Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TREATING THYMIC STROMAL
LYMPHOPOIETIN (TSLP)-MEDIATED CONDITIONS
FIELD OF THE INVENTION
The present invention relates generally to thymic stromal lymphopoietin (TSLP)
and
TSLP-mediated conditions, and more particularly to TSLP and TSLP receptor-
mediated
conditions (e.g., disorders of the immune system, allergic inflammation,
allergic airway
inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis,
atopic eczema,
allergic asthma, asthma, obstructive airways disease, chronic obstructive
pulmonary disease, and
food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-
mediated disorders,
and rhino-conjunctivitis). Particularly preferred aspects relate to modulation
(e.g., treating) of
TSLP and TSLP receptor-mediated conditions, by administering a therapeutic
composition
comprising at least one electrokinetically generated fluid (including gas-
enriched (e.g., oxygen
enriched) electrokinetically generated fluids) as disclosed herein, and in
further combination
therapy aspects with administration of such electrokinetic fluids in
combination with at least one
additional therapeutic agent.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to United States Provisional
Patent
Application Serial Nos. 61/107,480, and 61/107,453, both filed 22 October
2008, and to United
States Utility Patent Application Serial No. 12/258,210, filed 24 October
2008, both
incorporated here in by reference in their entirety.
BACKGROUND
Thymic stromal lymphopoietin (TSLP). Thymic stromal lymphopoietin (TSLP) is an
IL-7-like cytokine that triggers dendritic cell-mediated Th2-type inflammatory
responses and is
considered as a master switch for allergic inflammation. TSLP is an integral
growth factor to
both B and T cell development and maturation. Particularly, murine TSLP
supports B
lymphopoieses and is required for B cell proliferation. Murine TSLP plays a
crucial role in
controlling the rearrangement of the T cell receptor-gamma (TCRy) locus and
has a substantial
stimulatory effect on thymocytes and mature T cells. See, for example, Friend
et al., Exp.
Hematol., 22:321-328, 1994; Ray et al., Eur. J. Immunol., 26:10-16, 1996;
Candeias et al.,
Immunology Letters, 57:9-14, 1997.
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TSLP possesses cytokine activity similar to IL-7. For instance, TSLP can
replace IL-7 in
stimulating B cell proliferation responses (Friend et al., supra). Although
TSLP and IL-7
mediate similar effects on target cells, they appear to have distinct
signaling pathways and likely
vary in their biologic response. For Example, although TSLP modulates the
activity of STAT5,
it fails to activate any Janus family tyrosine kinase members (Levin et. al.,
J. Immunol.,
162:677-683, 1999).
TSLP effects on dendritic cells and TNF production. After human TSLP and the
human TSLP receptor were cloned in 2001, it was discovered that human TSLP
potently
activated immature CD1lc+ myeloid dendritic cells (mDCs) (see, e.g., Reche et
al., J. Immunol.,
167:336-343, 2001 and Soumelis et al., Nat. Immunol., 3:673-680, 2002). Th2
cells are
generally defined in immunology textbooks and literature as CD4+ T cells that
produce IL-4, IL-
5, IL-13, and IL-10. And Thl cells such as CD4+ T cells produce IFN-y and
sometimes TNF.
When TSLP-DCs are used to stimulate naive allogeneic CD4+ T cells in vitro, a
unique type of
Th2 cell is induced which produces the classical Th2 cytokines IL-4, IL-5, and
IL-13, and large
amounts of TNF, but little or no IL-10 or interferon-y (Reche et al., Supra)
(see also, e.g.,
Soumelis et al., Nat. Immunol., 3:673-680, 2002). TNF is not typically
considered a Th2
cytokine. However, TNF is prominent in asthmatic airways and genotypes that
correlate with
increased TNF secretion are associated with an increased asthma risk. See Shah
et al., Clin.
Exp. Allergy., 25:1038-1044, 1995 and Moffatt, M.F. and Cookson, W.O., Hum.
Mol. Genet.,
6:551-554, 1997.
TSLP induces human mDCs to express the TNF superfamily protein OX40L at both
the
mRNA and protein level (Ito et al., J. Exp. Med., 202:1213-1223). The
expression of OX40L by
TSLP-DCs is important for the elaboration of inflammatory Th2 cells. Thus,
TSLP-activated
DCs create a Th2-permissive microenvironment by up-regulating OX40L without
inducing the
production of Thl-polarizing cytokines. Id.
TSLP expression, allergen-specific responses and asthma. Early studies have
shown
that TSLP mRNA was highly expressed by human primary skin keratinocytes,
bronchial
epithelial cells, smooth muscle cells, and lung fibroblasts (Soumelis et al.,
Nat. Immunol., 3:673-
680, 2002). Because TSLP is expressed mainly in keratinocytes of the apical
layers of the
epidermis, this suggests that TSLP production is a feature of fully
differentiated keratinocytes.
TSLP expression in patients with atopic dermatitis was associated with
Langerhans cell
migration and activation in situ which suggests that TSLP may contribute
directly to the
activation of these cells which could subsequently migrate into the draining
lymph nodes and
prime allergen-specific responses. Id. In a more recent study, it was shown by
in situ
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hybridization that TSLP expression was increased in asthmatic airways and
correlated with both
the expression of Th2-attracting chemokines and with disease severity which
provided a link
between TSLP and asthma (Ying et al., J. Immunol., 174:8183-8190, 2005).
TSLP receptor (TSLPR) and allergic asthma. The TSLP receptor (TSLPR) is
approximately 50 kDa protein and has significant similarity to the common y-
chain. TSLPR is a
novel type 1 cytokine receptor, which, combined with IL-7Ra (CD127),
constitutes a TSLP
receptor complex as described, for example, in Pandey et al., Nat. Immunol.,
1:59-64, 2000.
TSLPR has a tyrosine residue near its carboxyl terminus, which can associate
with
phosphorylated STAT5 and mediate multiple biological functions when engaged
with TSLP
(Isaksen et al., J. Immunol., 168:3288-3294, 2002).
Human TSLPR is expressed by monocytes and CDllc+ dendritic cells, and TSLP
binding induces the expression of the TH2 cell-attracting chemokines CCL17 and
CCL22.
Furthermore, as stated above, the TSLPR-induced activation of dendritic cells
indirectly results
in the increased secretion of TH2 cytokines IL-4, -5 and -13, which may be
necessary for the
regulation of CD4+ T cell homeostasis. In mice, deficiency of TSLPR has no
effect on
lymphocyte numbers. However, a deficiency of TSLPR and common y-chain results
in fewer
lymphocytes as compared to mice deficient in the common y-chain alone. See
Reche et al., J.
Immunol., 167:336-343, 2001 and Soumelis et al., Nat. Immunol., 3:673-680,
2002.
Studies have found that TSLP and the TSLPR play a critical role in the
initiation of
allergic diseases in mice. In one study, it was demonstrated that mice
engineered to overexpress
TSLP in the skin developed atopic dermatitis which is characterized by
eczematous skin lesions
containing inflammatory infiltrates, a dramatic increase in circulating Th2
cells and elevated
serum IgE (Yoo et al., J. Exp. Med., 202:541-549, 2005). The study suggested
that TSLP may
directly activate DCs in mice. In another study, conducted by Li et al., the
group confirmed that
transgenic mice overexpressing TSLP in the skin developed atopic dermatitis
which solidifies
the link between TSLP and the development of atopic dermatitis.
