiments, the gas-enriched fluids and/or therapeutic
compositions of the present invention may decrease production
and/or secretion of pro-inflammatory molecules or cytokines or
increase the degradation of pro-inflammatory molecules or
cytokines, thereby alleviating or preventing at least one symptom
of inflammation.
[0172] Previous studies had shown a critical role of anti-MOG
antibodies in augmentation of demyelination and worsening of EAE
(experimental autoimmune encephalomyelitis), an animal model system
for the human autoimmune disorder of rheumatoid arthritis
(Linington, et al. 1992. J. Neuroimmunol. 40:219-224).
Additionally, antibodies against MOG have been implicated in the
pathogenesis of multiple sclerosis. (Berger et al. N. Engl. J. Med.
2003 Jul. 10; 349(2):139-45).
[0173] As set forth in FIG. 48 and Example 12, the inventive
gas-enriched fluid of the present invention amplifies the
lymphocyte response to an antigen for which an animal was
previously primed. As indicated in FIG. 48, lymphocyte
proliferation was greater for response to MOG challenge when
cultured in fluid reconstituted with the inventive gas-enriched
fluid comprising solvated electrons, when compared with
pressurized, oxygenated fluid (pressure pot) or control deionized
fluid.
Inventive Gas-Enriched Fluids and Solutions
[0174] Diffusing or enriching a fluid with another fluid may result
in a solution or suspension of the two fluids. In particular,
enriching a liquid with a gas (e.g. oxygen) may be beneficial for
certain applications, including therapeutic treatments. As utilized
herein, "fluid," may generally refer to a liquid, a gas, a vapor, a
mixture of liquids and/or gases, or any combination thereof, for
any particular disclosed embodiment. Furthermore, in certain
embodiments a "liquid" may generally refer to a pure liquid or may
refer to a gel, sol, emulsion, fluid, colloid, dispersion, or
mixture, as well as any combination thereof; any of which may vary
in viscosity.
[0175] In particular embodiments, the dissolved gas comprises
oxygen. In other particular embodiments, the dissolved gas
comprises nitric oxide. In still other embodiments, the dissolved
gas comprises ambient air.
[0176] There are several art-recognized methods of gas-enriching
fluids (such as oxygen-enriching water). For example, a turbine
aeration system can release air near a set of rotating blades of an
impeller, which mixes the air or oxygen with the water, or water
can be sprayed into the air to increase its oxygen content.
Additionally, other systems on the market inject air or oxygen into
the water and subject the water/gas to a large scale vortex.
Naturally occurring levels of oxygen in water are typically no more
than 10 ppm (parts per million), which is considered to be a level
of 10 ppm dissolved oxygen. Tests on certain devices have shown
that under ideal conditions, the device can attain upwards of
approximately 20 ppm dissolved oxygen, or twice the natural oxygen
levels of water. However, the water loses that high level of
dissolved oxygen very rapidly, and within minutes the water returns
to having the baseline of about 10 ppm dissolved oxygen.
[0177] In certain embodiments disclosed herein, a gas-enriched
fluid of the present invention provides a cosmetic and/or
therapeutic eye care benefit. Certain embodiments disclosed herein
relate to a cosmetic and/or therapeutic composition comprising a
gas-enriched fluid of the present invention, and optionally at
least one additional therapeutic agent, such as a pharmaceutical
drug, a metal, a peptide, a polypeptide, a protein, a nucleotide, a
carbohydrate or glycosylated protein, a fat (including oils or
waxes), or other agent that prevents or alleviates at least one
symptom of a condition or disease associated with eye
irritation.
[0178] Compositions and methods are disclosed for treating the eye,
or other organs and/or tissues in need thereof by topically
applying an effective amount of a composition comprising a
gas-enriched fluid.
[0179] As used herein, "treat," "treating," "treatment," and any
and all derivations thereof refer to using the compositions of the
present invention either prophylactically to prevent signs of an
eye condition or disease, or cosmetically or therapeutically to
ameliorate an existing condition or disease. The term "treating"
refers to, and includes, reversing, alleviating, inhibiting the
progress of, or preventing a disease, disorder or condition, or one
or more symptoms thereof; and "treatment" and "therapeutically"
refer to the act of treating, as defined herein.
[0180] A "therapeutically effective amount" is any amount of any of
the compounds utilized in the course of practicing the invention
provided herein that is sufficient to reverse, alleviate, inhibit
the progress of, or prevent a disease, disorder or condition, or
one or more symptoms thereof. In one particular embodiment, the
gas-enriched fluid of the present invention inhibits microbial
growth. In another particular embodiment, the gas-enriched fluid of
the present invention promotes apoptosis.
[0181] Microbial infections, particularly of Staphlycoccus,
Streptococcus, yeast, Serratia, E. coli, Pseudomonas aeruginosa,
and other microbial infections, can cause devastating infections in
the eyes if left untreated. Thus, in certain embodiments, the
gas-enriched fluid compositions and/or methods of the present
invention include anti-microbial agents, such as antifungal,
antibiotic, or other anti-microbial agents. Some examples of
anti-microbial agents that may be utilized with the gas-enriched
fluid compositions and/or methods include, but are not limited to,
amikacin, gentamicin, kanamycin, neomycin, netilmicin,
streptomycin, tobramycin, paromomycin, geldanamycin, herimycin,
loracarbef, ertapenem, imipenem/cilastatin, meropenem, cefadroxil,
cefazolin, cefalotin/cefalothin, cephalexin, cefaclor, cefamandole,
cefoxitin, cefuroxime, cefixime, cefdinir, cefditoren,
cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,
ceftizoxime, ceftriaxone, cefeprime, teicoplanin, vancomycin,
azithromycin, clarithromycin, dirithromycin, erythromycin,
roxithromycin, troleandomycin, telithromycin, spectinomycin,
aztreonam, amoxicillin, ampicillin, azlocillin, carbenicillin,
cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin,
penicillin, peperacillin, ticarcillin, bacitracin, colistin,
polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,
lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin,
mafenide, protosil, sulfacetamide, sulfamethizole, sulfanilamide,
sulfasalazine, sulfisoxazole, trimethoprim,
trimethoprim-sulfamethoxazole, demeclocycline, doxycycline,
minocycline, oxytetracycline, tetracycline, arsphenamine,
chloramphenicol, clindamycin, lincoamycin, ethambutol, fosfomycin,
fusidic acid, furazolidone, isoniazid, linezolid, metronidazole,
mupirocin, nitrofurantoin, platensimycin, pyrazinamide,
quinupristin/dalfopristin, rifampin/rifampicin, tinidazole,
miconazole, ketoconazole, clotrimazole, econazole, bifonazole,
butoconazole, fenticonazole, isoconazole, oxiconazole,
sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole,
isavuconazole, ravuconazole, posaconazole, voriconazole,
teronazole, terbinafine, amorolfine, naftifine, butenafine,
anidulafungin, caspofungin, micafungin, ciclopirox, flucytosine,
griseofulvin, Gentian violet, haloprogin, tolnaftate, undecylenic
acid, and others.
[0182] The gas-enriched fluid compositions and/or methods provided
herein may include any form suitable for topical application,
including a gas, aqueous or oil-based liquid, cream, lotion, balm,
oil- and fluid mixture, gel, sol, emulsion, microemulsion,
solution, suspension, or may be encapsulated in a liposome,
microsponge, polymer matrix, or other encapsulation technology
adapted to aid in the delivery of the gas-enriched fluid to the
areas of the eye in need thereof, or to enhance the stability of
the composition or effectiveness of the method employed. In yet
other embodiments, the gas-enriched fluid composition is formulated
for delivery by an instrument, such as ultrasound, to the affected
areas of the eye. In still other embodiments, the composition
further comprises an inert and physiologically-acceptable carrier
or diluent, enzymes, anti-microbial agents (anti-bacterial agents,
anti-fungal agents, etc.), vasoconstrictors (such as epinephrine,
naphazoline hydrochloride, tetrahydrozoline, etc.), acids (such as
boric acid, hydrochloric acid, etc.), bases (such as sodium
hydroxide, etc.), salts (such as sodium, potassium, calcium, etc.),
polymers, alcohols (such as polyvinyl alcohol), cellulose or
starch, dextrose, mannose, sucrose, or other carbohydrates;
glycoproteins, proteins, polypeptides or peptides, colors,
fragrances, preservatives (such as edentate disodium, chlorhexidine
gluconate, etc.), or a mixture thereof. In other related
embodiments, the composition further comprises an active
pharmaceutical drug or therapeutic drug substance or an active
cosmetic substance. In one particular embodiment, the
pharmaceutical or therapeutic drug comprises an antihistamine, such
as pheniramine maleate.
[0183] In addition to the other compositions described herein, the
present disclosure further contemplates combination therapies
comprising at least one embodiment of an inventive fluid in
combination with a therapeutic agent, including but not limited to:
ciclosporin, hyaluronic acid, carmellose, macrogol(s), dextran and
hyprolose, sodium and calcium, sodium and povidone, hypromellose,
carbomer and others. Some particular compositions containing these
agents include, but are not limited to: Restasis.TM., Hyalein.TM.,
Celluvisc.TM., Systane.TM., Tears Naturale.TM., Mytear.RTM.,
Refresh Tears.TM., Refresh.TM., Genteal.TM., Viscotears.TM.,
Artelac.TM., Opelead.TM., Opti-Free Express.TM. Soft Lens
Solutions, RENU.TM., Opti-Free.TM., Opti-Free Replenis.TM.,
AOSEPT.TM., RENU PLUS.TM., RENU MULTIPLUS.TM., SENSITIVE EYES.TM.,
BOSTON SIMPLUS.TM., LENSEPT.TM. OPTIFREE REPLENISH.TM., Opthalmic
Nonsteroidal Anti-Inflammatories such as ACULAR.TM. (KETOROLAC),
VOLTAREN.TM. (DICLOFENAC), NEVANAC.TM. (NEPAFENAC), XIBROM.TM.,
(BROMFENAC), ISTA PHARM.TM., (NIFLAN), PRANOPROFEN, INDOCOLLYRE,
INDOMETACIN; preparations for the treatment of non-specific
conjunctivitis such as PATANOL.TM. (OLOPATADINE), LIVOSTIN.TM.
(LEVOCABASTINE), ZADITEN.TM. (KETOTIFEN), PATADAY.TM.
(OLOPATADINE), OPTIVAR.TM. (AZELASTINE), RELESTAT.TM. (EPINASTINE),
OYXIN.TM. (TETRYZOLINE), INTAL (CROMOGLICIC ACID), RIZABEN
(TRANILAST), CLEAR EYES.TM. (NAPHAZOLINE), DACRYOSERUM.TM. (BORIC
ACID), Allergic eye care products, such as Naphcon-A.TM.,
Visine-A.TM., Opcon A.TM., Clear Eyes ACR.TM., All Clear AR.TM.,
Vasocon-A.TM.. Gas-enriched fluids produced in accordance with the
disclosed invention may also be used to decontaminate or wash away
contaminants from a tissue, intact and/or ex vivo. Contaminants can
be more thoroughly cleaned away by the gas-enriched fluids, which
may provide other therapeutic benefits.
[0184] Particular embodiments provided herein relate to a
diffuser-processed gas-enriched fluid as defined herein,
comprising: a fluid host material; an infusion material diffused
into the host material; and optionally, at least one cosmetic
and/or therapeutic agent dispersed in the host material. In certain
embodiments, the infusion material comprises oxygen micro-bubbles
in the host fluid, wherein the majority of the micro-bubbles are
less than 0.2 microns, or preferably less than 0.1 microns in
size.
[0185] Gas-enriched fluids produced in accordance with the
disclosed invention may also be used to decontaminate or wash away
contaminants from a tissue, intact and/or ex vivo. Contaminants can
be more thoroughly cleaned away by the gas-enriched fluids, which
may provide other therapeutic benefits.
[0186] There are several art-recognized methods of gas-enriching
liquids (such as oxygen-enriching water). For example, a turbine
aeration system can release air near a set of rotating blades of an
impeller, which mixes the air or oxygen with the water, or water
can be sprayed into the air to increase its oxygen content.
Additionally, other systems on the market inject air or oxygen into
the water and subject the water/gas to a large-scale vortex.
Naturally occurring levels of oxygen in water are typically no more
than 10 ppm (parts per million), which is considered to be a level
of 100% dissolved oxygen. Tests on certain devices have shown that
under ideal conditions, the device can attain upwards of
approximately 20 ppm, or twice the natural oxygen levels of water.
In certain embodiments, the oxygen level may be even higher.
[0187] Particular embodiments provided herein relate to a
diffuser-processed gas-enriched fluid as defined herein,
comprising: a fluid host material; an infusion material diffused
into the host material; and optionally, at least one cosmetic
and/or therapeutic agent dispersed in the host material. In certain
embodiments, the infusion material comprises oxygen micro-bubbles
in the host fluid, wherein the majority of the micro-bubbles are
less than 0.2 microns, or preferably less than 0.1 microns in size.
In certain embodiments, the dissolved oxygen level in the infused
fluid host material may be maintained at greater than about 30 ppm
at atmospheric pressure for at least 13 hours. In other particular
embodiments, the dissolved oxygen level in the infused fluid host
material may be maintained at greater than 40 ppm at atmospheric
pressure for at least 3 hours.
[0188] In additional embodiments, the infused fluid host material
further comprises a saline solution. In further embodiments, the
infused fluid host material maintains a dissolved oxygen level of
at least about 20 ppm to about 40 ppm for a period of at least 100
days, preferably at least 365 days within a sealed container at
atmospheric pressure. In certain embodiments, the infused fluid
host material may have a dissolved oxygen level of at least 50 ppm
at atmospheric pressure.
[0189] In certain embodiments, the infused fluid host material
exhibits Rayleigh scattering for a laser beam shining therethrough
for a selected period of time after the oxygen has been diffused
into therein.
[0190] Table 3 illustrates various partial pressure measurements
taken in a healing wound treated with an oxygen-enriched saline
solution and in samples of the gas-enriched oxygen-enriched saline
solution of the present invention.
TABLE-US-00003 TABLE 3 TISSUE OXYGEN MEASUREMENTS Probe Z082BO In
air: 171 mmHg 23.degree. C. Column Partial Pressure (mmHg) B1 32-36
B2 169-200 B3 20-180* B4 40-60 *wound depth minimal, majority
>150, occasional 20 s
[0191] In certain embodiments, the dissolved oxygen level in the
infused fluid host material may be maintained at greater than about
30 ppm at atmospheric pressure for at least about 13 hours. In
other particular embodiments, the dissolved oxygen level in the
infused fluid host material may be maintained at greater than about
40 ppm at atmospheric pressure for at least about 3 hours. In
further embodiments, the infused fluid host material maintains a
dissolved oxygen level of at least about 20 ppm for a period of at
least about 100 days within a sealed container at atmospheric
pressure. In certain embodiments, the infused fluid host material
may have a dissolved oxygen level of at least about 10 ppm, at
least about 15 ppm, at least about 20 ppm, at least about 25 ppm,
at least about 30 ppm, at least about 35 ppm, at least about 40
ppm, at least about 45 ppm, at least about 50 ppm, at least about
55 ppm, at least about 60 ppm, at least about 65 ppm, at least
about 70 ppm, at least about 75 ppm, at least about 77 ppm, at
least about 80 ppm, at least about 85 ppm, at least about 90 ppm,
at least about 95 ppm, at least about 100 ppm, or greater or any
value therebetween, at atmospheric pressure.
[0192] In certain embodiments, the infused fluid host material
exhibits Rayleigh scattering for a laser beam shining therethrough
for a selected period of time after the oxygen has been diffused
therein. In certain implementations, the infused fluid host
material includes solvated electrons created in the solution by the
process for diffusing the oxygen therein. In yet further
embodiments, the infused fluid host material containing oxygen
diffused therein is produced in a non-restricted flow through
manner.
[0193] By using the diffuser device described herein with respect
to exemplary embodiments illustrated in the corresponding figures,
an output fluid may be achieved having a gas diffused therein that
has a number of characteristics and provides a number of advantages
for use as a therapeutic composition. Solutions have been created
using freshwater, saline, oxygen, nitrogen and other components.
Experiments have indicated that oxygen bubbles produced within
saline solution are generally no greater than approximately 0.1
micron in size.
Bubble Size Measurements
[0194] Experimentation was performed to determine a size of the
bubbles of gas diffused within the fluid by the mixing device 100.
While experiments were not performed to measure directly the size
of the bubbles, experiments were performed that established that
the bubble size of the majority of the gas bubbles within the fluid
was smaller than 0.1 microns. In other words, the experiments
determined a size threshold value below which the sizes of the
majority of bubbles fall.
[0195] This size threshold value or size limit was established by
passing the output material 102 formed by processing a fluid and a
gas in the mixing device 100 through a 0.22 filter and a 0.1 micron
filter. In performing these tests, a volume of the first material
110, in this case, a fluid, and a volume of the second material
120, in this case, a gas, were passed through the mixing device 100
to generate a volume of the output material 102 (i.e., a fluid
having a gas diffused therein). Sixty milliliters of the output
material 102 was drained into a 60 ml syringe. The DO level of the
fluid within the syringe was then measured using an Orion 862a. The
Orion 862a is capable of measuring DO levels within a fluid. The
fluid within the syringe was injected through a 0.22 micron filter
into a 50 ml beaker. The filter comprised the Milipor Millex GP50
filter. The DO level of the material in the 50 ml beaker was then
measured. The experiment was performed three times to achieve the
results illustrated in Table 4 below.
TABLE-US-00004 TABLE 4 DO levels DO AFTER 0.22 MICRON DO IN SYRINGE
FILTER 42.1 ppm 39.7 ppm 43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm
[0196] As can be seen, the DO levels measured within the syringe
and the DO levels measured within the 50 ml beaker were not changed
drastically by passing the output material 102 through the 0.22
micron filter. The implication of this experiment is that the
bubbles of dissolved gas within the output material 102 are not
larger than 0.22 microns otherwise there would be a significantly
greater reduction in the DO levels in the output material 102
passed through the 0.22 micron filter.
[0197] A second test was performed in which the 0.1 micron filter
was substituted for the 0.22 micron filter. In this experiment,
saline solution was processed with oxygen in the mixing device 100
and a sample of the output material 102 was collected in an
unfiltered state. The DO level of the unfiltered sample was 44.7
ppm. The output material 102 was filtered using the 0.1 micron
filter and two additional samples were collected. The DO level of
the first sample was 43.4 ppm. The DO level of the second sample
was 41.4 ppm. Then, the filter was removed and a final sample was
taken from the unfiltered output material 102. The final sample had
a DO level of 45.4 ppm. These results were consistent with those
seen using the Millipore 0.2 micron filter. These results lead to
the conclusion that there is a trivial reduction in the DO levels
of the output material 102 passed through the 0.1 micron filter
providing an indication that the majority of the bubbles in the
processed saline solution are no greater than 0.1 micron in
size.
[0198] As appreciated in the art, the double-layer (interfacial)
(DL) appears on the surface of an object when it is placed into a
liquid. This object, for example, might be that of a solid surface
(e.g., rotor and stator surfaces), solid particles, gas bubbles,
liquid droplets, or porous body. In the mixing device 100, bubble
surfaces represent a significant portion of the total surface area
present within the mixing chamber that may be available for
electrokinetic double-layer effects. Therefore, in addition to the
surface area and retention time aspects discussed elsewhere herein,
the relatively small bubble sizes generated within the mixer 100
compared to prior art devices 10, may also contribute, at least to
some extent, to the overall electrokinetic effects and output fluid
properties disclosed herein. Specifically, in preferred
embodiments, as illustrated by the mixer 100, all of the gas is
being introduced via apertures on the rotor (no gas is being
introduced through stator apertures. Because the rotor is rotating
at a high rate (e.g., 3,400 rpm) generating substantial shear
forces at and near the rotor surface, the bubble size of bubbles
introduced via, and adjacent to the spinning rotor surface
apertures would be expected to be substantially (e.g., 2 to 3-times
smaller) smaller than those introduced via and near the stationary
stator. The average bubble size of the prior art device 10 may,
therefore, be substantially larger because at least half of the gas
is introduced into the mixing chamber from the stationary stator
apertures. Because the surface area of a sphere surface varies with
r.sup.2, any such bubble component of the electrokinetic surface
area of the mixing device 100 may be substantially greater than
that of the prior art diffusion device 10.
[0199] Therefore, without being bound by theory, not only does the
mixing chamber of the mixing device 100 have (i) a substantially
higher surface to volume ratio than that of the prior art device 10
(the prior art device 10 has a ratio of surface to volume of 10.9,
whereas the present mixer 100 has a surface to volume ratio of
39.4), along with (ii) a 7-fold greater dwell-time, but (iii) the
unique properties of the current output solutions may additionally
reflect a contribution from the substantially larger bubble surface
area in the mixing device 100. These distinguishing aspects reflect
distinguishing features of the present mixer 100, and likely each
contribute to the unique electrokinetic properties of the inventive
output materials/fluids.
[0200] Referring now to FIG. 30, there is illustrated the DO levels
in water enriched with oxygen in the mixing device 100 and stored
in a 500 ml thin-walled plastic bottle and a 1000 ml glass bottle
out to at least 365 days. Each of the bottles was capped and stored
at 65.degree. Fahrenheit. As can be seen in the Figure, the DO
levels of the oxygen-enriched fluid remained fairly constant out to
at least 365 days.
[0201] Referring to FIG. 31, there is illustrated the DO levels in
water enriched with oxygen in the mixing device 100 and stored in a
500 ml plastic thin-walled bottle and a 1000 ml glass bottle. Both
bottles were refrigerated at 39.degree. Fahrenheit. Again, DO
levels of the oxygen-enriched fluid remained steady and decreased
only slightly out to at least 365 days.
Compositions Comprising Hydrated (Solvated) Electrons Imparted to
the Inventive Compositions by the Inventive Processes
[0202] In certain embodiments as described herein (see under
"Double-layer"), the gas-enriched fluid is generated by the
disclosed electromechanical processes in which molecular oxygen is
diffused or mixed into the fluid and may operate to stabilize
charges (e.g., hydrated (solvated) electrons) imparted to the
fluid. Without being bound by theory or mechanism, certain
embodiments of the present invention relate to a oxygen-enriched
fluid (output material) comprising charges (e.g., hydrated
(solvated) electrons) that are added to the materials as the first
material is mixed with oxygen in the inventive mixer device to
provide the combined output material. According to particular
aspects, these hydrated (solvated) electrons (alternately referred
to herein as `solvated electrons`) are stabilized in the inventive
solutions as evidenced by the persistence of assayable effects
mediated by these hydrated (solvated) electrons. Certain
embodiments may relate to hydrated (solvated) electrons and/or
water-electron structures, clusters, etc., (See, for example, Lee
and Lee, Bull. Kor. Chem. Soc. 2003, v. 24, 6; 802-804; 2003).
[0203] Horseradish peroxidase (HRP) effects. Horseradish peroxidase
(HRP) is isolated from horseradish roots (Amoracia rusticana) and
belongs to the ferroprotoporphyrin group (Heme group) of
peroxidases. HRP readily combines with hydrogen peroxide or other
hydrogen donors to oxidize the pyrogallol substrate. Additionally,
as recognized in the art, HRP facilitates autoxidative degradation
of indole-3-acetic acid in the absence of hydrogen peroxide (see,
e.g., Heme Peroxidases, H. Brian Dunford, Wiley-VCH, 1999, Chapter
6, pages 112-123, describing that auto-oxidation involves a highly
efficient branched-chain mechanism; incorporated herein by
reference in its entirety). The HRP reaction can be measured in
enzymatic activity units, in which Specific activity is expressed
in terms of pyrogallol units. One pyrogallol unit will form 1.0 mg
purpurogallin from pyrogallol in 20 sec at pH 6.0 at 20.degree. C.
This purpurogallin (20 sec) unit is equivalent to approx. 18 .mu.M
units per min at 25.degree. C.
##STR00001##
[0204] It is known that Horseradish peroxidase enzyme catalyzes the
auto-oxidation of pyrogallol by way of facilitating reaction with
the molecular oxygen in a fluid. (Khajehpour et al., PROTEINS:
Struct, Funct, Genet. 53: 656-666 (2003)). It is also known that
oxygen binds the heme pocket of horseradish peroxidase enzyme
through a hydrophobic pore region of the enzyme (between Phe68 and
Phe142), whose conformation likely determines the accessibility of
oxygen to the interior. According to particular aspects, and
without being bound by mechanism, because surface charges on
proteins are known in the protein art to influence protein
structure, the solvated electrons present in the inventive
gas-enriched fluid may act to alter the conformation of the
horseradish peroxidase such that greater oxygen accessibility may
result. The greater accessibility of oxygen to the prosthetic heme
pocket of the horseradish peroxidase enzyme may in turn allow for
increased HRP reactivity, when compared with prior art oxygenated
fluids (pressure-pot, fine-bubbled).
[0205] In any event, according to particular aspects, production of
output material using the inventive methods and devices comprises a
process involving: an interfacial double layer that provides a
charge gradient; movement of the materials relative to surfaces
pulling charge (e.g., electrons) away from the surface by virtue of
a triboelectric effect, wherein the flow of material produces a
flow of solvated electrons. Moreover, according to additional
aspects, and without being bound by mechanism, the orbital
structure of diatomic oxygen creates charge imbalances (e.g., the
two unpaired electrons affecting the hydrogen bonding of the water)
in the hydrogen bonding arrangement within the fluid material
(water), wherein electrons are solvated and stabilized within the
imbalances.
[0206] Several chemical tests of the inventive oxygen-enriched
fluid for the presence of hydrogen peroxide were conducted as
described below, and none of these tests were positive (sensitivity
of 0.1 ppm hydrogen peroxide). Thus, the inventive oxygen-enriched
fluid of the instant application contain no, or less than 0.1 ppm
hydrogen peroxide.
[0207] According to particular aspects, despite the absence of
hydrogen peroxide, the inventive combination of oxygen-enrichment
and solvated electrons imparted by the double-layer effects and
configuration of the presently claimed devices may act to alter the
conformation and/or heme group accessibility of the horseradish
peroxidase.
Glutathione Peroxidase Study
[0208] The inventive oxygen-enriched output fluid material was
tested for the presence of hydrogen peroxide by testing the
reactivity with glutathione peroxidase using a standard assay
(Sigma). Water samples were tested by adding the enzymatic
reagents. Continuous spectrophotometric rate determination was made
at A.sub.340 nm, and room temperature (25.degree. Celsius). Samples
tested were: 1. deionized water (negative control), 2. inventive
oxygen-enriched fluid at low concentration, 3. inventive
oxygen-enriched fluid at high concentration, 4. hydrogen peroxide
(positive control). The hydrogen peroxide positive control showed a
strong reactivity, while none of the other fluids tested reacted
with the glutathione.
Device for Generating Gas-Enriched Fluids or Solutions
Description of the Related Art
[0209] FIG. 1 provides a partial block diagram, partial
cross-sectional view of a prior art device 10 for diffusing or
emulsifying one or two gaseous or liquid materials ("infusion
materials") into another gaseous or liquid material ("host
material") reproduced from U.S. Pat. No. 6,386,751, incorporated
herein by reference in its entirety. The device 10 includes a
housing configured to house a stator 30 and a rotor 12. The stator
30 encompasses the rotor 12. A tubular channel 32 is defined
between the rotor 12 and the stator 30. The generally cylindrically
shaped rotor 12 has a diameter of about 7.500 inches and a length
of about 6.000 inches providing a length to diameter ratio of about
0.8.
[0210] The rotor 12 includes a hollow cylinder, generally closed at
both ends. A gap exists between each of the first and second ends
of the rotor 12 and a portion of the housing 34. A rotating shaft
14 driven by a motor 18 is coupled to the second end of the rotor
12. The first end of the rotor 12 is coupled to an inlet 16. A
first infusion material passes through the inlet 16 and into the
interior of the rotor 12. The first infusion material passes from
the interior of the rotor 12 and into the channel 32 through a
plurality of openings 22 formed in the rotor 12.
[0211] The stator 30 also has openings 22 formed about its
circumference. An inlet 36 passes a second infusion material to an
area 35 between the stator 30 and the housing 34. The second
infusion material passes out of the area 35 and into the channel 32
through openings 22.
[0212] An external pump (not shown) is used to pump the host
material into a single inlet port 37. The host material passes
through a single inlet port 37 and into the channel 32 where it
encounters the first and second infusion materials, which enter the
channel 32 through openings 22. The infusion materials may be
pressurized at their source to prevent the host material from
passing through openings 22.
[0213] The inlet port 37, is configured and positioned such that it
is located along only a relatively small portion (<about 5%) of
the annular inlet channel 32, and is substantially parallel to the
axis of rotation of the rotor 12 to impart an axial flow toward a
portion of the channel 32 into the host material.
[0214] Unfortunately, before entering the tubular channel 32, the
host material must travel in tortuous directions other than that of
the axial flow (e.g., including in directions substantially
orthogonal thereto) and down into and between the gap formed
between the first end of the rotor 12 and the housing 34 (i.e.,
down a portion of the first end of the rotor adjacent to the inlet
16 between the end of the rotor 12 and the housing 34). The
non-axial and orthogonal flow, and the presence of the host
material in the gap between the first end of the rotor 12 and the
housing 34 causes undesirable and unnecessary friction. Further, it
is possible for a portion of the host material to become trapped in
eddy currents swirling between the first end of the rotor and the
housing. Additionally, in the device 10, the host material must
negotiate at least two right angles to enter any aspect of the
annual of the annular inlet of the tubular channel 32.
[0215] A single outlet port 40 is formed in the housing 34. The
combined host material and infusion material(s) exit the channel 32
via the outlet 40. The outlet port 40, which is also located along
only a limited portion (<about 5%) of the annular outlet of
tubular channel 32, is substantially parallel to the axis of
rotation of the rotor 12 to impart or allow for an axial flow of
the combined materials away from the limited portion of the annular
outlet of tubular channel 32 into the outlet port 40. An external
pump 42 is used to pump the exiting fluid through the outlet port
40.
[0216] Unfortunately, before exiting the channel 32, a substantial
portion of the exiting material must travel in a tortuous direction
other than that of the axial flow (e.g., including in directions
substantially orthogonal thereto) and down into and between the gap
formed between the second end of the rotor 12 and the housing 34
(i.e., down a portion of the second end of the rotor adjacent to
the shaft 14 between the end of the rotor 12 and the housing 34).
As mentioned above, the non-axial and orthogonal flow, and the
presence of the host material in the other gap between the end (in
this case, the second end) of the rotor 12 and the housing 34
causes additional undesirable and unnecessary friction. Further, it
is possible for a portion of the host material to become trapped in
eddy currents swirling between the second end of the rotor and the
housing. Additionally, in the device 10, a substantial portion of
the exiting combined material must negotiate at least two right
angles as it exits from the annular exit of the tubular channel 32
into the outlet port 40.
[0217] As is apparent to those of ordinary skill in the art, the
inlet port 37 imparts only an axial flow to the host material. Only
the rotor 21 imparts a circumferential flow into the host material.
Further, the outlet port 40 imparts or provides for only an axial
flow into the exiting material. Additionally, the circumferential
flow velocity vector is imparted to the material only after it
enters the annular inlet 37 of the tubular channel 32, and
subsequently the circumferential flow vector must be degraded or
eliminated as the material enters the exit port 40. There is,
therefore, a need for a progressive circumferential acceleration of
the material as it passes in the axial direction through the
channel 32, and a circumferential deceleration upon exit of the
material from the channel 32. These aspects, in combination with
the tortuous path that the material takes from the inlet port 37 to
the outlet port 40, create a substantial friction and flow
resistance over the path that is accompanied by a substantial
pressure differential (26 psi, at 60 gallons/min flow rate) between
the inlet 37 and outlet 40 ports, and these factors, inter alia,
combine to reduce the overall efficiency of the system.
Electrokinetically Oxygen-Enriched Aqueous Fluids and Solutions
[0218] FIG. 2 provides a block diagram illustrating some of the
components of a mixing device 100 and the flow of material into,
within, and out of the device. The mixing device 100 combines two
or more input materials to form an output material 102, which may
be received therefrom into a storage vessel 104. The mixing device
100 agitates the two or more input materials in a novel manner to
produce an output material 102 having novel characteristics. The
output material 102 may include not only a suspension of at least
one of the input materials in at least one of the other input
materials (e.g., emulsions) but also a novel combination (e.g.,
electrostatic combinations) of the input materials, a chemical
compound resulting from chemical reactions between the input
materials, combinations having novel electrostatic characteristics,
and combinations thereof.
[0219] The input materials may include a first material 110
provided by a source 112 of the first material, a second material
120 provided by a source 122 of the second material, and optionally
a third material 130 provided by a source 132 of the third
material. The first material 110 may include a liquid, such as
water, saline solution, chemical suspensions, polar liquids,
non-polar liquids, colloidal suspensions, cell growing media, and
the like. In some embodiments, the first material 110 may include
the output material 102 cycled back into the mixing device 100. The
second material 120 may consist of or include a gas, such as
oxygen, nitrogen, carbon dioxide, carbon monoxide, ozone, sulfur
gas, nitrous oxide, nitric oxide, argon, helium, bromine, and
combinations thereof, and the like. In preferred embodiments, the
gas is or comprises oxygen. The optional third material 130 may
include either a liquid or a gas. In some embodiments, the third
material 130 may be or include the output material 102 cycled back
into the mixing device 100 (e.g., to one or more of the pumps 210,
220 or 230, and/or into the chamber 310, and/or 330).
[0220] Optionally, the first material 110, the second material 120,
and the optional third material 130 may be pumped into the mixing
device 100 by an external pump 210, an external pump 220, and an
external pump 230, respectively. Alternatively, one or more of the
first material 110, the second material 120, and the optional third
material 130 may be stored under pressure in the source 112, the
source 122, and the source 132, respectively, and may be forced
into the mixing device 100 by the pressure. The invention is not
limited by the method used to transfer the first material 110, the
second material 120, and optionally, the third material 130 into
the mixing device 100 from the source 112, the source 122, and the
source 132, respectively.
[0221] The mixing device 100 includes a first chamber 310 and a
second chamber 320 flanking a mixing chamber 330. The three
chambers 310, 320, and 330 are interconnected and form a continuous
volume.
[0222] The first material 110 is transferred into the first chamber
310 and flows therefrom into the mixing chamber 330. The first
material 110 in the first chamber 310 may be pumped into the first
chamber 310 by an internal pump 410. The second material 120 is
transferred into the mixing chamber 330. Optionally, the third
material 130 may be transferred into the mixing chamber 330. The
materials in the mixing chamber 330 are mixed therein to form the
output material 102. Then, the output material 102 flows into the
second chamber 320 from which the output material 102 exits the
mixing device 100. The output material 102 in the mixing chamber
330 may be pumped into the second chamber 320 by an internal pump
420. Optionally, the output material 102 in the second chamber 320
may be pumped therefrom into the storage vessel 104 by an external
pump 430 (e.g., alone or in combination with the internal pump 410
and/or 420).
[0223] In particular aspects, a common drive shaft 500 powers both
the internal pump 410 and the internal pump 420. The drive shaft
500 passes through the mixing chamber 330 and provides rotational
force therein that is used to mix the first material 110, the
second material 120, and optionally, the third material 130
together. The drive shaft 500 is powered by a motor 510 coupled
thereto.
[0224] FIG. 3 provides a system 512 for supplying the first
material 110 to the mixing device 100 and removing the output
material 102 from the mixing device 100. In the system 512, the
storage vessel 104 of the output material 102 and the source 112 of
the first material 110 are combined. The external pump 210 is
coupled to the combined storage vessel 104 and source 112 by a
fluid conduit 514 such as hose, pipe, and the like. The external
pump 210 pumps the combined first material 110 and output material
102 from the combined storage vessel 104 and source 112 through the
fluid conduit 514 and into a fluid conduit 516 connecting the
external pump 210 to the mixing device 100. The output material 102
exits the mixing device 100 through a fluid conduit 518. The fluid
conduit 518 is coupled to the combined storage vessel 104 and
source 112 and transports the output material 102 exiting the
mixing device 100 to the combined storage vessel 104 and source
112. The fluid conduit 518 includes a valve 519 that establishes an
operating pressure or back pressure within the mixing device
100.
[0225] Referring to FIGS. 2 and 4-9 and 11, a more detailed
description of various components of an embodiment of the mixing
device 100 will be provided. The mixing device 100 is scalable.
Therefore, dimensions provided with respect to various components
may be used to construct an embodiment of the device or may be
scaled to construct a mixing device of a selected size.
[0226] Turning to FIG. 4, the mixing device 100 includes a housing
520 that houses each of the first chamber 310, the mixing chamber
330, and the second chamber 320. As mentioned above, the mixing
device 100 includes the drive shaft 500, which rotates during
operation of the device. Therefore, the mixing device 100 may
vibrate or otherwise move. Optionally, the mixing device 100 may be
coupled to a base 106, which may be affixed to a surface such as
the floor to maintain the mixing device 100 in a substantially
stationary position.
[0227] The housing 520 may be assembled from two or more housing
sections. By way of example, the housing 520 may include a central
section 522 flanked by a first mechanical seal housing 524 and a
second mechanical seal housing 526. A bearing housing 530 may be
coupled to the first mechanical seal housing 524 opposite the
central section 522. A bearing housing 532 may be coupled to the
second mechanical seal housing 526 opposite the central section
522. Optionally, a housing section 550 may be coupled to the
bearing housings 530.