Another set of studies demonstrated that TSLP is required for the initiation
of allergic
airway inflammation in mice in vivo. In one study, Zhou et al. demonstrated
that lung specific
expression of a TSLP transgene induced allergic airway inflammation (asthma)
which is
characterized by massive infiltration of leukocytes (including Th2 cells),
goblet cell hyperplasia,
and subepithelial fibrosis, and increased serum IgE levels (Zhou et al., Nat.
Immunol., 6:1047-
1053, 2005). However, in contrast, mice lacking the TSLPR failed to develop
asthma in
response to inhaled antigens (Zhou et al., supra and Al-Shami et al., J. Exp.
Med., 202:829-839,
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2005). Thus, these studies together demonstrate that TSLP is required for the
initiation of
allergic airway inflammation in mice.
Further, in a study conducted by Yong-Jun et al., it was demonstrated that
epithelial cell-
derived TSLP triggers DC-mediated inflammatory Th2 responses in humans which
suggest that
TSLP represents a master switch of allergic inflammation at the epithelial
cell-DC interface
(Yong-Jun et al., J. Exp. Med., 203:269-273, 2006).
In a recent study, it was shown that modulation of DCs function by inhibiting
TSLPR
lessened the severity in mice (Liyun Shi et al., Clin. Immunol., 129:202-210,
2008). In another
set of studies, it was demonstrated that TSLPR was not only expressed in DCs,
but also on
macrophages, mast cells, and CD4+ T cells (Rochman et al., J. Immunol.,
178:6720-6724, 2007
and Omori M. and Ziegler S., J. Immunol., 178:1396-1404, 2007). In order to
rule out the direct
effects of TSLPR neutralization on CD4+ T cells or other effector cells in
allergic inflammation,
Liyun Shi et al. performed experiments wherein OVA-loaded DCs were in vitro
treated with
anti-TSLPR before adoptive transfer to the airways of naive mice. It has
previously been found
that OVA-DCs triggered strong eosinophilic airway inflammation and accompanied
with
massive production of Th2 cytokines such as IL-4 and IL-5 (Sung et al., J.
Immunol., 166:1261-
1271 and Lambrecht et al., J. Clin. Invest., 106:551-559, 2000). However,
pretreating OVA-
DCs with anti-TSLPR resulted in a significant reduction of eosinophils and
lymphocyte
infiltration as well as IL-4 and IL-5 levels, further illuminating the role
that TSLPR plays in DC-
primed allergic disease. This result also supports that blocking of TSLPR on
DCs will aid in
controlling airway inflammation (Liyun Shi et al., supra).
There has been a growing body of experiments implicating the role of
TSLP/TSLPR in
various physiological and pathological processes. Physiological roles of TSLP
include
modulating the immune system, particularly in stimulating B and T cell
proliferation,
development, and maturation. TSLP plays a vital role in the pathobiology of
allergic asthma and
local antibody mediated blockade of TSLP receptor function to alleviate
allergic diseases. Thus,
interplay between TSLP and TSLP receptor is believed to be important in many
physiological
disease processes such as: allergic inflammation, skin lesions of patients
with atopic dermatitis
or atopic eczema, allergic asthma and asthma.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 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 normal saline (NS) had only a
marginal
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effect. Additionally, the non-electrokinetic control pressure pot fluid PP60
resulted in
approximately 50% reduction in DEP induced TSLP receptor expression.
Figure 2 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. Additionally, the non-electrokinetic control pressure pot fluid PP60
resulted in
approximately 30% reduction in DEP-induced cell surface attached MMP9 levels.
Figures 3 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.
Figures 4 A-C show, in relation to the experiments relating to Figures 3 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)).
Figures 5 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).
Figures 6 A-D show, in relation to the experiments relating to Figures 5 A-D,
the graphs
resulting from the subtraction of the CsCl current data (shown in Figure 5)
from the 20 mM
CaC12 (diamonds) and 40 mM CaC12 (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.).
Figures 7A and B demonstrate the results of a patch clamp experiment that
assessed the
effects of diluting the electrokinetically generated fluid (e.g., RNS-60) on
epithelial cell
membrane polarity and ion channel activity.
SUMMARY OF EXEMPLARY EMBODIMENTS
Particular aspect provide a method for treating a TSLP-mediated or TSLPR-
mediated
disease or condition, comprising administration to a mammal 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
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diameter of less than about 100 nanometers and stably configured in the ionic
aqueous fluid in
an amount sufficient for treating a TSLP-mediated or TSLPR-mediated disease or
condition. 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 certain method aspects, 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 certain 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.
In particular method aspects, the ionic aqueous solution comprises a saline
solution. In
certain aspects, the electrokinetically-altered aqueous fluid is
superoxygenated.
In particular method aspects, the electrokinetically-altered aqueous fluid
comprises a
form of solvated electrons.
In certain aspects, alteration of the electrokinetically-altered aqueous fluid
comprises
exposure of the fluid to hydrodynamically-induced, localized electrokinetic
effects. In certain
embodiments, exposure to the localized electrokinetic effects comprises
exposure to at least one
of voltage pulses and current pulses. In particular aspects, 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.
In particular aspects, the TSLP-mediated or TSLPR-mediated disease or
condition
comprises a disease or disorder of the immune system, including but not
limited to allergic
inflammation. In particular aspects, the allergic inflammation comprises at
least one of allergic
airway inflammation, DC-mediated inflammatory Th2 responses, atopic
dermatitis, atopic
eczema, asthma, obstructive airways disease, chronic obstructive pulmonary
disease, IgE-
mediated disorders, rhino-conjunctivitis and food allergies. In certain
embodiments, the TSLP-
mediated or TSLPR-mediated disease or condition comprises inflammatory
arthritis, for
example comprising at least one of rheumatoid arthritis and psoriasis.
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In certain aspects, the method further comprises combination therapy, wherein
at least
one additional therapeutic agent is administered to the patient. In particular
embodiments, the at
least one additional therapeutic agent is selected from the group consisting
of short-acting 13,-ag;mists, long-acting 132-agomsts, anticholrnergics,
corticosteroids, systemic corticosteroids.
mast cell stabilizers, leukotriene modifiers, methyixanthines, and
combinations thereof. In
certain aspects, the at least one additional therapeutic agent is selected
from the group consisting
of: bronchodilators consisting of Ok-agonists including albuterol,
levalhuterol, pirbuterol,
artformoterol, formoterol, salmetero , and anticholinergics such as
ipratropium and tiotropium;
corucosi;roids including beclomethasone, budesonide, f]unisohde. $luticasone,
rnornetasone,
triamcinoione, niethyprednisolone, prednisolone, prednisone; le rkotriene
modifiers including
rnontelukast, rafiriukast, and ziltniton; mast cell stabilizers including
cromolyn and nedocro.nril;
nmethvxanthines including theophyili e, combination drugs including
ipratropium and albuterol,
fluticasone and salmeterol, hudesonide and forraoterol; antihistamines
including hydroxyzine,
diphenhydramirle, 1oratadine, cetirizine, and hydrocortisone; immune system
modulating drug-,
including tacrolimus and phnecrolirnus: cyclosporine; azathioprine;
mycophenolatemofetil; and
corubinations thereof. In particular aspects, the at least one additional
therapeutic agent is a
TSLP and/or TSLPR antagonist, and in particular embodiments, the TSLP and/or
TSLPR
antagonist is 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.