[0228] Each of the bearing housings 530 and 532 may house a bearing
assembly 540 (see FIGS. 5 and 6). The bearing assembly 540 may
include any suitable bearing assembly known in the art including a
model number "202SZZST" manufactured by SKF USA Inc, of Kulpsville,
Pa., operating a website at www.skf.com.
[0229] Seals may be provided between adjacent housing sections. For
example, o-ring 560 (see FIG. 5) may be disposed between the
housing section 550 and the bearing housing 530, o-ring 562 (see
FIG. 5) may be disposed between the first mechanical seal housing
524 and the central section 522, and o-ring 564 (see FIG. 6) may be
disposed between the second mechanical seal housing 526 and the
central section 522.
Mixing Chamber 330
[0230] Turning now to FIG. 7, the mixing chamber 330 is disposed
inside the central section 522 of the housing 520 between the first
mechanical seal housing 524 and the second mechanical seal housing
526. The mixing chamber 330 is formed between two components of the
mixing device 100, a rotor 600 and a stator 700. The rotor 600 may
have a sidewall 604 with an inside surface 605 defining a generally
hollow inside portion 610 and an outside surface 606. The sidewall
604 may be about 0.20 inches to about 0.75 inches thick. In some
embodiments, the sidewall 604 is about 0.25 inches thick. However,
because the mixing device 100 may be scaled to suit a particular
application, embodiments of the device having a sidewall 604 that
is thicker or thinner than the values provided are within the scope
of the present teachings. The sidewall 604 includes a first end
portion 612 and a second end portion 614 and a plurality of
through-holes 608 formed between the first end portion 612 and the
second end portion 614. Optionally, the outside surface 606 of the
sidewall 604 may include other features such as apertures,
projections, textures, and the like. The first end portion 612 has
a relieved portion 616 configured to receive a collar 618 and the
second end portion 614 has a relieved portion 620 configured to
receive a collar 622.
[0231] The rotor 600 is disposed inside the stator 700. The stator
700 has a sidewall 704 with an inside surface 705 defining a
generally hollow inside portion 710 into which the rotor 600 is
disposed. The sidewall 704 may be about 0.1 inches to about 0.3
inches thick. In some embodiments, the sidewall 604 is about 1.5
inches thick. The stator 700 may be non-rotatably coupled to the
housing 520 in a substantially stationary position. Alternatively,
the stator 700 may integrally formed with the housing 520. The
sidewall 704 has a first end portion 712 and a second end portion
714. Optionally, a plurality of apertures 708 are formed in the
sidewall 704 of the stator 700 between the first end portion 712
and the second end portion 714. Optionally, the inside surface 705
of the sidewall 704 may include other features such as
through-holes, projections, textures, and the like.
[0232] The rotor 600 rotates with respect to the stationary stator
700 about an axis of rotation ".alpha." in a direction indicated by
arrow "C3" in FIG. 9. Each of the rotor 600 and the stator 700 may
be generally cylindrical in shape and have a longitudinal axis. The
rotor 600 has an outer diameter "D1" and the stator 700 may have an
inner diameter "D2." The diameter "D1" may range, for example, from
about 0.5 inches to about 24 inches. In some embodiments, the
diameter "D1" is about 3.04 inches. In some embodiments, the
diameter "D1" is about 1.7 inches. The diameter "D2," which is
larger than the diameter "D1," may range from about 0.56 inches to
about 24.25 inches. In some embodiments, the diameter "D2" is about
4 inches. Therefore, the mixing chamber 330 may have a ring-shaped
cross-sectional shape that is about 0.02 inches to about 0.125
inches thick (i.e., the difference between the diameter "D2" and
the diameter "D1"). In particular embodiments, the mixing chamber
330 is about 0.025 inches thick. The channel 32 between the rotor
12 and the stator 34 of prior art device 10 (see FIG. 1) has a
ring-shaped cross-sectional shape that is about 0.09 inches thick.
Therefore, in particular embodiments, the thickness of the mixing
chamber 330 is less than about one third of the channel 32 of the
prior art device 10.
[0233] The longitudinal axis of the rotor 600 may be aligned with
its axis of rotation ".alpha.." The longitudinal axis of the rotor
600 may be aligned with the longitudinal axis of the stator 700.
The rotor 600 may have a length of about 3 inches to about 6 inches
along the axis of rotation ".alpha.." In some embodiments, the
rotor 600 may have a length of about 5 inches along the axis of
rotation ".alpha.." The stator 700 may have a length of about 3
inches to about 6 inches along the axis of rotation ".alpha.." In
some embodiments, the stator 700 may have a length of about 5
inches along the axis of rotation ".alpha.."
[0234] While the rotor 600 and the stator 700 have been depicted as
having a generally cylindrical shape, those of ordinary skill in
the art appreciate that alternate shapes may be used. For example,
the rotor 600 and the stator 700 may be conically, spherically,
arbitrarily shaped, and the like. Further, the rotor 600 and the
stator 700 need not be identically shaped. For example, the rotor
600 may be cylindrically shaped and the stator 700 rectangular
shaped or vise versa.
[0235] The apertures 708 of the stator 700 and the through-holes
608 depicted in FIGS. 4-7 are generally cylindrically shaped. The
diameter of the through-holes 608 may range from about 0.1 inches
to about 0.625 inches. The diameter of the apertures 708 may range
from about 0.1 inches to about 0.625 inches. One or more of
apertures 708 of the stator 700 may have a diameter that differs
from the diameters of the other apertures 708. For example, the
apertures 708 may increase in diameter from the first end portion
712 of the stator 700 to the second end portion 714 of the stator
700, the apertures 708 may decrease in diameter from the first end
portion 712 of the stator 700 to the second end portion 714 of the
stator 700, or the diameters of the apertures 708 may vary in
another manner along the stator 700. One or more of through-holes
608 of the rotor 600 may have a diameter that differs from the
diameters of the other through-holes 608. For example, the
through-holes 608 may increase in diameter from the first end
portion 612 of the rotor 600 to the second end portion 614 of the
rotor 600, the through-holes 608 may decrease in diameter from the
first end portion 612 of the rotor 600 to the second end portion
614 of the rotor 600, or the diameters of the through-holes 608 may
vary in another manner along the rotor 600.
[0236] As described below with reference to alternate embodiments,
the apertures 708 and the through-holes 608 may have shapes other
than generally cylindrical and such embodiments are within the
scope of the present invention. For example, the through-holes 608
may include a narrower portion, an arcuate portion, a tapered
portion, and the like. Referring to FIG. 7, each of the
through-holes 608 includes an outer portion 608A, a narrow portion
608B, and a tapered portion 608C providing a transition between the
outer portion 608A and the narrow portion 608B. Similarly, the
apertures 708 may include a narrower portion, an arcuate portion, a
tapered portion, and the like.
[0237] FIG. 8 provides a non-limiting example of a suitable
arrangement of the apertures 708 of the stator 700 and the
through-holes 608 of the rotor 600. The apertures 708 of the stator
700 may be arranged in substantially parallel lateral rows "SLAT-1"
through "SLAT-6" substantially orthogonal to the axis of rotation
".alpha.." The apertures 708 of the stator 700 may also be arranged
in substantially parallel longitudinal rows "SLONG-1" through
"SLONG-7" substantially parallel with the axis of rotation
".alpha.." In other words, the apertures 708 of the stator 700 may
be arranged in a grid-like pattern of orthogonal rows (i.e., the
lateral rows are orthogonal to the longitudinal rows) having the
longitudinal rows "SLONG-1" through "SLONG-7" substantially
parallel with the axis of rotation ".alpha.."
[0238] Like the apertures 708 of the stator 700, the through-holes
608 of the rotor 600 may be arranged in substantially parallel
lateral rows "RLAT-1" through "RLAT-6" substantially orthogonal to
the axis of rotation ".alpha.." However, instead of being arranged
in a grid-like pattern of orthogonal rows, the through-holes 608 of
the rotor 600 may also be arranged in substantially parallel rows
"RLONG-1" through "RLONG-7" that extend longitudinally along a
helically path. Alternatively, the through-holes 608 of the rotor
600 may also be arranged in substantially parallel rows "RLONG-1"
through "RLONG-7" that extend longitudinally at an angle other than
parallel with the axis of rotation ".alpha.."
[0239] The apertures 708 of the stator 700 and the through-holes
608 of the rotor 600 may be configured so that when the rotor 600
is disposed inside the stator 700 the lateral rows "SLAT-1" to
"SLAT-6" at least partially align with the lateral rows "RLAT-1" to
"RLAT-6," respectively. In this manner, as the rotor 600 rotates
inside the stator 700, the through-holes 608 pass by the apertures
708.
[0240] The through-holes 608 in each of the lateral rows "RLAT-1"
to "RLAT-6" may be spaced apart laterally such that all of the
through-holes 608 in the lateral row align, at least partially,
with the apertures 708 in a corresponding one of the lateral rows
"SLAT-1" to "SLAT-6" of the stator 700 at the same time. The
longitudinally extending rows "RLONG-1" through "RLONG-6" may be
configured such that the through-holes 608 in the first lateral row
"RLAT-1" in each of the longitudinally extending rows passes
completely by the apertures 708 of the corresponding lateral row
"SLAT-1" before the through-holes 608 in the last lateral row
"RLAT-6" begin to partially align with the apertures 708 of the
corresponding last lateral row "SLAT-6" of the stator 700.
[0241] While, in FIG. 8, six lateral rows and six longitudinally
extending rows have been illustrated with respect to the rotor 600
and six lateral rows and seven longitudinally extending rows have
been illustrated with respect stator 700, it is apparent to those
of ordinary skill in the art that alternate numbers of lateral rows
and/or longitudinal rows may be used with respect to the rotor 600
and/or stator 700 without departing from the present teachings.
[0242] To ensure that only one pair of openings between
corresponding lateral rows will be coincident at any one time, the
number of apertures 708 in each of the lateral rows "SLAT-1" to
"SLAT-6" on the stator 700 may differ by a predetermined number
(e.g., one, two, and the like) the number of through-holes 608 in
each of the corresponding lateral rows "RLAT-1" to "RLAT-6" on the
rotor 600. Thus, for example, if lateral row "RLAT-1" has twenty
through-holes 608 evenly spaced around the circumference of rotor
600, the lateral row "SLAT-1" may have twenty apertures 708 evenly
spaced around the circumference of stator 700.
[0243] Returning to FIG. 7, the mixing chamber 330 has an open
first end portion 332 and an open second end portion 334. The
through-holes 608 formed in the sidewall 604 of the rotor 600
connect the inside portion 610 of the rotor 600 with the mixing
chamber 330.
[0244] The rotor 600 is rotated inside the stator 700 by the drive
shaft 500 aligned with the axis of rotation "a" of the rotor 600.
The drive shaft 500 may be coupled to the first end portion 612 and
the second end portion 614 of the rotor 600 and extend through its
hollow inside portion 610. In other words, a portion 720 of the
drive shaft 500 is disposed in the hollow inside portion 610 of the
rotor 600.
[0245] The collar 618 is configured to receive a portion 721 of the
drive shaft 500 disposed in the hollow inside portion 610 and the
collar 622 is configured to receive a portion 722 of the drive
shaft 500 disposed in the hollow inside portion 610.
[0246] The portion 721 has an outer diameter "D3" that may range
from about 0.5 inches to about 2.5 inches. In some embodiments, the
diameter "D3" is about 0.625 inches. The portion 722 has an outer
diameter "D4" that may be substantially similar to the diameter
"D3," although, this is not required. The diameter "D4" may range
from about 0.375 inches to about 2.5 inches.
[0247] The rotor 600 may be non-rotationally affixed to the portion
721 and the portion 722 of the drive shaft 500 by the collar 618
and the collar 622, respectively. By way of example, each of the
collars 618 and 622 may be installed inside relieved portions 616
and 620, respectively. Then, the combined rotor 600 and collars 618
and 622 may be heated to expand them. Next, the drive shaft 500 is
inserted through the collars 618 and 622 and the assembly is
allowed to cool. As the collars 618 and 622 shrink during cooling,
they tighten around the portions 722A and 722B of the drive shaft
500, respectively, gripping it sufficiently tightly to prevent the
drive shaft 500 from rotating relative to the rotor 600. The collar
618, which does not rotate with respect to either the portion 721
or the relieved portion 616, translates the rotation of the drive
shaft 500 to the first end portion 612 the rotor 600. The collar
622, which does not rotate with respect to either the portion 722
or the relieved portion 620, translates the rotation of the drive
shaft 500 to the second end portion 614 of the rotor 600. The drive
shaft 500 and the rotor 600 rotate together as a single unit.
[0248] The drive shaft 500 may have a first end portion 724 (see
FIG. 5) and a second end portion 726 (see FIG. 6). The first end
portion 724 may have a diameter "D5" of about 0.5 inches to about
1.75 inches. In particular embodiments, the diameter "D5" may be
about 1.25 inches. The second end portion 726 may have a diameter
"D6" that may be substantially similar to diameter "D5."
[0249] The second material 120 may be transported into the mixing
chamber 330 through one of the first end portion 724 and the second
end portion 726 of the rotating drive shaft 500. The other of the
first end portion 724 and the second end portion 726 of the drive
shaft 500 may be coupled to the motor 510. In the embodiment
depicted in FIGS. 5 and 6, the second material 120 is transported
into the mixing chamber 330 through the first end portion 724 and
the second end portion 726 of the drive shaft 500 is coupled to the
motor 510.
[0250] Turning to FIG. 5, the drive shaft 500 may have a channel
728 formed therein that extends from first end portion 724 into the
portion 720 disposed in the inside portion 610 of the rotor 600.
The channel 728 has an opening 730 formed in the first end portion
724. When the mixing device 100 is operating, the second material
120 is introduced into the channel 728 through the opening 730.
[0251] A valve 732 may be disposed inside a portion of the channel
728 located in the first end portion 724 of the drive shaft 500.
The valve 732 may restrict or otherwise control the backward flow
of the second material 120 from inside the hollow inside portion
610 through the channel 728 and/or the forward flow of the second
material 120 into the channel 728. The valve 732 may include any
valve known in the art including a check valve. A suitable check
valve includes a part number "CKFA1876205A," free flow forward
check valve, manufactured by The Lee Company USA having an office
in Bothell, Wash. and operating a website at www.theleeco.com.
[0252] The drive shaft 500 may include an aperture 740 located in
the inside portion 610 of the rotor 600 that connects the channel
728 with the inside portion 610 of the rotor 600. While only a
single aperture 740 is illustrated in FIG. 5, it is apparent to
those of ordinary skill in the art that multiple apertures may be
used to connect the channel 728 with the inside portion 610 of the
rotor 600.
[0253] Referring to FIG. 2, optionally, the external pump 220 may
pump the second material 120 into the mixing device 100. The pump
220 may include any suitable pump known in the art. By way of
non-limiting example, the pump 220 may include any suitable pump
known in the art including a diaphragm pump, a chemical pump, a
peristaltic pump, a gravity fed pump, a piston pump, a gear pump, a
combination of any of the aforementioned pumps, and the like. If
the second material 120 is a gas, the gas may be pressurized and
forced into the opening 730 formed in the first end portion 724 of
the drive shaft 500 by releasing the gas from the source 122.
[0254] The pump 220 or the source 122 is coupled to the channel 728
by the valve 732. The second material 120 transported inside the
channel 728 exits the channel 728 into the inside portion 610 of
the rotor 600 through the aperture 740. The second material 120
subsequently exits the inside portion 610 of the rotor 600 through
the through-holes 608 formed in the sidewall 608 of the rotor
600.
[0255] Referring to FIG. 5, the mixing device 100 may include a
seal assembly 750 coupled to the first end portion 724 of the drive
shaft 500. The seal assembly 750 is maintained within a chamber 752
defined in the housing 520. The chamber 752 has a first end portion
754 spaced across the chamber from a second end portion 756. The
chamber 752 also includes an input port 758 and an output port 759
that provide access into the chamber 752. The chamber 752 may be
defined by housing section 550 and the bearing housing 530. The
first end portion 754 may be formed in the housing section 550 and
the second end portion 756 may be adjacent to the bearing housing
530. The input port 758 may be formed in the bearing housing 530
and the output port 759 may be formed in the housing section
550.
[0256] The seal assembly 750 includes a first stationary seal 760
installed in the first end portion 754 of the chamber 752 in the
housing section 550 and the bearing housing 530. The first
stationary seal 760 extends around a portion 762 of the first end
portion 724 of the drive shaft 500. The seal assembly 750 also
includes a second stationary seal 766 installed in the second end
portion 756 of the chamber 752 in the bearing housing 530. The
second stationary seal 766 extends around a portion 768 of the
first end portion 724 of the drive shaft 500.
[0257] The seal assembly 750 includes a rotating assembly 770 that
is non-rotatably coupled to the first end portion 724 of the drive
shaft 500 between the portion 762 and the portion 768. The rotating
assembly 770 rotates therewith as a unit. The rotating assembly 770
includes a first seal 772 opposite a second seal 774. A biasing
member 776 (e.g., a spring) is located between the first seal 772
and the second seal 774. The biasing member 776 biases the first
seal 772 against the first stationary seal 760 and biases the
second seal 774 against the second stationary seal 766.
[0258] A cooling lubricant is supplied to the chamber 752 and
around rotating assembly 770. The lubricant enters the chamber 752
through the input port 758 and exits the chamber 752 through output
port 759. The lubricant may lubricate the bearing assembly 540
housed by the bearing housing 530. A chamber 570 may be disposed
between the bearing housing 530 and the mechanical seal housing
524. The bearing housing 530 may also include a second input port
759 connected to the chamber 570 into which lubricant may be
pumped. Lubricant pumped into the chamber 570 may lubricate the
bearing assembly 540. The seal assembly 750 may significantly, if
not greatly, reduce frictional forces within this portion of the
device caused by the rotation of the rotor 600 and may increase the
active life of the seals 770. The seals may include surfaces
constructed using silicon carbide.
[0259] Referring to FIG. 9, as the rotor 600 rotates about the axis
of rotation "a" in the direction indicated by arrow "C1," the rotor
expels the second material 120 into the mixing chamber 330. The
expelled bubbles, droplets, particles, and the like of the second
material 120 exit the rotor 600 and are imparted with a
circumferential velocity (in a direction indicated by arrow "C3")
by the rotor 600. The second material 120 may forced from the
mixing chamber 330 by the pump 220 (see FIG. 2), the centrifugal
force of the rotating rotor 600, buoyancy of the second material
120 relative to the first material 110, and a combination
thereof.
Motor 510
[0260] Returning to FIG. 6, the second end portion 726 of the drive
shaft 500 may be coupled to a rotating spindle 780 of a motor 510
by a coupler 900. The spindle 780 may have a generally circular
cross-sectional shape with a diameter "D7" of about 0.25 inches to
about 2.5 inches. In particular embodiments, the diameter "D7" may
be about 0.25 inches to about 1.5 inches. While in the embodiment
depicted in FIG. 6, the diameter "D5" of the first end portion 724
of the drive shaft 500 is substantially equal to the diameter "D7"
and the spindle 780, embodiments in which one of the diameter "D5"
and the diameter "D7" is larger than the other are within the scope
of the present invention.
[0261] Referring also to FIG. 4, it may be desirable to cover or
shield the coupler 900. In the embodiment illustrated in FIGS. 4
and 6, a drive guard 910 covers the coupler 900. The drive guard
910 may be generally U-shaped having a curved portion 914 flanked
by a pair of substantially linear portions 915 and 916. The distal
end of each of the substantially linear portions 915 and 916 of the
drive guard 910 may have a flange 918 and 919, respectively. The
drive guard 910 may be fastened by each of its flanges 918 and 919
to the base 106.
[0262] The motor 510 may be supported on the base 106 by a support
member 920. The support member 920 may be coupled to the motor 510
near the spindle 780. In the embodiment depicted, the support
member 920 includes a through-hole through which the spindle 780
passes. The support member 920 may be coupled to the motor 510
using any method known in the art, including bolting the support
member 920 to the motor 510 with one or more bolts 940.
[0263] The coupler 900 may include any coupler suitable for
transmitting a sufficient amount of torque from the spindle 780 to
the drive shaft 500 to rotate the rotor 600 inside to the stator
700. In the embodiment illustrated in FIGS. 4 and 6, the coupler
900 is a bellows coupler. A bellows coupler may be beneficial if
the spindle 780 and the drive shaft 500 are misaligned. Further,
the bellows coupler may help absorb axial forces exerted on the
drive shaft 500 that would otherwise be translated to the spindle
780. A suitable bellows coupler includes a model "BC32-8-8-A,"
manufactured by Ruland Manufacturing Company, Inc. of Marlborough,
Mass., which operates a website at www.ruland.com.
[0264] The motor 510 may rotate the rotor 600 at about 0.1
revolutions per minute ("rpm") to about 7200 rpm. The motor 510 may
include any motor suitable for rotating the rotor 600 inside to the
stator 700 in accordance with the present teachings. By way of
non-limiting example, a suitable motor may include a one-half
horsepower electric motor, operating at 230/460 volts and 3450 per
minute ("rpm"). A suitable motor includes a model "C4T34NC4C"
manufactured by LEESON Electric Corporation of Grafton, Wis., which
operates a website at www.leeson.com.
First Chamber 310
[0265] Turning to FIGS. 4 and 7, the first chamber 320 is disposed
inside the central section 522 of the housing 520 between the first
mechanical seal housing 524 and the first end portions 612 and 712
of the rotor 600 and the stator 700, respectively. The first
chamber 310 may be annular and have a substantially circular
cross-sectional shape. The first chamber 310 and the mixing chamber
330 form a continuous volume. A portion 1020 of the drive shaft 500
extends through the first chamber 310.
[0266] As may best be viewed in FIG. 4, the first chamber 310 has
an input port 1010 through which the first material 110 enters the
mixing device 100. The first material 110 may be pumped inside the
first chamber 310 by the external pump 210 (see FIG. 2). The
external pump 210 may include any pump known in the art for pumping
the first material 110 at a sufficient rate to supply the first
chamber 310.
[0267] The input port 1010 is oriented substantially orthogonally
to the axis of rotation ".alpha.." Therefore, the first material
110 enters the first chamber 310 with a velocity tangential to the
portion 1020 of the drive shaft 500 extending through the first
chamber 310. The tangential direction of the flow of the first
material 110 entering the first chamber 310 is identified by arrow
"T1." In the embodiment depicted in FIGS. 4 and 7, the input port
1010 may be offset from the axis of rotation ".alpha.." As is
apparent to those of ordinary skill in the art, the direction of
the rotation of the drive shaft 500 (identified by arrow "C1" in
FIG. 9), has a tangential component. The input port 1010 is
positioned so that the first material 110 enters the first chamber
310 traveling in substantially the same direction as the tangential
component of the direction of rotation of the drive shaft 500.
[0268] The first material 110 enters the first chamber 310 and is
deflected by the inside of the first chamber 310 about the portion
1020 of the drive shaft 500. In embodiments wherein the first
chamber 310 has a substantially circular cross-sectional shape, the
inside of the first chamber 310 may deflect the first material 110
in a substantially circular path (identified by arrow "C2" in FIG.
9) about the portion 1020 of the drive shaft 500. In such an
embodiment, the tangential velocity of the first material 110 may
cause it to travel about the axis of rotation ".alpha." at a
circumferential velocity, determined at least in part by the
tangential velocity.
[0269] Once inside the first chamber 310, the first material 110
may be pumped from the first chamber 310 into the mixing chamber
330 by the pump 410 residing inside the first chamber 310. In
embodiments that include the external pump 210 (see FIG. 2), the
external pump 210 may be configured to pump the first material 110
into the first chamber 310 at a rate at least as high as a rate at
which the pump 410 pumps the first material 110 from the first
chamber 310.
[0270] The first chamber 310 is in communication with the open
first end portion 332 of the mixing chamber 330 and the first
material 110 inside the first chamber 310 may flow freely into the
open first end portion 332 of the mixing chamber 330. In this
manner, the first material 110 does not negotiate any corners or
bends between the mixing chamber 330 and the first chamber 310. In
the embodiment depicted, the first chamber 310 is in communication
with the entire open first end portion 332 of the mixing chamber
330. The first chamber 310 may be filled completely with the first
material 110.
[0271] The pump 410 is powered by the portion 1020 of the drive
shaft 500 extending through the first chamber 310. The pump 410 may
include any pump known in the art having a rotating pump member
2022 housed inside a chamber (i.e., the first chamber 310) defined
by a stationary housing (i.e., the housing 520). Non-limiting
examples of suitable pumps include rotary positive displacement
pumps such as progressive cavity pumps, single screw pumps (e.g.,
Archimedes screw pump), and the like.
[0272] The pump 410 depicted in FIGS. 7 and 9, is generally
referred to as a single screw pump. In this embodiment, the pump
member 2022 includes a collar portion 2030 disposed around the
portion 1020 of the drive shaft 500. The collar portion 2030
rotates with the portion 1020 of the drive shaft 500 as a unit. The
collar portion 2030 includes one or more fluid displacement members
2040. In the embodiment depicted in FIGS. 7 and 9, the collar
portion 2030 includes a single fluid displacement member 2040
having a helical shape that circumscribes the collar portion 2030
along a helical path.
[0273] Referring to FIG. 9, the inside of the first chamber 310 is
illustrated. The pump 410 imparts an axial flow (identified by
arrow "A1" and arrow "A2") in the first material 110 inside the
first chamber 310 toward the open first end portion 332 of the
mixing chamber 330. The axial flow of the first material 110
imparted by the pump 410 has a pressure that may exceed the
pressure obtainable by the external pump of the prior art device 10
(see FIG. 1).
[0274] The pump 410 may also be configured to impart a
circumferential flow (identified by arrow "C2") in the first
material 110 as it travels toward the open first end portion 332 of
the mixing chamber 330. The circumferential flow imparted in the
first material 110 before it enters the mixing chamber 330 causes
the first material 110 to enter the mixing chamber 330 already
traveling in the desired direction at an initial circumferential
velocity. In the prior art device 10 depicted in FIG. 1, the first
material 110 entered the channel 32 of the prior art device 10
without a circumferential velocity. Therefore, the rotor 12 of the
prior art device 10 alone had to impart a circumferential flow into
the first material 110. Because the first material 110 is moving
axially, in the prior art device 10, the first material 110
traversed at least a portion of the channel 32 formed between the
rotor 12 and the stator 30 at a slower circumferential velocity
than the first material 110 traverses the mixing chamber 330 of the
mixing device 100. In other words, if the axial velocity of the
first material 110 is the same in both the prior art device 10 and
the mixing device 100, the first material 110 may complete more
revolutions around the rotational axis ".alpha." before traversing
the axial length of the mixing chamber 330, than it would complete
before traversing the axial length of the channel 32. The
additional revolutions expose the first material 110 (and combined
first material 110 and second material 120) to a substantially
larger portion of the effective inside surface 706 (see FIG. 7) of
the stator 700.
[0275] In embodiments including the external pump 210 (see FIG. 2),
the circumferential velocity imparted by the external pump 210
combined with the input port 1010 being oriented according to the
present teachings, may alone sufficiently increase the revolutions
of the first material 110 (and combined first material 110 and
second material 120) about the rotational axis "a." Further, in
some embodiments, the circumferential velocity imparted by the pump
210 and the circumferential velocity imparted by the pump 410
combine to achieve a sufficient number of revolutions of the first
material 110 (and combined first material 110 and second material
120) about the rotational axis ".alpha.." As is appreciated by
those of ordinary skill in the art, other structural elements such
as the cross-sectional shape of the first chamber 310 may
contribute to the circumferential velocity imparted by the pump
210, the pump 410, and a combination thereof.
[0276] In an alternate embodiment depicted in FIG. 10, the pump 410
may include one or more vanes 2042 configured to impart a
circumferential flow in the first material 110 as it travels toward
the open first end portion 332 of the mixing chamber 330.
Second Chamber 320
[0277] Turning now to FIGS. 4 and 7, the second chamber 320 is
disposed inside the central section 522 of the housing 520 between
the second mechanical seal housing 526 and the second end portions
614 and 714 of the rotor 600 and the stator 700, respectively. The
second chamber 320 may be substantially similar to the first
chamber 310. However, instead of the input port 1010, the second
chamber 320 may include an output port 3010. A portion 3020 of the
drive shaft 500 extends through the second chamber 320.
[0278] The second chamber 320 and the mixing chamber 330 form a
continuous volume. Further, the first chamber 310, the mixing
chamber 330, and the second chamber 320 form a continuous volume.
The first material 110 flows through the mixing device 100 from the
first chamber 310 to the mixing chamber 330 and finally to the
second chamber 320. While in the mixing chamber 330, the first
material 110 is mixed with the second material 120 to form the
output material 102. The output material 102 exits the mixing
device 100 through the output port 3010. Optionally, the output
material 102 may be returned to the input port 1010 and mixed with
an additional quantity of the second material 120, the third
material 130, or a combination thereof.
[0279] The output port 3010 is oriented substantially orthogonally
to the axis of rotation ".alpha." and may be located opposite the
input port 1010 formed in the first chamber 310. The output
material 102 enters the second chamber 320 from the mixing chamber
330 having a circumferential velocity (in the direction indicated
by arrow "C3" in FIG. 9) imparted thereto by the rotor 600. The
circumferential velocity is tangential to the portion 3020 of the
drive shaft 500 extending through the second chamber 320. In the
embodiment depicted in FIGS. 4, 6, and 7, the output port 3010 may
be offset from the axis of rotation "a." The output port 3010 is
positioned so that the output material 102, which enters the second
chamber 320 traveling in substantially the same direction in which
the drive shaft 500 is rotating (identified in FIG. 9 by arrow
"C1"), is traveling toward the output port 3010.
[0280] The output material 102 enters the second chamber 320 and is
deflected by the inside of the second chamber 320 about the portion
3020 of the drive shaft 500. In embodiments wherein the second
chamber 320 has a substantially circular cross-sectional shape, the
inside of the second chamber 320 may deflect the output material
102 in a substantially circular path about the portion 3020 of the
drive shaft 500.
[0281] Referring to FIG. 2, optionally, the output material 102 may
be pumped from inside the second chamber 320 by the external pump
430. The external pump 430 may include any pump known in the art
for pumping the output material 102 at a sufficient rate to avoid
limiting throughput of the mixing device 100. In such an
embodiment, the external pump 430 may introduce a tangential
velocity (in a direction indicated by arrow "T2" in FIGS. 4 and 11)
to at least a portion of the output material 102 as the external
pump 430 pumps the output material 102 from the second chamber 320.
The tangential velocity of the portion of the output material 102
may cause it to travel about the axis of rotation ".alpha." at a
circumferential velocity, determined in part by the tangential
velocity.
Pump 420
[0282] Turning to FIGS. 6 and 7, the pump 420 residing inside the
second chamber 320 may pump the output material 102 from the second
chamber 320 into the output port 3010 and/or from the mixing
chamber 330 into the second chamber 320. In embodiments that
include the external pump 430, the external pump 430 may be
configured to pump the output material 102 from the second chamber
320 at a rate at least as high as a rate at which the pump 420
pumps the output material 102 into the output port 3010.
[0283] The second chamber 320 is in communication with the open
second end portion 334 of the mixing chamber 330 and the output
material 102 inside the mixing chamber 330 may flow freely from the
open second end portion 334 into the second chamber 320. In this
manner, the output material 102 does not negotiate any corners or
bends between the mixing chamber 330 and the second chamber 320. In
the embodiment depicted, the second chamber 320 is in communication
with the entire open second end portion 334 of the mixing chamber
330. The second chamber 320 may be filled completely with the
output material 102.
[0284] The pump 420 is powered by the portion 3020 of the drive
shaft 500 extending through the second chamber 320. The pump 420
may be substantially identical to the pump 410. Any pump described
above as suitable for use as the pump 410 may be used for the pump
420. While the pump 410 pumps the first material 110 into the
mixing chamber 330, the pump 420 pumps the output material 102 from
the mixing chamber 330. Therefore, both the pump 410 and the pump
420 may be oriented to pump in the same direction.
[0285] As is appreciated by those of ordinary skill in the art, the
first material 110 may differ from the output material 102. For
example, one of the first material 110 and the output material 102
may be more viscous than the other. Therefore, the pump 410 may
differ from the pump 420. The pump 410 may be configured to
accommodate the properties of the first material 110 and the pump
420 may be configured to accommodate the properties of the output
material 102.
[0286] The pump 420 depicted in FIGS. 6 and 7, is generally
referred to as a single screw pump. In this embodiment, the pump
member 4022 includes a collar portion 4030 disposed around the
portion 3020 of the drive shaft 500. The collar portion 4030
rotates with the portion 3020 of the drive shaft 500 as a unit. The
collar portion 4030 includes one or more fluid displacement members
4040. The collar portion 4030 includes a single fluid displacement
member 4040 having a helical shape that circumscribes the collar
portion 4030 along a helical path.
[0287] Referring to FIG. 11, the inside of the second chamber 320
is illustrated. The pump 420 imparts an axial flow (identified by
arrow "A3" and arrow "A4") in the output material 102 inside the
second chamber 320 away from the open second end portion 334 of the
mixing chamber 330.
[0288] The pump 420 may be configured to impart a circumferential
flow (identified by arrow "C4") in the output material 102 as it
travels away from the open second end portion 334 of the mixing
chamber 330. The circumferential flow imparted in the output
material 102 may help reduce an amount of work required by the
rotor 600. The circumferential flow also directs the output
material 102 toward the output port 3010.
[0289] In an alternate embodiment, the pump 420 may have
substantially the same configuration of the pump 410 depicted in
FIG. 10. In such an embodiment, the one or more vanes 2042 are
configured to impart a circumferential flow in the output material
102 as it travels away from the open second end portion 334 of the
mixing chamber 330.
[0290] As is apparent to those of ordinary skill, various
parameters of the mixing device 100 may be modified to obtain
different mixing characteristics. Exemplary parameters that may be
modified include the size of the through-holes 608, the shape of
the through-holes 608, the arrangement of the through-holes 608,
the number of through-holes 608, the size of the apertures 708, the
shape of the apertures 708, the arrangement of the apertures 708,
the number of apertures 708, the shape of the rotor 600, the shape
of the stator 700, the width of the mixing chamber 330, the length
of the mixing chamber 330, rotational speed of the drive shaft 500,
the axial velocity imparted by the internal pump 410, the
circumferential velocity imparted by the internal pump 410, the
axial velocity imparted by the internal pump 420, the
circumferential velocity imparted by the internal pump 420, the
configuration of disturbances (e.g., texture, projections,
recesses, apertures, and the like) formed on the outside surface
606 of the rotor 600, the configuration of disturbances (e.g.,
texture, projections, recesses, apertures, and the like) formed on
the inside surface 706 of the stator 700, and the like.
Alternate Embodiment
[0291] Referring to FIG. 12, a mixing device 5000 is depicted. The
mixing device 5000 is an alternate embodiment of the mixing device
100. Identical reference numerals have been used herein to identify
components of the mixing device 5000 that are substantially similar
corresponding components of the mixing device 100. Only components
of the mixing device 5000 that differ from the components of the
mixing device 100 will be described.
[0292] The mixing device 5000 includes a housing 5500 for housing
the rotor 600 and the stator 5700. The stator 5700 may be
non-rotatably coupled by its first end portion 5712 and its second
end portion 5714 to the housing 5500. A chamber 5800 is defined
between the housing 5500 and a portion 5820 of the stator 5700
flanked by the first end portion 5712 and the second end portion
5714. The housing 5500 includes an input port 5830 which provides
access into the chamber 5800. The input port 5830 may be oriented
substantially orthogonally to the axis of rotation "a." however,
this is not a requirement.
[0293] The stator 5700 includes a plurality of through-holes 5708
that connect the chamber 5800 and the mixing chamber 330 (defined
between the rotor 600 and the stator 5700). An external pump 230
may be used to pump the third material 130 (which may be identical
to the second material 120) into the chamber 5800 via the input
port 5830. The third material 130 pumped into the chamber 5800 may
enter the mixing chamber 330 via the through-holes 5708 formed in
the stator 5700. The third material 130 may forced from the channel
5800 by the pump 230, buoyancy of the third material 130 relative
to the first material 110, and a combination thereof. As the rotor
600 rotates, it may also draw the third material 130 from the
channel 5800 into the mixing chamber 330. The third material 130
may enter the mixing chamber 330 as bubbles, droplets, particles,
and the like, which are imparted with a circumferential velocity by
the rotor 600.
Alternate Embodiment
[0294] An alternate embodiment of the mixing device 100 may be
constructed using a central section 5900 depicted in FIG. 13 and a
bearing housing 5920 depicted in FIG. 14. FIG. 13 depicts the
central section 5900 having in its interior the stator 700 (see
FIG. 7). Identical reference numerals have been used herein to
identify components associated with the central section 5900 that
are substantially similar corresponding components of the mixing
device 100. Only components of the central section 5900 that differ
from the components of the central section 522 will be described.
The central section 5900 and the stator 700 are both constructed
from a conductive material such as a metal (e.g., stainless steel).
The input port 1010 and the output port 3010 are both constructed
from a nonconductive material such as plastic (e.g., PET, Teflon,
nylon, PVC, polycarbonate, ABS, Delrin, polysulfone, etc.).