In particular method aspects, modulation of at least one of cellular membrane
potential
and cellular membrane conductivity comprises altering at least one of cellular
membrane
structure or function comprising altering at least one of a conformation,
ligand binding activity,
and a catalytic activity of a membrane associated protein or constituent. 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, integrins, etc. In certain embodiments, the
transmembrane receptor
comprises a G-Protein Coupled Receptor (GPCR). In particular aspects, the G-
Protein Coupled
Receptor (GPCR) interacts with a G protein a subunit, for example, wherein the
G protein a
subunit comprises at least one selected from the group consisting of Gas ,
Gai, Gaq, and Ga12,
and in certain embodiments the at least one G protein a subunit is Gaq.
In particular method aspects, modulation of at least one of cellular membrane
potential
and cellular membrane conductivity comprises modulating whole-cell
conductance, for example,
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wherein modulating whole-cell conductance comprises modulating at least one of
a linear and a
non-linear voltage-dependent contribution of the whole-cell conductance.
In certain method aspects, modulation of at least one of cellular membrane
potential and
cellular membrane conductivity comprises modulation of a calcium dependant
cellular
messaging pathway or system. In certain method aspects, modulation of at least
one of cellular
membrane potential and cellular membrane conductivity comprises modulation of
phospholipase
C activity. In certain method aspects, modulation of at least one of cellular
membrane potential
and cellular membrane conductivity comprises modulation of adenylate cyclase
(AC) activity.
In certain method aspects, modulation of at least one of cellular membrane
potential and cellular
membrane conductivity comprises modulation of intracellular signal
transduction associated
with at least one condition or symptom selected from the group consisting of
diseases or
disorders of the immune system, allergic inflammation, allergic airway
inflammation, DC-
mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma,
obstructive
airways disease, chronic obstructive pulmonary disease, IgE-mediated
disorders, rhino-
conjunctivitis, food allergies, inflammatory arthritis, rheumatoid arthritis
and psoriasis.
Particular method aspects comprise administration of the electrokinetic fluid
to a cell
network or layer, and further comprise modulation of an intercellular junction
therein. In certain
embodiments, the intracellular junction comprises at least one selected from
the group consisting
of tight junctions, gap junctions, zona adherens and desmosomes. In particular
aspects, the cell
network or layers comprise at least one selected from the group consisting of
pulmonary
epithelium, bronchial epithelium, and intestinal epithelium.
In certain method aspects, the electrokinetically altered aqueous fluid is
oxygenated,
wherein 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.
In certain method aspects, the electrokinetically altered aqueous fluid
comprises at least
one of solvated electrons, and electrokinetically modified or charged oxygen
species, for
example, wherein 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 certain aspects, the electrokinetically altered aqueous
fluid comprises a form
of solvated electrons stabilized by molecular oxygen.
In certain aspects, the ability of the electrokinetically-altered fluid to
modulate at least
one of cellular membrane potential and cellular membrane conductivity persists
for at least two,
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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.
In certain aspects, the amount of oxygen present in charge-stabilized oxygen-
containing
nanostructures of the electrokinetically-altered 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.
In particular aspects, treating comprises administration by at least one of
topical,
inhalation, intranasal, and intravenous.
DETAILED DESCRIPTION
Provided ar=e methods for treating a TSLP-mediated or TSLPR-mediated disease
or
condition, comprising administration 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 a TSLP-mediated or TSLPR-
mediated disease
or condition. The charge-stabilized oxygen-containing nanostructures are
preferably stably
configured in the fluid in an amount sufficient to provide for modulation of
cellular membrane
potential and/or conductivity. Certain aspects comprising r )du labor or down-
reguuiation of
TSLP expression and/or activity have utility for treating TSLP-mediated or
TSLPR-mediated
diseases or conditions as disclosed herein (e.g., disorders of the immune
system, allergic
inflammation, allergic airway inflammation, DC-mediated inflammatory Th2
responses, atopic
dermatitis, atopic eczema, asthma, obstructive airways disease, chronic
obstructive pulmonary
disease, and food allergies, inflammatory arthritis, rheumatoid arthritis,
psoriasis, IgE-mediated
disorders, and rhino-conjunctivitis).
Electrokinetically-generated fluids:
"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
W02008/052143, 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).
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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:
In particular aspects, the electrokinetically altered aqueous fluid comprise
an ionic
aqueous solution of charge- stabilized oxygen-containing nano structures
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.
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.
In particular aspects, the electrokinetically altered aqueous fluids are
suitable to modulate
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.
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.
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).
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.
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."
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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.
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., Gas , Gai, Gaq , and Ga12).
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).
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 herein.
In particular aspects, the electrokinetically altered aqueous fluids display
synergy with
Albuterol, and with Budesonide as shown herein
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.
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.
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 herein.
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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).
Therefore, further aspects provide said electrokinetically-generated solutions
and
methods of producing an electrokinetically altered oxygenated aqueous fluid or
solution,
comprising: providing a flow of a fluid material between two spaced surfaces
in relative motion
and defining a mixing volume therebetween, wherein the dwell time of a single
pass of the
flowing fluid material within and through the mixing volume is greater than
0.06 seconds or
greater than 0.1 seconds; and introducing oxygen (02) into the flowing fluid
material within the
mixing volume under conditions suitable to dissolve at least 20 ppm, at least
25 ppm, at least 30,
at least 40, at least 50, or at least 60 ppm oxygen into the material, and
electrokinetically alter
the fluid or solution. In certain aspects, the oxygen is infused into the
material in less than 100
milliseconds, less than 200 milliseconds, less than 300 milliseconds, or less
than 400
milliseconds. In particular embodiments, the ratio of surface area to the
volume is at least 12, at
least 20, at least 30, at least 40, or at least 50.
Yet further aspects, provide a method of producing an electrokinetically
altered
oxygenated aqueous fluid or solution, comprising: providing a flow of a fluid
material between
two spaced surfaces defining a mixing volume therebetween; and introducing
oxygen into the
flowing material within the mixing volume under conditions suitable to infuse
at least 20 ppm,
at least 25 ppm, at least 30, at least 40, at least 50, or at least 60 ppm
oxygen into the material in
less than 100 milliseconds, less than 200 milliseconds, less than 300
milliseconds, or less than
400 milliseconds. In certain aspects, the dwell time of the flowing material
within the mixing
volume is greater than 0.06 seconds or greater than 0.1 seconds. In particular
embodiments, the
ratio of surface area to the volume is at least 12, at least 20, at least 30,
at least 40, or at least 50.
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
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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.
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., transition metal-based salts (e.g.,
Cr, Fe, Co, Ni, Cu, Zn,
etc.,), along with any suitable anion/counterion components). Particular
aspects comprise mixed
salt based electrokinetic fluids (e.g., Na, K, Ca, Mg, etc., in various
combinations and
concentrations). In particular aspects, the inventive electrokinetically-
altered fluids comprise
standard saline (e.g., approx. 0.9% NaCl, or about 0.15 M NaC1). 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 pS/cm, at least 40
S/cm, at least 80
pS/cm, at least 100 pS/cm, at least 150 pS/cm, at least 200 pS/cm, at least
300 pS/cm, or at least
500 pS/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 nano
structures) as
disclosed herein.
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 as, for
example, described
above, and/or 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.
Exemplary preferred embodiments:
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Particular aspect provide a method for treating a TSLP-mediated or TSLPR-
mediated
disease or condition, comprising administration to a mammal 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 a TSLP-mediated or TSLPR-mediated disease or
condition. 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 certain method aspects, 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 certain 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.
In particular method aspects, the ionic aqueous solution comprises a saline
solution. In
certain aspects, the electrokinetically-altered aqueous fluid is
superoxygenated.
In particular method aspects, the electrokinetically-altered aqueous fluid
comprises a
form of solvated electrons.
In certain aspects, alteration of the electrokinetically-altered aqueous fluid
comprises
exposure of the fluid to hydrodynamically-induced, localized electrokinetic
effects. In certain
embodiments, exposure to the localized electrokinetic effects comprises
exposure to at least one
of voltage pulses and current pulses. In particular aspects, 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.
In particular aspects, the TSLP-mediated or TSLPR-mediated disease or
condition
comprises a disease or disorder of the immune system, including but not
limited to allergic
inflammation. In particular aspects, the allergic inflammation comprises at
least one of allergic
airway inflammation, DC-mediated inflammatory Th2 responses, atopic
dermatitis, atopic
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eczema, asthma, obstructive airways disease, chronic obstructive pulmonary
disease, IgE-
mediated disorders, rhino-conjunctivitis and food allergies. In certain
embodiments, the TSLP-
mediated or TSLPR-mediated disease or condition comprises inflammatory
arthritis, for
example comprising at least one of rheumatoid arthritis and psoriasis.
In certain aspects, the method further comprises combination therapy, wherein
at least
one additional therapeutic agent is administered to the patient. In particular
embodiments, the at
least one additional therapeutic agent is selected from the group consisting
of short-acting 132_
agonists, long-acting 132,,-agonists, anticholinergics, corticosteroids,
systemic corticosteroids,
nmast cell stabilizers, leukotriene nmodifier_=s, rrr_ethylxanffiines, and
combinations thereof. In
certain aspects, the at least one additional therapeutic agent is selected
from the group consisting
of: bronchodilators consisting of Bz-agon_ists including a.lbuter_=ol,
leval_buterol, pirburterol,
art{ormote.rol, forrnoterol, sa(meterol, and antic_holinergics such as
ipratropiuln and tiotropium;
corticosteroids including beclomethasone, budesonide, flunisolide.
ffirticasone, mometasone.
triameinolone, methyprednisolone, prednisolone, prednisone; leukotriene
modifiers including
montelukast. zafirlukast, and zileuton; mast cell stabilizers including
cromolyn and nedocromil;
rnethylxanthines including theoplrylli e, cornhination drugs including
ipratropium and alburterol,
fluticasone and salmeterol, hudesonide and formoterol; antihistamines
including hydroxyzine.
diphenhydranmir_ae, lora.tadine, cetirizine, and hydrocort_sone: immune system
nniodirlating drugs
including tacrolimus and pirnecrolimus; cyclosporine; azathioprine;
mycophenolatemofetil; and
cornhinations thereof. In particular aspects, the at least one additional
therapeutic agent is a
TSLP and/or TSLPR antagonist, and in particular embodiments, the TSLP and/or
TSLPR
antagonist is 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.
In particular method aspects, modulation of at least one of cellular membrane
potential
and cellular membrane conductivity comprises altering at least one of cellular
membrane
structure or function comprising altering at least one of a conformation,
ligand binding activity,
and a catalytic activity of a membrane associated protein or constituent. 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, integrins, etc. In certain embodiments, the
transmembrane receptor
comprises a G-Protein Coupled Receptor (GPCR). In particular aspects, the G-
Protein Coupled
Receptor (GPCR) interacts with a G protein a subunit, for example, wherein the
G protein a
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subunit comprises at least one selected from the group consisting of Gas ,
Gai, Gaq, and Ga12,
and in certain embodiments the at least one G protein a subunit is Gaq.
In particular method aspects, modulation of at least one of cellular membrane
potential
and cellular membrane conductivity comprises modulating whole-cell
conductance, for example,
wherein modulating whole-cell conductance comprises modulating at least one of
a linear and a
non-linear voltage-dependent contribution of the whole-cell conductance.
In certain method aspects, modulation of at least one of cellular membrane
potential and
cellular membrane conductivity comprises modulation of a calcium dependant
cellular
messaging pathway or system. In certain method aspects, modulation of at least
one of cellular
membrane potential and cellular membrane conductivity comprises modulation of
phospholipase
C activity. In certain method aspects, modulation of at least one of cellular
membrane potential
and cellular membrane conductivity comprises modulation of adenylate cyclase
(AC) activity.
In certain method aspects, modulation of at least one of cellular membrane
potential and cellular
membrane conductivity comprises modulation of intracellular signal
transduction associated
with at least one condition or symptom selected from the group consisting of
diseases or
disorders of the immune system, allergic inflammation, allergic airway
inflammation, DC-
mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma,
obstructive
airways disease, chronic obstructive pulmonary disease, IgE-mediated
disorders, rhino-
conjunctivitis, food allergies, inflammatory arthritis, rheumatoid arthritis
and psoriasis.
Particular method aspects comprise administration of the electrokinetic fluid
to a cell
network or layer, and further comprise modulation of an intercellular junction
therein. In certain
embodiments, the intracellular junction comprises at least one selected from
the group consisting
of tight junctions, gap junctions, zona adherens and desmosomes. In particular
aspects, the cell
network or layers comprise at least one selected from the group consisting of
pulmonary
epithelium, bronchial epithelium, and intestinal epithelium.
In certain method aspects, the electrokinetically altered aqueous fluid is
oxygenated,
wherein 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.
In certain method aspects, the electrokinetically altered aqueous fluid
comprises at least
one of solvated electrons, and electrokinetically modified or charged oxygen
species, for
example, wherein 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
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at least 20 ppm. In certain aspects, the electrokinetically altered aqueous
fluid comprises a form
of solvated electrons stabilized by molecular oxygen.
In certain aspects, the ability of the electrokinetically-altered fluid to
modulate 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.
In certain aspects, the amount of oxygen present in charge-stabilized oxygen-
containing
nanostructures of the electrokinetically-altered 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.
In particular aspects, treating comprises administration by at least one of
topical,
inhalation, intranasal, and intravenous.
Exemplary relevant Molecular Interactions:
Conventionally, quantum properties are thought to belong to elementary
particles of less
than 10-10 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.
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
water 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 dilution 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.
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.
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A simplified protonated water cluster forming a nanoscale cage is shown in
Applicants'
previous patent application: WO 2009/055729. A protonated water cluster
typically takes the
form of H(H20)1. 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 may be caught in the resulting structures. The chemistry of the semi-
bound nanocage
allows the oxygen 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.
Fluids processed by the mixing device have been shown previously via
experiments to
exhibit different structural characteristics that are consistent with an
analysis of the fluid in the
context of a cluster structure. See, for example, WO 2009/055729.
Charge-stabilized nanostructures (e.g., charge stabilized oxygen-containing
nanostructures):
As described previously in Applicants' WO 2009/055729, "Double Layer Effect,"
"Dwell Time," "Rate of Infusion," and "Bubble size Measurements," the
electrokinetic mixing
device creates, in a matter of milliseconds, a unique non-linear fluid dynamic
interaction of the
first material and the second material 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 using a specially designed mixing device
comprising
insulated rotor and stator features.
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, 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
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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
Example 23 from WO 2009/055729 and as disclosed herein).
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, 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.
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, 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).
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.
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),
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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_'_ ions to form an electrical
double layer) and
nonadsorptive ions (e.g., Cl- 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_'_ 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_'_ 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).
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.