[0295] An electrical contact 5910 is coupled to the central section
5900 and configured to deliver a charge thereto. The central
section 5900 conducts an electrical charge applied to the
electrical contact 5910 to the stator 700. In further embodiments,
the central section 5900 may be constructed from a nonconductive
material. In such embodiments, the electrical contact 5910 may pass
through the central section 5900 and coupled to the stator 700. The
electric charge applied by the electrical contact 5910 to the
stator 700 may help facilitate redox or other chemical reactions
inside the mixing chamber 330.
[0296] Optionally, insulation (not shown) may be disposed around
the central section 5900 to electrically isolate it from the
environment. Further, insulation may be used between the central
section 5900 and the first and second mechanical seals 524 and 526
that flank it to isolate it electrically from the other components
of the mixing device.
[0297] Turning now to FIG. 14, the bearing housing 5920 will be
described. The bearing housing 5920 is disposed circumferentially
around the portion 726 of the drive shaft 500. An electrical
contact 5922 is coupled to the bearing housing 5920. A rotating
brush contact 5924 provides an electrical connection between the
drive shaft 500 and the electrical contact 5922.
[0298] In this embodiment, the drive shaft 500 and the rotor 600
are both constructed from a conductive material such as a metal
(e.g., stainless steel). The bearing housing 5920 may be
constructed from either a conductive or a nonconductive material.
An electrical charge is applied to the drive shaft 500 by the
electrical contact 5922 and the rotating brush contact 5924. The
electrical charge is conducted by the drive shaft 500 to the rotor
600.
[0299] The alternate embodiment of the mixing device 100
constructed using the central section 5900 depicted in FIG. 13 and
the bearing housing 5920 depicted in FIG. 14 may be operated in at
least two ways. First, the electrical contacts 5910 and 5922 may be
configured not to provide an electrical charge to the stator 700
and the rotor 600, respectively. In other words, neither of the
electrical contacts 5910 and 5922 are connected to a current
source, a voltage source, and the like.
[0300] Alternatively, the electrical contacts 5910 and 5922 may be
configured to provide an electrical charge to the stator 700 and
the rotor 600, respectively. For example, the electrical contacts
5910 and 5922 may be coupled to a DC voltage source (not shown)
supplying a steady or constant voltage across the electrical
contacts 5910 and 5922. The negative terminal of the DC voltage
source may be coupled to either of the electrical contacts 5910 and
5922 and the positive terminal of the DC voltage source may be
coupled to the other of the electrical contacts 5910 and 5922. The
voltage supplied across the electrical contacts 5910 and 5922 may
range from about 0.0001 volts to about 1000 volts. In particular
embodiments, the voltage may range from about 1.8 volts to about
2.7 volts. By way of another example, a pulsed DC voltage having a
duty cycle of between about 1% to about 99% may be used.
[0301] While the above examples of methods of operating the mixing
device apply a DC voltage across the electrical contacts 5910 and
5922, as is apparent to those of ordinary skill in the art, a
symmetrical AC voltage or non symmetrical AC voltage having various
shapes and magnitudes may be applied across the electrical contacts
5910 and 5922 and such embodiments are within the scope of the
present invention.
Mixing Inside the Mixing Chamber 330
[0302] As mentioned above, in the prior art device 10 (shown in
FIG. 1), the first material 110 entered the channel 32 between the
rotor 12 and the stator 30 via a single limited input port 37
located along only a portion of the open second end of the channel
32. Likewise, the output material 102 exited the channel 32 via a
single limited output port 40 located along only a portion of the
open first end of the channel 32. This arrangement caused
undesirable and unnecessary friction. By replacing the single
limited inlet port 37 and the single limited outlet port 40 with
the chambers 310 and 320, respectively, friction has been reduced.
Moreover, the first material 110 does not negotiate a corner before
entering the mixing chamber 330 and the output material 102 does
not negotiate a corner before exiting the mixing chamber 330.
Further, the chambers 310 and 320 provide for circumferential
velocity of the material prior to entering, and after exiting the
channel 32.
[0303] Accordingly, pressure drop across the mixing device 100 has
been substantially reduced. In the embodiments depicted in FIGS. 2,
4-9, and 11, the pressure drop between the input port 1010 and the
output port 3010 is only approximately 12 psi when the mixing
device 100 is configured to produce about 60 gallons of the output
material 102 per minute. This is an improvement over the prior art
device 10 depicted in FIG. 1, which when producing about 60 gallons
of output material per minute was at least 26 psi. In other words,
the pressure drop across the mixing device 100 is less than half
that experienced by the prior art device 10.
[0304] According to additional aspects, the inclusion of pumps 410
and 420, which are powered by the drive shaft 500, provides a
configuration that is substantially more efficient in mixing
materials and that requires less energy than the external pumps
used in the prior art.
Micro-Cavitation
[0305] During operation of the mixing device 100, the input
materials may include the first material 110 (e.g., a fluid) and
the second material 120 (e.g., a gas). The first material 110 and
the second material 120 are mixed inside the mixing chamber 330
formed between the rotor 600 and the stator 700. Rotation of the
rotor 600 inside the stator 700 agitates the first material 110 and
the second material 120 inside the mixing chamber 330. The
through-holes 608 formed in the rotor 600 and/or the apertures 708
formed in the stator 700 impart turbulence in the flow of the first
material 110 and the second material 120 inside the mixing chamber
330.
[0306] Without being limited by theory, the efficiency and
persistence of the diffusion of the second material 120 into the
first material 110 is believed to be caused in part by
micro-cavitation, which is described in connection with FIGS.
15-17. Whenever a material flows over a smooth surface, a rather
laminar flow is established with a thin boundary layer that is
stationary or moving very slowly because of the surface tension
between the moving fluid and the stationary surface. The
through-holes 608 and optionally, the apertures 708, disrupt the
laminar flow and can cause localized compression and decompression
of the first material 110. If the pressure during the decompression
cycle is low enough, voids (cavitation bubbles) will form in the
material. The cavitation bubbles generate a rotary flow pattern
5990, like a tornado, because the localized area of low pressure
draws the host material and the infusion material, as shown in FIG.
15. When the cavitation bubbles implode, extremely high pressures
result. As two aligned openings (e.g., one of the apertures 708 and
one of the through-holes 608) pass one another, a succussion (shock
wave) occurs, generating significant energy. The energy associated
with cavitation and succussion mixes the first material 110 and the
second material 120 together to an extremely high degree, perhaps
at the molecular level.
[0307] The tangential velocity of the rotor 600 and the number of
openings that pass each other per rotation may dictate the
frequency at which the mixing device 100. It has been determined
that operating the mixing device 100 within in the ultrasonic
frequency range can be beneficial in many applications. It is
believed that operating the mixing device 100 in the ultrasonic
region of frequencies provides the maximum succession shock energy
to shift the bonding angle of the fluid molecule, which enables it
to transport an additional quantity of the second material 120
which it would not normally be able to retain. When the mixing
device 100 is used as a diffuser, the frequency at which the mixing
device 100 operates appears to affect the degree of diffusion,
leading to much longer persistence of the second material 120
(infusion material) in the first material 110 (host material).
[0308] Referring now to FIG. 15, an alternate embodiment of the
rotor 600, rotor 6000 is provided. The cavitations created within
the first material 110 in the mixing chamber 330 may be configured
to occur at different frequencies along the length of the mixing
chamber 330. The frequencies of the cavitations may be altered by
altering the number and/or the placement of the through-holes 6608
along the length of the rotor 600. Each of the through-holes 6608
may be substantially similar to the through-holes 608 (discussed
above).
[0309] By way of non-limiting example, the rotor 6000 may be
subdivided into three separate exemplary sections 6100, 6200, and
6300. The through-holes 6608 increase in density from the section
6100 to the section 6200, the number of holes in the section 6100
being greater than the number of holes in the section 6200. The
through-holes 6608 also increase in density from the section 6200
to the section 6300, the number of holes in the section 6200 being
greater than the number of holes in the section 6300. Each of the
sections 6100, 6200, and 6300 create succussions within their
particular area at a different frequency due to the differing
numbers of through-holes 6608 formed therein.
[0310] By manufacturing the rotor 6000 with a desired number of
through-holes 6608 appropriately arranged in a particular area, the
desired frequency of the succussions within the mixing chamber 330
may be determined. Similarly, the desired frequency of the
cavitations may be determined by a desired number of apertures 708
appropriately arranged in a particular area upon the stator 700
within which the rotor 600 rotates. Further, the desired frequency
(or frequencies) of the succussions within the mixing chamber 330
may be achieved by selecting both a particular number and
arrangement of the apertures 708 formed in the stator 700 and a
particular number and arrangement of the through-holes 608 formed
in the rotor 600.
[0311] FIGS. 19-21, depict various alternative arrangements of the
apertures 708 formed in the stator 700 and the through-holes 608
formed in the rotor 600 configured to achieve different results
with respect to the cavitations created. FIG. 19 illustrates a
configuration in which the apertures 708 and the through-holes 608
are aligned along an axis 7000 that is not parallel with any line
(e.g., line 7010) drawn through the axis of rotation ".alpha." of
the rotor 600. In other words, if the rotor 600 has a cylindrical
shape, the axis 7000 does not pass through the center of the rotor
600. Thus, the first material 110 within the mixing chamber 330
will not be oriented perpendicularly to the compressions and
decompressions created by the apertures 708 and the through-holes
608. The compressions and decompressions will instead have a force
vector that has at least a component parallel to the
circumferential flow (in the direction of arrow "C3" of FIG. 9) of
first material 110 within the mixing chamber 330.
[0312] Relative alignment of the apertures 708 and the
through-holes 608 may also affect the creation of cavitations in
the mixing chamber 330. FIG. 20 illustrates an embodiment in which
the apertures 708 are in registration across the mixing chamber 330
with the through-holes 608. In this embodiment, rotation of the
rotor 600 brings the through-holes 608 of the rotor into direct
alignment with the apertures 708 of the stator 700. When in direct
alignment with each other, the compressive and decompressive forces
created by the apertures 708 and the through-holes 608 are directly
aligned with one another.
[0313] In the embodiment depicted in FIG. 21, the apertures 708 and
the through-holes 608 are offset by an offset amount "X" along the
axis of rotation ".alpha..". By way of non-limiting example, the
offset amount "X" may be determined as a function of the size of
the apertures 708. For example, the offset amount "X" may be
approximately equal to one half of the diameter of the apertures
708. Alternatively, the offset amount "X" may be determined as a
function of the size of the through-holes 608. For example, the
offset amount "X" may be approximately equal to one half of the
diameter of the through-holes 608. If features (e.g., recesses,
projections, etc.) other than or in addition to the through-holes
608 and the apertures 708 are included in either the rotor 600 or
the stator 700, the offset amount "X" may be determined as a
function of the size of such features. In this manner, the
compressive and decompressive forces caused by the apertures 708 of
the stator 700 and the through-holes 608 of the rotor 600 collide
at a slight offset causing additional rotational and torsional
forces within the mixing chamber 330. These additional forces
increase the mixing (e.g., diffusive action) of the second material
120 into the first material 110 within the mixing chamber 330.
[0314] Referring now to FIGS. 22-25, non-limiting examples of
suitable cross-sectional shapes for the apertures 708 and the
through-holes 608 are provided. The cross-sectional shape of the
apertures 708 and/or the through-holes 608 may be square as
illustrated in FIG. 22, circular as illustrated in FIG. 23, and the
like.
[0315] Various cross-sectional shapes of apertures 708 and/or the
through-holes 608 may be used to alter flow of the first material
110 as the rotor 600 rotates within the stator 700. For example,
FIG. 24 depicts a teardrop cross-sectional shape having a narrow
portion 7020 opposite a wide portion 7022. If the through-holes 608
have this teardrop shape, when the rotor 600 is rotated (in the
direction generally indicated by the arrow "F"), the forces exerted
on the first material 110, the second material 120, and optionally
the third material 130 within the mixing chamber 330 increase as
the materials pass from the wide portion 7022 of the teardrop to
the narrow portion 7020.
[0316] Additional rotational forces can be introduced into the
mixing chamber 330 by forming the apertures 708 and/or the
through-holes 608 with a spiral configuration as illustrated in
FIG. 25. Material that flows into and out of the apertures 708
and/or the through-holes 608 having the spiral configuration
experience a rotational force induced by the spiral configuration.
The examples illustrated in FIGS. 22-25 are provided as
non-limiting illustrations of alternate embodiments that may be
employed within the mixing device 100. By application of ordinary
skill in the art, the apertures 708 and/or the through-holes 608
may be configured in numerous ways to achieve various succussive
and agitative forces appropriate for mixing materials within the
mixing chamber 330.
Double Layer Effect
[0317] The mixing device 100 may be configured to create the output
material 102 by complex and non-linear fluid dynamic interaction of
the first material 110 and the second material 120 with complex,
dynamic turbulence providing complex mixing that further favors
electrokinetic effects (described below). The result of these
electrokinetic effects may be observed within the output material
102 as charge redistributions and redox reactions, including in the
form of solvated electrons that are stabilized within the output
material.
[0318] Ionization or dissociation of surface groups and/or
adsorption of ions from a liquid cause most solid surfaces in
contact with the liquid to become charged. Referring to FIG. 26, an
electrical double layer ("EDL") 7100 forms around exemplary surface
7110 in contact with a liquid 7120. In the EDL 7100, ions 7122 of
one charge (in this case, negatively charged ions) adsorb to the
surface 7120 and form a surface layer 7124 typically referred to as
a Stern layer. The surface layer 7124 attracts counterions 7126 (in
this case, positively charged ions) of the opposite charge and
equal magnitude, which form a counterion layer 7128 below the
surface layer 7124 typically referred to as a diffuse layer. The
counterion layer 7128 is more diffusely distributed than the
surface layer 7124 and sits upon a uniform and equal distribution
of both ions in the bulk material 7130 below. For OH- and H+ ions
in neutral water, the Gouy-Chapman model would suggest that the
diffuse counterion layer extends about one micron into the
water.
[0319] According to particular aspects, the electrokinetic effects
mentioned above are caused by the movement of the liquid 7120 next
to the charged surface 7110. Within the liquid 7120 (e.g., water,
saline solution, and the like), the adsorbed ions 7122 forming the
surface layer 7124 are fixed to the surface 7120 even when the
liquid 7120 is in motion (for example, flowing in the direction
indicated by arrow "G"); however, a shearing plane 7132 exists
within the diffuse counterion layer 7128 spaced from the surface
7120. Thus, as the liquid 7120 moves, some of the diffuse
counterions 7126 are transported away from the surface 7120, while
the absorbed ions 7122 remain at the surface 7120. This produces a
so-called `streaming current.`
[0320] Within the mixing chamber 330, the first material 110, the
second material 120, and optionally, the third material 130 are
subject to an electromagnetic field created by the inside surface
705 of the stator 700 and/or the outside surface 606 of the rotor
600, a voltage between the inside surface 705 and the outside
surface 606, and/or an electrokinetic effect (e.g., streaming
current) caused by at least one EDL formed in the first material
110. The at least one EDL may be introduced into the first material
110 by at least one of the inside surface 705 of the stator 700 and
the outside surface 606 of the rotor 600.
[0321] Movement of the first material 110 through the mixing
chamber 330 relative to surface disturbances (e.g., the
through-holes 608 and apertures 708) creates cavitations in the
first material 110 within the mixing chamber 330, which may diffuse
the second material 120 into the first material 110. These
cavitations may enhance contact between of the first material 110
and/or the second material 120 with the electric double layer
formed on the inside surface 705 of the stator 700 and/or the
electric double layer formed on the outside surface 606 of the
rotor 600. Larger surface to volume ratios of the mixing chamber,
an increased dwell time of the combined materials within the mixing
chamber, and further in combination with a smaller average bubble
size (and hence substantially greater bubble surface area) provide
for effectively imparting EDL-mediated effects to the inventive
output materials.
[0322] In embodiments in which the inside surface 705 and the
outside surface 606 are constructed from a metallic material, such
as stainless steel, the motion of the liquid 7120 and/or the
streaming current(s) facilitate redox reactions involving H.sub.2O,
OH-, H+, and O.sub.2 at the inside surface 705 and the outside
surface 606.
[0323] Referring to FIG. 27, without being limited by theory, it is
believed a section 7140 of the mixing chamber 330 between the
inside surface 705 and the outside surface 606 may be modeled as a
pair of parallel plates 7142 and 7144. If the first material 110 is
a liquid, the first material 110 enters the section 7140 through an
inlet "IN" and exits the section 7140 through an outlet "OUT." The
inlet "IN" and the outlet "OUT" restrict the flow into and out of
the section 7140.
[0324] Referring to FIG. 28, the area between the parallel plates
7142 and 7144 has a high surface area to volume ratio. Hence, a
substantial portion of the counterion layer 7128 (and counterions
7126) may be in motion as the first material 110 moves between the
plates 7142 and 7144. The number of counterions 7126 in motion may
exceed the number allowed to enter the section 7140 by the inlet
"IN" and the number allowed to exit the section 7140 by the outlet
"OUT." The inlet "IN" and the outlet "OUT" feeding and removing the
first material 110 from the section 7140, respectively, have far
less surface area (and a lower surface area to volume ratio) than
the parallel plates 7142 and 7144 and thereby reduce the portion of
the counterions 7126 in motion in the first material 110 entering
and leaving the section 7140. Therefore, entry and exit from the
section 7140 increases the streaming current locally. While a
background streaming current (identified by arrow "BSC") caused by
the flowing first material 110 over any surface is always present
inside the mixing device 100, the plates 7142 and 7144 introduce an
increased "excess" streaming current (identified by arrow "ESC")
within the section 7140.
[0325] Without a conductive return current (identified by arrow
"RC") in the plates 7142 and 7144 in the opposite direction of the
flow of the first material 110, an excess charge 7146 having the
same sign as the adsorbing ions 7122 would accumulate near the
inlet "IN," and an excess charge 7148 having the same sign as the
counterion 7126 would accumulate near the at outlet "OUT." Because
such accumulated charges 7146 and 7148, being opposite and
therefore attracted to one another, cannot build up indefinitely
the accumulated charges seek to join together by conductive means.
If the plates 7142 and 7144 are perfectly electrically insulating,
the accumulated charges 7146 and 7148 can relocate only through the
first material 110 itself. When the conductive return current
(identified by arrow "RC") is substantially equivalent to the
excess streaming current (identified by arrow "ESC") in the section
7140, a steady-state is achieved having zero net excess streaming
current, and an electrostatic potential difference between the
excess charge 7146 near the inlet "IN," and the excess charge 7148
near the outlet "OUT" creating a steady-state charge separation
therebetween.
[0326] The amount of charge separation, and hence the electrostatic
potential difference between the excess charge 7146 near the inlet
"IN," and the excess charge 7148 near the outlet "OUT," depends on
additional energy per unit charge supplied by a pump (e.g., the
rotor 600, the internal pump 410, and/or the external pump 210) to
"push" charge against the opposing electric field (created by the
charge separation) to produce a liquid flow rate approximating a
flow rate obtainable by a liquid without ions (i.e., ions 7122 and
7126). If the plates 7142 and 7144 are insulators, the
electrostatic potential difference is a direct measure of the EMF
the pump (e.g., the rotor 600, the internal pump 410 and/or the
external pump 210) can generate. In this case, one could measure
the electrostatic potential difference using a voltmeter having a
pair of leads by placing one of the leads in the first material 110
near the inlet "IN," and the other lead in the first material 110
near the outlet "OUT."
[0327] With insulating plates 7142 and 7144, any return current is
purely an ion current (or flow of ions), in that the return current
involves only the conduction of ions through the first material
110. If other conductive mechanisms through more conductive
pathways are present between the excess charge 7146 near the inlet
"IN," and the excess charge 7148 near the outlet "OUT," the return
current may use those more conductive pathways. For example,
conducting metal plates 7142 and 7144 may provide more conductive
pathways; however, these more conductive pathways transmit only an
electron current and not the ion current.
[0328] As is appreciated by those of ordinary skill, to transfer
the charge carried by an ion to one or more electrons in the metal,
and vise versa, one or more oxidation-reduction reactions must
occur at the surface of the metal, producing reaction products.
Assuming the first material 110 is water (H.sub.2O) and the second
material 120 is oxygen (O.sub.2), a non-limiting example of a redox
reaction, which would inject negative charge into the conducting
plates 7142 and 7144 includes the following known half-cell
reaction:
O.sub.2+H.sub.2O.fwdarw.O.sub.3+2H.sup.++2e.sup.-,
[0329] Again, assuming the first material 110 is water (H.sub.2O)
and the second material 120 is oxygen (O.sub.2), a non-limiting
example of a redox reaction includes the following known half-cell
reaction, which would remove negative charge from the conducting
plates 7142 and 7144 includes the following known half-cell
reaction:
2H.sup.++e.sup.-.fwdarw.H.sub.2,
[0330] With conducting metal plates 7142 and 7144, most of the
return current is believed to be an electron current, because the
conducting plates 7142 and 7144 are more conductive than the first
material 110 (provided the redox reactions are fast enough not to
be a limiting factor). For the conducting metal plates 7142 and
7144, a smaller charge separation accumulates between the inlet
"IN" and the outlet "OUT," and a much smaller electrostatic
potential exists therebetween. However, this does not mean that the
EMF is smaller.
[0331] As described above, the EMF is related to the energy per
unit charge the pump provides to facilitate the flow of the first
material 110 against the opposing electric field created by the
charge separation. Because the electrostatic potential is smaller,
the pump may supply less energy per unit charge to cause the first
material 110 to flow. However, the above example redox reactions do
not necessarily occur spontaneously, and thus may require a work
input, which may be provided by the pump. Therefore, a portion of
the EMF (that is not reflected in the smaller electrostatic
potential difference) may be used to provide the energy necessary
to drive the redox reactions.
[0332] In other words, the same pressure differentials provided by
the pump to push against the opposing electric field created by the
charge separation for the insulating plates 7142 and 7144, may be
used both to "push" the charge through the conducting plates 7142
and 7144 and drive the redox reactions.
[0333] Referring to FIG. 29, an experimental setup for an
experiment conducted by the inventors is provided. The experiment
included a pair of substantially identical spaced apart 500 ml
standard Erlenmeyer flasks 7150 and 7152, each containing a volume
of deionized water 7153. A rubber stopper 7154 was inserted in the
open end of each of the flasks 7150 and 7152. The stopper 7154
included three pathways, one each for a hollow tube 7156, a
positive electrode 7158, and a negative electrode 7160. With
respect to each of the flasks 7150 and 7152, each of the hollow
tube 7156, the positive electrode 7158, and the negative electrode
7160 all extended from outside the flask, through the stopper 7154,
and into the deionized water 7153 inside the flask. The positive
electrode 7158 and the negative electrode 7160 were constructed
from stainless steel. The hollow tubes 7156 in both of the flasks
7150 and 7152 had an open end portion 7162 coupled to a common
oxygen supply 7164. The positive electrode 7158 and the negative
electrode 7160 inserted into the flask 7152 where coupled to a
positive terminal and a negative terminal, respectively, of a DC
power supply 7168. Exactly the same sparger was used in each
flask.
[0334] Oxygen flowed through the hollow tubes 7156 into both of the
flasks 7150 and 7152 at a flow rate (Feed) of about 1 SCFH to about
1.3 SCFH (combined flow rate). The voltage applied across the
positive electrode 7158 and the negative electrode 7160 inserted
into the flask 7152 was about 2.55 volts. This value was chosen
because it is believed to be an electrochemical voltage value
sufficient to affect all oxygen species. This voltage was applied
continuously over three to four hours during which oxygen from the
supply 7164 was bubbled into the deionized water 7153 in each of
the flasks 7150 and 7152.
[0335] Testing of the deionized water 7153 in the flask 7150 with
HRP and pyrogallol gave an HRP-mediated pyrogallol reaction
activity, consistent with the properties of fluids produced with
the alternate rotor/stator embodiments described herein. The HRP
optical density was about 20% higher relative to pressure-pot or
fine-bubbled solutions of equivalent oxygen content. The results of
this experiment indicate that mixing inside the mixing chamber 330
involves a redox reaction. According to particular aspects, the
inventive mixing chambers provide for output materials comprising
added electrons that are stabilized by either oxygen-rich water
structure within the inventive output solutions, or by some form of
oxygen species present due to the electrical effects within the
process.
[0336] Additionally, the deionized water 7153 in both of the flasks
7150 and 7152 was tested for both ozone and hydrogen peroxide
employing industry standard colorimetric test ampules with a
sensitivity of 0.1 ppm for hydrogen peroxide and 0.6 ppm for ozone.
There was no positive indication of either species up to the
detection limits of those ampules.
Dwell Time
[0337] Dwell time is an amount of time the first material 110, the
second material 120, and optionally the third material 130 spend in
the mixing chamber 330. The ratio of the length of the mixing
chamber 330 to the diameter of the mixing chamber 330 may
significantly affect dwell time. The greater the ratio, the longer
the dwell time. As mentioned in the Background Section, the rotor
12 of the prior art device 10 (see FIG. 1) had a diameter of about
7.500 inches and a length of about 6.000 inches providing a length
to diameter ratio of about 0.8. In contrast, in particular
embodiments, the length of the mixing chamber 330 of the mixing
device 100 is about 5 inches and the diameter "D1" of the rotor 600
is about 1.69 inches yielding a length to diameter ratio of about
2.95.
[0338] Dwell time represents the amount of time that the first
material 110, the second material 120, and optionally the third
material 130 are able to interact with the electrokinetic phenomena
described herein. The prior art device 10 is configured to produce
about 60 gallons of the output material 102 per minute and the
mixing device 100 is configured to produce about 0.5 gallons of the
output material 102 per minute, the prior art device 10 (see FIG.
1) had a fluid dwell time of about 0.05 seconds, whereas
embodiments of the mixing device 100 have a substantially greater
(about 7-times greater) dwell time of about 0.35 seconds. This
longer dwell time allows the first material 110, the second
material 120, and optionally the third material 130 to interact
with each other and the surfaces 606 and 705 (see FIG. 7) inside
the mixing chamber 330 for about 7 times longer than was possible
in the prior art device 10. In additional embodiments, the dwell
time is at least 1.5-times, at least 2-times, at least 3-times, at
least 4-times, at least 5-times, at least 6-times, at least 7-times
or greater, than was possible in the prior art device 10.
[0339] With reference to Table 5 below, the above dwell times were
calculated by first determining the flow rate for each device in
gallons per second. In the case of the prior art device 10 was
configured to operate at about 60 gallons of output material per
minute, while the mixing device 100 is configured to operate over a
broader range of flow rate, including at an optimal range of about
0.5 gallons of output material per minute. The flow rate was then
converted to cubic inches per second by multiplying the flow rate
in gallons per second by the number of cubic inches in a gallon
(i.e., 231 cubic inches). Then, the volume (12.876 cubic inches) of
the channel 32 of the prior art device 10 was divided by the flow
rate of the device (231 cubic inches/second) to obtain the dwell
time (in seconds) and the volume (0.673 cubic inches) of the mixing
chamber 330 of the mixing device 100 was divided by the flow rate
(1.925 cubic inches/second) of the device (in cubic inches per
second) to obtain the dwell time (in seconds).
TABLE-US-00005 TABLE 5 Inventive device can accommodate a range of
dwell times, including a substantially increased (e.g., 7-times)
dwell time relative to prior art devices. Flow Flow Flow Rate
Volume Rate Rate Cubic Mixing Dwell Gallons/ Gallons/ Inches/
Chamber Time Device Minute Second Second (Cubic Inches) (Seconds)
Prior art 60 1.000 231.000 12.876 0.056 device 10 Mixing 2 0.033
7.700 0.673 0.087 device 100 Mixing 0.5 0.008 1.925 0.673 0.350
device 100
Rate of Infusion
[0340] Particular aspects of the mixing device 100 provide an
improved oxygen infusion rate over the prior art, including over
prior art device 10 (see FIG. 1). When the first material 110 is
water and the second material 120 is oxygen, both of which are
processed by the mixing device 100 in a single pass (i.e., the
return block of FIG. 2 is set to "NO") at or near 20.degree.
Celsius, the output material 102 has a dissolved oxygen level of
about 43.8 parts per million. In certain aspects, an output
material having about 43.8 ppm dissolved oxygen is created in about
350 milliseconds via the inventive flow through the inventive non
pressurized (non-pressure pot) methods. In contrast, when the first
material 110 (water) and the second material 120 (oxygen) are both
processed in a single pass at or near 20.degree. Celsius by the
prior art device 10, the output material had dissolved oxygen level
of only 35 parts per million in a single pass of 56
milliseconds.
Output Material 102
[0341] When the first material 110 is a liquid (e.g., freshwater,
saline, GATORADE.RTM., and the like) and the second material 120 is
a gas (e.g., oxygen, nitrogen, and the like), the mixing device 100
may diffuse the second material 120 into the first material 110.
The following discusses results of analyses performed on the output
material 102 to characterize one or more properties of the output
material 102 derived from having been processed by the mixing
device 100.
[0342] When the first material 110 is saline solution and the
second material 120 is oxygen gas, experiments have indicated that
a vast majority of oxygen bubbles produced within the saline
solution are no greater than 0.1 micron in size.
Decay of Dissolved Oxygen Levels
[0343] Referring now to FIG. 30, there is illustrated the DO levels
in water processed with oxygen in the mixing device 100 and stored
in a 500 ml thin-walled plastic bottle and a 1000 ml glass bottle.
Each of the bottles was capped and stored at 65.degree. Fahrenheit.
Point 7900 is the DO level at bottling. Line 7902 illustrates the
Henry's Law equilibrium state (i.e., the amount of dissolved oxygen
that should be within the water at 65.degree. Fahrenheit), which is
a DO level of slightly less than 10 ppm. Points 7904 and 7906
represent the DO levels within the water in the plastic bottle at
65 days and 95 days respectively. As can be seen at point 7904,
when the plastic bottle is opened approximately 65 days after
bottling, the DO level within the water is approximately 27.5 ppm.
When the bottle is opened approximately 95 days after bottling, as
indicated at point 7906, the DO level is approximately 25 ppm.
Likewise, for the glass bottle, the DO level is approximately 40
ppm at 65 days as indicated at point 7908 and is approximately 41
ppm at 95 days as illustrated at point 7910. Thus, FIG. 30
indicates the DO levels within both the plastic bottle and the
glass bottle remain relatively high at 65.degree. Fahrenheit.
[0344] Referring to FIG. 31, there is illustrated the DO levels in
water enriched with oxygen in the mixing device 100 and stored in a
500 ml plastic thin-walled bottle and a 1000 ml glass bottle. Both
bottles were refrigerated at 39 degrees Fahrenheit. Again, DO
levels of the oxygen-enriched fluid remained steady and decreased
only slightly out to at least 365 days.
Molecular Interactions
[0345] Conventionally, quantum properties are thought to belong to
elementary particles of less than 10.sup.-10 meters, while the
macroscopic world of our everyday life is referred to as classical,
in that it behaves according to Newton's laws of motion.
[0346] Recently, molecules have been described as forming clusters
that increase in size with dilution. These clusters measure several
micrometers in diameter, and have been reported to increase in size
non-linearly with dilution. Quantum coherent domains measuring 100
nanometers in diameter have been postulated to arise in pure water,
and collective vibrations of water molecules in the coherent domain
may eventually become phase locked to electromagnetic field
fluctuations, providing for stable oscillations in water, providing
a form of `memory` in the form of excitation of long lasting
coherent oscillations specific to dissolved substances in the watet
that change the collective structure of the water, which may in
turn determine the specific coherent oscillations that develop.
Where these oscillations become stabilized by magnetic field phase
coupling, the water, upon diluction may still carry `seed` coherent
oscillations. As a cluster of molecules increases in size, its
electromagnetic signature is correspondingly amplified, reinforcing
the coherent oscillations carried by the water.
[0347] Despite variations in the cluster size of dissolved
molecules and detailed microscopic structure of the water, a
specificity of coherent oscillations may nonetheless exist. One
model for considering changes in properties of water is based on
considerations involved in crystallization.
[0348] With reference to FIG. 36, a simplified protonated water
cluster forming a nanoscale cage 8700 is shown. A protonated water
cluster typically takes the form of H.sup.+ (H.sub.20).sub.n. Some
protonated water clusters occur naturally, such as in the
ionosphere. Without being bound by any particular theory, and
according to particular aspects, other types of water clusters or
structures (clusters, nanocages, etc) are possible, including
structures comprising oxygen and stabilized electrons imparted to
the inventive output materials. Oxygen atoms 8704 may be caught in
the resulting structures 8700. The chemistry of the semi-bound
nanocage allows the oxygen 8704 and/or stabilized electrons to
remain dissolved for extended periods of time. Other atoms or
molecules, such as medicinal compounds, can be caged for sustained
delivery purposes. The specific chemistry of the solution material
and dissolved compounds depend on the interactions of those
materials.
[0349] Fluids processed by the mixing device 100 have been shown
via experiments to exhibit different structural characteristics
that are consistent with an analysis of the fluid in the context of
a cluster structure.
[0350] Water processed through the mixing device 100 has been
demonstrated to have detectible structural differences when
compared with normal unprocessed water. For example, processed
water has been shown to have more Rayleigh scattering than is
observed in unprocessed water. In the experiments that were
conducted, samples of processed and unprocessed water were prepared
(by sealing each in a separate bottle), coded (for later
identification of the processed sample and unprocessed sample), and
sent to an independent testing laboratory for analysis. Only after
the tests were completed were the codes interpreted to reveal which
sample had been processed by the mixing device 100.
[0351] At the laboratory, the two samples were placed in a laser
beam having a wavelength of 633 nanometers. The fluid had been
sealed in glass bottles for approximately one week before testing.
With respect to the processed sample, Sample B scattered light
regardless of its position relative to the laser source. However,
Sample A did not. After two to three hours following the opening of
the bottle, the scattering effect of Sample B disappeared. These
results imply the water exhibited a memory causing the water to
retain its properties and dissipate over time. These results also
imply the structure of the processed water is optically different
from the structure of the unprocessed fluid. Finally, these results
imply the optical effect is not directly related to DO levels
because the DO level at the start was 45 ppm and at the end of the
experiment was estimated to be approximately 32 ppm.
Charge-Stabilized Nanostructures (e.g., Charge Stabilized
Oxygen-Containing Nanostructures):
[0352] As described herein above under "Double Layer Effect,"
"Dwell Time," "Rate of Infusion," and "Bubble size Measurements,"
the mixing device 100 creates, in a matter of milliseconds, a
unique non-linear fluid dynamic interaction of the first material
110 and the second material 120 with complex, dynamic turbulence
providing complex mixing in contact with an effectively enormous
surface area (including those of the device and of the
exceptionally small gas bubbles of less that 100 nm) that provides
for the novel electrokinetic effects described herein.
Additionally, feature-localized electrokinetic effects
(voltage/current) were demonstrated herein (see working Example 20)
using a specially designed mixing device comprising insulated rotor
and stator features.
[0353] As well-recognized in the art, charge redistributions and/or
solvated electrons are known to be highly unstable in aqueous
solution. According to particular aspects, Applicants'
electrokinetic effects (e.g., charge redistributions, including, in
particular aspects, solvated electrons) are surprisingly stabilized
within the output material (e.g., saline solutions, ionic
solutions). In fact, as described herein, the stability of the
properties and biological activity of the inventive electrokinetic
fluids (e.g., RNS-60 or Solas) can be maintained for months in a
gas-tight container, indicating involvement of dissolved gas (e.g.,
oxygen) in helping to generate and/or maintain, and/or mediate the
properties and activities of the inventive solutions.
Significantly, as described in the working Examples herein, the
charge redistributions and/or solvated electrons are stably
configured in the inventive electrokinetic ionic aqueous fluids in
an amount sufficient to provide, upon contact with a living cell
(e.g., mammalian cell) by the fluid, modulation of at least one of
cellular membrane potential and cellular membrane conductivity
(see, e.g., cellular patch clamp working Examples 23 and 24).
[0354] As described herein under "Molecular Interactions," to
account for the stability and biological compatibility of the
inventive electrokinetic fluids (e.g., electrokinetic saline
solutions), Applicants have proposed that interactions between the
water molecules and the molecules of the substances (e.g., oxygen)
dissolved in the water change the collective structure of the water
and provide for nanoscale cage clusters, including nanostructures
comprising oxygen and/or stabilized electrons imparted to the
inventive output materials. Without being bound by mechanism, and
according to the properties and activities described herein, the
configuration of the nanostructures in particular aspects is such
that they: comprise (at least for formation and/or stability and/or
biological activity) dissolved gas (e.g., oxygen); enable the
electrokinetic fluids (e.g., RNS-60 or Solas saline fluids) to
modulate (e.g., impart or receive) charges and/or charge effects
upon contact with a cell membrane or related constituent thereof;
and in particular aspects provide for stabilization (e.g.,
carrying, harboring, trapping) solvated electrons in a
biologically-relevant form.
[0355] According to particular aspects, and as supported by the
present disclosure, in ionic or saline (e.g., standard saline,
NaCl) solutions, the inventive nanostructures comprise charge
stabilized nanostrutures (e.g., average diameter less that 100 nm)
that may comprise at least one dissolved gas molecule (e.g.,
oxygen) within a charge-stabilized hydration shell. According to
additional aspects, and as described elsewhere herein, the
charge-stabilized hydration shell may comprise a cage or void
harboring the at least one dissolved gas molecule (e.g., oxygen).
According to further aspects, by virtue of the provision of
suitable charge-stabilized hydration shells, the charge-stabilized
nanostructure and/or charge-stabilized oxygen containing
nano-structures may additionally comprise a solvated electron
(e.g., stabilized solvated electron).