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
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.
According to particular aspects of the present invention, Applicants' novel
electrokinetic
device and methods provide for novel electrokinetically-altered fluids
comprising significant
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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.
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.
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.
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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.
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.).
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 charge-stabilized oxygen-containing nanostructures can provide for
formation of
hydrophobic nanobubbles upon contact with a hydrophobic surface.
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 nanobubbles (e.g., hydrophobid nanobubbles) of about 20 nm x
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^l.atm/mol.K; T=295K; V=pr2h=4.7x10-22 L, where r=10x10-9 m,
h=1.5x10-9
m, and n=1.95x10-22 moles).
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%,
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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
nm.
In certain aspects, the inventive electrokinetic fluids comprise solvated
electrons. In
further aspects, the inventive electrokinetic fluids comprises charge-
stabilized nanostructures
10 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.
By contrast, relative to the inventive electrokinetic fluids, control pressure
pot
oxygenated fluids (non-electrokinetic fluids) and the like do not comprise
such electrokinetically
generated 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
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.
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Table 1 illustrates various partial pressure measurements taken in a healing
wound
treated with an oxygen-enriched saline solution (Table 1) and in samples of
the gas-enriched
oxygen-enriched saline solution of the present invention.
TABLE 1
TISSUE OXYGEN MEASUREMENTS
Probe Z082BO
In air: 171 mmHg 23 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
TSLP and TSLP-mediated conditions
TSLP and TSLPR agonists/antagonists: An agent that has affinity for and
stimulates
physiologic activity at cell receptors normally stimulated by naturally
occurring substances, thus
triggering a biochemical response. A TSLP receptor agonist has affinity for
the TSLP receptor
and stimulates an activity induced by the binding of TSLP with its receptor.
For example, a
TSLP/TSLP receptor agonist is a molecule that binds to the TSLP receptor and
induces
intracellular signaling. In contrast, an "antagonist" is an agent that
inhibits activity of a cell
receptor normally stimulated by a naturally occurring substance. Accordingly,
a TSLP/TSLP
receptor antagonist binds to TSLP or to the TSLP receptor and inhibits binding
of TSLP to the
TSLP receptor and/or inhibits an activity normally induced by binding of TSLP
with its
receptor. For example, a TSLP/TSLP receptor antagonist can bind to TSLP or to
the TSLP
receptor and diminish or prevent binding, for example, by blocking binding, of
TSLP to the
TSLP receptor. Alternatively, a TSLP/TSLP receptor antagonist can bind to the
TSLP receptor
and diminish or prevent downstream signaling that would normally be induced by
the binding of
TSLP with its receptor. Agonists and antagonists can include a variety of
classes of molecules
including polypeptides, such as ligand-like polypeptides, antibodies, and
fragments or
subsequences thereof. Agonists and antagonists can also include fusion
polypeptides, antibodies,
peptides (such as peptides of less than about 20 amino acids in length), and
small molecules.
Exemplary antagonists include: neutralizing antibodies specific for TSLP and
the TSLP
receptor, soluble TSLP receptor molecules, and TSLP receptor fusion proteins,
such as TSLPR-
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WO 2010/048425 PCT/US2009/061710
immunoglobulin Fc molecules or polypeptides that encode components of more
than one
receptor chain, that thereby mimic a physiological receptor heterodimer or
higher order
oligomer. If the receptor is includes more than one polypeptide chain, a
single chain fusion can
be utilized.
Antibody: A polypeptide ligand comprising at least a light chain or heavy
chain
immunoglobulin variable region which specifically recognizes and binds an
epitope (e.g., as an
antigen, such as TSLP or a fragment thereof, or a TSLP receptor of a fragment
thereof). This
includes intact immunoglobulins and the variants and portions of them well
known in the art,
such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins
("scFv"), and disulfide
stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a
light chain variable
region of an immunoglobulin and a heavy chain variable region of an
immunoglobulin are
bound by a linker, while in dsFvs, the chains have been mutated to introduce a
disulfide bond to
stabilize the association of the chains. The term also includes genetically
engineered forms such
as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate
antibodies (e.g.,
bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995
(Pierce Chemical Co.,
Rockford, Ill.); Kuby, J., Immunology, 3`d Ed., W.H. Freeman & Co., New York,
1997.
Typically, an immunoglobulin has a heavy and a light chain. Each heavy and
light chain
contains a constant region and a variable region, (the regions are also known
as "domains"). In
combination, the heavy and the light chain variable regions specifically bind
the antigen. Light
and heavy chain variable regions contain a "framework" region interrupted by
three
hypervariable regions, also called "complementarity-determining regions" or
"CDRs". The
extent of the framework region and CDRs has been defined (see, Kabat et al.,
Sequences of
Proteins of Immunological Interest, U.S. Department of Health and Human
Services, 1991,
which is hereby incorporated by reference). The Kabat database is now
maintained online. The
sequences of the framework regions of different light or heavy chains are
relatively conserved
within a species. The framework region of an antibody, that is the combined
framework regions
of the constituent light and heavy chains, serves to position and align the
CDRs in three-
dimensional space. Antibodies include monoclonal antibodies, humanized
antibodies, etc.
TSLP antagonists include small molecule antagonists, antibodies to TSLP,
antibodies to
the TSLP receptor, and TSLP receptor fusion proteins, such as TSLPR-
immunoglobulin Fc
molecules or polypeptides that encode components of more than one receptor
chain, that thereby
mimic a physiological receptor heterodimer or higher order oligomer, amongst
others. TSLP has
been shown to bind directly to a type I cytokine receptor superfamily member
(which are also
known as hematopoietin receptor superfamily members), TSLPR. TSLPR has been
cloned. The
CA 02741336 2011-04-20
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functional high-affinity receptor for TSLP has been demonstrated to include
two polypeptides,
TSLPR and the IL-7 receptor alpha chain. Thus, both TSLP and IL-7 shares IL-
7Ralpha as a
component of their receptors. However, these receptors are distinctive in that
the TSLP receptor
additionally contains TSLPR whereas the IL-7 receptor additionally contains
the common
cytokine receptor gamma chain, which is a signal-transducing component of
various cytokine
receptors. TSLPR (and Fc fusions of this receptor chain) are described, for
example, in
Published U.S. Patent Application No. 2002/0160949, which is incorporated
herein by
reference.
Antibodies to TSLP polypeptides are known in the art. In addition, anti-TSLPR
antibodies are commercially available (R & D Systems, Minneapolis, Minn., cat.
no. MAB981;
DNAX Research, Inc., Palo Alto, Calif.). Antibodies are also prepared against
TSLP receptor or
TSLP by immunization with specified epitopes, such as regions of increased
antigenicity
determined by the Welling plot of Vector NTI® Suite (Informax, Inc,
Bethesda, Md.). The
sequence of the TSLP receptor, and regions of increased antigenicity in human
TSLP receptor
are disclosed in U.S. Patent Publication No. 2003/0186875. Pharmaceutical
compositions (see
above) generally include a therapeutically effective amount of a TSLP
antagonist, and can also
include additional agents. The preparation of pharmaceutical compositions is
disclosed above.
Indications
TSLP and TSLPR are believed to have roles in many types of allergic conditions
including, but not limited to, disorders of the immune system, allergic
inflammation, allergic
airway inflammation, DC-mediated inflammatory Th2 responses, atopic
dermatitis, atopic
eczema, allergic asthma, asthma, obstructive airways disease, chronic
obstructive pulmonary
disease (COPD), food allergies, inflammatory arthritis, rheumatoid arthritis,
psoriasis, IgE-
mediated disorders, and rhino-conjunctivitis. TSLP involvement in DC-mediated
inflammatory
Th2 responses has been shown in several publications including a recent review
by Ziegler and
Liu (Ziegler and Liu, Nat. Immunol., 7:709-714, 2005, which is incorporated by
reference in its
entirety).
Allergic airway inflammation. A recent study demonstrated that TSLP is
required for the
initiation of allergic airway inflammation in mice in vivo (Zhou et al., Nat.
Immunol., 6:1047-
1053, 2005). In this study, Zhou et al. demonstrated that lung specific
expression of a TSLP
transgene induced allergic airway inflammation (asthma) which is characterized
by massive
infiltration of leukocytes (including Th2 cells), goblet cell hyperplasia, and
subepithelial
fibrosis, and increased serum IgE levels. In addition, a recent study showed
that allergen
challenge caused a rapid accumulation of TSLP in the airways of asthmatic mice
(Liyun Shi et
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al., Clin. Immunol., 129:202-210, 2008). These results indicate that TSLP
plays an important
role in the pathogenesis of allergic airway inflammation. Here Applicants show
that the
inventive electrokinetically-altered fluids significantly downregulated TSLP.
According to
certain embodiments, the inventive electrokinetically-altered fluids, have
substantial utility for
treating allergic airway inflammation and similar conditions.
Allergic inflammation. A recent review summarizes and describes the results
from
studies investing the role of TSLP in allergic inflammation e.g. allergic skin
inflammation.
(Ziegler and Liu, 2008). In particular, studies have shown that normal skin or
nonlesional skin
in patients with atopic dermatitis has no detectable TSLP protein, whereas,
the skin taken from
acute and chronic atopic dermatitis lesions has high expression of TSLP. In
another study, mice
lacking TSLPR were constructed and examined for effects on allergic skin
inflammation. (He et
al., PNAS, 105:11875-11880, 2008, which is incorporated by reference in its
entirety). He et al.,
discovered that skin inflammation due to an allergen in mice lacking TSLPR was
significantly
reduced than when compared to wildtype. In yet another study, it was
demonstrated that mice
engineered to overexpress TSLP in the skin developed atopic dermatitis which
is characterized
by eczematous skin lesions containing inflammatory infiltrates, a dramatic
increase in
circulating Th2 cells and elevated serum IgE (Yoo et al., J. Exp. Med.,
202:541-549, 2005). The
study suggested that TSLP may directly activate DCs in mice. In another study,
conducted by
Li et al., the group confirmed that transgenic mice overexpressing TSLP in the
skin developed
atopic dermatitis which solidifies the link between TSLP and the development
of atopic
dermatitis. These results indicate that TSLP plays an important role in the
pathogenesis of
allergic inflammation, e.g. allergic skin inflammation (e.g., atopic
dermatitis and eczema). Here
Applicants show that the inventive electrokinetically-altered fluids
significantly downregulated
TSLP. According to certain embodiments, the inventive electrokinetically-
altered fluids, have
substantial utility for treating allergic inflammation, e.g. allergic skin
inflammation (e.g., atopic
dermatitis and eczema) and similar conditions.
Psoriasis. A recent study showed that TSLP had substantially higher expression
in skin
biopsies taken from patients with acute psoriasis (Guttman-Yassky, et al., J.
Allergy and Clinical
Immunology 119:1210-1217, 2007). This result indicates that TSLP plays an
important role in
the pathogenesis of psoriasis. Herein, Applicants show that the inventive
electrokinetically-
altered fluids significantly downregulated TSLP. According to certain
embodiments, therefore,
the inventive electrokinetically-altered fluids, have substantial utility for
treating psoriasis and
similar conditions.
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Allergic asthma. Recently, a study showed that allergen challenge caused a
rapid
accumulation of TSLP in the airways of asthmatic mice. (Liyun Shi et al.,
Clin. Immunol.,
129:202-210, 2008). In the same study, it was shown that modulation of DCs
function by
inhibiting TSLPR lessened the severity in mice. These results indicate that
TSLP plays an
important role in the pathogenesis of allergic asthma. Herein, Applicants show
that the
inventive electrokinetically-altered fluids significantly downregulated TSLP.
According to
certain embodiments, therefore, the inventive electrokinetically-altered
fluids, have substantial
utility for treating allergic asthma and similar conditions.
Obstructive airways disease. A recent study demonstrated that COPD is
associated with
elevated bronchial mucosal expression of TSLP (Ying et al., J Immunol,
181:2790-2798, 2008).
COPD is a type of obstructive airways diseases. This results indicate that
TSLP plays an
important role in the pathogenesis of obstructive airways disease, e.g. COPD.
Herein,
Applicants show that the inventive electrokinetically-altered fluids
significantly downregulated
TSLP. According to certain embodiments, therefore, the inventive
electrokinetically-altered
fluids, have substantial utility for treating obstructive airways disease,
e.g. COPD.
Food allergies. Dendritic cells of the intestines were found to stimulate
naive T cells,
skewing them to a TH2 response in an OX40L dependent manner. (Blazquez AB,
Berin MC.
Gastrointestinal dendritic cells promote Th2 skewing via OX40L. J Immunol,
180:4441-4450,
2008, which is incorporated by reference in its entirety). In addition, a
recent review discusses
the presence of TSLP in the intestines and its role in regulation of immune
homeostasis. (Iliev
ID, Matteoli G, Rescigno M. The yin and yang of intestinal epithelial cells in
controlling
dendritic cell function. J Exp Med; 204:2253-2257, 2007, which is incorporated
by reference in
its entirety). These results indicate that TSLP has a role in food allergies.
Herein, Applicants
show that the inventive electrokinetically-altered fluids significantly
downregulated TSLP.
According to certain embodiments, therefore, the inventive electrokinetically-
altered fluids, have
substantial utility for treating food allergies and similar conditions.
Inflammatory arthritis. A recent study found increased levels of TSLP in
synovial fluid
specimens derived from patients with rheumatoid arthritis (RA) when compared
with synovial
fluid obtained from patients with other forms of arthritis. (Koyama et al.
Biochem and Biophyis
Res Comm., 357:99-104, 2007, which is incorporated by reference in its
entirety). The same
study found that use of an anti-TSLP neutralizing antibody ameliorated a TNF-a-
dependent
experimental arthritis induced by anti-type II collagen antibody in mice.
These results indicate
that TSLP is a significant player in inflammatory arthritis such as RA.
Herein, Applicants show
that the inventive electrokinetically-altered fluids significantly
downregulated TSLP. According
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to certain embodiments, therefore, the inventive electrokinetically-altered
fluids, have
substantial utility for treating inflammatory arthritis and similar
conditions, e.g. RA.
Allergic rhinitis. In a recent research study, Mou et al., discovered that
TSLP was
present at both the mRNA and protein levels in the nasal mucosa of all
patients tested that were
suffering from allergic rhinitis (AR). (Mou et al., Acta Oto-laryngologica,
129:297-301, 2009,
which is incorporated by reference in its entirety). In addition, TSLP levels
were tightly
correlated with the severity of AR. These results indicate that TSLP plays an
important role in
the pathogenesis of AR and/or rhino-conjunctivitis. Herein, Applicants show
that the inventive
electrokinetically-altered fluids significantly downregulated TSLP in a
relevant model system.