[0356] Without being bound by mechanism or particular theory, after
the present priority date, charge-stabilized microbubbles
stabilized by ions in aqueous liquid in equilibrium with ambient
(atmospheric) gas have been proposed (Bunkin et al., Journal of
Experimental and Theoretical Physics, 104:486-498, 2007;
incorporated herein by reference in its entirety). According to
particular aspects of the present invention, Applicants' novel
electrokinetic fluids comprise a novel, biologically active form of
charge-stabilized oxygen-containing nanostructures, and may further
comprise novel arrays, clusters or associations of such
structures.
[0357] According to the charge-stabilized microbubble model, the
short-range molecular order of the water structure is destroyed by
the presence of a gas molecule (e.g., a dissolved gas molecule
initially complexed with a nonadsorptive ion provides a short-range
order defect), providing for condensation of ionic droplets,
wherein the defect is surrounded by first and second coordination
spheres of water molecules, which are alternately filled by
adsorptive ions (e.g., acquisition of a `screening shell of
Na.sup.+ ions to form an electrical double layer) and nonadsorptive
ions (e.g., Cl.sup.- ions occupying the second coordination sphere)
occupying six and 12 vacancies, respectively, in the coordination
spheres. In under-saturated ionic solutions (e.g., undersaturated
saline solutions), this hydrated `nucleus` remains stable until the
first and second spheres are filled by six adsorptive and five
nonadsorptive ions, respectively, and then undergoes Coulomb
explosion creating an internal void containing the gas molecule,
wherein the adsorptive ions (e.g., Na.sup.+ ions) are adsorbed to
the surface of the resulting void, while the nonadsorptive ions (or
some portion thereof) diffuse into the solution (Bunkin et al.,
supra). In this model, the void in the nanostructure is prevented
from collapsing by Coulombic repulsion between the ions (e.g.,
Na.sup.+ ions) adsorbed to its surface. The stability of the
void-containing nanostrutures is postulated to be due to the
selective adsorption of dissolved ions with like charges onto the
void/bubble surface and diffusive equilibrium between the dissolved
gas and the gas inside the bubble, where the negative (outward
electrostatic pressure exerted by the resulting electrical double
layer provides stable compensation for surface tension, and the gas
pressure inside the bubble is balanced by the ambient pressure.
According to the model, formation of such microbubbles requires an
ionic component, and in certain aspects collision-mediated
associations between particles may provide for formation of larger
order clusters (arrays) (Id).
[0358] The charge-stabilized microbubble model suggests that the
particles can be gas microbubbles, but contemplates only
spontaneous formation of such structures in ionic solution in
equilibrium with ambient air, is uncharacterized and silent as to
whether oxygen is capable of forming such structures, and is
likewise silent as to whether solvated electrons might be
associated and/or stabilized by such structures.
[0359] According to particular aspects, the inventive
electrokinetic fluids comprising charge-stabilized nanostructures
and/or charge-stabilized oxygen-containing nanostructures are novel
and fundamentally distinct from the postulated non-electrokinetic,
atmospheric charge-stabilized microbubble structures according to
the microbubble model. Significantly, this conclusion is in
unavoidable, deriving, at least in part, from the fact that control
saline solutions do not have the biological properties disclosed
herein, whereas Applicants' charge-stabilized nanostructures
provide a novel, biologically active form of charge-stabilized
oxygen-containing nanostructures.
[0360] According to particular aspects of the present invention,
Applicants' novel electrokinetic device and methods provide for
novel electrokinetically-altered fluids comprising significant
quantities of charge-stabilized nanostructures in excess of any
amount that may or may not spontaneously occur in ionic fluids in
equilibrium with air, or in any non-electrokinetically generated
fluids. In particular aspects, the charge-stabilized nanostructures
comprise charge-stabilized oxygen-containing nanostructures. In
additional aspects, the charge-stabilized nanostrutures are all, or
substantially all charge-stabilized oxygen-containing
nanostructures, or the charge-stabilized oxygen-containing
nanostructures the major charge-stabilized gas-containing
nanostructure species in the electrokinetic fluid.
[0361] According to yet further aspects, the charge-stabilized
nanostructures and/or the charge-stabilized oxygen-containing
nanostructures may comprise or harbor a solvated electron, and
thereby provide a novel stabilized solvated electron carrier. In
particular aspects, the charge-stabilized nanostructures and/or the
charge-stabilized oxygen-containing nanostructures provide a novel
type of electride (or inverted electride), which in contrast to
conventional solute electrides having a single organically
coordinated cation, rather have a plurality of cations stably
arrayed about a void or a void containing an oxygen atom, wherein
the arrayed sodium ions are coordinated by water hydration shells,
rather than by organic molecules. According to particular aspects,
a solvated electron may be accommodated by the hydration shell of
water molecules, or preferably accommodated within the
nanostructure void distributed over all the cations. In certain
aspects, the inventive nanostructures provide a novel `super
electride` structure in solution by not only providing for
distribution/stabilization of the solvated electron over multiple
arrayed sodium cations, but also providing for association or
partial association of the solvated electron with the caged oxygen
molecule(s) in the void--the solvated electron distributing over an
array of sodium atoms and at least one oxygen atom. According to
particular aspects, therefore, `solvated electrons` as presently
disclosed in association with the inventive electrokinetic fluids,
may not be solvated in the traditional model comprising direct
hydration by water molecules. Alternatively, in limited analogy
with dried electride salts, solvated electrons in the inventive
electrokinetic fluids may be distributed over multiple
charge-stabilized nanostructures to provide a `lattice glue` to
stabilize higher order arrays in aqueous solution.
[0362] In particular aspects, the inventive charge-stabilized
nanostructures and/or the charge-stabilized oxygen-containing
nanostructures are capable of interacting with cellular membranes
or constituents thereof, or proteins, etc., to mediate biological
activities. In particular aspects, the inventive charge-stabilized
nanostructures and/or the charge-stabilized oxygen-containing
nanostructures harboring a solvated electron are capable of
interacting with cellular membranes or constituents thereof, or
proteins, etc., to mediate biological activities.
[0363] In particular aspects, the inventive charge-stabilized
nanostructures and/or the charge-stabilized oxygen-containing
nanostructures interact with cellular membranes or constituents
thereof, or proteins, etc., as a charge and/or charge effect donor
(delivery) and/or as a charge and/or charge effect recipient to
mediate biological activities. In particular aspects, the inventive
charge-stabilized nanostructures and/or the charge-stabilized
oxygen-containing nanostructures harboring a solvated electron
interact with cellular membranes as a charge and/or charge effect
donor and/or as a charge and/or charge effect recipient to mediate
biological activities.
[0364] In particular aspects, the inventive charge-stabilized
nanostructures and/or the charge-stabilized oxygen-containing
nanostructures are consistent with, and account for the observed
stability and biological properties of the inventive electrokinetic
fluids, and further provide a novel electride (or inverted
electride) that provides for stabilized solvated electrons in
aqueous ionic solutions (e.g., saline solutions, NaCl, etc.).
[0365] In particular aspects, the charge-stabilized
oxygen-containing nanostructures substantially comprise, take the
form of, or can give rise to, charge-stabilized oxygen-containing
nanobubbles. In particular aspects, charge-stabilized
oxygen-containing clusters provide for formation of relatively
larger arrays of charge-stabilized oxygen-containing
nanostructures, and/or charge-stabilized oxygen-containing
nanobubbles or arrays thereof. In particular aspects, the the
charge-stabilized oxygen-containing nanostructures can provide for
formation of hydrophobic nanobubbles upon contact with a
hydrophobic surface (see elsewhere herein under EXAMPLE 25).
[0366] In particular aspects, the charge-stabilized
oxygen-containing nanostructures substantially comprise at least
one oxygen molecule. In certain aspects, the charge-stabilized
oxygen-containing nanostructures substantially comprise at least 1,
at least 2, at least 3, at least 4, at least 5, at least 10 at
least 15, at least 20, at least 50, at least 100, or greater oxygen
molecules. In particular aspects, charge-stabilized
oxygen-containing nanostructures comprise or give rise to
nanobubles (e.g., hydrophobid nanobubbles) of about 20 nm.times.1.5
nm, comprise about 12 oxygen molecules (e.g., based on the size of
an oxygen molecule (approx 0.3 nm by 0.4 nm), assumption of an
ideal gas and application of n=PV/RT, where P=1 atm, R=0.082 057
I.atm/mol.K; T=295K; V=pr.sup.2h=4.7.times.10.sup.-22 L, where
r=10.times.10.sup.-9 m, h=1.5.times.10.sup.-9 m, and
n=1.95.times.10.sup.-22 moles).
[0367] In certain aspects, the percentage of oxygen molecules
present in the fluid that are in such nanostructures, or arrays
thereof, having a charge-stabilized configuration in the ionic
aqueous fluid is a percentage amount selected from the group
consisting of greater than: 0.1%, 1%; 2%; 5%; 10%; 15%; 20%; 25%;
30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%;
and greater than 95%. Preferably, this percentage is greater than
about 5%, greater than about 10%, greater than about 15% f, or
greater than about 20%. In additional aspects, the substantial size
of the charge-stabilized oxygen-containing nanostructures, or
arrays thereof, having a charge-stabilized configuration in the
ionic aqueous fluid is a size selected from the group consisting of
less than: 100 nm; 90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm;
20 nm; 10 nm; 5 nm; 4 nm; 3 nm; 2 nm; and 1 nm. Preferably, this
size is less than about 50 nm, less than about 40 nm, less than
about 30 nm, less than about 20 nm, or less than about 10 nm.
[0368] In certain aspects, the inventive electrokinetic fluids
comprise solvated electrons. In further aspects, the inventive
electrokinetic fluids comprises charge-stabilized nanostructures
and/or charge-stabilized oxygen-containing nanostructures, and/or
arrays thereof, which comprise at least one of: solvated
electron(s); and unique charge distributions (polar, symmetric,
asymmetric charge distribution). In certain aspects, the
charge-stabilized nanostructures and/or charge-stabilized
oxygen-containing nanostructures, and/or arrays thereof, have
paramagnetic properties.
[0369] By contrast, relative to the inventive electrokinetic
fluids, control pressure pot oxygenated fluids (non-electrokinetic
fluids) and the like do not comprise such charge-stabilized
biologically-active nanostructures and/or biologically-active
charge-stabilized oxygen-containing nanostructures and/or arrays
thereof, capable of modulation of at least one of cellular membrane
potential and cellular membrane conductivity.
Systems for Making Gas-Enriched Fluids
[0370] The presently disclosed system and methods allow gas (e.g.
oxygen) to be enriched stably at a high concentration with minimal
passive loss. This system and methods can be effectively used to
enrich a wide variety of gases at heightened percentages into a
wide variety of fluids. By way of example only, deionized water at
room temperature that typically has levels of about 2-3 ppm (parts
per million) of dissolved oxygen can achieve levels of dissolved
oxygen ranging from at least about 5 ppm, at least about 10 ppm, at
least about 15 ppm, at least about 20 ppm, at least about 25 ppm,
at least about 30 ppm, at least about 35 ppm, at least about 40
ppm, at least about 45 ppm, at least about 50 ppm, at least about
55 ppm, at least about 60 ppm, at least about 65 ppm, at least
about 70 ppm, at least about 75 ppm, at least about 80 ppm, at
least about 85 ppm, at least about 90 ppm, at least about 95 ppm,
at least about 100 ppm, or any value greater or therebetween using
the disclosed systems and/or methods. In accordance with a
particular exemplary embodiment, oxygen-enriched water may be
generated with levels of about 30-60 ppm of dissolved oxygen.
[0371] Table 6 illustrates various partial pressure measurements
taken in a healing wound treated with an oxygen-enriched saline
solution (Table 6) and in samples of the gas-enriched
oxygen-enriched saline solution of the present invention.
TABLE-US-00006 TABLE 6 Various partial pressure measurements taken
in a healing wound treated with an inventive salinesolution. TISSUE
OXYGEN MEASUREMENTS Probe Z082BO In air: 171 mmHg 23.degree. C.
Column Partial Pressure (mmHg) B1 32-36 B2 169-200 B3 20-180* B4
40-60 *wound depth minimal, majority >150, occasional 20 s
Cosmetic and/or Therapeutic Application and Administration
[0372] In particular exemplary embodiments, the gas-enriched fluid
of the present invention may function as a cosmetic and/or
therapeutic composition alone or in combination with another
cosmetic and/or therapeutic agent such that the therapeutic
composition prevents or alleviates at least one symptom of an
eye-related disease or condition. The therapeutic compositions of
the present invention include compositions that are able to be
administered to a subject in need thereof. As used herein,
"subject," may refer to any living creature, preferably an animal,
more preferably a mammal, and even more preferably a human.
[0373] In certain embodiments, the composition formulation may also
comprise at least one additional agent selected from the group
consisting of: carriers, adjuvants, emulsifying agents, suspending
agents, sweeteners, flavorings, perfumes, and binding agents.
[0374] As used herein, "pharmaceutically acceptable carrier" and
"carrier" generally refer to a non-toxic, inert solid, semi-solid
or liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. Some non-limiting examples of materials
which can serve as pharmaceutically acceptable carriers are sugars
such as lactose, glucose and sucrose; starches such as corn starch
and potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such as propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol, and phosphate
buffer solutions, as well as other non-toxic compatible lubricants
such as sodium lauryl sulfate and magnesium stearate, as well as
coloring agents, releasing agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the composition, according to the judgment of
the formulator. In particular aspects, such carriers and excipients
may be gas-enriched fluids or solutions of the present
invention.
[0375] The pharmaceutically acceptable carriers described herein,
for example, vehicles, adjuvants, excipients, or diluents, are
well-known to those who are skilled in the art. Typically, the
pharmaceutically acceptable carrier is chemically inert to the
therapeutic agents and has no detrimental side effects or toxicity
under the conditions of use. The pharmaceutically acceptable
carriers can include polymers and polymer matrices, nanoparticles,
microbubbles, and the like.
[0376] In addition to the therapeutic gas-enriched fluid of the
present invention, the therapeutic composition may further comprise
inert diluents such as additional non-gas-enriched water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. As is appreciated by
those of ordinary skill, a novel and improved formulation of a
particular therapeutic composition, a novel gas-enriched
therapeutic fluid, and a novel method of delivering the novel
gas-enriched therapeutic fluid may be obtained by replacing one or
more inert diluents with a gas-enriched fluid of identical,
similar, or different composition. For example, conventional water
may be replaced or supplemented by a gas-enriched fluid produced by
infusing oxygen into water or deionized water to provide
gas-enriched fluid.
[0377] Certain embodiments provide for therapeutic compositions
comprising a gas-enriched fluid of the present invention, a
pharmaceutical composition or other therapeutic agent or a
pharmaceutically acceptable salt or solvate thereof, and at least
one pharmaceutical carrier or diluent. These pharmaceutical
compositions may be used in the prophylaxis and treatment of the
foregoing diseases or conditions and in therapies as mentioned
above. Preferably, the carrier must be pharmaceutically acceptable
and must be compatible with, i.e. not have a deleterious effect
upon, the other ingredients in the composition. The carrier may be
a solid or liquid and is preferably formulated as a unit dose
formulation, for example, a tablet which may contain from 0.05 to
95% by weight of the active ingredient.
[0378] While the compositions and/or methods disclosed herein
generally relate to topical application, the most suitable means of
administration for a particular subject will depend on the nature
and severity of the disease or condition being treated or the
nature of the therapy being used, as well as the nature of the
therapeutic composition or additional therapeutic agent.
[0379] Formulations suitable for topical application include
liquids (aqueous or oil based), ointments, creams, lotions, pastes,
gels (such as hydrogels), sprays, dispersible powders and granules,
emulsions, sprays or aerosols using flowing propellants (such as
liposomal sprays, nasal drops, nasal sprays, and the like) and
oils. Suitable carriers for such formulations include petroleum
jelly, lanolin, polyethylene glycol (such as PEG 3000, PEG 5000, or
other), alcohols, and combinations thereof.
[0380] Formulations of the invention may be prepared by any
suitable method, typically by uniformly and intimately admixing the
gas-enriched fluid optionally with an active compound with liquids
or finely divided solid carriers or both, in the required
proportions and then, if necessary, shaping the resulting mixture
into the desired shape.
[0381] Ointments, pastes, foams, occlusions, creams, gels, sols,
suspensions, and patches also can contain excipients, such as
starch, tragacanth, cellulose derivatives, silicones, bentonites,
silica acid, and talc, or mixtures thereof. Powders and sprays also
can contain excipients such as lactose, talc, silica acid, aluminum
hydroxide, and calcium silicates, or mixtures of these substances.
Solutions of nanocrystalline antimicrobial metals can be converted
into aerosols or sprays by any of the known means routinely used
for making aerosol pharmaceuticals. In general, such methods
comprise pressurizing or providing a means for pressurizing a
container of the fluid, usually with an inert carrier gas, and
passing the pressurized gas through a small orifice. Sprays can
additionally contain customary propellants, such as nitrogen,
carbon dioxide, or other inert gases. In addition, microspheres or
nanoparticles may be employed with the gas-enriched therapeutic
compositions or fluids of the present invention in any of the
routes required to administer the compounds to a subject.
[0382] The topical formulations can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials, and can be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid excipient, or gas-enriched fluid,
immediately prior to use. Extemporaneous fluids, and/or suspensions
can be prepared from sterile powders, granules, and tablets.
[0383] The dose administered to a subject, especially an animal,
particularly a human, in the context of the present invention
should be sufficient to effect a cosmetic and/or therapeutic
response in the animal over a reasonable time frame. One skilled in
the art will recognize that dosage will depend upon a variety of
factors including a condition of the animal, the body weight of the
animal, the desired outcome, as well as the condition being
treated. A suitable dose is that which will result in a
concentration of the cosmetic and/or therapeutic composition in a
subject which is known to affect the desired response. The dosage
administered will, of course, vary depending upon known factors,
such as the pharmacodynamic characteristics of the particular agent
and its mode and route of administration; the age, health and
weight of the recipient; the nature and extent of the symptoms; the
kind of concurrent treatment; the frequency of treatment; and the
effect desired. A daily dosage of active ingredient can be expected
to be about 0.0001 to 1000 milligrams (mg) per kilogram (kg) of
body weight, with the preferred dose being 0.01 to about 30 mg/kg.
According to certain aspects daily dosage of active ingredient may
be 0.00001 liters to 10 liters, with the preferred dose being from
about 0.0001 liters to 1 liter.
[0384] Dosage forms (compositions suitable for administration)
contain from about 1 mg to about 500 mg of active ingredient per
unit. In these pharmaceutical compositions, the active ingredient
will ordinarily be present in an amount of about 0.5-95% weight
based on the total weight of the composition.
[0385] The size of the dose also will be determined by the route,
timing and frequency of administration as well as the existence,
nature, and extent of any adverse side effects that might accompany
the administration of the therapeutic composition and the desired
physiological effect.
[0386] The gas-enriched fluids of the present invention may be used
to improve existing drug delivery compositions and methods. The
diffuser processed fluids may be formulated, alone or together with
one or more therapeutic agents, in suitable dosage unit
formulations. In various embodiments, these formulations may
include conventional non-toxic pharmaceutically acceptable
carriers, adjuvants, emulsifying and suspending agents, sweetening,
flavoring, perfuming agents, and vehicles appropriate for the
particular route taken into the body of the patient.
[0387] In certain embodiments, the gas-enriched fluid disclosed
herein may comprise a wetting, irrigation or soaking fluid to apply
to organs, such as the eye, or into which organs and/or tissues may
be placed. One or more therapeutic agents may be dissolved in the
gas-enriched fluid or placed in the tissue before wetting,
irrigating or soaking it in the gas-enriched fluid. The
gas-enriched fluid may also be applied to the eye or other organ or
tissue in combination with a preexisting medication, thereby
creating a gas-enriched therapeutic fluid, to increase the efficacy
of the medication. The gas-enriched fluid may also be used to
dissolve a powder, thereby creating a gas-enriched therapeutic
fluid and drug delivery method. Alternatively, the inventive
gas-enriched fluid may comprise infused ingredients of these
patches, gels, creams, lotions, ointments, pastes, solutions,
sprays, aqueous or oily suspensions, emulsions, and the like.
[0388] Topical drug delivery, including drug delivery by
administration to the eye or surfaces of the eye is also within the
scope of this invention. Particular aspects provide for therapeutic
eye care products for delivery to the eye or to a portion thereof.
For example, the gas-enriched diffuser processed fluid may comprise
eye ointments, eye drops, therapeutic solutions, irrigation fluids,
pharmaceuticals (prescription and over the counter eye drops and
other medications, steroid eye drops, carteol, a non-selective beta
blocker, ointment for dry eye, ocular vitamins), and the like. For
example, the gas-enriched fluid may replace common carriers such as
saline and water used in eye ointments, eye drops, therapeutic
solutions, irrigation fluids and the like. Alternatively, the
inventive gas-enriching diffuser may be used to infuse one or more
of the ingredients of eye ointments, eye drops, therapeutic
solution, irrigation fluids and the like into a fluid, thereby
producing a novel gas-enriched fluid.
[0389] People use contact lenses to correct refractive disorders of
the eye such as myopia, hyperopia and astigmatism. Lack of oxygen
available to the cornea, due to coverage by the contact lens,
results in an increase in microbial infections in contact lens
wearers. Thus, certain embodiments herein relate to manufacture of
a contact lens utilizing the inventive gas-enriched fluid.
[0390] Pseudomonas aeruginosa, and other microbial infections, can
cause devastating infections in the human eye and has been
associated with neonatal ophthalmia. Pseudomonas can colonize the
ocular epithelium by means of a fimbrial attachment to sialic acid
receptors. If the environment is conducive, and particularly if the
host's immune system is compromised, the bacteria can proliferate
rapidly via production of enzymes such as elastase, alkaline
protease, and exotoxin A. Infection with Pseudomonas can cause loss
of use or loss of the entire eye.
[0391] An oxygen-enriched fluid may be used to formulate an
eye-drop that patients can use daily to optimize contact lens wear
and thereby provide enhanced levels of oxygen to the
oxygen-deprived contact-lens covered cornea 3508. The gas-enriched
fluid produced in accordance with the disclosed systems and/or
methods may be used in the manufacture, storage or care of contact
lenses. Heightened levels of dissolved gas may increase the potency
or safety of fluids, such as solutions used to store, clean, or
moisten contact lenses.
[0392] According to various embodiments of the present invention,
the gas-enriched inventive fluids disclosed can be used in the
manufacture, storage or care of contact lenses (e.g., saline
solutions and other contact lens storage and wetting solutions).
The introduction and retention of gases (such as oxygen) to the
contact lens may be implemented during the manufacture and use of
contact lenses. Additionally, saline solutions and other contact
lens storage and wetting solutions may be produced using
gas-enriched fluids described herein.
[0393] Contact lenses are typically formed of soft polymer
substances and may generally be divided into the categories of
hydrophilic and hydrophobic lenses. Hydrophilic contact lenses have
a water content in excess of ten percent while hydrophobic lenses
have water content of less than ten percent. The oxygen
permeability of a contact lens depends largely on the specific
polymer used to form the lens. The oxygen permeability may be
increased by using a gas-enriched fluid to hydrate the polymer when
the lens is created. Contact lenses may be made from a variety of
commercially available materials, such as hydrophilic polymers
(e.g., hydrogels) or poly(methyl methacrylate). A typical hydrogel
polymer composition may consist of a reaction product of
hydrophilic methacrylamide as well as an acrylic monomer, which may
contain a zwitterionic monomer, such as a sulfobetaine, for
example, N-(3-sulfopropyl)-Nmethacryloxyethyl-N,N-dimethylammonium
betaine (SPE), in order to improve the water retention
capability.
[0394] A variety of oxygen-enriched liquids may be used as eye
drops or contact lens solutions. Typical contact lens solutions are
made for rinsing, cleaning and disinfecting the contact lenses,
including but not limited to saline solutions and other contact
lens storage and wetting solutions.
[0395] The gas-enriched (i.e. oxygenated) solution may be packaged
in a bottle or other sealed container provided with a pipette or
eye dropper for use in various ocular applications. One such
application is that of a gas-enriched (i.e. oxygenated) saline
solution for use as eye drops or artificial tears. The moisturizing
eye drops may be applied directly to the eye using the eye dropper
and applying two or three drops per application directly to the eye
to alleviate dry eyes, redness, allergic reactions, and to provide
additional moisture to the corneal region of the eye.
[0396] In addition to gas-enriching artificial tears to further
supply a gas (e.g. oxygen) to the corneal region, it is also
possible to gas-enrich other fluids such as medications that might
be applied topically to the surface of the eye. This may be
particularly useful to patients that have recently undergone
surgery on the cornea or other areas of the eye to improve vision
(e.g., laser keratotomy, LASIK, cataract surgery of all types,
extracapsular surgery, phacoemulsification, vitreoretinal surgery,
intraocular lenses and delivery systems, pharmaceuticals,
prescription and over the counter eye drops and other medications,
steroid eye drops, carteol, a non-selective beta blocker, ointments
for dry eye, ocular vitamins and so forth) or to alleviate or
lessen the effects of glaucoma.
[0397] It is common to prescribe various antibiotics,
anti-inflammatory and pain relieving agents which are applied as
drops in solution directly to the eye itself following these and
other surgical procedures. By using gas-enriched (i.e. oxygenated)
aqueous solutions to increase oxygen diffusion into the surface of
the eye, it is believed that faster healing may occur and that
recovery time may be reduced.
[0398] Aging eyes often become dry as a result of lowered tear
production due to problems with the tear ducts. This problem is
particularly marked in menopausal women. The most immediate
patho-physiological problem produced by lower tear volume in these
patients is the lack of dissolved oxygen from the air which has
only a smaller volume to diffuse to reach the eye. Using a
gas-enriched fluid (such as oxygen-enriched saline) may be an
effective tear supplement. In addition to possible applications in
the form of artificial tears or gas-enriched medicines, the
gas-enriching process may also be applied to contact lens solutions
such as saline solutions. A contact lens is normally stored in a
solution to keep the semi-permeable polymer membrane moist and
flexible. As shown here, a contact lens which has been stored in a
lens solution is normally disposed just above the cornea of the
eye. The contact lens will normally float just above the surface of
the cornea on a layer of solution which may comprise the
gas-enriched (i.e., oxygenated) saline solution in which the lens
has been stored. The gas-enriched (i.e., oxygenated) saline
solution should increase the amount of gas (e.g., dissolved oxygen)
near the cornea of the eye and allow the eye to absorb greater
amounts of oxygen than is usually possible with a contact lens in
place.
Surgery:
[0399] Particular embodiments provide methods for improved surgery,
including eye surgery procedures, that involve irrigation of
tissues (e.g., eye tissues) including but not limited to laser
keratomay, LASIK, intraLASIK, cataract surgery, extracapsular
surgery, phacoemulsification, intracapsular surgery, vitreoretinal
surgery, intraocular lenses and others.
[0400] Over 1 million cataract surgeries are performed in the
United States each year. Typically, cataract surgeries involve
removing the lens, and possibly receiving an artificial lens. There
are three types of surgery to remove lenses that have a cataract.
In extracapsular surgery, the lens is removed while leaving the
outer covering of the lens in place; in phacoemulsification, the
lens is fragmented by ultrasonic vibrations and simultaneously
irrigated and aspirated, leaving the back half of the lens capsule
in place.
[0401] Complications of eye surgeries can occur, including high
pressure in the eye; blood collection inside the eye; infection
inside the eye; artificial lens damage or dislocation; drooping
eyelids; retinal detachment; severe bleeding inside the eye;
swelling or clouding of the cornea; blindness; and loss of vision
or loss of the entire eye itself. Infection and general tissue
health continues to be a challenge in eye surgeries. Certain
embodiments provided herein relate to compositions and/or methods
for improved eye surgery procedures that involve irrigation, and/or
moistening, etc., of eye tissues.
[0402] According to certain embodiments described herein,
compositions and/or methods are provided for improved surgery,
including eye surgery procedures that involve irrigation,
moistening, etc., of eye tissues. Gas-enriched fluids or solutions
of the present invention can be used, for example, for keeping the
tissue surface (e.g., ocular surface) moist and/or sterile prior
to, during, and/or subsequent to the surgical procedure.
Enhancing Biological Tissue Growth:
[0403] In other related embodiments, the gas-enriched fluid
compositions and/or methods described herein can be used in
relation to enhancing biological cell and/or tissue growth of cell
(including stem cells), tissue and organ cultures, whether
naturally occurring, or genetically modified. In addition,
gas-enriched fluid compositions and/or methods disclosed herein can
be used for artificial blood and surgical procedures requiring
artificial blood (such as coronary bypass surgery and shock-trauma
procedures). Similarly, oxygen-rich solutions may be used to
perfuse one or more solid organs, such as liver, kidney, heart,
eye, hand, foot, brain, and others after harvesting and prior to
(e.g. in transit) and during transplantation. Use of gas-enriched
(particularly oxygenated) fluids in accordance with the disclosed
embodiments may lead to longer storage time and more successful
transplant rates.
[0404] In certain embodiments, the inventive fluid may be used for
transporting or storing organs, organ samples, test subjects and/or
other living tissues that require or could benefit from the
gas-enriched fluids and/or methods disclosed herein. The
gas-enriched (oxygenated) fluid may contain an organ or other
living tissue(s). In certain embodiments, the container may be
insulated or provided with a portable refrigeration unit (not
shown) and may further include various impellers or other
circulators for moving the oxygen-enriched solution on and about
all the surfaces of the living tissues which are being stored 20 or
transported for transplantation. In this manner, it is believed
that living tissues will be better preserved with less cell damage
prior to and during organ or tissue transplantation.
[0405] Further embodiments may include systems further comprising a
circulation pump which draws fluid and combines the fluid with gas
(such as oxygen) from the supply using a diffuser constructed in
accordance with the present invention. The pump, diffuser, and
other components of the transportation and storage system may be
provided with a portable power supply in the form of one or more
batteries or a hydrogen fuel cell.
[0406] By storing and transporting organs and other living tissues
in a gas-enriched storage medium, it is possible to reduce damage
to cells and living tissues outside the body and to supply these
tissues to transplantation candidates in a healthier condition. By
using gas-enriched fluids as a storage and transportation medium it
is possible to promote life and health in transplant recipients by
introducing higher levels of dissolved gas(es) (such as oxygen),
slowing down cell decomposition during storage and transportation,
and further increasing the probability of a successful organ
transplant.
[0407] Gas-enriched fluids, such as water, produced in accordance
with the disclosed embodiments may also be used to decontaminate or
wash away contaminants from the eye of a subject, such as a person
or animal. The gas-enriched fluid compositions and/or methods
disclosed herein provide high levels of gas(es) (such as oxygen) to
the surface being cleaned (e.g. the eye), which may be particularly
therapeutic.
EXAMPLES
Example 1
Dissolved Oxygen Stability
[0408] As indicated in FIG. 30, there is illustrated the dissolved
oxygen levels in a 500 ml thin-walled plastic bottle and a 1000 ml
glass bottle which were each capped and stored at 65 degrees
Fahrenheit.
[0409] As can be seen, when the plastic bottle is opened
approximately 65 days after bottling, the dissolved oxygen level
within the water is approximately 27.5 ppm. When a second bottle is
opened at approximately 95 days after bottling, the dissolved
oxygen level is approximately 25 ppm. Likewise, for the glass
bottle, the dissolved oxygen level is approximately 40 ppm at 65
days and is approximately 41 ppm at 95 days. Thus, this chart
indicates that the dissolved oxygen levels within both plastic and
glass bottles are maintained at relatively high rates at 65.degree.
Fahrenheit when the oxygen is diffused within the fluid using the
described system and method.
Example 2
Decayed Oxygen Content in Balanced Salt Solution
[0410] FIG. 33 illustrates the dissolved oxygen retention of a 500
ml balanced salt solution that originally had a dissolved oxygen
level of 5 ppm. Following enrichment of the solution at standard
temperature and pressure with the diffuser of the present
invention, the dissolved oxygen level was approximately 41 ppm. The
solution was kept in an amber glass bottle. After an hour, the
dissolved oxygen level was 40 ppm; 36 ppm after two hours; 34 ppm
after three hours; and slightly more than 30 ppm after
approximately four and a half hours. The final measurement was
taken shortly before six hours, at which point the dissolved oxygen
level was approximately 28 ppm.
Example 3
Microbubble Size
[0411] Experiments were performed with a gas-enriched fluid by
using the diffuser of the present invention in order to determine a
gas microbubble size limit. The microbubble size limit was
established by passing the gas enriched fluid through 0.22 and 0.1
micron filters. In performing these tests, a volume of fluid passed
through the diffuser of the present invention and generated a
gas-enriched fluid. Sixty milliliters of this fluid was drained
into a 60 ml syringe. The dissolved oxygen level of the fluid
within the syringe was then measured by Winkler titration. The
fluid within the syringe was injected through a 0.22 micron
Millipore Millex GP50 filter and into a 50 ml beaker. The dissolved
oxygen rate of the material in the 50 ml beaker was then measured.
The experiment was performed three times to achieve the results
illustrated in Table 7 below.
TABLE-US-00007 TABLE 7 DO AFTER 0.22 MICRON DO IN SYRINGE FILTER
42.1 ppm 39.7 ppm 43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm
[0412] As can be seen, the dissolved oxygen levels that were
measured within the syringe and the dissolved oxygen levels within
the 50 ml beaker were not significantly changed by passing the
diffused material through a 0.22 micron filter, which implies that
the microbubbles of dissolved gas within the fluid are not larger
than 0.22 microns.
[0413] A second test was performed in which a batch of saline
solution was enriched with the diffuser of the present invention
and a sample of the output solution was collected in an unfiltered
state. The dissolved oxygen level of the unfiltered sample was 44.7
ppm. A 0.1 micron filter was used to filter the oxygen-enriched
solution from the diffuser of the present invention and two
additional samples were taken. For the first sample, the dissolved
oxygen level was 43.4 ppm. For the second sample, the dissolved
oxygen level was 41.4 ppm. Finally, the filter was removed and a
final sample was taken from the unfiltered solution. In this case,
the final sample had a dissolved oxygen level of 45.4 ppm. These
results were consistent with those in which the Millipore 0.22
micron filter was used. Thus, the majority of the gas bubbles or
microbubbles within the saline solution are approximately less than
0.1 microns in size.
Example 4
Sparging Effects
[0414] FIGS. 34 and 35 illustrate the sparging affects of the
diffuser of the present invention on a fluid passing therethrough.
The sparging of oxygen-enriched water occurred in an 8 gallon tank
at standard temperature and pressure. As indicated, initially the
oxygen-enriched water had a dissolved oxygen level of approximately
42 ppm. After 2 minutes of running through the diffuser, the
nitrogen had sparged the oxygen-enriched water such that the
dissolved oxygen level was then slightly more than 20 ppm. At 6
minutes, the dissolved oxygen level was approximately 6 ppm. The
dissolved oxygen level of the oxygen-enriched water reached a
minimum value slightly greater than zero (0) at approximately 14
minutes after the beginning of the process. These figures
illustrate the manner in which nitrogen may be diffused into water
to sparge the oxygen from the water. However, any gas could be used
within any fluid to sparge one gas from the other and diffuse the
other gas into the fluid. The same experiment could utilize any
host fluid material, and any fluid infusion material.
Example 5
Rayleigh Effects
[0415] Fluids processed through the diffuser device described
herein exhibit differences within the structure of the water when
compared with normal unprocessed water. Gas-enriched water made by
embodiments disclosed herein has been shown to have more Rayleigh
scattering compared to unprocessed water.
[0416] In experiments conducted, samples of gas-enriched and
non-enriched water were prepared and sent for optical analysis. The
purpose of these tests was to determine whether there are any gross
optical differences between normal (unprocessed) deionized water
and water enriched by the diffuser device of the present
invention.
[0417] The two samples, were coded to maintain their identities in
secrecy, and only after the tests were completed were the samples
identified. The two samples were placed in a laser beam of 633
nanometers according to the diagram illustrated in FIG. 37A. Sample
B, which was gas-enriched fluid according to certain embodiments
disclosed herein, exhibited scattered light regardless of its
position relative to the laser source. The Sample B fluid had been
sealed in glass bottles for approximately one week. After two to
three hours of opening the bottle, the scattering effect
disappeared. Thus, the structure of the gas-enriched fluid is
optically different from the structure of the unprocessed fluid.
The optical effect is not directly related to dissolved oxygen
levels since the dissolved oxygen level at the start was
approximately 45 ppm and at the end of the experiment was estimated
to be approximately 32 ppm. Results are shown in FIG. 37B.
Example 6
Generation of Solvated Electrons
[0418] Additional evidence has also suggested that the enriching
process generated by the diffuser device of the present invention
results in solvated electrons within the gas-enriched fluid. Due to
the results of the polarographic dissolved oxygen probes, it is
believed that the diffused fluid exhibits an electron capture
effect and thus the fluid may include solvated electrons within the
gas-enriched material.
[0419] There are two fundamental techniques for measuring dissolved
oxygen levels electrically: galvanic measuring techniques and
polarographic measurements. Each process uses an electrode system
wherein the dissolved oxygen levels within the solution being
tested react with a cathode of the probe to produce a current.
Dissolved oxygen level sensors consist of two electrodes, an anode
and a cathode, which are both immersed in electrolyte within the
sensor body. An oxygen permeable membrane separates the anode and
cathode from the solution being tested. Oxygen diffuses across the
membrane and interacts with the internal components of the probe to
produce an electrical current. The cathode is a hydrogen electrode
and carries negative potential with respect to the anode. The
electrolyte solution surrounds the electrode pair and is contained
by the membrane. When no oxygen is present, the cathode is
polarized by hydrogen and resists the flow of current. When oxygen
passes through the membrane, the cathode is depolarized and
electrons are consumed. The cathode electrochemically reduces the
oxygen to hydroxyl ions according to the following equation:
O.sub.2+2H.sub.2O+4E.sup.-=40H.sup.-
[0420] When performing dissolved oxygen level measurements of a
gas-enriched solution according to the systems of the present
invention, an overflow condition has been repeatedly experienced
wherein the dissolved oxygen meter displays a reading that is
higher than the meter is capable of reading. However, evaluation of
the gas-enriched solution by Winkler Titration indicates lower
dissolved oxygen (DO) level for the solution than indicated by the
probe. Typically, a DO probe (such as the Orion 862 used in these
experiments) has a maximum reading of 60 ppm. However, when the
meter is left in gas-enriched water of the present invention, it
overflows.
[0421] Without wishing to be bound by any particular mechanism of
action, the mechanism of the meter responds to electrons where the
oxygen reacts. However, according to electron spin resonance, no
free ions are present in the fluid. Thus, the fluid presumably
contains solvated electrons stabilized by the oxygen species that
is also present in the fluid.
Example 7
In Vitro Wound Healing
[0422] The effects of a gas-enriched fluid (enriched with oxygen)
were tested for the ability of cultured human epidermal
keratinocytes to seal a wound.
[0423] Human epidermal keratinocytes were isolated from neonatal
foreskins that were obtained from routine circumcision and
de-identified. Foreskins were washed twice in PBS and incubated in
2.4 U/mL Dispase II in order to separate the dermis from the
epidermis. The epidermis was incubated with 0.25% trypsin/1 mM
EDTA, neutralized with soy bean trypsin inhibitor, agitated, and
passed through a 70 um sieve to separate the cells. Next, the cell
suspension was centrifuged and resuspended in cell culture medium
(M154) supplemented with 0.07 mM CaCl.sub.2, and human keratinocyte
growth supplements (0.2% hydrocortisone, 0.2 ng/mL human epidermal
growth factor) and penicillin/streptomycin, amphoteracin antibiotic
cocktail. The keratinocyte cell suspensions were plated onto
uncoated 12-well culture dishes and the medium replaced after 24
hours, and every 48 hours after the initial seeding.
[0424] Upon reaching cellular confluence, linear scratches were
made with a sterile p1000 pipette tip, which resulted in a uniform
cell-free wound. The monolayers were washed several times with
Dulbecco's PBS in order to remove any cellular debris. The wound
monolayers were then incubated in the following media: i) the
complete growth media (as described above in this Example); ii) the
complete growth media diluted 1:1 with a sheared version of saline
without oxygen (control fluid that was processed using the
disclosed diffuser device but without adding a gas); and iii) the
complete growth media diluted 1:1 with oxygen-enriched saline. Each
study was done in triplicate.
[0425] Prior to incubation, the wells were filled with the
respective media and sealed by placing a 25.times.25 mm glass
coverslip on top of each well. At 6, 12, 24, and 48 hours
post-wounding, oxygen measurements were made, and cultures were
imagined.
[0426] Six hours post-wounding, the edges of the wounds in the
saline and gas-enriched media were more ruffled than those in the
media control that was processed with the diffuser device disclosed
herein, but without the addition of a gas. Twelve hours
post-wounding the edges of the wounds in all three media appeared
uneven, with keratinocytes along the borders migrating toward the
center of the wounds. Quantification of migrating keratinocytes
revealed approximately the same level of keratinocyte migration in
the saline and gas-enriched media. Results of the experiment are
shown in FIGS. 40A and 44B.
Example 8
Improved Wound Healing
[0427] A study was performed to determine the improved healing
characteristics of wounds that were exposed to an oxygen-enriched
saline solution that was processed according to embodiments
disclosed herein. In this experiment, bandages were placed on
porcine dermal excision biopsy wounds. The bandages soaked in
oxygen-enriched saline solution or a control group of bandages
soaked in a saline solution that was not oxygen-enriched.
Microscopically, several factors were evaluated by the study
including: 1) epidermalization; 2) neovascularization; 3) epidermal
differentiation; 4) mast cell migration; and 5) mitosis.
[0428] Externally, the wounds appeared to heal at varying rates.
The wounds treated with the oxygen-enriched saline solution showed
an increase in wound healing at days 4 through 11. However, both
wounds seemed to complete healing at approximately the same time.
The study showed that between days 3 and 11, the new epidermis in
wounds treated with the oxygen-enriched saline solution migrated at
two to four times as fast as the epidermis of the wounds treated
with the normal saline solution. The study also showed that between
15 and 22 days, the wound treated by the oxygen-enriched saline
solution differentiated at a more rapid rate as evidenced by the
earlier formation of more mature epidermal layers. At all stages,
the thickening that occurs in the epidermis associated with normal
healing did not occur within the wounds treated by the
oxygen-enriched saline solution.
[0429] Without wishing to be bound by any particular theory, it is
believed that the oxygen-enriched saline solution may increase the
localized level of NO within the wounds. NO modulates growth
factors, collagen deposition, inflammation, mast cell migration,
epidermal thickening, and neovascularization in wound healing.
Furthermore, nitric oxide is produced by an inducible enzyme that
is regulated by oxygen.
[0430] Thus, while not wishing to be bound to any particular
theory, the inventive gas-enriched fluid may stimulate NO
production, which is in accordance with the spectrum of wound
healing effects seen in these experiments.
[0431] The epidermis of the healing pigs experienced earlier
differentiation in the oxygen-enriched saline group at days 15
through 22. In the case of mast cell migration, differences also
occurred in early and late migration for the oxygen-enriched
solution. A conclusive result for the level of mitosis was
unascertainable due to the difficulty in staining.
[0432] Referring now to FIG. 41A through 41F, various illustrations
compare the wound healing results of the porcine epidermal tissues
with or without oxygen-enriched saline solution. Thus, the healing
of the control wound and of the wound using the oxygen-enriched
saline solution was followed for days 1, 4 and 16. FIG. 41A
illustrates the wound healing for the control wound on day 1. As
can be seen, the wound shows epidermal/dermal thickening and a loss
of contour. FIG. 41B illustrates the wound healing on day 1 for the
wound treated using the oxygen-enriched saline solution. The wound
shows normal epidermal/dermal thickness and normal contouring is
typical on a new wound.
[0433] Referring now to FIGS. 41C and 41D, there are illustrated
the wound healing for the control wound on day 4 and the wound
healing for the wound treated with the oxygen-enriched saline
solution on day 4. For the control wound illustrated in FIG. 41C,
the wound shows a 600 micron epidermal spur. In the wound treated
with the oxygen-enriched saline solution in FIG. 41D, there is
illustrated a 1200 micron epidermal spur. Thus, in the first 4 days
of the experiment, the epidermal spur created in the wound treated
using the oxygen-enriched saline solution shows an epidermal growth
rate of twice of that of the wound that was not treated with the
oxygen-enriched saline solution.
[0434] Referring now to FIG. 41E, there is illustrated the control
wound at day 16. The wound shows less differentiated epidermis with
loss of epidermal/dermal contour than that illustrated by the wound
treated with the oxygen-enriched saline solution illustrated in
FIG. 41F. FIG. 41F shows more differentiated epidermis and more
normal epidermal/dermal contouring in the wound.
[0435] Thus, as illustrated with respect to FIGS. 41A through 41F,
the wound treated with the oxygen-enriched saline solution shows
much greater healing characteristics than the untreated wound and
shows a greater differentiated epidermis with more normal
epidermal/dermal contour.
Example 9
Glutathione Peroxidase Study
[0436] The inventive oxygen-enriched fluid was tested for the
presence of hydrogen peroxide by testing the reactivity with
glutathione peroxidase using a standard assay (Sigma). Water
samples were tested by adding the enzyme cocktail and inverting.
Continuous spectrophotometric rate determination was made at
A.sub.340 nm, and room temperature (25 degrees Celsius). Samples
tested were: 1. deionized water (negative control), 2. inventive
oxygen-enriched fluid at low concentration, 3. inventive
oxygen-enriched fluid at high concentration, 4. hydrogen peroxide
(positive control). The hydrogen peroxide positive control showed a
strong reactivity, while none of the other fluids tested reacted
with the glutathione peroxidase.
Example 10
Electrokinetically Generated Superoxygenated Fluids and Solas were
Shown to Provide for Synergistic Prolongation Effects (e.g.,
Suppression of Bronchoconstriction) with Albuterol In Vivo in an
Art-Recognized Animal Model of Human Bronchoconstriction (Human
Asthma Model)
Experiment 1
[0437] In an initial experiment, sixteen guinea pigs were evaluated
for the effects of bronchodilators on airway function in
conjunction with methacholine-induced bronchoconstriction.
Following determination of optimal dosing, each animal was dosed
with 50 .mu.g/mL to deliver the target dose of 12.5 .mu.g of
albuterol sulfate in 250 .mu.L per animal.
[0438] The study was a randomized blocked design for weight and
baseline PenH values. Two groups (A and B) received an
intratracheal instillation of 250 .mu.L of 50 .mu.g/mL albuterol
sulfate in one or two diluents: Group A was deionized water that
had passed through the inventive device, without the addition of
oxygen, while Group B was inventive gas-enriched water. Each group
was dosed intratracheally with solutions using a Penn Century
Microsprayer. In addition, the animals were stratified across BUXCO
plethysmograph units so that each treatment group is represented
equally within nebulizers feeding the plethysmographs and the
recording units.
[0439] Animals that displayed at least 75% of their baseline PenH
value at 2 hours following albuterol administration were not
included in the data analyses. This exclusion criteria is based on
past studies where the failure to observe bronchoprotection with
bronchodilators can be associated with dosing errors. As a result,
one animal from the control group was dismissed from the data
analyses.
[0440] Once an animal had greater than 50% bronchoconstriction, the
animal was considered to be not protected. As set forth in Table 8
below, 50% of the Group B animals (shaded) were protected from
bronchoconstriction out to 10 hours (at which time the test was
terminated).
TABLE-US-00008 TABLE 8 Bronchoconstriction Protection as Measured
with Methacholine Challenge Group A Percent protection from
bronchoconstriction by animal number Time (hours) 1 2 3 4 5 6 7 0
100.00 100.00 100.00 100.00 100.00 100.00 100.00 2 20.81 23.82
32.89 11.56 7.91 24.95 20.15 6 15.67 9.96 8.53 8.40 81.66 75.60
91.97 10 173.92 130.34 95.45 68.14 57.85 103.95 69.03 Group B
Percent protection from bronchoconstriction by animal number Time
(hours) 1 ##STR00002## 3 ##STR00003## ##STR00004## ##STR00005## 7
##STR00006## 0 100.00 ##STR00007## 100.00 ##STR00008## ##STR00009##
##STR00010## 100.00 ##STR00011## 2 15.85 ##STR00012## 17.88
##STR00013## ##STR00014## ##STR00015## 21.33 ##STR00016## 6 211.57
##STR00017## 68.79 ##STR00018## ##STR00019## ##STR00020## 118.26
##STR00021## 10 174.54 ##STR00022## 88.16 ##STR00023## ##STR00024##
##STR00025## 123.47 ##STR00026##
Experiment 2
A Bronchoconstriction Evaluation of RDC1676 with Albuterol Sulfate
in Male Hartley Guinea Pigs
[0441] An additional set of experiments was conducted using a
larger number of animals to evaluate the protective effects of the
inventive electrokinetically generated fluids (e.g, RDC1676-00,
RDC1676-01, RDC1676-02 and RDC1676-03) against methacholine-induced
bronchoconstriction when administered alone or as diluents for
albuterol sulfate in male guinea pigs.
Materials:
[0442] Guinea Pigs (Cavia porcellus) were Hartley albino,
Crl:(HA)BR from Charles River Canada Inc. (St. Constant, Quebec,
Canada). Weight: Approximately 325.+-.50 g at the onset of
treatment. Number of groups was 32, with 7 male animals per group
(plus 24 spares form same batch of animals). Diet; All animals had
free access to a standard certified pelleted commercial laboratory
diet (PMI Certified Guinea Pig 5026; PMI Nutrition International
Inc.) except during designated procedures.
Methods:
[0443] Route of administration was intratracheal instillation via a
Penn Century Microsprayer and methacholine challenge via whole body
inhalation. The intratracheal route was selected to maximize lung
exposure to the test article/control solution. Whole body
inhalation challenge has been selected for methacholine challenge
in order to provoke an upper airway hypersensitivity response (i.e.
bronchoconstriction).
[0444] Duration of treatment was one day.
[0445] Table 9 shows the experimental design. All animals were
subjected to inhalation exposure of methacholine (500 .mu.g/ml), 2
hours following TA/Control administration. All animals received a
dose volume of 250 .mu.l. Therefore, albuterol sulfate was diluted
(in the control article and the 4 test articles) to concentrations
of 0, 25, 50 and 100 .mu.g/ml.
[0446] Thirty minutes prior to dosing, solutions of albuterol
sulfate of 4 different concentrations (0, 25, 50 and 100 .mu.g/ml)
was made up in a 1 Ox stock (500 .mu.g/mL) in each of these four
test article solutions (RDC1676-00, RDC1676-01, RDC1676-02; and
RDC1676-03). These concentrations of albuterol sulfate were also
made up in non-electrokinetically generated control fluid (control
1). The dosing solutions were prepared by making the appropriate
dilution of each stock solution. All stock and dosing solutions
were maintained on ice once prepared. The dosing was completed
within one hour after the test/control articles are made. A
solution of methacholine (500 .mu.g/ml) was prepared on the day of
dosing.
[0447] Each animal received an intratracheal instillation of test
or control article using a Penn Century microsprayer. Animals were
food deprived overnight and were anesthetized using isoflurane, the
larynx was visualized with the aid of a laryngoscope (or suitable
alternative) and the tip of the microsprayer was inserted into the
trachea. A dose volume of 250 .mu.l/animal of test article or
control was administered.
[0448] The methacholine aerosol was generated into the air inlet of
a mixing chamber using aeroneb ultrasonic nebulizers supplied with
air from a Buxco bias flow pump. This mixing chamber in turn fed
four individual whole body unrestrained plethysmographs, each
operated under a slight negative pressure maintained by means of a
gate valve located in the exhaust line. A vacuum pump was used to
exhaust the inhalation chamber at the required flow rate.
[0449] Prior to the commencement of the main phase of the study, 12
spare animals were assigned to 3 groups (n=4/group) to determine
the maximum exposure period at which animals may be exposed to
methacholine to induce a severe but non-fatal acute
bronchoconstriction. Four animals were exposed to methacholine (500
.mu.g/mL) for 30 seconds and respiratory parameters were measured
for up to 10 minutes following commencement of aerosol.
Methacholine nebulizer concentration and/or exposure time of
aerosolization was adjusted appropriately to induce a severe but
non-fatal acute/reversible bronchoconstriction, as characterized by
a transient increase in penes.
[0450] Once prior to test article administration (Day -1) and again
at 2, 6, 10, 14, 18, 22 and 26 hours postdose, animals were placed
in the chamber and ventilatory parameters (tidal volume,
respiratory rate, derived minute volume) and the enhanced pause
Penh were measured for a period of 10 minutes using the Buxco
Electronics BioSystem XA system, following commencement of aerosol
challenge to methacholine. Once animals were within chambers
baseline, values were recorded for 1-minute, following which
methacholine, nebulizer concentration of 500 ug/mL were
aerosoloized for 30 seconds, animals were exposed to the aerosol
for further 10 minutes during which time ventilatory paramaters
were continuously assessed. Penh was used as the indicator of
bronchoconstriction; Penh is a derived value obtained from peak
inspiratory flow, peak expiratory flow and time of expiration.
Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory
time/time to expire 65% of expiratory volume -1).
[0451] Animals that did not display a severe acute
broncoconstriction during the predose methacholine challenge were
replaced. Any animal displaying at least 75% of their baseline
PenhPenes value at 2 hours post dose were not included in the data
analysis. The respiratory parameters were recorded as 20 second
means.
[0452] Data considered unphysiological was excluded from further
analysis.
[0453] Changes in Penh were plotted over a 15 minute period and
Penh value was expressed as area under the curve. Numerical data
was subjected to calculation of group mean values and standard
deviations (as applicable).
TABLE-US-00009 TABLE 9 Experimental design; 7 male guinea pigs per
group. Albuterol Albuterol Albuterol Albuterol Group ID (0
.mu.g/animal) (6/25 .mu.g/animal) (12.5 .mu.g/animal) (25
.mu.g/animal) 1 (control 1) 7 males 7 males 7 males 7 males
(ambient oxygen) 5 (RDC1676-00 7 males 7 males 7 males 7 males
(Solas) 6 (RDC1676-01 7 males 7 males 7 males 7 males (20 ppm
oxygen) 7 (RDC1676-02 7 males 7 males 7 males 7 males (40 ppm
oxygen) 8 (RDC1676-03 7 males 7 males 7 males 7 males (60 ppm
oxygen)
Results:
[0454] As shown in FIG. 107A-D, in the absence of Albuterol,
administration of the inventive electrokinetically generated fluids
had no apparent effect on mean percent baseline PenH values, when
measured over a 26 hour period.
[0455] Surprisingly, however, as shown in FIG. 108A-D,
administration of albuterol (representative data for the 25 .mu.g
albuterol/animal groups are shown) formulated in the inventive
electrokinetically generated fluids (at all oxygen level values
tested; ambient (FIG. 108-A), 20 ppm (FIG. 108-B), 40 ppm (FIG.
108-C) and 60 ppm (FIG. 108-D)) resulted in a striking prolongation
of anti-broncoconstrictive effects of albuterol, compared to
control fluid. That is, the methacholine results showed a
prolongation of the bronchodilation of albuterol out to at least 26
hours. FIGS. 108 A-D shows that there were consistent differences
at all oxygen levels between RDC1676 and the normal saline control.
Combining all 4 RDC1676 fluids, the p value for the overall
treatment difference from normal saline was 0.03.
[0456] According to particular aspects of the present invention,
therefore, the inventive electrokinetically generated solutions
provide for synergistic prolongation effects with Albuterol, thus
providing for a decrease in a patient's albuterol usage, enabling
more efficient cost-effective drug use, fewer side effects, and
increasing the period over which a patient may be treated and
responsive to treatment with albuterol.
Example 11
A Cytokine Profile was Determined
[0457] Mixed lymphocytes were obtained from a single healthy
volunteer donor. Buffy coat samples were washed according to
standard procedures to remove platelets. Lymphocytes were plated at
a concentration of 2.times.10.sup.6 per plate in RPMI media (+50 mm
HEPES) diluted with either inventive gas-enriched fluid or
distilled water (control). Cells were stimulated with 1
microgram/mL T3 antigen, or 1 microgram/mL phytohemagglutinin (PHA)
lectin (pan-T cell activator), or unstimulated (negative control).
Following 24-hour incubation, cells were checked for viability and
the supernatants were extracted and frozen.
[0458] The supernatants were thawed, centrifuged, and tested for
cytokine expression using a XMAP.RTM. (Luminex) bead lite protocol
and platform.
[0459] Two million cells were plated into 6 wells of a 24-well
plate in full RPMI+50 mm Hepes with either inventive
oxygen-enriched fluid (water) (wells 1, 3, and 5) or distilled
water (2, 4 and 6) (10X RPMI diluted into water to make 1.times.).
Cells were stimulated with 1 ug/ml T3 antigen (wells 1 and 2) or
PHA (wells 3 and 4). Control wells 5 and 6 were not stimulated.
After 24 hours, cells were checked for viability and supernatants
were collected and frozen. Next, the supernatants were thawed and
spun at 8,000 g to pellet. The clarified supernatants were assayed
for the cytokines listed using a LUMINEX BEAD LITE.TM. protocol and
platform. The numerical data is tabulated in Table 10, and the
corresponding bar graphs are depicted in FIG. 38. Notably,
IFN-.gamma. level was higher in the inventive gas-enriched culture
media with T3 antigen than in the control culture media with T3
antigen, while IL-8 was lower in the inventive gas-enriched culture
media with T3 antigen than in the control culture media with T3
antigen. Additionally, IL-6, IL-8, and TNF-.alpha. levels were
lower in the inventive gas-enriched media with PHA, than in the
control media with PHA, while IL-1.beta. levels were lower in the
inventive gas-enriched fluid with PHA when compared with control
media with PHA. In the inventive gas-enriched media alone,
IFN-.gamma. levels were higher than in control media.
TABLE-US-00010 TABLE 10 Sample IFN Il-10 Il-12p40 Il-12p70 Il-2
Il-4 Il-5 Il-6 Il-8 Il-ib IP-10 TNFa 1 0 0 0 2.85 0 0 7.98 20.3
1350 7.56 11500 15.5 2 0 0 0 3.08 0 0 8 15.2 8940 3.68 4280 7.94 3
0 581 168 3.15 0 0 8 16400 2200 3280 862 13700 4 0 377 56.3 4.22 0
0 8.08 23800 22100 33600 558 16200 5 0 0 0 2.51 0 0 7.99 24 1330
7.33 5900 8.55 6 0 0 0 2.77 0 0 8 5.98 3210 4.68 3330 0
Example 12
Myelin Oligodendrocyte Glycoprotein (MOG)
[0460] As set forth in FIG. 48, lymphocyte proliferation in
response to MOG antigenic peptide was increased when cultured in
the presence of the inventive gas-enriched fluid when compared to
pressurized, oxygenated fluid (pressure pot) or deionized control
fluid. Thus, the inventive gas-enriched fluid amplifies the
lymphocyte proliferative response to an antigen to which the cells
were previously primed.
[0461] Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG
35-55) (M-E-V-G-W-Y-R-S-P-F-S-R-O-V-H-L-Y-R-N-G-K) (SEQ ID NO:1;
see publication US20080139674, incorporated by reference herein,
including for purposes of this SEQ ID NO:1) corresponding to the
known mouse sequence was synthesized. Next, 5.times.10.sup.5 spleen
cells were removed from MOG T cell receptor transgenic mice
previously immunized with MOG, and were cultured in 0.2 ml TCM
fluid reconstituted with inventive gas-enriched fluid, pressurized
oxygenated water (pressure pot water) or with control deionized
water. Splenocytes were cultured with MOG p 35-55 for 48 or 72
hours, respectively. Cultures were pulsed with 1Ci [3H]-thymidine
and harvested 16 hours later. Mean cpm of [3H] thymidine
incorporation was calculated for triplicate cultures. Results are
shown in FIG. 48.
Example 13
Cytokine Expression
[0462] In particular aspects, human mixed lymphocytes were
stimulated with T3 antigen or PHA in inventive electrokinetic
fluid, or control fluid, and changes in IL-1.beta., IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13,
IL-17, Eotaxin, IFN-.gamma., GM-CSF, MIP-1.beta., MCP-1, G-CSF,
FGFb, VEGF, TNF-.alpha., RANTES, Leptin, TNF-.beta., TFG-.beta.,
and NGF were evaluated. As can be seen from FIG. 38,
pro-inflammatory cytokines (IL-1.beta., TNF-.alpha., IL-6, and
GM-CSF), chemokines (IL-8, MIP-1.alpha., RANTES, and Eotaxin),
inflammatory enzymes (iNOS, COX-2, and MMP-9), allergen responses
(MHC class II, CD23, B7-1, and B7-2), and Th2 cytokines (IL-4,
IL-13, and IL-5) tested were reduced in test fluid versus control
fluid. By contrast, anti-inflammatory cytokines (e.g.,
IL1R-.alpha., TIMPs) tested were increased in test fluid versus
control fluid.
[0463] To expand on these data, Applicants used an art recognized
model system involving ovalbumin sensitization, for assessing
allergic hypersensitivity reactions. The end points studied were
particular cytologic and cellular components of the reaction as
well as serologic measurements of protein and LDH. Cytokine
analysis was performed, including analysis of Eotaxin, IL-1A,
IL-1B, KC, MCP-1, MCP-3, MIP-1A, RANTES, TNF-A, and VCAM.
[0464] Briefly, male Brown Norway rats were injected
intraperitoneally with 0.5 mL Ovalbumin (OVA) Grade V (A5503-1G,
Sigma) in solution (2.0 mg/mL) containing aluminum hydroxide (A1
(OH).sub.3) (200 mg/mL) once each on days 1, 2, and 3. The study
was a randomized 2.times.2 factorial arrangement of treatments (4
groups). After a two week waiting period to allow for an immune
reaction to occur, the rats were either exposed or were treated for
a week with either RDC1676-00 (sterile saline processed through the
Revalesio proprietary device), and RDC1676-01 (sterile saline
processed through the Revalesio proprietary device with additional
oxygen added). At the end of the 1 week of treatment for once a
day, the 2 groups were broken in half and 50% of the rats in each
group received either Saline or OVA challenge by inhalation.
[0465] Specifically, fourteen days following the initial
serialization, 12 rats were exposed to RDC 1676-00 by inhalation
for 30 minutes each day for 7 consecutive days. The air flow rate
through the system was set at 10 liters/minute. A total of 12 rats
were aligned in the pie chamber, with a single port for nebulized
material to enter and evenly distribute to the 12 sub-chambers of
the Aeroneb.
[0466] Fifteen days following initial sensitization, 12 rats were
exposed to RDC 1676-01 by ultrasonic nebulization for 30 minutes
each day for 7 consecutive days. The air flow was also set for 10
liters/minute, using the same nebulizer and chamber. The RDC
1676-00 was nebulized first and the Aeroneb chamber thoroughly
dried before RDC 1676-01 was nebulized.
[0467] Approximately 2 hours after the last nebulization treatment,
6 rats from the RDC 1676-00 group were re-challenged with OVA (1%
in saline) delivered by intratreacheal instillation using a Penn
Century Microsprayer (Model 1A-1B). The other 6 rats from the RDC
1676-00 group were challenged with saline as the control group
delivered by way of intratreacheal instillation. The following day,
the procedure was repeated with the RDC 1676-01 group.
[0468] Twenty four hours after re-challenge, all rats in each group
were euthanized by overdose with sodium pentobarbital. Whole blood
samples were collected from the inferior vena-cava and placed into
two disparate blood collection tubes: Qiagen PAXgene.TM. Blood RNA
Tube and Qiagen PAXgene.TM. Blood DNA Tube. Lung organs were
processed to obtain bronchoalveolar lavage (BAL) fluid and lung
tissue for RT-PCR to assess changes in markers of cytokine
expression known to be associated with lung inflammation in this
model. A unilateral lavage technique was to be employed in order to
preserve the integrity of the 4 lobes on the right side of the
lung. The left "large" lobe was lavaged, while the 4 right lobes
were tied off and immediately placedinot TRI-zol.TM., homogenized,
and sent to the lab for further processing.
[0469] BAL analysis. Lung lavage was collected and centrifuged for
10 minutes at 4.degree. C. at 600-800 g to pellet the cells. The
supernatants were transferred to fresh tubes and frozen at
-80.degree. C. Bronchial lavage fluid ("BAL") was separated into
two aliquots. The first aliquot was spun down, and the supernatant
was snap frozen on crushed dry ice, placed in -80.degree. C., and
shipped to the laboratory for further processing. The amount of
protein and LDH present indicates the level of blood serum protein
(the protein is a serum component that leaks through the membranes
when it's challenged as in this experiment) and cell death,
respectively. The proprietary test side showed slight less protein
than the control.
[0470] The second aliquot of bronchial lavage fluid was evaluated
for total protein and LDH content, as well as subjected to
cytological examination. The treated group showed total cells to be
greater than the saline control group. Further, there was an
increase in eosinophils in the treated group versus the control
group. There were also slightly different polymorphonuclear cells
for the treated versus the control side.
[0471] Blood Analysis.
[0472] Whole blood was analyzed by transfer of 1.2-2.0 mL blood
into a tube, and allowing it to clot for at least 30 minutes. The
remaining blood sample (approximately 3.5-5.0 mL) was saved for RNA
extraction using TRI-zol.TM. or PAXgene.TM.. Next, the clotted
blood sample was centrifuged for 10 minutes at 1200 g at room
temperature. The serum (supernatant) was removed and placed into
two fresh tubes, and the serum was stored at -80.degree. C.
[0473] For RNA extraction utilizing Tri-Reagent (TB-126, Molecular
Research Center, Inc.), 0.2 mL of whole blood or plasma was added
to 0.75 mL of TRI Reagent BD supplemented with 20 .mu.L of 5N
acetic acid per 0.2 mL of whole blood or plasma. Tubes were shaken
and stored at -80.degree. C. Utilizing PAXgene.TM., tubes were
incubated for approximately two hours at room temperature. Tubes
were then placed on their side and stored in the -20.degree. C.
freezer for 24 hours, and then transferred to -80.degree. C. for
long term storage.
[0474] Luminex analysis. By Luminex platform, a microbead analysis
was utilized as a substrate for an antibody-related binding
reaction which is read out in luminosity units and can be compared
with quantified standards. Each blood sample was run as 2 samples
concurrently. The units of measurement are luminosity units and the
groups are divided up into OVA challenged controls, OVA challenged
treatment, and saline challenged treatment with proprietary
fluid.
[0475] For Agilant gene array data generation, lung tissue was
isolated and submerged in TRI Reagent (TR118, Molecular Research
Center, Inc.). Briefly, approximately 1 mL of TRI Reagent was added
to 50-100 mg of tissue in each tube. The samples were homogenized
in TRI Reagent, using glass-Teflon.TM. or Polytron.TM. homogenizer.
Samples were stored at -80.degree. C.
Blood Samples:
[0476] FIGS. 49-58 show the results of whole blood sample
evaluations.
[0477] Exemplary FIG. 49 shows the basic luminosity data
presentation format for the blood sample data. Letters designating
the identity of the measured cytokine (in this case KC) are at the
top right of each data figure. The data is presented both as data
points (upper graph) and bar graphs (lower graph) of the individual
samples. In either case, the graphs are divided, from left to
right, in four groups. The first 2 groups (RDC1676-00 OVA and
RDC1676-01 OVA, respectively) were those that were re-challenged
with OVA by inhalation, whereas the last two groups (RDC1676-00 OVA
and RDC1676-01 OVA, respectively) where those that were
re-challenged with saline control only. Again, the suffix 00
represents saline treatment and suffix 01 represents inventive
electrokinetic fluid treated groups.
[0478] Each blood sample was split into 2 samples and the samples
were run concurrently. The units of measure are units of luminosity
and the groups, going from left to right are: OVA challenged
controls; OVA challenged inventive electrokinetic fluid treatment;
followed by saline challenged saline treatment; and saline
challenged inventive electrokinetic fluid treatment. To facilitate
review, both the RDC1676-01 groups are highlighted with gray shaded
backdrops, whereas the control saline treatment groups have
unshaded backdrops.
[0479] Generally, in comparing the two left groups, while the
spread of the RDC1676-01 group data is somewhat greater, particular
cytokine levels in the RDC1676-01 group as a whole are less than
the samples in the control treated group; typically about a 30%
numerical difference between the 2 groups. Generally, in comparing
the right-most two groups, the RDC1676-01 group has a slightly
higher numerical number compared to the RDC1676-00 group.
[0480] FIG. 50 shows analysis of RANTES (IL-8 super family) in
blood sample data according to particular exemplary aspects.
Luminosity units for the leftmost two groups (the OVA challenged
groups) indicate that generally values in the RDC1676-01 treated
group were less than the RDC1676-00 control group as shown by the
dot plot in the upper graph portion which again shows a 30-35%
differential between the two groups, whereas in the saline only
exposed groups the cytokine level values where roughly the same, or
perhaps slightly increased in the RDC1676-01 treated group.
[0481] FIG. 51 shows analysis of MCP-1 in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0482] FIG. 52 shows analysis of TNF alpha in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0483] FIG. 53 shows analysis of MIP-1 alpha in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0484] FIG. 54 shows analysis of IL-1 alpha in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0485] FIG. 55 shows analysis of Vcam in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0486] FIG. 56 shows analysis of IL-1 beta in blood sample data
according to particular exemplary aspects. Luminosity units for the
leftmost two groups (the OVA challenged groups) indicate that
generally values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
[0487] FIGS. 57 and 58 show analysis of Eotaxin and MCP-3,
respectively, in blood sample data according to particular
exemplary aspects. In each case, luminosity units for the leftmost
two groups (the OVA challenged groups) indicate that generally
values in the RDC1676-01 treated group were less than the
RDC1676-00 control group as shown by the dot plot in the upper
graph portion, whereas in the saline only exposed groups the
cytokine level values where roughly the same, or perhaps slightly
increased in the RDC1676-01 treated group.
Bronchial Lavage Samples:
[0488] FIGS. 59-68 show the corresponding results of
bronchoalveolar lavage fluid (BAL) sample evaluations.
[0489] FIG. 59 shows analysis of KC in BAL data according to
particular exemplary aspects. In this instance the response level,
coupled with sampling variability, was inconclusive with respect to
a difference between the RDC1676-01 and RDC1676-00-treated groups;
that is, KC showed relatively little difference between the 2
groups, but the units of luminosity were very small.
[0490] Likewise, FIG. 60 shows analysis of RANTES in BAL data
according to particular exemplary aspects, and showing marked
variability in the RDC1676-01 group with one reading being markedly
higher than the others, skewing the results.
[0491] Likewise, FIG. 61 shows analysis of TNF alpha in BAL data
according to particular exemplary aspects, and showing relatively
little significance in the way of difference between the RDC1676-01
and RDC1676-00-treated groups.
[0492] FIG. 62 shows analysis of MCP-1 in BAL data according to
particular exemplary aspects, and showing relatively little
significance in the way of difference between the RDC1676-01 and
RDC1676-00-treated groups.
[0493] FIGS. 63 through 68 show analysis of MIP1-A, IL-1 alpha,
Vcam, IL-1 beta, MCP-3, and Eotaxin, respectively, in BAL data
according to particular exemplary aspects, and showing relatively
little significance in the way of difference between the RDC1676-01
and RDC1676-00-treated groups.
[0494] In summary, this standard assay of inflammatory reaction to
a known sensitization produced, at least in the blood samples, a
marked clinical and serologic affect. Additionally, while
significant numbers of control animals were physiologically
stressed and nearly dying in the process, none of the RDC1676-01
treated group showed such clinical stress effects. This was
reflected then in the circulating levels of cytokines, with
approximately 30% differences between the RDC1676-01-treated and
the RDC1676-01-treated groups in the OVA challenged groups. By
contrast, there were small and fairly insignificant changes in
cytokine, cellular and serologic profiles between the
RDC1676-01-treated and the RDC1676-01-treated groups in the non-OVA
challenged groups., which likely merely represent minimal baseline
changes of the fluid itself.
Example 14
Bradykinin B2 Receptor Affinity Binding
[0495] A Bio-Layer Interferometry biosensor, Octet Rapid Extended
Detection (RED) (forteBio.TM.) was utilized in order to examine
membrane receptor affinity binding of Bradykinin ligand with the
Bradykinin B2 receptor. The biosensor system consists of a polished
fiber optic embedded into a polypropylene hub with a
sensor-specific chemistry at the tip. The biosensor set-up has a
layer of molecules attached to the tip of an optic fiber that
creates an interference pattern at the detector. Any change in the
number of molecules bound causes a measured shift in the pattern of
light.
[0496] As shown in FIG. 69 the Bradykinin B2 membrane receptor was
immobilized onto aminopropylsilane (APS) biosensor. The sample
plate set up was designated in FIG. 69 and analyzed in FIG. 70.
Next, the binding of Bradykinin to the immobilized receptor was
assessed according to the sample set up as designated in FIG. 71.
Results of Bradykinin binding are shown in FIG. 72. Bradykinin
binding to the receptor was further titrated according to the
set-up as designated in FIG. 73.
[0497] As indicated in FIG. 74, Bradykinin binding to the B2
receptor was concentration dependent, and binding affinity was
increased in the proprietary gas-enriched saline fluid of the
instant disclosure compared to normal saline. Stabilization of
Bradykinin binding to the B2 receptor is shown in FIG. 75.
Example 15
A Regulatory T-Cell Assay was Used to Show Effects of the Inventive
Electrokinetically Generated Fluids in Modulation of T-Cell
Proliferation and Elaboration of Cytokines (II-10) and Other
Proteins (e.g., GITR, Granzyme A, XCL1, pStat5, and Foxp3)) in
Regulatory T-Cell Assays, and of, for Example, Tryptase in PBMC
[0498] The ability of particular embodiments disclosed herein to
regulate T cells was studied by irradiating antigen presenting
cells, and introducing antigen and T cells. Typically, these
stimulated T cells proliferate. However, upon the introduction of
regulatory T cells, the usual T cell proliferation is
suppressed.
Methods:
[0499] Briefly, FITC-conjugated anti-CD25 (ACT-1) antibody used in
sorting was purchased from DakoCytomation (Chicago, Ill.). The
other antibodies used were as follows: CD3 (HIT3a for soluble
conditions), GITR (PE conjugated), CD4 (Cy-5 and FITC-conjugated),
CD25 (APC-conjugated), CD28 (CD28.2 clone), CD127-APC, Granzyme A
(PE-conjugated), FoxP3 (BioLegend), Mouse IgG1 (isotype control),
and XCL1 antibodies. All antibodies were used according to
manufacturer's instructions.