According to certain embodiments, therefore, the inventive electrokinetically-
altered fluids, have
substantial utility for treating AR, allergic rhino-conjunctivitis and similar
conditions.
Methods of Treatment
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.
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.
Certain embodiments herein relate to therapeutic compositions and methods of
treatment
for a subject by preventing or alleviating at least one symptom of
inflammation associated with
certain conditions or diseases. Many conditions or diseases associated with
inflammation
disorders have been treated with steroids, methotrexate, immunosuppressive
drugs including
cyclophosphamide, cyclosporine, azathioprine and leflunomide, nonsteroidal
anti-inflammatory
agents such as aspirin, acetaminophen and COX-2 inhibitors, gold agents and
anti-malarial
treatments.
Routes and Forms of Administration
As used herein, "subject," may refer to any living creature, preferably an
animal, more
preferably a mammal, and even more preferably a human.
In particular exemplary embodiments, the gas-enriched fluid of the present
invention
may function as a therapeutic composition alone or in combination with another
therapeutic
agent such that the therapeutic composition prevents or alleviates at least
one symptom of
inflammation. The therapeutic compositions of the present invention include
compositions that
are able to be administered to a subject in need thereof. In certain
embodiments, the therapeutic
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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.
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.
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.
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
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different composition. For example, conventional water may be replaced or
supplemented by a
gas-enriched fluid produced by mixing oxygen into water or deionized water to
provide gas-
enriched fluid.
In certain embodiments, the inventive gas-enriched fluid may be combined with
one or
more therapeutic agents and/or used alone. In particular embodiments,
incorporating the gas-
enriched fluid may include replacing one or more solutions known in the art,
such as deionized
water, saline solution, and the like with one or more gas-enriched fluid,
thereby providing an
improved therapeutic composition for delivery to the subject.
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 that
may contain from
0.05 to 95% by weight of the active ingredient.
Possible administration routes include oral, sublingual, buccal, parenteral
(for example
subcutaneous, intramuscular, intra-arterial, intraperitoneally,
intracisternally, intravesically,
intrathecally, or intravenous), rectal, topical including transdermal,
intravaginal, intraoccular,
intraotical, intranasal, inhalation, and injection or insertion of implantable
devices or materials.
Administration Routes
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. In certain
embodiments, oral or topical administration is preferred.
Formulations suitable for oral administration may be provided as discrete
units, such as
tablets, capsules, cachets, syrups, elixirs, chewing gum, "lollipop"
formulations,
microemulsions, solutions, suspensions, lozenges, or gel-coated ampules, each
containing a
predetermined amount of the active compound; as powders or granules; as
solutions or
suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-
oil emulsions.
Formulations suitable for transmucosal methods, such as by sublingual or
buccal
administration include lozenges patches, tablets, and the like comprising the
active compound
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and, typically a flavored base, such as sugar and acacia or tragacanth and
pastilles comprising
the active compound in an inert base, such as gelatin and glycerine or sucrose
acacia.
Formulations suitable for parenteral administration typically comprise sterile
aqueous
solutions containing a predetermined concentration of the active gas-enriched
fluid and possibly
another therapeutic agent; the solution is preferably isotonic with the blood
of the intended
recipient. Additional formulations suitable for parenteral administration
include formulations
containing physiologically suitable co-solvents and/or complexing agents such
as surfactants
and cyclodextrins. Oil-in-water emulsions may also be suitable for
formulations for parenteral
administration of the gas-enriched fluid. Although such solutions are
preferably administered
intravenously, they may also be administered by subcutaneous or intramuscular
injection.
Formulations suitable for urethral, rectal or vaginal administration include
gels, creams,
lotions, aqueous or oily suspensions, dispersible powders or granules,
emulsions, dissolvable
solid materials, douches, and the like. The formulations are preferably
provided as unit-dose
suppositories comprising the active ingredient in one or more solid carriers
forming the
suppository base, for example, cocoa butter. Alternatively, colonic washes
with the gas-
enriched fluids of the present invention may be formulated for colonic or
rectal administration.
Formulations suitable for topical, intraoccular, intraotic, or intranasal
application include
ointments, creams, pastes, 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, polyethyleneglycols, alcohols, and combinations
thereof. Nasal or
intranasal delivery may include metered doses of any of these formulations or
others. Likewise,
intraotic or intraocular may include drops, ointments, irritation fluids and
the like.
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.
For example a tablet may be prepared by compressing an intimate mixture
comprising a
powder or granules of the active ingredient and one or more optional
ingredients, such as a
binder, lubricant, inert diluent, or surface active dispersing agent, or by
molding an intimate
mixture of powdered active ingredient and a gas-enriched fluid of the present
invention.
Suitable formulations for administration by inhalation include fine particle
dusts or mists
which may be generated by means of various types of metered dose pressurized
aerosols,
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nebulisers, or insufflators. In particular, powders or other compounds of
therapeutic agents may
be dissolved or suspended in a gas-enriched fluid of the present invention.
For pulmonary administration via the mouth, the particle size of the powder or
droplets is
typically in the range 0.5-10 M, preferably 1-5 M, to ensure delivery into
the bronchial tree.
For nasal administration, a particle size in the range 10-500 M is preferred
to ensure retention
in the nasal cavity.
Metered dose inhalers are pressurized aerosol dispensers, typically containing
a
suspension or solution formulation of a therapeutic agent in a liquefied
propellant. In certain
embodiments, as disclosed herein, the gas-enriched fluids of the present
invention may be used
in addition to or instead of the standard liquefied propellant. During use,
these devices
discharge the formulation through a valve adapted to deliver a metered volume,
typically from
10 to 150 L, to produce a fine particle spray containing the therapeutic
agent and the gas-
enriched fluid. Suitable propellants include certain chlorofluorocarbon
compounds, for
example, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane and
mixtures thereof.
The formulation may additionally contain one or more co-solvents, for example,
ethanol
surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and
suitable flavoring agents.
Nebulisers are commercially available devices that transform solutions or
suspensions of the
active ingredient into a therapeutic aerosol mist either by means of
acceleration of a compressed
gas (typically air or oxygen) through a narrow venturi orifice, or by means of
ultrasonic
agitation. Suitable formulations for use in nebulisers consist of another
therapeutic agent in a
gas-enriched fluid and comprising up to 40% w/w of the formulation, preferably
less than 20%
w/w. In addition, other carriers may be utilized, such as distilled water,
sterile water, or a dilute
aqueous alcohol solution, preferably made isotonic with body fluids by the
addition of salts,
such as sodium chloride. Optional additives include preservatives, especially
if the formulation
is not prepared sterile, and may include methyl hydroxy-benzoate, anti-
oxidants, flavoring
agents, volatile oils, buffering agents and surfactants.
Suitable formulations for administration by insufflation include finely
comminuted
powders that may be delivered by means of an insufflator or taken into the
nasal cavity in the
manner of a snuff. In the insufflator, the powder is contained in capsules or
cartridges, typically
made of gelatin or plastic, which are either pierced or opened in situ and the
powder delivered
by air drawn through the device upon inhalation or by means of a manually-
operated pump. The
powder employed in the insufflator consists either solely of the active
ingredient or of a powder
blend comprising the active ingredient, a suitable powder diluent, such as
lactose, and an
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optional surfactant. The active ingredient typically comprises from 0.1 to 100
w/w of the
formulation.