[0500] CD4+ T cells were isolated from peripheral whole blood with
CD4+ Rosette Kit (Stemcell Technologies). CD4+ T cells were
incubated with anti-CD127-APC, anti-CD25-PE and anti-CD4-FITC
antibodies. Cells were sorted by flow cytometry using a FACS Aria
into CD4+CD25hiCD127lo/nTreg and CD4+CD25- responder T cells.
[0501] Suppression assays were performed in round-bottom 96 well
microtiter plates. 3.75.times.103 CD4+CD25neg responder T cells,
3.75.times.103 autologous T reg, 3.75.times.104 allogeneic
irradiated CD3-depleted PBMC were added as indicated. All wells
were supplemented with anti-CD3 (clone HIT3a at 5.0 ug/ml). T cells
were cultured for 7 days at 37.degree. C. in RPMI 1640 medium
supplemented with 10% fetal bovine serum. Sixteen hours before the
end of the incubation, 1.0 mCi of .sup.3H-thymidine was added to
each well. Plates were harvested using a Tomtec cell harvester and
.sup.3H-thymidine incorporation determined using a Perkin Elmer
scintillation counter. Antigen-presenting cells (APC) consisted of
peripheral blood mononuclear cells (PBMC) depleted of T cells using
StemSep human CD3+ T cell depletion (StemCell Technologies)
followed by 40 Gy of irradiation.
[0502] Regulatory T cells were stimulated with anti-CD3 and
anti-CD28 conditions and then stained with Live/Dead Red viability
dye (Invitrogen), and surface markers CD4, CD25, and CD127. Cells
were fixed in the Lyze/Fix PhosFlow.TM. buffer and permeabilized in
denaturing Permbuffer III.RTM.. Cells were then stained with
antibodies against each particular selected molecule.
[0503] Statistical analysis was performed using the GraphPad Prism
software. Comparisons between two groups were made by using the
two-tailed, unpaired Student's t-test. Comparisons between three
groups were made by using 1-way ANOVA. P values less than 0.05 were
considered significant (two-tailed). Correlation between two groups
were determined to be statistically significant via the Spearman
coefficient if the r value was greater than 0.7 or less than -0.7
(two-tailed).
Results:
[0504] As indicated in FIG. 76, regulatory T cell proliferation was
studied by stimulating cells with diesel exhaust particulate matter
(PM, from EPA). The x-axis of FIG. 76 shows activated autologous
CD4+ effector T cells (responder cells) as a solid black bar, and
regulatory T cells alone in the gray bar (shown for confirmation of
anergy) which were mixed at a 1:1 ratio as shown in the white bar.
The y axis shows proliferation as measured by uptake of
.sup.3H-thymidine. As shown from left to right along the x-axis,
"PM" indicates diesel exhaust derived Particulate Matter, "PM+Rev"
indicates PM plus a gas-enriched electrokinetically generated fluid
(Rev) of the instant disclosure, "Solis" indicates an
electrokinetically generated fluid of the instant disclosure and
device that is not gas-enriched beyond ambient atmosphere, only (no
PM added), "Rev" indicates Rev alone (no PM added) as defined
above, "Media" indicates the cell growth media alone control (minus
PM; no Rev, no Solis), and "Saline Con" indicates the saline
control (minus PM; no Rev, no Solis), "V" indicates verapamil, and
"P" indicates propanolol, and "DT" is DT390 at 1:50.
[0505] As shown in FIG. 77, cells stimulated with PM (no Rev, no
Solis) resulted in a decrease in secreted IL-10, while cells
exposed to PM in the presence of the fluids of the instant
disclosure ("PM+Rev") resulted in a maintained or only slightly
decreased production of IL-10 relative to the Saline and Media
controls (no PM). Furthermore, Diphtheria toxin (DT390, a truncated
diphtheria toxin molecule; 1:50 dilution of std. commercial
concentration) was titrated into inventive fluid samples, and
blocked the Rev-mediated effect of increase in IL-10 in FIG. 77.
Note that treatment with Rev alone resulted in higher IL-10 levels
relative to Saline and Media controls.
[0506] Likewise, similar results, shown in FIGS. 78-82, were
obtained with GITR, Granzyme A, XCL1, pStat5, and Foxp3,
respectively. In Figures, "NSC" is the same as "Solis" (no PM).
[0507] FIG. 83 shows AA PBMC data, obtained from an allergic asthma
(AA) profile of peripheral blood mononuclear cells (PBMC)
evaluating tryptase. The AA PBMC data was consistent with the above
T-regulatory cell data, as cells stimulated with particulate matter
(PM) showed high levels of tryptase, while cells treated with PM in
the presence of the fluids of the instant disclosure ("PM+Rev")
resulted in significantly lower tryptase levels similar to those of
the Saline and Media controls. Consistent with the data from
T-regulatory cells, exposure to DT390 blocked the Rev-mediated
effect on tryptase levels, resulting in an elevated level of
tryptase in the cells as was seen for PM alone (minus Rev, no Rev,
no Solis). Note that treatment with Rev alone resulted in lower
tryptase levels relative to Saline and Media controls.
[0508] In summary, the data of FIG. 76, showing a decreased
proliferation in the presence of PM and Rev relative to PM in
control fluid (no Rev, no Solis), indicates that the inventive
electrokinetically generated fluid Rev improved regulatory T-cell
function as shown by relatively decreased proliferation in the
assay. Moreover, the evidence of this Example and FIGS. 76-83,
indicate that beta blockade, GPCR blockade and Ca channel blockade
affects the activity of Revera on Treg function.
Example 16
Treatment of Primary Bronchial Epithelial Cells (BEC) with the
Inventive Electrokinetically Generated Fluids Resulted in Reduced
Expression and/or Activity of Two Key Proteins of the Airway
Inflammatory Pathways, MMP9 and TSLP
[0509] Overview. As shown in Example 14 above (e.g., FIG. 75,
showing Stabilization of Bradykinin binding to the B2 receptor
using Bio-Layer Interferometry biosensor, Octet Rapid Extended
Detection (RED) (forteBio.TM.)), Bradykinin binding to the B2
receptor was concentration dependent, and binding affinity was
increased in the electrokinetically generated fluid (e.g., Rev;
gas-enriched electrokinetically generated fluid) of the instant
disclosure compared to normal saline. Additionally, as shown in
Example 15 in the context of T-regulatory cells stimulated with
diesel exhaust particulate matter (PM, standard commercial source),
the data showed a decreased proliferation of T-regulatory cells in
the presence of PM and Rev relative to PM in control fluid (no Rev,
no Solis) (FIG. 76), indicating that the inventive
electrokinetically generated fluid Rev improved regulatory T-cell
function; e.g., as shown by relatively decreased proliferation in
the assay. Moreover, exposure to the inventive fluids resulted in a
maintained or only slightly decreased production of IL-10 relative
to the Saline and Media controls (no PM). Likewise, in the context
of the allergic asthma (AA) profiles of peripheral blood
mononuclear cells (PBMC) stimulated with particulate matter (PM),
the data showed that exposure to the fluids of the instant
disclosure ("PM+Rev") resulted in significantly lower tryptase
levels similar to those of the Saline and Media controls.
Additionally, the Diphtheria toxin (DT390, a truncated diphtheria
toxin molecule; 1:50 dilution of std. commercial concentration)
effects shown in Example 15 and FIGS. 76-83, indicate that beta
blockade, GPCR blockade and Ca channel blockade affects the
activity of the electrokinetically generated fluids on Treg and
PBMC function. Furthermore, the data of Example 18 shows that,
according to additional aspects, upon exposure to the inventive
fluids, tight junction related proteins were upregulated in lung
tissue. FIGS. 85-89 show upregulation of the junction adhesion
molecules JAM 2 and 3, GJA1, 3, 4 and 5 (junctional adherins), OCLN
(occludin), claudins (e.g., CLDN 3, 5, 7, 8, 9, 10), TJP1 (tight
junction protein 1), respectively. Furthermore, as shown in the
patch clamp studies of Example 23, the inventive electrokinetically
generated fluids (e.g., RNS-60) affect modulation of whole cell
conductance (e.g., under hyperpolarizing conditions) in Bronchial
Epithelial Cells (BEC; e.g., Calu-3), and according to additional
aspects, modulation of whole cell conductance reflects modulation
of ion channels.
[0510] In this Example, Applicants have extended these discoveries
by conducting additional experiments to measure the effects of
production of two key proteins of the airway inflammatory pathways.
Specifically, MMP9 and TSLP were assayed in primary bronchial
epithelial cells (BEC).
Materials and Methods:
[0511] Commercially available primary human bronchial epithelial
cells (BEC) (HBEpC-c from Promocell, Germany) were used for these
studies. Approximately 50,000 cells were plated in each well of a
12 well plate until they reached .about.80% confluence. The cells
were then treated for 6 hours with normal saline, control fluid
Solas or the test fluid Revera 60 at a 1:10 dilution (100 ul in 1
ml of airway epithelial growth medium) along with the diesel
exhaust particulate matter (DEP or PM) before being lifted for FACS
analysis, as described in Example 8 herein. Both MMP9 and TSLP
receptor antibodies were obtained from BD Biosciences and used as
per manufacturer's specifications.
Results:
[0512] In FIGS. 115 and 116, DEP represents cells exposed to diesel
exhaust particulate matter (PM, standard commercial source) alone,
"NS" represents cells exposed to normal saline alone, "DEP-ENS"
represent cells treated with particulate matter in the presence of
normal saline, "Revera 60" refers to cells exposed only to the test
material, "DEP+Revera 60" refer to cells treated with particulate
matter in the presence of the test material Revera 60. In addition,
"Solas" and "DEP+Solas" represents cells exposed to the control
fluid Solas alone or in combination with the particulate matter,
respectively.
[0513] FIG. 115 shows that the test material Revera 60 reduces DEP
induced TSLP receptor expression in bronchial epithelial cells
(BEC) by approximately 90%. Solas resulted in a 55% reduction in
TSLP receptor expression, while Normal saline failed to produce
similar level of reduction in TSLP receptor expression
(approximately 20% reduction). The effect of the inventive solution
in reducing TSLP receptor expression is a significant discovery in
view of recent findings showing that TSLP plays a pivotal role in
the pathobiology of allergic asthma and local antibody mediated
blockade of TSLP receptor function alleviated allergic disease
(Liu, Y J, Thymic stromal lymphopoietin: Master switch for allergic
inflammation, J Exp Med 203:269-273, 2006; A1-Shami et al., A role
for TSLP in the development of inflammation in an asthma model, J
Exp Med 202:829-839, 2005; and Shi et al., Local blockade of TSLP
receptor alleviated allergic disease by regulating airway dendritic
cells, Clin Immunol. 2008, Aug. 29. (Epub ahead of print)).
[0514] Likewise, FIG. 116 shows the effect of Revera 60, Solas and
normal saline on the DEP-mediated increase in MMP 9. Specifically,
Revera 60 inhibited the DEP-induced cell surface bound MMP9 levels
in bronchial epithelial cells by approximately 80%, and Solas had
an inhibitory effect of approximately 70%, whereas normal saline
(NS) had a marginal effect of about 20% reduction. MMP-9 is one of
the major proteinases involved in airway inflammation and bronchial
remodeling in asthma. Recently, it has been demonstrated that the
levels of MMP-9 are significantly increased in patients with stable
asthma and even higher in acute asthmatic patients compared with
healthy control subjects. MMP-9 plays a crucial role in the
infiltration of airway inflammatory cells and the induction of
airway hyperresponsiveness indicating that MMP-9 may have an
important role in inducing and maintaining asthma (Vignola et al.,
Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1
ratio correlates with airflow obstruction in asthma and chronic
bronchitis, Am J Respir Crit Care Med 158:1945-1950, 1998; Hoshino
et al., Inhaled corticosteroids decrease subepithelial collagen
deposition by modulation of the balance between matrix
metalloproteinase-9 and tissue inhibitor of metalloproteinase-1
expression in asthma, J Allergy Clin Immunol 104:356-363, 1999;
Simpson et al., Differential proteolytic enzyme activity in
eosinophilic and neutrophilic asthma, Am J Respir Crit Care Med
172:559-565,2005; Lee et al., A murine model of toluene
diisocyanate-induced asthma can be treated with matrix
metalloproteinase inhibitor, J Allergy Clin Immunol 108:1021-1026,
2001; and Lee et al., Matrix metalloproteinase inhibitor regulates
inflammatory cell migration by reducing ICAM-1 and VCAM-1
expression in a murine model of toluene diisocyanate-induced
asthma, J Allergy Clin Immunol 2003; 111:1278-1284).
[0515] According to additional aspects, therefore, the inventive
electrokinetically generated fluids have substantial therapeutic
utility for modulating (e.g., reducing) TSLP receptor expression
and/or for inhibiting expression and/or activity of MMP-9,
including, for example, for treatment of inflammation and
asthma.
Example 17
The Inventive Electrokinetically Generated Fluids were Shown to
have a Synergistic Anti-Inflammatory Effect with Budesonide in an
Art-Recognized Animal Model for Allergic Asthma
[0516] This working Example describes experiments performed to
assess the airway anti-inflammatory properties of the inventive
electrokinetically generated fluids (e.g., RDC-1676-03) in a Brown
Norway rat ovalbumin sensitization model. The Brown Norway rat is
an art-recognized model for determining the effects of a test
material on airway function and this strain has been widely used,
for example, as a model of allergic asthma. Airway pathology and
biochemical changes induced by ovalbumin sensitization in this
model resemble those observed in man (Elwood et al., J Allergy Clin
Immuno 88:951-60, 1991; Sirois & Bissonnette, Clin Exp Immunol
126:9-15, 2001). The inhaled route was selected to maximize lung
exposure to the test material or the control solution. The
ovalbumin-sensitized animals were treated with budesonide alone or
in combination with the test material RDC 1676-03 for 7 days prior
to ovalbumin challenge. 6 and 24 hours following the challenge,
total blood count and levels of several pro and anti-inflammatory
cytokines as well as various respiratory parameters were measured
to estimate any beneficial effect of administering the test
material on various inflammatory parameters.
Materials and Methods:
[0517] Brown Norway rats of strain Bn/Crl were obtained from
Charles River Kingston, weighing approximately 275+50 g at the
onset of the experiment. All animal studies were conducted with the
approval by PCS-MTL Institutional Animal Care and Use Committee.
During the study, the use and care of animals were conducted
according to guidelines of the USA National Research Council as
well as Canadian Council of Animal Care.
[0518] Sensitization.
[0519] On day 1 of the experiment, animals (14 animals in each
treatment group) were sensitized by administration of a 1 ml
intraperitoneal injection of a freshly prepared solution of 2 mg
ovalbumin/100 mg Aluminum Hydroxide per 1 ml of 0.9% Sodium
Chloride, followed by repeat injection on day 3.
[0520] Treatment.
[0521] Fifteen days following the initial sensitization, animals
were subjected to nebulized exposure to control (Normal saline) or
test solutions (electrokinetically generated fluids RDC1676-00,
RDC1676-02 and RDC-1676-03), either administered alone or in
combination with Budesonide, once daily for 15 minutes for 7
consecutive days. Animals were dosed in a whole body chamber of
approximately 20 L, and test atmosphere was generated into the
chamber air inlet using aeroneb ultrasonic nebulizers supplied with
air from a Buxco bias flow pump. The airflow rate was set at 10
liters/min.
[0522] Ovalbumin Challenge.
[0523] On day 21, 2 hours following treatment with the test
solutions, all animals were challenged with 1% ovalbumin nebulized
solution for 15 minutes (in a whole body chamber at airflow 2
L/min).
[0524] Sample collection.
[0525] At time points of 6 and 24 hours after the ovalbumin
challenge, blood samples were collected for total and differential
blood cell counts as well as for measuring levels of various pro
and anti-inflammatory cytokines. In addition, Immediately after and
at 6 and 24 hours following ovalbumin challenge the enhanced pause
Penh and tidal volume were measured for a period of 10 minutes
using the Buxco Electronics BioSystem XA system.
Results:
[0526] Eosinophil Count: As expected, and shown in FIG. 109,
treatment with Budesonide ("NS+Budesonide 750 .mu.g/Kg"; densely
crosshatched bar graph) reduced the total eosinophil count in the
challenged animals relative to treatment with the normal saline
"NS" alone control (open bar graph). Additionally, while treatment
with the inventive fluid "RDC1676-03" alone (lightly crosshatched
bar graph) did not significantly reduce the eosinophil count, it
nonetheless displayed a substantial synergy with Budesonide in
reducing the eosinophil count ("RDC1676-03+Budesonide 750
.mu.g/Kg", solid dark bar graph). Similarly, in FIG. 110, the
Eosinophil % also reflected a similar trend. While RDC1676-03
(lightly crosshatched graph bar) or Budesonide 750 ug/kg (densely
crosshatched bar graph) alone did not have a significant effect on
Eosinophil % count in the challenged animals, the two in
combination reduced the Eosinophil significantly (solid dark bar
graph).
[0527] Therefore, FIGS. 109 and 110 show, according to particular
aspects of the present invention that the inventive
electrokinetically generated fluids (e.g., RDC1676-03) were
demonstrated to have a substantial synergistic utility in
combination with Budesonide to significantly reduce eosinophil
count ("Eosinophil %" and total count) in an art-recognized rat
model for human allergic asthma.
Respiratory Parameters:
[0528] FIGS. 111 A-C and 112 A-C demonstrate the observed effect of
the test fluids on Penh and tidal volume as measured immediately, 6
and 24 hours after the ovalbumin challenge. Penh is a derived value
obtained from peak inspiratory flow, peak expiratory flow and time
of expiration and lowering of penh value reflects a favorable
outcome for lung function.
Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory
time/time to expire 65% of expiratory volume-1).
[0529] As evident from FIGS. 111 A-C, treatment with Budesonide (at
both 500 and 750 ug/kg) alone or in combination with any of the
test fluids failed to significantly affect the Penh values
immediately after the challenge. However, 6 hours after the
challenge, animals treated with RDC1676-03 alone or in combination
with Budesonide 500 or 750 ug/kg demonstrated a significant drop in
Penh values. Although the extent of this drop was diminished by 24
hours post challenge, the trend of a synergistic effect of
Budesonide and RDC fluid was still observed at this time point.
[0530] Tidal volume is the volume of air drawn into the lungs
during inspiration from the end-expiratory position, which leaves
the lungs passively during expiration in the course of quiet
breathing. As shown in FIGS. 112 A-C, animals treated with
Budesonide alone showed no change in tidal volumes immediately
after the challenge. However, RDC1676-03 alone had a significant
stimulatory effect on tidal volume even at this early time point.
And again, RDC1676-03 in combination with Budesonide (both 500 and
750 ug/kg) had an even more pronounced effect on Tidal volume
measurements at this time point. Six hours after the challenge,
RDC1676-03 alone was sufficient to cause a significant increase in
tidal volume and addition of Budesonide to the treatment regimen
either alone or in combination had no added effect on tidal volume.
Any effect observed at these earlier time points were, however,
lost by the 24 hours time point.
[0531] Taken together, these data demonstrate that RDC1676-03 alone
or in combination with Budesonide provided significant relief to
airway inflammation as evidenced by increase in tidal volume and
decrease in Penh values at 6 hours post challenge.
Cytokine Analysis:
[0532] To analyze the mechanism of the effects seen on the above
discussed physiological parameters, a number of pro as well as
anti-inflammatory cytokines were measured in blood samples
collected at 6 and 24 hours after the challenge, immediately
following the physiological measurements.
[0533] FIGS. 113A and 113B clearly demonstrate that Rev 60 (or
RDC1676-03) alone lowered the blood level of eotaxin significantly
at both 6 and 24 hours post challenge. Budesonide 750 ug/kg also
reduced the blood eotaxin levels at both of these time points,
while Budesonide 250 ug/kg only had a notable effect at the later
time point. However, the test solution Rev 60 alone showed effects
that are significantly more potent (in reducing blood eotaxin
levels) than both concentrations of Budesonide, at both time
points. Eotaxin is a small C-C chemokine known to accumulate in and
attract eosinophils to asthmatic lungs and other tissues in
allergic reactions (e.g., gut in Crohn's disease). Eotaxin binds to
a G protein coupled receptor CCR3. CCR3 is expressed by a number of
cell types such as Th2 lymphocytes, basophils and mast cells but
expression of this receptor by Th2 lymphocyte is of particular
interest as these cells regulate eosinophil recruitment. Several
studies have demonstrated increased production of eotaxin and CCR3
in asthmatic lung as well as establishing a link between these
molecules and airway hyperresponsiveness (reviewed in Eotaxin and
the attraction of eosinophils to the asthmatic lung, Dolores M
Conroy and Timothy J Williams Respiratory Research 2001,
2:150-156). It is of particular interest to note that these studies
completely agree with the results in FIGS. 109 and 110 on
eosinophil counts.
[0534] Taken together these results strongly indicate that
treatment with RDC1676-03 alone or in combination with Budesonide
can significantly reduce eosinophil total count and % in blood 24
hours after the ovalbumin challenge. This correlates with a
significant drop in eotaxin levels in blood observed as early as 6
hours post challenge.
[0535] Blood levels of two major key anti-inflammatory cytokines,
IL10 and Interferon gamma are also significantly enhanced at 6
hours after challenge as a result of treatment with Rev 60 alone or
in combination with Budesonide. FIGS. 113C and 113D show such
effects on Interferon gamma and IL 10, respectively. It is evident
from these figures that Rev 60 alone or Rev 60 in combination with
Budesonide 250 ug/kg significantly increased the blood level of
IL10 in the challenged animals up to 6 hrs post challenge.
Similarly, Rev 60 alone or in combination with Budesonide 250 or
750 ug/kg significantly increased the blood level of IFN gamma at 6
hours post challenge. Increase in these anti-inflammatory cytokines
may well explain, at least in part, the beneficial effects seen on
physiological respiratory parameters seen 6 hours post challenge.
The effect on these cytokines was no longer observed at 24 hour
post challenge (data not shown).
[0536] Rantes or CCL5 is a cytokine expressed by circulating T
cells and is chemotactic for T cells, eosinophils and basophils and
has an active role in recruiting leukocytes into inflammatory
sites. Rantes also activates eosinophils to release, for example,
eosinophilic cationic protein. It changes the density of
eosinophils and makes them hypodense, which is thought to represent
a state of generalized cell activation. It also is a potent
activator of oxidative metabolism specific for eosinophils.
[0537] As shown in FIG. 114, systemic levels of Rantes was reduced
significantly at 6 hours, but not at 24 hours post challenge in
animals treated with Rev 60 alone or in combination of Budesonide
250 or 750 ug/kg. Once again, there is a clear synergistic effect
of Budesonide 750 ug/kg and Rev 60 that is noted in this set of
data. A similar downward trend was observed for a number of other
pro-inflammatory cytokines, such as KC or IL8, MCP3, IL1b, GCSF,
TGFb as well as NGF, observed either at 6 or at 24 hours post
challenge, in animals treated with Rev60 alone or in combination
with Budesonide.
Example 18
The Inventive Therapeutic Fluids have Substantial Utility for
Modulating Intercellular Tight Junctions
[0538] According to particular aspects, the inventive diffuser
processed therapeutic fluids have substantial utility for
modulating intercellular tight junctions, including those relating
with pulmonary and systemic delivery and bioavailability of
polypeptides, including the exemplary polypeptide salmon calcitonin
(sCT).
[0539] Example Overview.
[0540] Salmon calcitonin (sCT) is a 32 amino acid peptide with a
molecular weight of 3,432 Daltons. Pulmonary delivery of calcitonin
has been extensively studied in model systems (e.g., rodent model
systems, rat model systems, etc) to investigate methods to enhance
pulmonary drug delivery (e.g., intratracheal drug delivery).
According to particular exemplary aspects, the inventive diffuser
processed therapeutic fluid has substantial utility for modulating
(e.g., enhancing) intercellular tight junctions, for example those
associated with pulmonary and systemic delivery and bioavailability
of sCT in a rat model system.
Methods:
[0541] Intratracheal Drug Delivery.
[0542] According to particular embodiments, sCT is formulated in
the inventive therapeutic fluid and administered to rats using an
intratracheal drug delivery device. In certain aspects, a Penn
Century Micro-Sprayer device designed for rodent intratracheal drug
delivery is used, allowing for good lung delivery, but, as
appreciated in the art, with relatively low alveolar deposition
resulting in poor systemic bioavailability of peptides. According
to particular aspects, this art-recognized model system was used to
confirm that the inventive diffuser processed therapeutic fluid has
substantial utility for modulating (e.g., enhancing) intercellular
tight junctions, including those associated with pulmonary and
systemic delivery and bioavailability of polypeptides.
[0543] Animal Groups and Dosing.
[0544] In certain aspects, rats are assigned to one of 3 groups
(n=6 per group): a) sterile saline; b) base solution without 02
enrichment (`base solution`); or c) inventive diffuser processed
therapeutic fluid (`inventive enriched based solution`). The
inventive enriched based solution is formed, for example by
infusing oxygen in 0.9% saline. Preferably, the base solution
comprises about 0.9% saline to minimize the potential for
hypo-osmotic disruption of epithelial cells. In certain
embodiments, sCT is separately reconstituted in the base solution
and the inventive enriched based solution and the respective
solutions are delivered to respective animal groups by
intratracheal instillation within 60 minutes (10 .mu.g sCT in 200
.mu.L per animal).
[0545] Assays.
[0546] In particular aspects, blood samples (e.g., 200 .mu.l) are
collected and placed into EDTA coated tubes prior to dosing and at
5, 10, 20, 30, 60, 120 and 240 minutes following dosing. Plasma is
harvested and stored at .ltoreq.-70.degree. C. until assayed for
sCT using an ELISA.
[0547] For Agilant gene array data generation, lung tissue was
isolated and submerged in TRI Reagent (TR118, Molecular Research
Center, Inc.). Briefly, approximately 1 mL of TRI Reagent was added
to 50-100 mg of tissue in each tube. The samples were homogenized
in TRI Reagent, using glass-Teflon.TM. or Polytron.TM. homogenizer.
Samples were stored at -80.degree. C.
Results:
[0548] Enhancement of Tight Junctions.
[0549] FIG. 84 shows that RDC1676-01 (sterile saline processed
through the instant proprietary device with additional oxygen
added; gas-enriched electrokinetically generated fluid (Rev) of the
instant disclosure) decreased systemic delivery and bioavailability
of sCT. According to particular aspects, the decreased systemic
delivery results from decreased adsorption of sCT, most likely
resulting from enhancement of pulmonary tight junctions. RDC1676-00
signifies sterile saline processed according to the presently
disclosed methods, but without oxygenation.
[0550] Additionally, according to particular aspects, tight
junction related proteins were upregulated in lung tissue. FIGS.
85-89 show upregulation of the junction adhesion molecules JAM 2
and 3, GJA1, 3, 4 and 5 (junctional adherins), OCLN (occludin),
claudins (e.g., CLDN 3, 5, 7, 8, 9, 10), TJP1 (tight junction
protein 1), respectively.
Example 19
The Inventive Therapeutic Fluids have Substantial Utility for
Modulating Nitric Oxide Levels
[0551] According to particular aspects, the inventive diffuser
processed therapeutic fluids have substantial utility for
modulating nitric oxide levels, and/or related enzymes. FIGS. 90-94
show data obtained from human foreskin keratinocytes exposed to
RDC1676-01 (sterile saline processed through the instant
proprietary device with additional oxygen added; gas-enriched
electrokinetically generated fluid (Rev) of the instant disclosure)
showing up-regulation of NOS1 and 3, and Nostrin, NOS3. By
contrast, data obtained from rat lung tissue (tissue of above
Example entitled "Cytokine Expression") shows down regulation of
NOS2 and 3, Nostrin and NOS1AP with Rev (FIGS. 93, 94).
Example 20
(Localized Electrokinetic Effects (Voltage/Current) were
Demonstrated Using a Specially Designed Mixing Device Comprising
Insulated Rotor and Stator Features
[0552] In this Example, feature-localized electrokinetic effects
(voltage/current) were demonstrated using a specially designed
mixing device comprising insulated rotor and stator features.
[0553] Overview.
[0554] As discussed in detail herein above under "Double Layer
Effect" (see also FIGS. 26 and 28) The mixing device 100 may be
configured to create the output material 102 by complex and
non-linear fluid dynamic interaction of the first material 110 and
the second material 120 with complex, dynamic turbulence providing
complex mixing that further favors electrokinetic effects.
According to particular aspects, the result of these electrokinetic
effects may be present within the output material 102 as charge
redistributions and redox reactions, including in the form of
solublized electrons that are stabilized within the output
material.
[0555] In addition to general surface-related double layer effects
in the mixing chamber, Applicants additionally reasoned that
localized electrokinetic effects may be imparted by virtue of the
feature-induced microcavitation and fluid acceleration and
deceleration in the vicinity of the features. The studies of this
Example were thus performed to further investigate and confirm said
additional electrokinetic aspects.
Materials:
[0556] A test device similar to the inventive mixing devices
described herein was constructed, comprising a stainless steel
rotor 12 having two features 18 (disposed at 180 degrees), and a
stator 14 with a single feature 16 positioned to be rotationally
opposable to the rotor features 18 and stator features 16.
Significantly, the rotor and stator features, in each case, are
insulated from the respective rotor and stator bodies (FIG. 95).
The device was machined to provide for a consistent rotor:stator
gap 20 of 0.020 inches to conform with the devices disclosed
elsewhere herein. There is a rotating contact (not shown) at the
end of the rotor shaft (not shown) that provides an electrical path
for the rotor surface and for the insulated rotor features.
Likewise the stator has a similar insulated feature 16 (FIG. 95),
wherein the stator inner surface and the insulated stainless steel
feature are connected to respective contacts on the stator
exterior.
[0557] An operational amplifier (OpAmp) circuit (M) 22 is connected
between the contacts. The operational amplifier (OpAmp) circuit was
constructed to provide for collection of very low voltage
measurements by taking advantage of the high input impedance of
such amplifiers. The outputs of the OpAmp are fed to the inputs of
an oscilloscope (e.g., a battery powered laptop running an
oscilloscope application with a Pico Scope 3000.TM.).
[0558] To eliminate the introduction of any ambient noise (e.g., RF
radiation from wireless network signals and from the 60 Hz power
line) during testing of the device, a fine copper mesh, RF-shielded
compartment (approx. three by four by four feet) was constructed to
provide a Faraday cage. This configuration provided for excellent
signal to noise ratios during experimental testing, as interfering
signals from 60 Hz AC noise (e.g., of approximately two volts) and
high frequency RF was reduced well below the signals of interest.
Using a battery powered laptop running an oscilloscope application
with a Pico Scope 3000 enabled detection of the 30 mV signals (as
in FIG. 96) created by the features of the test device. In
addition, a variable speed DC motor was positioned outside the
Faraday cage and coupled to the rotatable test device via a
non-metallic shaft to effectively isolate the motor noise away from
the test device.
Methods:
[0559] The OpAmp circuit was used to measure voltage potential
between the contacts connecting the stator inner surface 12 and the
insulated stator feature 16. With the particular circuit
arrangement, only a potential was measured. The rotational speed of
the device could be varied between about 700 to about 2800 rpm
(with the data of FIG. 96 being measured with the device running at
about 1800 rpm).
[0560] To avoid any extraneous voltage generation due to a pump or
peristaltic pump, fluid flow through the device was accomplished
using inert nitrogen or air or argon acting on fluid in tanks
connected to the device. There was no perceptible voltage
contribution from the flow mechanism, and typically air was used as
the pumping force to provide for fluid flow through the device.
[0561] Fluid flow rate through the device was about 1 L/min.
[0562] An initial set of non-rotational experiments was conducted
by directing fluid flow through the device chamber but without
rotation of the rotor in order to assess the presence of any
voltage between the stator body 12 and the isolated feature 16.
Separate experiments were conducted for both flow directions.
[0563] An additional set of rotational experiments was then
conducted with the same fluid flow rate, and with the device rotor
rotating at various speeds from about 300 to about 1800 rpm. For
any given experiment, the flow rate and rotational speed were held
constant.
Results:
[0564] With respect to the non-rotational experiments, with fluid
flowing through the device in either direction without any rotor
rotation there was only a barely perceptible voltage (e.g., 1 to 2
mV)) between the body of the stator and the insulated feature.
[0565] With respect to the rotational experiments, and with
reference to FIG. 96, it can be seen that voltage pulses (potential
pulses), temporally correlating (in this case at about 1800 rpm)
with rotational alignment of opposing rotor stator features, were
measurable with the OpAmp in the operating test device. Moreover,
such periodic voltage pulses, correlating with feature alignments,
could be observed over a range from about 250 or 300 rpm to about
1800. Additionally, with or without fluid flow, such voltage pulses
were observed in the rotational experiments as long as the
cavity/fluid chamber of the device was filled with fluid. According
to particular aspects, and without being bound by mechanism, rapid,
violent compression (e.g., cavitation), acceleration and
deceleration of fluid flow in the vicinity of the repetitive
rotationally aligned features created the respective local voltage
pulses that correlate exactly with the rotational period,
providing, at least in part, for electrokinetically generated fluid
according to the present invention. Additional experiments revealed
that the amplitude (peak shape and height) of the voltage pulses
increased with increasing rotational velocity, being initially
observable at about 250 to 300 rpm in this particular test device,
and increasing up to at least about 2800 rpm. The magnitude of the
violent acceleration and deceleration, etc., of fluid flow in the
vicinity of the rotationally aligned features would be expected to
generally increase with increasing rotational velocity; at least
until a maximum was reached reflecting physical limits imposed by
the geometry, configuration and/or flow rate of the device.
According to additional aspects, because localized voltage spikes
are present, localized current flow (e.g., current pulses) is
generated in the vicinity of the features, providing, at least in
part, for electrokinetically generated fluid according to the
present invention (e.g., without being bound by mechanism,
providing for electrochemical reactions as discussed elsewhere
herein).
[0566] According to additional aspects, and without being bound by
mechanism, such feature-localized effects (e.g., voltage pulses and
current and/or currents pulses) contribute to generation of the
electrokinetically generated fluids in combination with more
general surface-related double layer and streaming current effects
discussed elsewhere herein above under "Double Layer Effect" (see
also FIGS. 26 and 28).
Example 21
Relative to Non-Electrokinetically Generated Control Fluids, the
Inventive Electrokinetically Generated Fluids were Shown to
Differentially Affect Line Widths in .sup.13C NMR Analysis of the
Dissolved Solute .alpha.,.alpha.-Trehalose
[0567] Overview.
[0568] Applicants data disclosed elsewhere herein support utility
and mechanism wherein the inventive electrokinetically generated
fluids mediate regulation or modulation of intracellular signal
transduction by modulation of at least one of cellular membranes,
membrane potential/conductance, membrane proteins (e.g., membrane
receptors such as G protein coupled receptors), calcium dependant
cellular signaling systems, and intercellular junctions (e.g.,
tight junctions, gap junctions, zona adherins and desmasomes).
Specifically, using a variety of art-recognized biological test
systems and assays, Applicants data shows, relative to control
fluids, differential effects of the inventive fluid on, for
example: regulatory T cell proliferation; cytokine and protein
levels (e.g, IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,
tyrptase, tight junction related proteins, TSLP receptor, MMP9,
etc.); binding of Bradykinin ligand with the Bradykinin B2
receptor; expression of TSLP receptor, whole cell conductance; etc.
Moreover, the Diphtheria toxin (DT390) effects shown herein
indicate that beta blockade (beta 2 adrenergic receptor), and/or
GPCR blockade and/or Ca channel blockade affects the activity of
the electrokinetically generated fluids on, for example, Treg and
PBMC function.
[0569] Taken together these effects indicate that the inventive
electrokinetically generated fluids are not only fundamentally
distinguished from prior art fluids, but also that they provide for
novel compositions and substantial utilities such as those
presently disclosed and claimed herein.
[0570] In this Example.
[0571] Applicants have in this Example performed nuclear magnetic
resonance (NMR) studies to further characterize the fundamental
nature of the inventive electrokinetically generated fluids.
Specifically, Applicants have analyzed the .sup.13C NMR spectra of
.alpha.,.alpha.-Trehalose dissolved in the electrokinetically
generated fluid, compared to dissolution in non-electrokinetically
generated fluid. Trehalose (shown below with carbons numbered for
reference) is a cosmotrophic solute and is known, for example to
protect against protein denaturation, membrane desiccation,
organism viability upon freezing, etc. Applicants, given the data
summarized above, reasoned that .alpha.,.alpha.-Trehalose might
provide an effective tool to further probe the properties/structure
of the inventive electrokinetically generated fluids. Applicants
reasoned that NMR-related `chemical shifts` and effects on `line
widths` could be used to assess properties of the inventive fluids.
For these studies, a non-superoxygenated inventive
electrokinetically generated fluid (referred to herein as "Solas")
was employed to minimize the possibility that paramagnetic
impurities, such as dissolved oxygen, might act to counter or
otherwise mask the effects being analyzed.
##STR00027##
Materials and Methods:
[0572] Solution Preparation.
[0573] The Phosphate (sodium salt) and D-(+)-Trehalose dihydrate
(T9531-10G, reduced metal content) and 99.9% D2O containing 1% DSS
were purchased from Sigma. The "Normal Saline" is 0.9% Sodium
Chloride, pH 5.6 (4.5-7.0), from Hospira. The 0.25 M
.alpha.,.alpha.-Trehalose solutions were prepared by dissolving
0.949 g trehalose into 965 .mu.L Normal Saline and 35 mL Phoshate
Buffered Saline (100 mM Phosphate Buffer in 0.9% NaCl preparted in
such a way that when 35 .mu.L of this buffer are added to 1.0 mL
trehalose solution the pH becomes 6.93).
[0574] Nuclear Magnetic Resonance Spectra Collection.
[0575] Spectra were collected at the University of Washington NMR
facility using either an 500 MHz or 300 MHz Bruker Avance series
instrument fitted with a Bruker BBO: X {1H} probe and running
XWINNMR 3.5. .sup.13C NMR spectra were collected at 125.7 MHz or
75.46 MHz using a 14000 Hz or 7900 Hz sweep width using 64K or 128K
data points and 128 or 256 scans. The resulting FIDs were
zero-filled twice and processed with a 1.0 Hz line broadening
factor. Temperature was controlled using the Bruker Biospin
Variable Temperature unit. External deuterium locking was employed
by placing 99.9% D2O+1% DSS+a trace of acetone in a coaxial NMR
insert tube, purchased from Wilmad. The NMR data was processed
using the iNMR software v. 2.6.4 from Mestrelab Research.
Results:
[0576] Sample Spectra.
[0577] FIG. 97A-C shows expansions of six .sup.13C-NMR spectra
overlaid on top of each other such that the DSS signals line up at
-2.04 ppm. The DSS signals are shown at the far right of the
figure, and the acetone methyl signal is shown near 30.9 ppm. The
remaining signals correspond to the 6 carbons of trehalose as shown
in the .alpha.,.alpha.-Trehalose structure above. As can be seen,
the carbon signals in the Solas solutions show small chemical
shifts (generally upfield) compared to the control solutions.
[0578] Line Width Measurements.
[0579] TABLE 11 below shows the measured .sup.13C NMR line widths
for the six carbons of trehalose and the methyl carbon of acetone
at 3 different temperatures for Solas Saline (an inventive
electrokinetically generated fluid). The corresponding Normal
Saline samples represent non-electrokinetic control solutions at
each temperature. In the Solas solutions, the line widths are
significantly different from the line widths in the control
solution for each carbon atom. The smaller linewidths in the Solas
solutions at lower temperatures likely result from a faster
tumbling rate of the trehalose molecule as a whole (including any
solvated water molecules) compared to the control solutions.
TABLE-US-00011 TABLE 11 .sup.13C NMR Line Widths for
.alpha.,.alpha.-Trehalose in Solas & Normal Saline.sup.a,b Test
Fluid (Temp. degrees K) C-1 C-2 C-3 C-4 C-5 C-6 Acetone Solas (277)
8.4 8.22 8.3 8.15 8.3 11.1 5.1 Normal (269.9) 15.4 16.1 15.8 14.9
15.4 21.7 5.1 Solas (293) 9.52 8.7 9.28 9 8.9 11.25 5.63 Normal
(292.9) 10.33 10.23 10.23 9.93 10.23 13.13 5.63 Solas (310) 2.28
2.03 2.18 2.19 2 2.55 0.67 Normal (309.9) 1.17 0.99 1.1 1.02 0.97
1.42 0.67 .sup.a1.0 Hz was subtracted from all line width values
due to the 1.0 Hz line broadening used during processing. In
addition, line width values were normalized relative to the acetone
signal in the external reference tube in order to compensate for
magnetic field inhomogeneities. This was done by subtracting from
the Normal Saline line widths the amount by which the acetone peak
was broadened in the corresponding Solas Saline spectra.
.sup.bError in line width measurements estimated to be within
+/-0.30 Hz
[0580] The .sup.13C NMR line widths for .alpha.,.alpha.-Trehalose
in Solas and normal saline, in each case normalized with respect to
the Acetone line, are shown graphically in FIG. 97A. In conclusion,
the NMR data for .sup.13C NMR line widths for
.alpha.,.alpha.-Trehalose in Solas and normal saline indicate that
there is a property of the inventive solution which alters solute
tumbling.
[0581] Taken together with the biological activities summarize
above and elsewhere herein, these .sup.13C NMR line width effects
indicate that the inventive electrokinetically generated fluids are
not only fundamentally distinguished from prior art fluids in terms
of solute interactions, but also that they provide for novel
compositions and substantial utilities such as those presently
disclosed and claimed herein.
Example 22
Relative to Non-Electrokinetically Generated Control Fluids, the
Inventive Electrokinetically Generated Fluids Produced Differential
Square Wave Voltametry Profiles and Displayed Unique
Electrochemical Properties Under Stripping Polarography
[0582] Overview.
[0583] Applicants' data disclosed elsewhere herein support utility
and mechanism wherein the inventive electrokinetically generated
fluids mediate regulation or modulation of intracellular signal
transduction by modulation of at least one of cellular membranes,
membrane potential/conductance, membrane proteins (e.g., membrane
receptors such as G protein coupled receptors), calcium dependant
cellular signaling systems, and intercellular junctions (e.g.,
tight junctions, gap junctions, zona adherins and desmasomes).
Specifically, using a variety of art-recognized biological test
systems and assays. Applicants data shows, relative to control
fluids, differential effects of the inventive fluid on, for
example: regulatory T cell proliferation; cytokine and protein
levels (e.g, IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,
tyrptase, tight junction related proteins, TSLP receptor, MMP9,
etc.); binding of Bradykinin ligand with the Bradykinin B2
receptor; expression of TSLP receptor, whole cell conductance; etc.
Moreover, the Diphtheria toxin (DT390) effects shown herein
indicate that beta blockade (beta 2 adrenergic receptor), and/or
GPCR blockade and/or Ca channel blockade affects the activity of
the electrokinetically generated fluids on, for example, Treg and
PBMC function.
[0584] Taken together these effects indicate that the inventive
electrokinetically generated fluids are not only fundamentally
distinguished from prior art fluids, but also that they provide for
novel compositions and substantial utilities such as those
presently disclosed and claimed herein.
[0585] In this Example.
[0586] Applicants have, in this Example, performed voltametry
studies to further characterize the fundamental nature of the
inventive electrokinetically generated fluids. Voltametry is
frequently used to determine the redox potential or measure kinetic
rates and constants of fluids. The common characteristic of all
voltametric methods is that they involve the application of a
potential to an electrode and the resultant current flowing is
monitored through an electrochemical cell. The applied potential
produces a change in the concentration of an electroactive species
at the electrode surface by electrochemically reducing or oxidizing
the species.
[0587] Specifically, Applicants have utilized voltametric methods
(i.e., square wave voltametry and stripping polarography) to
further characterize fundamental differences between control saline
fluid and the inventive electrokinetically generated test fluids
(e.g., Solas and Revera). Applicants, given the biological and
membrane effects data summarized above, reasoned that square wave
voltametry and stripping polarography would provide an effective
means to further characterize the unique properties of the
inventive electrokinetically generated fluids.
[0588] Applicants further reasoned that differences in current at
specific voltages, production of different concentrations of an
electroactive redox compound, creation of new redox compounds, and
possession of unique electrochemical properties could be used to
assess and characterize properties of the inventive fluids. For
these studies, both a superoxygenated electrokinetically generated
fluid (Revera), and a non-superoxygenated inventive
electrokinetically generated fluid (Solas) were used.
Materials and Methods:
[0589] Materials and Solution Preparation.
[0590] The experiments were conducted on an EG & G SMDE 303A
polarographer (Princeton Applied Research). The electrolyte, NaOH,
used in the square wave voltametry experiment, was purchased from
Sigma. A 10 mL sample of the inventive fluid solution was prepared
by adding 100 .mu.L of NaOH to 9.9 mL of Revera Saline to make a
0.18 molar solution. With regards to the stripping polarography
experiment, no extra electrolyte was utilized.
[0591] Square Wave Voltametry.
[0592] As stated above, voltametry is used to determine the redox
potential or measure kinetic rates and constants in fluids. In the
square wave voltametry experiment, a potential of 0.0 to
approximately -1.75 V was applied to an electrode and the resultant
current flowing through the electrochemical cell was monitored.
[0593] Stripping Polarography.
[0594] The stripping polarography method is similar to the square
wave voltametry method. However, no electrolyte was utilized as
stated above and also involved a pre-step. In the pre-step, the
static mercury drop electrode was held for 30 seconds at -1.1 V to
amalgamate any compounds whose reduced form was soluble in mercury.
Then, the potentials between -1.1 V and 0.0 V were scanned and the
resultant current flowing through the electrochemical cell was
monitored. A linear scan into the negative potentials on this
amalgam provided a sensitive measurement of these compounds.
Results:
[0595] Square Wave Voltametry.
[0596] As evident from FIG. 98, the current profiles at -0.14V,
-0.47V, -1.02V and -1.36V differ between the various tested agents.
According to particular aspects, the differences in current
generated at the various specific voltages indicate at least one of
a different concentration of an electroactive redox compound and/or
a new or unique electroactive redox compound, and/or a change in
the diffusion-limiting electrical double layer surrounding the
mercury drop.
[0597] Stripping Polarography.
[0598] FIG. 99 shows that the inventive electrokinetically
generated fluids, Revera and Solas, show unique spectra with
pronounced peaks at -0.9 volts that are not present in the
non-electrokinetically generated blank and saline control fluids.
Additionally, the spectra of the non-electrokinetically generated
blank and saline control fluids show characteristic peaks at -0.19
and -0.3 volts that are absent in the spectra for the
electrokinetically generated Solas and Revera fluids.
[0599] According to particular aspects, therefore, these results
show unique electrochemical properties of the inventive
electrokinetically generated Solas and Revera fluids compared to
non-electrokinetically generated Saline control fluid. According to
additional aspects, the results indicate the presence or generation
of at least one of a different concentration of an electroactive
redox compound and a new and/or unique electroactive redox compound
in electrokinetically generated versus non-electrokinetically
generated fluids.
[0600] On top of the various biological data presented elsewhere
herein, this differential voltametry data, particularly when
considered along with the differential effects on whole cell
conductance, .sup.13C NMR line-width analysis, and the mixing
device feature-localized effects (e.g., voltage pulses and current
and/or currents pulses) indicate that the inventive
electrokinetically generated fluids are not only fundamentally
distinguished from prior art fluids, but also provide for novel
compositions and substantial utilities such as those presently
disclosed and claimed herein.
Example 23
Patch Clamp Analysis Conducted on Bronchial Epithilial Cells (BEC)
Perfused with Inventive Electrokinetically Generated Fluid (RNS-60)
Revealed that Exposure to RNS-60 Resulted in a Decrease in Whole
Cell Conductance, and Stimulation with a cAMP Stimulating
"Cocktail", which Dramatically Increased the Whole-Cell
Conductance, and Also Increased the Drug-Sensitive Portion of the
Whole-Cell Conductance, which was Ten-Times Higher than that
Observed Under Basal Conditions
[0601] In this Example, patch clamp studies were performed to
further confirm the utility of the inventive electrokinetically
generated fluids to modulate intracellular signal transduction by
modulation of at least one of membrane structure, membrane
potential or membrane conductivity, membrane proteins or receptors,
ion channels, and calcium dependant cellular messaging systems.
[0602] Overview.
[0603] As shown in Example 14 above (e.g., FIG. 75, showing
Stabilization of Bradykinin binding to the B2 receptor using
Bio-Layer Interferometry biosensor, Octet Rapid Extended Detection
(RED) (forteBio.TM.)), Bradykinin binding to the B2 receptor was
concentration dependent, and binding affinity was increased in the
electrokinetically generated fluid (e.g., Rev; gas-enriched
electrokinetically generated fluid) of the instant disclosure
compared to normal saline. Additionally, as shown in Example 15 in
the context of T-regulatory cells stimulated with particulate
matter (PM), the data showed a decreased proliferation of
T-regulatory cells in the presence of PM and Rev relative to PM in
control fluid (no Rev, no Solis) (FIG. 76), indicating that the
inventive electrokinetically generated fluid Rev improved
regulatory T-cell function; e.g., as shown by relatively decreased
proliferation in the assay. Moreover, exposure to the inventive
fluids resulted in a maintained or only slightly decreased
production of IL-10 relative to the Saline and Media controls (no
PM). Likewise, in the context of the allergic asthma (AA) profiles
of peripheral blood mononuclear cells (PBMC) stimulated with
particulate matter (PM), the data showed that exposure to the
fluids of the instant disclosure ("PM+Rev") resulted in
significantly lower tryptase levels similar to those of the Saline
and Media controls. Additionally, the Diphtheria toxin (DT390)
effects shown in Example 15 and FIGS. 76-83, indicate that beta
blockade, GPCR blockade and Ca channel blockade affects the
activity of the electrokinetically generated fluids on Treg and
PBMC function. Furthermore, the data of Example 18 shows that,
according to additional aspects, upon expose to the inventive
fluids, tight junction related proteins were upregulated in lung
tissue. FIGS. 85-89 show upregulation of the junction adhesion
molecules JAM 2 and 3, GJA1,3,4 and 5 (junctional adherins), OCLN
(occludin), claudins (e.g., CLDN 3, 5, 7, 8, 9, 10), TJP1 (tight
junction protein 1), respectively.
[0604] Patch clamp studies were performed to further investigate
and confirm said utilities.
Materials and Methods:
[0605] The Bronchial Epithelial line Calu-3 was used in Patch clamp
studies. Calu-3 Bronchial Epithelial cells (ATCC #HTB-55) were
grown in a 1:1 mixture of Ham's F12 and DMEM medium that was
supplemented with 10% FBS onto glass coverslips until the time of
the experiments. In brief, a whole cell voltage clamp device was
used to measure effects on Calu-3 cells exposed to the inventive
electrokinetically generated fluids (e.g., RNS-60;
electrokinetically treated normal saline comprising 60 ppm
dissolved oxygen; sometimes referred to as "drug" in this
Example).
[0606] Patch clamping techniques were utilized to assess the
effects of the test material (RNS-60) on epithelial cell membrane
polarity and ion channel activity. Specifically, whole cell voltage
clamp was performed upon the Bronchial Epithelial line Calu-3 in a
bathing solution consisting of: 135 mM NaCl, 5 mM KC, 1.2 mM CaCl2,
0.8 mM MgCl2, and 10 mM HEPES (pH adjusted to 7.4 with N-methyl
D-Glucamine). Basal currents were measured after which RNS-60 was
perfused onto the cells.
[0607] More specifically, patch pipettes were pulled from
borosilicate glass (Garner Glass Co, Claremont, Calif.) with a
two-stage Narishige PB-7 vertical puller and then fire-polished to
a resistance between 6-12 Mohms with a Narishige MF-9 microforge
(Narishige International USA, East Meadow, N.Y.). The pipettes were
filled with an intracellular solution containing (in mM): 135 KC,
10 NaCl, 5 EGTA, 10 Hepes, pH was adjusted to 7.4 with NMDG
(N-Methyl-D-Glucamine).
[0608] The cultured Calu-3 cells were placed in a chamber
containing the following extracellular solution (in mM): 135 NaCl,
5 KC, 1.2 CaCl2, 0.5 MgCl2 and 10 Hepes (free acid), pH was
adjusted to 7.4 with NMDG.
[0609] Cells were viewed using the 40.times.DIC objective of an
Olympus IX71 microscope (Olympus Inc., Tokyo, Japan). After a
cell-attached gigaseal was established, a gentle suction was
applied to break in, and to attain the whole-cell configuration.
Immediately upon breaking in, the cell was voltage clamped at -120,
-60, -40 and 0 mV, and was stimulated with voltage steps between
.+-.100 mV (500 ms/step). After collecting the whole-cell currents
at the control condition, the same cell was perfused through bath
with the test fluid comprising same exatracelluar solutes and pH as
for the above control fluid, and whole-cell currents at different
holding potentials were recorded with the same protocols.
[0610] Electrophysiological data were acquired with an Axon Patch
200B amplifier, low-pass filtered at 10 kHz, and digitized with
1400A Digidata (Axon Instruments, Union City, Calif.). The pCLAMP
10.0 software (Axon Instruments) was used to acquire and to analyze
the data. Current (I)-to-voltage (V) relationships (whole cell
conductance) were obtained by plotting the actual current value at
approximately 400 msec into the step, versus the holding potential
(V). The slope of the IN relationship is the whole cell
conductance.
[0611] Drugs and Chemicals. Whenever indicated, cells were
stimulated with a cAMP stimulatory cocktail containing 8-Br-cAMP
(500 mM), IBMX (isobutyl-1-methylxanthie, 200 mM) and forskolin (10
mM). The cAMP analog 8-Br-cAMP (Sigma Chem. Co.) was used from a 25
mM stock in H.sub.2O solution. Forskolin (Sigma) and IBMX (Sigma)
were used from a DMSO solution containing both 10 mM Forskolin and
200 mM IBMX stock solution.
Patch Clamp Results:
[0612] FIG. 100 shows whole-cell currents under basal (no cAMP)
conditions, with a protocol stepping from zero mV holding potential
to +/-100 mV. Representative tracings are the average of n=12
cells. The tracings on the left are the control, followed by the
whole-cell tracings while perfusing the test solution (middle). The
tracings on the right are the composite delta obtained by
subtraction of the test average values, from those under control
conditions. The whole-cell conductance, obtained from the
current-to-voltage relationships is highly linear under both
conditions, and reflects a modest, albeit significant change in
conductance due to the test conditions. The contribution to the
whole-cell conductance, i.e., the component inhibited by the drug
(inventive electrokinetically generated fluid) is also linear, and
the reversal potential is near zero mV. There is a decrease in the
whole cell conductance under hyperpolarizing conditions.
[0613] FIG. 101 shows whole-cell currents under basal conditions,
with a protocol stepping from -40 mV holding potential to .+-.100
mV. Representative tracings are the average of n=12 cells. The
tracings on the left are the control, followed by the whole-cell
tracings while perfusing the test solution (middle). The tracings
on the right are the composite delta obtained by subtraction of the
test average values, from those under control conditions. The
whole-cell conductance obtained from the current-to-voltage
relationships is highly linear under both conditions, and reflects
a modest, albeit significant change in conductance due to the test
conditions. The contribution to the whole-cell conductance, i.e.,
the component inhibited by the drug (inventive electrokinetically
generated fluid) is also linear, and the reversal potential is near
zero mV. Values are comparatively similar to those obtained with
the zero mV protocol.
[0614] FIG. 102 shows whole-cell currents under basal conditions,
with a protocol stepping from -60 mV holding potential to .+-.100
mV. Representative tracings are the average of n=12 cells. The
tracings on the left are the control, followed by the whole-cell
tracings while perfusing the test solution (middle). The tracings
on the right are the composite delta obtained by subtraction of the
test average values, from those under control conditions. The
whole-cell conductance obtained from the current-to-voltage
relationships is highly linear under both conditions, and reflects
a minor, albeit significant change in conductance due to the test
conditions. The contribution to the whole-cell conductance, i.e.,
the component inhibited by the drug is also linear, and the
reversal potential is near zero mV. Values are comparatively
similar to those obtained with the zero mV protocol.
[0615] FIG. 103 shows whole-cell currents under basal conditions,
with a protocol stepping from -120 mV holding potential to .+-.100
mV. Representative tracings are the average of n=12 cells. The
tracings on the left are the control, followed by the whole-cell
tracings while perfusing the test solution (middle). The tracings
on the right are the composite delta obtained by subtraction of the
test average values, from those under control conditions. The
whole-cell conductance obtained from the current-to-voltage
relationships is highly linear under both conditions, and reflects
a minor, albeit significant change in conductance due to the test
conditions. The contribution to the whole-cell conductance, i.e.,
the component inhibited by the drug is also linear, and the
reversal potential is near zero mV. Values are comparatively
similar to those obtained with the zero mV protocol.
[0616] FIG. 104 shows whole-cell currents under cAMP-stimulated
conditions, obtained with protocols stepping from various holding
potentials to .+-.100 mV. Representative tracings are the average
of n=5 cells. The tracings on the left are the control, followed by
the whole-cell tracings after cAMP stimulation, followed by
perfusion with the drug-containing solution. The tracings on the
right are the composite delta obtained by subtraction of the test
average values in drug+cAMP, from those under control conditions
(cAMP alone). The tracings on the Top are those obtained from
voltage protocol at zero mV, and the ones below, at -40 mV. The
whole-cell conductance obtained from the current-to-voltage
relationships is highly linear under all conditions, and reflects a
change in conductance due to the test conditions.
[0617] FIG. 105 shows whole-cell currents under cAMP-stimulated
conditions, obtained with protocols stepping from various holding
potentials to .+-.100 mV. Representative tracings are the average
of n=5 cells. The tracings on the left are the control, followed by
the whole-cell tracings after cAMP stimulation, followed by
perfusion with the drug-containing solution. The tracings on the
right are the composite delta obtained by subtraction of the test
average values in drug+cAMP, from those under control conditions
(cAMP alone). The tracings on the Top are those obtained from
voltage protocol at -60 mV, and the ones below, at -120 mV. The
whole-cell conductance, obtained from the current-to-voltage
relationships, is highly linear under all conditions, and reflects
a change in conductance due to the test conditions.
[0618] FIG. 106 shows the effect of holding potential on
cAMP-activated currents. The effect of the drug (the inventive
electrokinetically generated fluids; RNS-60; electrokinetically
treated normal saline comprising 60 ppm dissolved oxygen) on the
whole-cell conductance was observed under different voltage
protocols (0, -40, -60, -120 mV holding potentials). Under basal
conditions, the drug-sensitive whole-cell current was identical at
all holding potentials (voltage-insensitive contribution, Top Left
panel). In the cAMP-activated conditions, however, the
drug-sensitive currents were much higher, and sensitive to the
applied voltage protocol. The current-to-voltage relationships are
highly nonlinear. This is further observed in the subtracted
currents (Bottom panel), where the contribution of the whole cell
conductance at zero mV was further subtracted for each protocol
(n=5).
[0619] Summary of Example.
[0620] According to particular aspects, therefore, the data
indicate that there is a modest but consistent effect of the drug
(the inventive electrokinetically generated fluids; RNS-60;
electrokinetically treated normal saline comprising 60 ppm
dissolved oxygen) under basal conditions. To enhance the effect of
the drug on the whole-cell conductance, experiments were also
conducted by perfusing the drug after stimulation with a cAMP
stimulating "cocktail", which dramatically increased the whole-cell
conductance. Interestingly, this protocol also increased the
drug-sensitive portion of the whole-cell conductance, which was
ten-times higher than that observed under basal conditions.
Additionally, in the presence of cAMP stimulation, the drug showed
different effects with respect to the various voltage protocols,
indicating that the electrokinetically generated fluids affect a
voltage-dependent contribution of the whole-cell conductance. There
was also a decrease in a linear component of the conductance,
further suggesting at least a contribution of the drug to the
inhibition of another pathway (e.g., ion channel, voltage gated
cation channels, etc.).
[0621] In particular aspects, and without being bound by mechanism,
Applicants' data are consistent with the inventive
electrokinetically generated fluids (e.g., RNS-60;
electrokinetically treated normal saline comprising 60 ppm
dissolved oxygen) producing a change either on a channel(s), being
blocked or retrieved from the plasma membrane.
Example 24
Patch Clamp Analysis Conducted on Calu-3 Cells Perfused with
Inventive Electrokinetically Generated Fluids (RNS-60 and Solas)
Revealed that (i) Exposure to RNS-60 and Solas Resulted in
Increases in Whole Cell Conductance, (ii) that Exposure of Cells to
the RNS-60 Produced an Increase in a Non-Linear Conductance,
Evident at 15 Min Incubation Times, and (iii) that Exposure of
Cells to the RNS-60 Produced an Effect of RNS-60 Saline on Calcium
Permeable Channels
[0622] Overview.
[0623] In this Example, patch clamp studies were performed to
further confirm the utilities, as described herein, of the
inventive electrokinetically generated slaine fluids (RNS-60 and
Solas), including the utility to modulate whole-cell currents. Two
sets of experiments were conducted.
[0624] The summary of the data of the first set of experiments
indicates that the whole cell conductance (current-to-voltage
relationship) obtained with Solas saline is highly linear for both
incubation times (15 min, 2 hours), and for all voltage protocols.
It is however evident, that longer incubation (2 hours) with Solas
increased the whole cell conductance. Exposure of cells to the
RNS-60 produced an increase in a non-linear conductance, as shown
in the delta currents (Rev-Sol subtraction), which is only evident
at 15 min incubation time. The effect of the RNS-60 on this
non-linear current disappears, and is instead highly linear at the
two-hour incubation time. The contribution of the non-linear whole
cell conductance, as previously observed, was voltage sensitive,
although present at all voltage protocols.
[0625] The summary of data of the second set of experiments
indicates that there is an effect of the RNS-60 saline on a
non-linear current, which was made evident in high calcium in the
external solution. The contribution of the non-linear whole cell
conductance, although voltage sensitive, was present in both
voltage protocols, and indicates an effect of RNS-60 saline on
calcium permeable channels.
First Set of Experiments (Increase of Conductance; and Activation
of a Non-Linear Voltage Regulated Conductance)
Methods for First Set of Experiments:
[0626] See EXAMPLE 23 for general patch clamp methods. In the
following first set of experiments, patch clamp studies were
performed to further confirm the utility of the inventive
electrokinetically generated saline fluids (RNS-60 and Solas) to
modulate whole-cell currents, using Calu-3 cells under basal
conditions, with protocols stepping from either zero mV holding
potential, -120 mV, or -60 mV.
[0627] The whole-cell conductance in each case was obtained from
the current-to-voltage relationships obtained from cells incubated
for either 15 min or two hours, to further confirm the results of
EXAMPLE 23. In this study, groups were obtained at a given time,
for either Solas or RNS-60 saline solutions. The data obtained are
expressed as the mean.+-.SEM whole cell current for 5-9 cells.
Results:
[0628] FIGS. 117 A-C show the results of a series of patch clamping
experiments that assessed the effects of the electrokinetically
generated fluid (e.g., RNS-60 and Solas) on epithelial cell
membrane polarity and ion channel activity at two time-points (15
min (left panels) and 2 hours (right panels)) and at different
voltage protocols (A, stepping from zero mV; B, stepping from -60
mV; and C, stepping from -120 mV). The results indicate that the
RNS-60 (filled circles) has a larger effect on whole-cell
conductance than Solas (open circles). In the experiment similar
results were seen in the three voltage protocols and at both the 15
minute and two-hour incubation time points.
[0629] FIGS. 118 A-C show graphs resulting from the subtraction of
the Solas current data from the RNS-60 current data at three
voltage protocols ("Delta currents") (A, stepping from zero mV; B,
stepping from -60 mV; and C, stepping from -120 mV) and the two
time-points (15 mins (open circles) and 2 hours (filled circles)).
These data indicated that at the 15 minute time-point with RNS-60,
there is a non-linear voltage-dependent component that is absent at
the 2 hour time point.
[0630] As in previous experiments, data with "Normal" saline gave a
very consistent and time-independent conductance used as a
reference. The present results were obtained by matching groups
with either Solas or RNS-60 saline, and indicate that exposure of
Calu-3 cells to the RNS-60 saline under basal conditions (without
cAMP, or any other stimulation), produces time-dependent effect(s),
consistent with the activation of a voltage-regulated conductance
at shorter incubation times (15 min). This phenomenon was not as
apparent at the two-hour incubation point. As described elsewhere
herein, the linear component is more evident when the conductance
is increased by stimulation with the cAMP "cocktail". Nonetheless,
the two-hour incubation time showed higher linear conductance for
both the RNS-60 and the Solas saline, and in this case, the RNS-60
saline doubled the whole cell conductance as compared to Solas
alone. This evidence indicates that at least two contributions to
the whole cell conductance are affected by the RNS-60 saline,
namely the activation of a non-linear voltage regulated
conductance, and a linear conductance, which is more evident at
longer incubation times.
Second Set of Experiments (Effect on Calcium Permeable
Channels)
Methods for Second Set of Experiments:
[0631] See EXAMPLE 23 for general patch clamp methods. In the
following second set of experiments, yet additional patch clamp
studies were performed to further confirm the utility of the
inventive electrokinetically generated saline fluids (RNS-60 and
Solas) to modulate whole-cell currents, using Calu-3 cells under
basal conditions, with protocols stepping from either zero mV or
-120 mV holding potentials.
[0632] The whole-cell conductance in each case was obtained from
the current-to-voltage relationships obtained from cells incubated
for 15 min with either saline. To determine whether there is a
contribution of calcium permeable channels to the whole cell
conductance, and whether this part of the whole cell conductance is
affected by incubation with RNS-60 saline, cells were patched in
normal saline after the incubation period (entails a high NaCl
external solution, while the internal solution contains high KC).
The external saline was then replaced with a solution where NaCl
was replaced by CsCl to determine whether there is a change in
conductance by replacing the main external cation. Under these
conditions, the same cell was then exposed to increasing
concentrations of calcium, such that a calcium entry step is made
more evident.
Results:
[0633] FIGS. 119 A-D show the results of a series of patch clamping
experiments that assessed the effects of the electrokinetically
generated fluid (e.g., Solas (panels A and B) and RNS-60 (panels C
and D)) on epithelial cell membrane polarity and ion channel
activity using different external salt solutions and at different
voltage protocols (panels A and C show stepping from zero mV,
whereas panels B and D show stepping from -120 mV). In these
experiments one time-point of 15 minutes was used. For Solas
(panels A and B) the results indicate that: 1) using CsCl (square
symbols) instead of NaCl as the external solution, increased whole
cell conductance with a linear behavior when compared to the
control (diamond symbols); and 2) CaCl.sub.2 at both 20 mM
CaCl.sub.2 (circle symbols) and 40 mM CaCl.sub.2 (triangle symbols)
increased whole cell conductance in a non-linear manner. For RNS-60
(panels C and D), the results indicate that: 1) using CsCl (square
symbols) instead of NaCl as the external solution had little effect
on whole cell conductance when compared to the control (diamond
symbols); and 2) CaCl.sub.2 at 40 mM (triangle symbols) increased
whole cell conductance in a non-linear manner.
[0634] FIGS. 120 A-D show the graphs resulting from the subtraction
of the CsCl current data (shown in FIG. 119) from the 20 mM
CaCl.sub.2 (diamond symbols) and 40 mM CaCl.sub.2 (square symbols)
current data at two voltage protocols (panels A and C, stepping
from zero mV; and B and D, stepping from -120 mV) for Solas (panels
A and B) and RNS-60 (panels C and D). The results indicate that
both Solas and RNS-60 solutions activated a calcium-induced
non-linear whole cell conductance. The effect was greater with
RNS-60 (indicating a dosage responsiveness), and with RNS-60 was
only increased at higher calcium concentrations. Moreover, the
non-linear calcium dependent conductance at higher calcium
concentration was also increased by the voltage protocol.
[0635] The data of this second set of experiments further indicates
an effect of RNS-60 saline and Solas saline for whole cell
conductance data obtained in Calu-3 cells. The data indicate that
15-min incubation with either saline produces a distinct effect on
the whole cell conductance, which is most evident with RNS-60, and
when external calcium is increased, and further indicates that the
RNS-60 saline increases a calcium-dependent non-linear component of
the whole cell conductance.
[0636] The accumulated evidence suggests activation by Revalesio
saline of ion channels, which make different contributions to the
basal cell conductance.
[0637] Taken together with Applicants' other data (e.g., the data
of Applicants other working Examples) particular aspects of the
present invention provide compositions and methods for modulating
intracellular signal transduction, including modulation of at least
one of membrane structure, membrane potential or membrane
conductivity, membrane proteins or receptors, ion channels, lipid
components, or intracellular components with are exchangeable by
the cell (e.g., signaling pathways, such as calcium dependant
cellular signaling systems, comprising use of the inventive
electrokinetically generated solutions to impart electrochemical
and/or conformational changes in membranous structures (e.g.,
membrane and/or membrane proteins, receptors or other membrane
components) including but not limited to GPCRs and/or g-proteins.
According to additional aspects, these effects modulate gene
expression, and may persist, dependant, for example, on the half
lives of the individual messaging components, etc.
Example 25
Atomic Force Microscopy (AFM) Measurements of the Inventive
Electrokinetic Fluid (RNS-60) Indicated the Presence and/or
Formation of Hydrophobic Surface Nanobubbles that were
Substantially Smaller that Those Present in Control `Pressure Pot`
(PNS-60) Fluid
[0638] Overview.
[0639] Applicants used Atomic Force Microscopy (AFM) measurements
to characterize hydrophobic nanobubbles in the inventive
electrokinetic fluid (RNS-60).
Materials and Methods:
[0640] AFM Studies.
[0641] AFM studies were preformed at an art-recognized Nanotech
User Facility (NTUF). For AFM studies, a very small and sensitive
needle is dipped into a droplet of water placed onto a hydrophobic
surface. The needle then scans over the water/surface interface at
rates such as 1 mm.sup.2 in .about.15 minutes. The needle records
any imperfections in the surface geometry, and is sensitive enough
to record the presence of small bubbles.
[0642] The Silicon substrate upon which the water droplets were
placed was prepared using
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane), and the resulting
hydrophobic surface causes water to bead up with contact angles of
approximately 95 degrees. This coating is used in many AFM studies,
in part, because it is particularly durable.
[0643] Solution Preparation.
[0644] Two test solutions were studied: RNS-60 and PNS-60. RNS-60
is an inventive electrokinetic fluid comprising 60 ppm oxygen,
whereas PNS-60 is a non-electrokinetic control fluid comprising 60
ppm oxygen prepared by conventional exposure to a pressurized
oxygen head (i.e., pressure pot oxygenated fluid). Each test
solution was initially buffered by addition of a small amount of
neutral phosphate buffer (pH 7) solution, and approximately 60-70
uL of each buffered test solution (approximately 22.degree. C.) was
placed onto a previously prepared silica plate.
Results:
[0645] Under AFM, the RNS-60 droplet displayed a distribution of
about 20 hydrophobid nanobubbles in a 1 mm.sup.2 area, having
dimensions of -20 nm wide and -1.5 nm tall or smaller (FIG. 121 A).
By contrast, under AFM, the PNS-60 droplet displayed approx 5
hydrophobic nanobubbles in a 1 mm.sup.2 area, having dimensions of
-60 nm wide and -5 nm tall (FIG. 121 B). The PNS-60 droplet,
therefore, had much fewer and much larger hydrophobic nanobubbles
compared to the RNS60 droplet.
[0646] According to particular aspects, therefore, there is a
substantial difference in the size and distribution of hydrophobic
surface nanobubbles between the RNS-60 and PNS-60 test solutions,
where the nanobubbles are either initially present in, and/or
formed within the test fluids during AFM measurement.
[0647] As discussed elsewhere herein, according to particular
aspects of the present invention, the inventive electrokinetically
altered fluids comprise an ionic aqueous solution of
charge-stabilized oxygen-containing nanostructures substantially
having an average diameter of less than about 100 nanometers and
stably configured in the ionic aqueous fluid in an amount
sufficient to provide, upon contact of a living cell by the fluid,
modulation of at least one of cellular membrane potential and
cellular membrane conductivity.
[0648] Applicants point out, however, that the hydrophobic bubbles
(forming on a hydrophobic surface), such as those observed in AFM
experiments are likely fundamentally different from inventive
biologically-active charge-stabilized nanostructure disclosed
herein. According to particular aspects therefore, while the AFM
experiments in this working Example support, based on the size and
distribution hydrophobic bubble formation, that the inventive
electrokinetic fluids (e.g., RNS-60) are fundamentally distinct
from non-electrokinetic control fluids, the hydrophobic bubbles are
likely distinct from and/or derived from the inventive
charge-stablilized oxygen-containing nanostrutures described in
detail elsewhere herein. In any event, relative to the inventive
electrokinetic fluids, control pressure pot oxygenated fluids do
not comprise charge-stabilized oxygen-containing nanostructures
capable of modulation of at least one of cellular membrane
potential and cellular membrane conductivity.
Example 26
Analysis of Inventive Fluid on Growth Inhibition of Pseudomonas on
Plates
[0649] Applying gas-enriched saline solution of the present
invention limits Pseudomonas growth. These tests were performed
using an inventive oxygen-enriched saline solution.
[0650] Two test strains of Pseudomonas (ATCC accession no. 10145
and ATCC accession no. 27853) were prepared from fresh 24-hour
cultures to a McFarland 1 concentration (approximately
3.times.10.sup.8 microorganisms/mL). Each of the bacterial aliquots
(1 mL) was serially diluted in 10 fold dilutions in 9 mL of
broth-saline made from 1 part TSB broth and 9 parts sterile saline.
The bacterial concentrations tested were 10.sup.7, 10.sup.6,
10.sup.5, 10.sup.4, 10.sup.3, and 10.sup.2. A negative control tube
(no bacteria and no gas-enriched fluid of the present invention)
was prepared. The positive control tubes (containing no
gas-enriched fluid, normal saline and each of the 6 bacterial
concentrations) were included in each set of tubes for testing of
each bacterial strain.
[0651] Using the 10 fold dilutions of each bacterial strain, a set
of 36 tubes was inoculated as follows: [0652] Six tubes received 1
mL of each of the dilutions; then 4 mL of each of the test
gas-enriched fluids were added to each tube (bringing the total
volume to 5 mL). The gas-enriched fluids were labeled as 50 ppm, 40
ppm, 30 ppm, 20 ppm, 10 ppm, and normal saline. The positive
control was normal saline, against which all of the other tubes
were measured for growth.
[0653] All tubes of each 36-tube experiment set for each
Pseudomonas species were measured serially at 2 hour intervals
within a 24 hour period after initial incubation at 35.degree. C.
Between readings of each set, the tubes were returned to 35.degree.
C. for continued incubation. Readings were performed using a
calibrated spectrophotometer set at OD.sub.540.
[0654] The results of these tests indicate that the gas-enriched
fluid of the present invention positively inhibits Pseudomonas
strains at several bacterial dilutions within 4 to 12 hours after
the start of incubation at 35.degree. C., as well as later (16-24
hours) for both strains tested.
[0655] Specifically, the highest positive inhibition was found
during 16 to 24 hours for both strains, at 30 or 50 concentration.
The positive inhibition varies slightly depending on the
concentration of test solutions, and concentration of bacterial
samples tested.
TABLE-US-00012 TABLE 11 Time at positive inhibition of Pseudomonas
Time (hours) 2 4 6 8 10 12 16 18 20 22 24 ATCC 27853 0 1 3 1 2 2 5
0 5 7 6 % + -- 3 10 3 6 6 16 -- 16 23 20 ATCC 10145 0 4 3 4 3 3 2 1
5 6 8 % + -- 13 10 13 10 10 6 3 16 20 26 % + = number +/30 (6
bacterial concentrations .times. 5 solution concentrations)
Example 27
MIC Studies on Pseudomonas Growth Inhibition in Tubes
[0656] Minimum inhibitory concentration (MIC) test solutions in
two-fold dilutions were prepared in a base of broth-saline mixture
made from 1 part TSB broth and 4 parts CFU-NS. The negative control
tube (containing no bacteria and no gas-enriched fluid) and the
positive control tube (containing no gas-enriched fluid and
bacteria) were included in each set of tubes tested. The fluid
dilutions were: 50, 25, 12.5, 6.25, 3.12, 1.55, and 0.7 ppm,
respectively. Following preparation of the tubes for each bacterial
strain, pH was measured on samples from the two solutions.
CFU-gas-enriched fluid pH was about 6.8-7.2, while CFU-NS was pH
about 6.2.
[0657] Following preparation of the 2 sets of covered tubes at
35.degree. C. for 18 hours, visual inspection of all tubes revealed
that the first tube labeled 50 in each set showed no growth and all
other tubes (except the negative control tube) showed moderate
growth.
[0658] The tubes labeled 50 and showed no growth upon visual
inspection from the MIC studies were further tested for minimum
bactericidal concentrations (MBC). Samples were taken from each of
these two tubes using sterile calibrated disposable loops of 0.1 mL
capacity and were immediately streaked on BA plates to detect
growth or inhibition of growth on the challenge bacteria. Following
18 hours incubation at 35.degree. C., samples collected from each
of the tubes showing no visual growth (tube 50 from ATCC 10145 and
tube 50 from ATCC 27853) both showed growth at very high levels
when streaked and plated on BA plates. The numbers of colonies were
too numerous to count. Thus, a slight delay in growth was
experienced with the tube 50 gas-enriched fluids.
Example 28
Analysis of Inventive Fluid on Growth Inhibition of Pseudomonas in
Dressings
[0659] Aquacel dressings were tested dry and hydrated with either
test fluids 52 (pH 7.2-7.8), 50 (pH 6.0-6.2), 42 (pH 7.2), 34 (pH
7.2), 25 (pH 6.8), and 10 (pH 6.2), or normal saline (pH 6.2),
against Pseudomonas strains ATCC accession no. 10145 and ATCC
accession no. 27853 A first application of the gas-enriched test
fluids or normal saline fluid to the dressings (0.4 mL) was
followed by a second application 12 hours later (0.25 mL).
[0660] Results revealed a 3-4 mm clear area of inhibition around
one of the three dressing pieces (1 cm square) treated with sterile
25 fluid applied to the three Pseudomonas ATCC accession no. 10145
seeded plates, and a 3-4 mm clear area of inhibition around one of
the three dressing pieces (1 cm square) treated with sterile 25
fluid applied to one of the three Pseudomonas ATCC accession no.
27853 seeded plates. A clear area of less than 1-2 mm of inhibition
was around 2 sides of the dry dressing on both strains. No zones of
inhibition were detected around the other test or control
dressings.
Example 29
Analysis of Inventive Fluid on Growth Inhibition of Microorganisms
in Dressings
[0661] Blood Agar plates were used and wound dressings used were
Promogram Prisma matrix dressing (A) and hydrofiber dressing (B).
The sterile dressings were tested with microorganisms from a 24
hour culture at a cell density with McFarland equivalence turbidity
standards of 3.0.times.10.sup.8/mL.
[0662] Test microorganisms were Staphlycoccus aureaus,
Staphylococcus epidermidis, Pseudomonas aeruginosa, E. coli, and
Candida albicans.
[0663] Dressings of each type were hydrated with approximately 0.8
mL of either gas-enriched fluid or normal saline, and left for
approximately 30 minutes. Dry dressings were used as controls.
Plates were then grown for 24 hours and results recorded.
[0664] As viewed in the cell plates, the hydrofiber dressing (B)
had no effect on growth of test microorganisms whether dry or
hydrated with either fluid. The Prisma dressing (A) hydrated with
gas-enriched fluid showed a zone of partial inhibition with
colonies for Staph. aureaus, a 1 mm zone of inhibition for Staph.
epidermidis, a 2-3 mm zone of inhibition with an added halo effect
extending out an additional 2-3 mm for Pseudomonas, a 2 mm zone of
inhibition for Candida, and no effect for E. coli.
[0665] The dry control hydrofiber dressing (A) had a zone of 2-3 mm
of inhibition for Staph. auereaus, Staph. epidermidis, Pseudomonas,
and a partial inhibition of about 1-2 mm for E. coli and Candida,
with break-through colonies observed.
[0666] The normal saline control hydrofiber dressing (A) showed a 2
mm zone of partial inhibition for Staph. auereaus, Staph.
epidermidis, and Pseudomonas. Colonies were observed grown into E.
coli and Candida test strains.
Example 30
MIC/MBC Testing with Tobramycin
[0667] The gas-enriched fluids of the present invention were tested
randomly against three Pseudomonas strains, PA 01, PA 14, and PA K.
Each bacterial culture was prepared from fresh 24-hour cultures to
McFarland 1 concentration (approximately 3.times.10.sup.8
microorganisms/mL). Each of the bacterial samples (1 mL) were then
serially diluted in 10 fold dilutions to 9 mL of a broth-saline
mixture made from 1 part MH broth and 9 parts sterile saline. The
bacterial concentrations tested were 10.sup.5, 10.sup.4, 10.sup.3,
and 10.sup.2. Negative control tubes (containing no bacteria and no
gas-enriched saline solution, and no Tobramycin), as well as
positive control tubes (containing test bacteria concentrations and
normal saline but no Tobramycin, and no gas-enriched fluid), were
run side-by-side with the test solutions. Cells were incubated at
35.degree. C. Results are shown in Table 13 below. Sheared normal
saline was passed through the gas-enriching diffuser device of the
present invention without the addition of a gas to the fluid.
TABLE-US-00013 TABLE 13 Betterkill rate with Tobramycin and
Gas-enriched fluid than Tobramycin Alone Dissolved Gas PA Fluid
Description (Oxygen, ppm) PA01 14 PAK A Gas-enriched Saline 33.4
Yes Yes No B Gas-enriched saline 23.8 No Yes No C Gas-enriched
saline 49.6 Yes Yes Yes D Normal saline 9.6 No No No E Gas-enriched
saline 42.8 Yes Yes Yes F Sheared normal saline 14.1 Yes Yes Yes
(no gas enrichment)
[0668] Test results shown in FIG. 39 illustrate the results of
testing Pseudomonas growth inhibition with deionized water, an
oxygen-enriched fluid of the present invention, as well as normal
saline.
Example 31
Analysis of Corneal Fibroblast Proliferation
[0669] Sterile gas-enriched water (with oxygen content of
approximately 50 ppm), or standard sterile deionized water was
utilized in preparing cell culture media. A human corneal stromal
fibroblast cell line was plated in 24 well tissue culture plates at
a density of 1.times.10.sup.5 cells/cm.sup.2 and cells were tested
for viability using a standard Live/Dead assay from Molecular
Probes, Inc. Viability tests were conducted at 1 or 3 days of
continuous culture with media changes twice per day. Viable and
non-viable cells were counted in 10 random 20.times. fields (0.2
mm.sup.2 area/field) in each well. The percentage of dead cells was
then calculated. Cells in the inventive gas-enriched media had
fewer dead cells than in standard culture media. Results are shown
in FIG. 46.
Example 32
Assessment of Gas-Enriched Fluid on Keratoconjunctivitis Sicca
[0670] According to particular aspects of the present invention,
upon the initial visitation, subjects are be examined, and systemic
and ophthalmic histories reviewed, including current use of
concomitant medications and/or therapies. Slit lamp examination is
conducted by way of assessment of corneal and conjunctival
staining, assessment of tear volume, and assessment of bulbar
conjunctival erythema. Tearscope examination ise conducted by
assessment of inferior tear meniscus height, and stability and
thickness of tear film. Treatment is dispensed based on
randomization into the following groups:
[0671] a. Treatment group: Gas-Enriched fluid instilled four times
per day for 6 weeks.
[0672] b. Control group: non-preserved saline instilled four times
per day for 6 weeks.
[0673] Upon the second visitation following a 2 week interval (+/-2
days), each subject completes a dry eye questionnaire, and the
subject's systemic and ophthalmic history, as well as any change in
medications and/or therapies will be reviewed. Slit lamp
examination is conducted by assessment of corneal and conjunctival
staining, assessment of tear volume, and assessment of bulbar
conjunctival erythema. Tearscope examination is conducted by
assessment of inferior tear meniscus height as well as stability
and thickness of tear film.
[0674] Upon the third examination following a 4 week interval (+/-2
days), each subject completes a dry eye questionnaire, and the
subject's system and ophthalmic history is reviewed, as well as any
change in medications and/or therapies. Slit lamp examination is
conducted by assessment of corneal and conjunctival staining,
assessment of tear volume, and assessment of bulbar conjunctival
erythema. Tearscope examination is conducted by assessment of
inferior tear meniscus height and stability and thickness of tear
film.
[0675] Upon the fourth examination following a 6 week interval
(+/-2 days), each subject completes a dry eye questionnaire, and
the subject's system and ophthalmic history is reviewed, as well as
any change in medications and/or therapies. Slit lamp examination
is conducted by assessment of corneal and conjunctival staining,
assessment of tear volume, and assessment of bulbar conjunctival
erythema. Tearscope examination is conducted by assessment of
inferior tear meniscus height and stability and thickness of tear
film. In addition, a treatment cross-over is evaluated according to
previous treatment solutions collected, new treatment dispensed,
and patients counseled to begin using new therapy immediately.
[0676] Upon the fifth examination following a 2 week interval (+/-2
days), each subject completes a dry eye questionnaire, and the
subject's system and ophthalmic history is reviewed, as well as any
change in medications and/or therapies. Slit lamp examination is
conducted by assessment of corneal and conjunctival staining,
assessment of tear volume, and assessment of bulbar conjunctival
erythema. Tearscope examination is conducted by assessment of
inferior tear meniscus height and stability and thickness of tear
film.
[0677] Upon the sixth examination following a 4 week interval (+/-2
days), each subject completes a dry eye questionnaire, and the
subject's system and ophthalmic history is reviewed, as well as any
change in medications and/or therapies. Slit lamp examination is
conducted by assessment of corneal and conjunctival staining,
assessment of tear volume, and assessment of bulbar conjunctival
erythema. Tearscope examination is conducted by assessment of
inferior tear meniscus height and stability and thickness of tear
film.
[0678] Upon the seventh examination following a 6 week interval
(+/-2 days), each subject completes a dry eye questionnaire, and
the subject's system and ophthalmic history is reviewed, as well as
any change in medications and/or therapies. Slit lamp examination
is conducted by assessment of corneal and conjunctival staining,
assessment of tear volume, and assessment of bulbar conjunctival
erythema. Tearscope examination is conducted by assessment of
inferior tear meniscus height and stability and thickness of tear
film, and subjects are exited from the study.
[0679] No intitiation or occasional use of new topically applied
medication or systemic medication ios allowed by subjects,
specifically including but not limited to antihistamines,
aspirin-related drugs, steroids, Paquenil.TM. or
psychoactive/anti-depressant medication. Subjects currently using
this medication on a continual basis are allowed. Additionally,
subjects with a history of glaucoma, anterior membrane dystrophy,
active trichiasis or any eyelid globe malposition abnormality, e.g.
entropion, ectropion, etc. are allowed to participate.
Keratoconjunctivitis sicca secondary to the destruction of
conjunctival goblet cells as with vitamin A deficiency or scarring
such as that with cicatricial pemphigoid, libal stem cell
deficiency, graft vs. host disease, alkali burns, Stevens-Johnson
syndrome, trachoma, or irradiation are allowed to participate.
Finally, uncontrolled systemic disease or the presence of any
significant illness or condition that could, in the judgment of the
investigator, interfere with interpretation of the study results
(such as Sjogren's syndrome and related autoimmune diseases are
allowed) are allowed. In the event that a subject becomes pregnant,
the study immediately terminates for that subject.
Example 33
Adjunct Therapy for Keratoconjunctivitis Sicca
[0680] According to additional aspects of the present invention,
the treatment dispensed in this study is used as an adjunct to all
habitual topical and oral medications being used. All patients
enrolled continue taking medications as prescribed previously.
[0681] Following recruitment, test subjects are randomized to
either the Gas-enriched saline treatment group or the non-preserved
saline (control) group. Subjects are masked as to the treatment
therapy they are receiving (treatment gas-enriched or control). The
clinical observer performing the objective measurements is masked
to the treatment group as well.
[0682] Sample size calculations and statistical methods to detect
significant changes in the outcome measures are derived from
previous comparable clinical trials. In order to detect a
significant change compared to control with a power of 0.8, a
minimum of 25 subjects is required. For all datasets, normality and
equal variance assumption testing is performed using the
Kolmogorov-Smirnov Test and the Levene median tests. The results of
all outcome measures are compared, significance (p<0.05) for
each treatment group compared to baseline values with one-way
repeated measure analysis of variance and subsequently with a post
hoc multiple comparison test (Student-Newman-Keuls test) using
Sigma Stat Software (Systat Software, Inc., San Jose, Calif.). In
addition, the test and control groups are compared with two-way
repeated measure analysis of variance.
Example 34
Clinical Assessments of Dry Eyes
[0683] According to additional aspects of the present invention,
clinical screening procedures are effectuated, based on health
history of both eyes by observing redness, pain, irritation, visual
change, and compliance. As a subjective assessment, each subject
are asked to complete the Ocular Surface Disease Index Dry Eye
Questionnaire at each visit. The OSDI uses 12 questions to address
the subject's symptoms and visual impact. For each question, the
index utilizes a grading scale of 0-4 as follows: 0 if none of the
time; 1 if some of the time; 2 if half of the time; 3 if most of
the time; 4 if all of the time.
[0684] As a subjective assessment, each subject is asked to
complete a Snellen VA measurement at 20 feet, as well as undergo a
slit lamp examination to assess corneal staining with sodium
fluorescein, conjunctival staining with lissamine green and bulbar
conjunctival erythema. All grading is using standard grading scales
of 0-4 as described below. In addition, tearscope examination using
Keeler Tearscope is used to measure the height of the inferior tear
meniscus, tear break up time and thickness of tear film. In the
absence of any historical or clinical (subjective/objective)
abnormalities, participation in the study is continued.
Table 14: Brief Noninvasive Clinical Assessment Schedule Study for
Visits 1-7
[0685] 1. Vision Snellen distance acuity at 20 feet, Room #6, B1,
Aston Center [0686] 2. Questionnaire Ocular Surface Disease Index
[0687] 3. Ocular Surface Corneal staining (0-4) [0688] Bulbar
conjunctival staining (0-4) [0689] Bulbar conjunctival erythema
(0-4) [0690] 4. Tear Film Height of inferior tear meniscus* [0691]
Tear break up time* [0692] Tear film thickness* *Height of inferior
tear meniscus, tear break up time and tear film thickness is
assessed using a Tearscope. Corneal staining: The extent of corneal
staining by sodium fluorescein is graded according to the scale
below for each quadrant: 1 central, 2 superior, 3 temporal, 4
nasal, 5 inferior. 0: No staining
1: Trace
2: Mild
3: Moderate
4: Severe
[0693] Conjunctival staining: The extent of bulbar conjunctival
staining by lissamine green is graded according to the scale below
for each quadrant as show in FIG. 122A: 0: No staining
1: Trace
2: Mild
3: Moderate
4: Severe
[0694] Erythema: The extent of conjunctival erythema is assessed
according to the scale below for each quadrant as show in FIG.
122B: 0: No redness [0695] 1: Trace [0696] 2: Mild [0697] 3:
Moderate [0698] 4: Severe
[0699] Tearscope: A Keeler Tearscope (Keeler, Broomall, Pa.) is
used to analyze tear interference patterns using white light. For
image acquisition, a digital camera, or other image capturing
device, is coupled to a computer will be used to record changes in
tear interference patterns. The digitally recorded images are
analyzed to assess the lipid tear pattern, the stability of the
tear film, and the tear film thickness.
[0700] Stability of the tear film is assessed non-invasively by
measurements of tear break up time. For tear film thickness, a
computer generated (ADOBE Photoshop, ADOBE Systems, San Jose,
Calif.) thickness chart using a map-color intensity scale is used
to covert light reflectance into nm.
Example 35
Therapy for Allergy Conjunctiva
[0701] Allergen challenge will be done using our standard protocol
to confirm the allergic subject's level of reactivity according to
Table 15
TABLE-US-00014 TABLE 15 Allergen challenge protocol Allergen
Relative Concentration Quantitative Concentration Cat Dander Low
2500 AU/ml Medium 5000 AU/ml High 10,000 AU/ml Dust Mite Low 2500
AU/ml Medium 5000 AU/ml High 10,000 AU/ml Grass Pollen Low 10,000
PNU/ml Medium 20,000 PNU/ml High 40,000 PNU/ml Ragweed Pollen Low
10,000 PNU/ml Medium 20,000 PNU/ml High 40,000 PNU/ml
[0702] According to the concentration chart, the investigator
determines the allergen threshold dose starting with the lowest
dose of allergen and titrating to the medium and then highest dose
at 15-minute intervals until a positive response is achieved as
follows:
[0703] 1. one drop of allergen in the required concentration is
instilled into the conjunctival sac of each eye.
[0704] 2. At 5, 10, 15, and 20 minutes following challenge, the eye
symptom assessments (itching and redness only) is performed.
[0705] 3. For the symptom of itching in each eye, if a score of
greater than or equal to 2+ on the severity scale is obtained using
the Low concentration of allergen on at least one of the time
points (5, 10, 15, and 20 minutes), this is considered a positive
response. Both eyes do not need to obtain a positive response at
the same time point.
[0706] 4. For the symptom of redness in each eye, if a score of
greater than or equal to 2+ on the severity scale is obtained using
the Low concentration of allergen on at least one of the time
points (5, 10, 15, and 20 minutes), this is considered a positive
response. Both eyes do not need to obtain a positive response at
the same time point.
[0707] 5. If a negative response is obtained using the Low
concentration of allergen, i.e. less than 2+ on the itching and
redness severity scale, then the challenge is repeated as above at
the medium, and if necessary, at the high concentration of the
allergen. Once a positive result is obtained, then this
concentration is recorded as the appropriate allergen dose, and
used at all subsequent visits.
[0708] Standardized extracts of cat dander, dust mite, grass pollen
or ragweed pollen is used. Three different concentrations of
allergen may be used in order to establish the lowest concentration
which elicits a moderate conjunctival allergic response.
Conjunctival erythema, edema, and itching is graded on a scale of 0
to 4 according to the following scale:
Edema:
0=None.
[0709] 1=Mild: slight lower or upper lid swelling. 2=Moderate:
noticeable swelling of lower and/or upper lids. 3=Severe: visible
swelling; difficult for patient to open eyes. 4=Very Severe: eyes
are swollen shut.
Erythema:
0=None.
[0710] 1=Mild: slightly dilated blood vessels; color of vessels is
typically pink. 2=Moderate: more apparent dilation of blood
vessels: vessel color is more intense (redder) 3=Severe: numerous
and obvious dilated blood vessels; the color is deep red. 4=Very
Severe: large, numerous dilated blood vessels characterized by
severe deep red color. Itching, as evaluated by patient
0=None.
[0711] 1=Mild: an intermittent tickle sensation involving more than
just the corner of the eye. 2=Moderate: a mild continuous itch (can
be localized) without desire to rub. 3=Severe: a severe itch with
the desire to rub. 4=Very Severe: an incapacitating itch with an
irresistible urge to rub.
Example 36
Assessment of Allergic Conjunctivitis
[0712] According to additional aspects of the present invention,
the allergen concentration established during Visit 1 is used.
Evaluation of conjunctival erythema, edema, and itching is measured
before and after allergen challenge. In order to determine the
rapidity of onset, evaluations are done before and after
instillation of active eye drops and vehicle. In one group of 20
subjects, eyes are randomized to receive either one drop of active
eye drop or one drop of vehicle twice a day for two days, and again
10 minutes before allergen challenge. In a second group of 20
subjects, eyes are randomized to receive either one drop of active
eye drop, or one drop of vehicle, 10 minutes before allergen
challenge. The allergen determined at Visit 1 or 2 is instilled in
both eyes. Erythema, edema, and itching is evaluated at 5 minute
intervals for 20 minutes following instillation of the
allergen.
[0713] In order to determine the duration of action, evaluations
are done before and after instillation of active eyedrops and
vehicle. In the first group of 20 subjects, eyes are randomized to
receive either one drop of active eye drop or one drop of vehicle
twice a day for two days, and again 8 hours before allergen
challenge. In the second group of 20 subjects, eyes are randomized
to receive either one drop of active eye drop, or one drop of
vehicle, 8 hours before allergen challenge. Subjects leave the
clinic and return 8 hours later. When the subject returns, allergen
is instilled in both eyes. Erythema, edema, and itching are
evaluated at 5 minute intervals for 20 minutes following
instillation of allergen.
[0714] Assessments will be conducted as follows:
Visit 1 (Day 0):
[0715] Obtain informed consent. Obtain medical, ocular, and
allergic history (to include concomitant medications). If female of
childbearing potential, perform urine pregnancy test. Perform
baseline slit lamp examination. Perform baseline allergen challenge
titrating as needed to obtain positive response (2+ itching and
erythema). Instruct patient regarding post-challenge use of rescue
medications. Schedule patient to return in one week.
Visit 2 (day 7+/-2)
[0716] Update history and query for adverse events. Update
medication history. Perform slit lamp exam. Evaluation of
conjunctival erythema, edema, and itching before and after allergen
challenge on a scale of 0-4. Perform allergen challenge with
threshold dose determined at Visit 1. Randomization: Group I (20
subjects) eyes will be randomized to receive either one drop of
active eye drop or one drop of vehicle twice a day for two days
prior to visit 3. Instruct Group I subjects regarding use of study
eye drops. Instruct patient regarding post-challenge use of rescue
medications. Schedule patient to return in one week.
Visit 3 (Day 14+/-2)
[0717] Update history and query for adverse events. Update
medication history. Perform slit lamp exam. Evaluation of
conjunctival erythema, edema, and itching before and after
instillation of active eye drops and vehicle on a scale of 0-4.
Randomization: Group I: (20 subjects) eyes receive an additional
drop of active eye drop or an additional drop of vehicle 10 minutes
before allergen challenge. Group II: (20 subjects) eyes are
randomized to receive either one drop of active eye drop or one of
the vehicle 10 minutes before challenge. Instill study eyedrops for
Groups I and II. Perform allergen challenge with threshold dose
determined at Visits 1 and 2 ten minutes after instillation of
study eyedrops. Record findings at 5 minute intervals for 20
minutes. Evaluate which drop was preferred by the subject.
Randomization: Group I (20 subjects) eyes are randomized to receive
either one drop of active eye drop or one drop of vehicle twice a
day for two days prior to visit 4 and again 8 hours prior to
allergen challenge. Group II (20 subjects) eyes are randomized to
receive either one drop of active eye drop or one drop of vehicle 8
hours before allergen challenge. Instruct patient regarding use of
study eye drops. Instruct patient regarding post-challenge use of
rescue medications. Schedule patient to return in one week.
Visit 4 (Day 21+/-)
[0718] Update history and query for adverse events. Update
medication history. Perform slit lamp exam. Evaluation of
conjunctival erythema, edema, and itching before and after
instillation of active eye drops and vehicle on a scale of 0-4.
Perform allergen challenge with threshold dose determined at Visits
1 and 2 eight hours after instillation of study eyedrops. Record
findings at 5 minute intervals for 20 minutes. Evaluate which drop
was preferred by the subject. Instruct patient regarding
post-challenge use of rescue medications. Exit patient from
study.
Example 26
RNS-60 was Shown by Fluorescence-Activated Cell Sorting (FACS)
Analysis to have a Pronounced Effect on Expression of Cell Surface
Receptors: CD193 (CCR3); CD154 (CD40L); CD11B; and CD3
[0719] Overview.
[0720] Applicants used Fluorescence-Activated Cell Sorting (FACS)
analysis to compare the levels of expression of cell surface
receptors, CD193 (CCR3); CD154 (CD40L); CD11B; and CD3, on white
blood cells incubagted with either the inventive electrokinetic
fluid (RNS-60) or normal saline control fluid.
Methods:
[0721] Ficoll-hypaque separated PBMC (apheresis--All Cells)
preincubated approximately 1 hour in 30% solutions of RNS60 or
Normal Saline (NS);
[0722] PBMC activated with 2 .mu.g/ml of PHA-L for 24 or 40
hours;
[0723] Cells collected and washed into blocking/staining buffer,
stained and fixed; and
[0724] Cells were analyzed by flow cytometry.
Results:
[0725] With respect to CD193 (CCR3), the receptor is substantially
down-regulated in the presence of RNS-60 when compared to the level
of the receptor expression in the normal saline contol. This down
regulation affects the phosphorylation of MAPK p38 which in turn
down-regulates eotaxin (e.g., see Example 13) which in turn down
regulates IL 5 and as well alters eosinophil counts (e.g., see
Example 13), which is one of the factors that, that example, alters
the bronchoconstrictive response.
[0726] As discussed above in Example 13 in the context of the
ovalbumin challenge model, RNS-60 decreased the serum eotaxin
levels in the OVA challenged groups when compared to the effect of
normal saline. Therefore, according to particular aspects, RNS-60
has the potential to decrease both the ligand eotaxin and its
receptor CCR3.
[0727] The CD154 (CD40L), CD11B, and CD3 receptors all were
down-regulated in the presence of RNS-60 when compared to the level
of the receptor expression in normal saline. Thus, according to
particular aspects, RNS-60 has the potential to decrease the CD154
(CD40L), CD11B, and CD3 receptors.
Example 27
RNS60, but not Normal Saline (NS), Attenuated the Activation of
NF.kappa.B in MBP-Primed T Cells
[0728] This Example shows that RNS60, but not normal saline (NS),
attenuated the activation of NF.kappa.B in MBP-primed T cells.
According to particular aspects, therefore, the present
electrokinetically-generated fluids have substantial utility for
treating inflammation and inflammation-mediated conditions and
diseases, including but not limited to, diabetes and related
metabolic disorders, insulin resistance, neurodegenerative diseases
(e.g., M. S., Parkinson's, Alzheimer's, etc), asthma, cystic
fibrosis, vascular/coronary disease, retinal and/or macular
degeneration, digestive disorders (e.g., inflammatory bowel
disease, ulcerative colitis, Crohn's, etc.).
Overview:
[0729] It is increasing clear that inhibition of insulin receptor
signaling pathways is a central mechanism through which
inflammatory and stress responses mediate insulin resistance (see,
e.g., review by Wellen & Hotamisligil, The Journal of Clinical
Investigation, 115:1111-1119, 2005).
[0730] Overlap of Metabolic and Immune Pathways.
[0731] Several serine/threonine kinases are activated by
inflammatory or stressful stimuli and contribute to inhibition of
insulin signaling, including JNK, inhibitor of NF-.kappa.B kinase
(IKK), and PKC-.theta. (Zick, Y. 2003. Role of Ser/Thr kinases in
the uncoupling of insulin signaling. Int. J. Obes. Relat. Metab.
Disord. 27(Suppl. 3):556-560). Again, the activation of these
kinases in obesity highlights the overlap of metabolic and immune
pathways; these are the same kinases, particularly IKK and JNK,
that are activated in the innate immune response by Toll-like
receptor (TLR) signaling in response to LPS, peptidoglycan,
double-stranded RNA, and other microbial products (Medzhitov, R.
2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol.
1:135-145). Hence it is likely that components of TLR signaling
pathways will also exhibit strong metabolic activities.
[0732] PKC and IKK are activated by cellular lipid metabolites. Two
other inflammatory kinases that play a large role in counteracting
insulin action, particularly in response to lipid metabolites, are
IKK and PKC-.theta.. Lipid infusion has been demonstrated to lead
to a rise in levels of intracellular fatty acid metabolites, such
as diacylglycerol (DAG) and fatty acyl CoAs. This rise is
correlated with activation of PKC-.theta. and increased Ser307
phosphorylation of IRS-1 (Yu, C., et al. 2002. Mechanism by which
fatty acids inhibit insulin activation of insulin receptor
substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase
activity in muscle. J. Biol. Chem. 277:50230-50236). PKC-.theta.
may impair insulin action by activation of another serine/threonine
kinase, IKK.beta., or JNK (Perseghin, G., Petersen, K., and
Shulman, G. I. 2003. Cellular mechanism of insulin resistance:
potential links with inflammation. Int. J. Obes. Relat. Metab.
Disord. 27(Suppl. 3):S6-S11). Other PKC isoforms have also been
reported to be activated by lipids and may also participate in
inhibition of insulin signaling (Schmitz-Peiffer, C. 2002. Protein
kinase C and lipid-induced insulin resistance in skeletal muscle.
Ann. N. Y. Acad. Sci. 967:146-157).
[0733] IKK.beta. can impact insulin signaling by activating
NF-.kappa.B. IKK.beta. can impact on insulin signaling through at
least 2 pathways. First, it can directly phosphorylate IRS-1 on
serine residues (Yin, M. J., Yamamoto, Y., and Gaynor, R. B. 1998.
The anti-inflammatory agents aspirin and salicylate inhibit the
activity of I.kappa.B kinase-.beta.. Nature. 396:77-80, Gao, Z., et
al. 2002. Serine phosphorylation of insulin receptor substrate 1 by
inhibitor kappa B kinase complex. J. Biol. Chem.
277:48115-48121).
[0734] Second, it can phosphorylate inhibitor of NF-.kappa.B
(I.kappa.B), thus activating NF-.kappa.B, a transcription factor
that, among other targets, stimulates production of multiple
inflammatory mediators, including TNF-.alpha. and IL-6 (Shoelson,
S. E., Lee, J., and Yuan, M. 2003. Inflammation and the
IKK.beta./I.kappa.B/NF-.kappa.B axis in obesity- and diet-induced
insulin resistance. Int. J. Obes. Relat. Metab. Disord. 27(Suppl.
3):549-552). Mice heterozygous for IKK.beta. are partially
protected against insulin resistance due to lipid infusion,
high-fat diet, or genetic obesity (Yuan, M., et al. 2001. Reversal
of obesity- and diet induced insulin resistance with salicylates or
targeted disruption of IKK.beta. Science. 293:1673-1677; Kim, J.
K., et al. 2001. Prevention of fat-induced insulin resistance by
salicylate. J. Clin. Invest. 108:437-446;
doi:10.1172/JCI200111559).
[0735] Moreover, inhibition of IKK.beta. in human diabetics by
high-dose aspirin treatment also improves insulin signaling,
although at this dose, it is not clear whether other kinases are
also affected (Hundal, R. S., et al. 2002. Mechanism by which
high-dose aspirin improves glucose metabolism in type 2 diabetes.
J. Clin. Invest. 109:1321-1326. doi:10.1172/JCI200214955). Recent
studies have also begun to tease out the importance of IKK in
individual tissues or cell types to the development of insulin
resistance. Activation of IKK in liver and myeloid cells appears to
contribute to obesity-induced insulin resistance, though this
pathway may not be as important in muscle (Cai, D., et al. 2005.
Local and systemic insulin resistance resulting from hepatic
activation of IKK.beta. and NF-.kappa.B. Nat. Med. 11:183-190;
Arkan, M. C., et al. 2005. IKK.beta. links inflammation to
obesity-induced insulin resistance. Nat. Med. 11:191-198; and Rohl,
M., et al. 2004. Conditional disruption of I.kappa.B kinase 2 fails
to prevent obesity-induced insulin resistance. J. Clin. Invest.
113:474-481; doi:10.1172/JCI200418712).
[0736] Methods.
[0737] For the electrophoretic mobility shift assay (EMSA), T cells
isolated from MBP-immunized mice were re-primed with MBP and after
24 h, cells received different concentrations of RNS60 and NS.
After 2 h of treatment, DNA-binding activity of NF-.kappa.B was
monitored in nuclear extracts by EMSA.
[0738] For the luciferase experiments, T cells isolated from
MBP-immunized mice were transfected with PBIIX-Luc, an NF-.kappa.B
dependent reporter construct, followed by repriming with MBP. After
24 h of MBP priming, cells were treated with different
concentrations of RNS60 and NS for 2 h followed by assay of
luciferase activity in total cell extracts by a luciferase assay
kit (Promega). In other cases, MBP-primed T cells were also
stimulated with 30 nM PMA for 1 h. In these cases, PMA was added
after 1 h of pretreatment with RNS60 and NS. Results are mean+SD of
three different experiments.
[0739] Results.
[0740] The results show that RNS60, but not normal saline (NS),
attenuated the activation of NF-.kappa.B in MBP-primed T cells in a
dose-responsive manner.
[0741] According to particular aspects, therefore, the disclosed
electrokinetically-generated fluids have substantial utility for
treating inflammation and inflammation-mediated conditions and
diseases, including but not limited to, diabetes and related
metabolic disorders, insulin resistance, neurodegenerative diseases
(e.g., M. S., Parkinson's, Alzheimer's, etc), asthma, cystic
fibrosis, vascular/coronary disease, retinal and/or macular
degeneration, digestive disorders (e.g., inflammatory bowel
disease, ulcerative colitis, Crohn's, etc.).
INCORPORATION BY REFERENCE
[0742] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications, and non-patent publications referred to in
this specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0743] It should be understood that the drawings and detailed
description, herein are to be regarded in an illustrative rather
than a restrictive manner, and are not intended to limit the
invention to the particular forms and examples disclosed. On the
contrary, the invention includes any further modifications,
changes, rearrangements, substitutions, alternatives, design
choices, and embodiments apparent to those of ordinary skill in the
art, without departing from the spirit and scope of this invention,
as defined by the following claims. Thus, it is intended that the
following claims be interpreted to embrace all such further
modifications, changes, rearrangements, substitutions,
alternatives, design choices, and embodiments.
[0744] The foregoing described embodiments depict different
components contained within, or connected with, different other
components. It is to be understood that such depicted architectures
are merely exemplary, and that in fact many other architectures can
be implemented which achieve the same functionality. In a
conceptual sense, any arrangement of components to achieve the same
functionality is effectively "associated" such that the desired
functionality is achieved. Hence, any two components herein
combined to achieve a particular functionality can be seen as
"associated with" each other such that the desired functionality is
achieved, irrespective of architectures or intermedial components.
Likewise, any two components so associated can also be viewed as
being "operably connected," or "operably coupled," to each other to
achieve the desired functionality.
[0745] While particular embodiments of the present invention have
been shown and described, it will be obvious to those skilled in
the art that, based upon the teachings herein, changes and
modifications may be made without departing from this invention and
its broader aspects and, therefore, the appended claims are to
encompass within their scope all such changes and modifications as
are within the true spirit and scope of this invention.
Furthermore, it is to be understood that the invention is solely
defined by the appended claims. It will be understood by those
within the art that, in general, terms used herein, and especially
in the appended claims (e.g., bodies of the appended claims) are
generally intended as "open" terms (e.g., the term "including"
should be interpreted as "including but not limited to," the term
"having" should be interpreted as "having at least," the term
"includes" should be interpreted as "includes but is not limited
to," etc.). It will be further understood by those within the art
that if a specific number of an introduced claim recitation is
intended, such an intent will be explicitly recited in the claim,
and in the absence of such recitation no such intent is present.
For example, as an aid to understanding, the following appended
claims may contain usage of the introductory phrases "at least one"
and "one or more" to introduce claim recitations. However, the use
of such phrases should not be construed to imply that the
introduction of a claim recitation by the indefinite articles "a"
or "an" limits any particular claim containing such introduced
claim recitation to inventions containing only one such recitation,
even when the same claim includes the introductory phrases "one or
more" or "at least one" and indefinite articles such as "a" or "an"
(e.g., "a" and/or "an" should typically be interpreted to mean "at
least one" or "one or more"); the same holds true for the use of
definite articles used to introduce claim recitations. In addition,
even if a specific number of an introduced claim recitation is
explicitly recited, those skilled in the art will recognize that
such recitation should typically be interpreted to mean at least
the recited number (e.g., the bare recitation of "two recitations,"
without other modifiers, typically means at least two recitations,
or two or more recitations). Accordingly, the invention is not
limited except as by the appended claims.
* * * * *
References