In addition to the ingredients specifically mentioned above, the formulations
of the
present invention may include other agents known to those skilled in the art,
having regard for
the type of formulation in issue. For example, formulations suitable for oral
administration may
include flavoring agents and formulations suitable for intranasal
administration may include
perfumes.
The therapeutic compositions of the invention can be administered by any
conventional
method available for use in conjunction with pharmaceutical drugs, either as
individual
therapeutic agents or in a combination of therapeutic agents.
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 0.001 to 1000
milligrams (mg)
per kilogram (kg) of body weight, with the preferred dose being 0.1 to about
30 mg/kg.
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.
Ointments, pastes, foams, occlusions, creams and gels 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
solution, 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,
and 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 therapeutic compounds to a subject.
The injection-use 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
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requiring only the addition of the sterile liquid excipient, or gas-enriched
fluid, immediately
prior to use. Extemporaneous injection solutions and suspensions can be
prepared from sterile
powders, granules, and tablets. The requirements for effective pharmaceutical
carriers for
injectable compositions are well known to those of ordinary skill in the art.
See, for example,
Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa.,
Banker and
Chalmers, Eds., 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel,
4th ed.,
622-630 (1986).
Formulations suitable for topical administration include lozenges comprising a
gas-
enriched fluid of the invention and optionally, an additional therapeutic and
a flavor, usually
sucrose and acacia or tragacanth; pastilles comprising a gas-enriched fluid
and optional
additional therapeutic agent in an inert base, such as gelatin and glycerin,
or sucrose and acacia;
and mouth washes or oral rinses comprising a gas-enriched fluid and optional
additional
therapeutic agent in a suitable liquid carrier; as well as creams, emulsions,
gels and the like.
Additionally, formulations suitable for rectal administration may be presented
as
suppositories by mixing with a variety of bases such as emulsifying bases or
water-soluble
bases. Formulations suitable for vaginal administration may be presented as
pessaries, tampons,
creams, gels, pastes, foams, or spray formulas containing, in addition to the
active ingredient,
such carriers as are known in the art to be appropriate.
Suitable pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences,
Mack Publishing Company, a standard reference text in this field.
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 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 the condition of the animal, the
body weight of the
animal, as well as the condition being treated. A suitable dose is that which
will result in a
concentration of the therapeutic composition in a subject that is known to
affect the desired
response.
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.
It will be appreciated that the compounds of the combination may be
administered: (1)
simultaneously by combination of the compounds in a co-formulation or (2) by
alternation, i.e.
delivering the compounds serially, sequentially, in parallel or simultaneously
in separate
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pharmaceutical formulations. In alternation therapy, the delay in
administering the second, and
optionally a third active ingredient, should not be such as to lose the
benefit of a synergistic
therapeutic effect of the combination of the active ingredients. According to
certain
embodiments by either method of administration (1) or (2), ideally the
combination should be
administered to achieve the most efficacious results. In certain embodiments
by either method of
administration (1) or (2), ideally the combination should be administered to
achieve peak plasma
concentrations of each of the active ingredients. A one pill once-per-day
regimen by
administration of a combination co-formulation may be feasible for some
patients suffering from
inflammatory neurodegenerative diseases. According to certain embodiments
effective peak
plasma concentrations of the active ingredients of the combination will be in
the range of
approximately 0.001 to 100 M. Optimal peak plasma concentrations may be
achieved by a
formulation and dosing regimen prescribed for a particular patient. It will
also be understood
that the inventive fluids and a glucocorticoid steroid (e. g., Budesonide) or
the physiologically
functional derivatives of any thereof, whether presented simultaneously or
sequentially, may be
administered individually, in multiples, or in any combination thereof. In
general, during
alternation therapy (2), an effective dosage of each compound is administered
serially, where in
co-formulation therapy (1), effective dosages of two or more compounds are
administered
together.
The combinations of the invention may conveniently be presented as a
pharmaceutical
formulation in a unitary dosage form. A convenient unitary dosage formulation
contains the
active ingredients in any amount from 1 mg to 1 g each, for example but not
limited to, 10 mg to
300 mg. The synergistic effects of the inventive fluid in combination with,
for example, a
glucocorticoid steroid (e. g., Budesonide) may be realized over a wide ratio,
for example 1:50 to
50:1 (inventive fluid: a glucocorticoid steroid (e. g., Budesonide)). In one
embodiment the ratio
may range from about 1:10 to 10:1. In another embodiment, the weight/weight
ratio of inventive
fluid to a glucocorticoid steroid (e. g., Budesonide) in a co-formulated
combination dosage
form, such as a pill, tablet, caplet or capsule will be about 1, i.e. an
approximately equal amount
of inventive fluid and a glucocorticoid steroid (e. g., Budesonide). In other
exemplary co-
formulations, there may be more or less inventive fluid and a glucocorticoid
steroid (e. g.,
Budesonide). In one embodiment, each compound will be employed in the
combination in an
amount at which it exhibits anti-inflammatory activity when used alone. Other
ratios and
amounts of the compounds of said combinations are contemplated within the
scope of the
invention.
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A unitary dosage form may further comprise inventive fluid and, for example, a
glucocorticoid steroid (e. g., Budesonide), or physiologically functional
derivatives of either
thereof, and a pharmaceutically acceptable carrier.
It will be appreciated by those skilled in the art that the amount of active
ingredients in
the combinations of the invention required for use in treatment will vary
according to a variety
of factors, including the nature of the condition being treated and the age
and condition of the
patient, and will ultimately be at the discretion of the attending physician
or health care
practitioner. The factors to be considered include the route of administration
and nature of the
formulation, the animal's body weight, age and general condition and the
nature and severity of
the disease to be treated.
It is also possible to combine any two of the active ingredients in a unitary
dosage form
for simultaneous or sequential administration with a third active ingredient.
The three-part
combination may be administered simultaneously or sequentially. When
administered
sequentially, the combination may be administered in two or three
administrations. According to
certain embodiments the three-part combination of inventive fluid and a
glucocorticoid steroid
(e. g., Budesonide) may be administered in any order.
According to particular aspects, the inventive electrokinetically-altered
fluids, have
substantial utility for treating the TSLP and/or TSLPR-mediated conditions,
including but not
limited to the exemplary genus of indications disclosed herein. According to
additional aspects,
the inventive electrokinetically-altered fluids, have utility for treating
various subgenera of the
exemplary genus, wherein at least one indication of the genus is excluded from
each of said
subgenera.
EXAMPLE 1
(Synergistic effects of inventive electrokinetically-altered fluids and
Albuterol were
demonstrated)
Overview. The inventive electrokinetically-altered fluids provided 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)) and
thus
provides for a decrease in a patient's albuterol usage. The results disclosed
in this Example are
also disclosed in Applicants' WO 2009/055729.
First experiment. In a first 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
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50 g/mL to deliver the target dose of 12.5 g of albuterol sulfate in 250 L
per animal. The
study was a randomized blocked design for weight and baseline PenH values. Two
groups (A
and B) received an intratracheal instillation of 250 pL of 50 pg/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.
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. Once an animal had greater than 50% bronchoconstriction, the animal
was considered
to be not protected. The results indicate that 50% of the Group B animals were
protected from
bronchoconstriction out to 10 hours (at which time the test was terminated).
Second experiment. 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 and methods. Guinea Pigs (Caviaporcellus) 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. 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). Duration of treatment was one day.
Experimental design. All animals were subjected to inhalation exposure of
methacholine
(500 g/ml), 2 hours following TA/Control administration. All animals received
a dose volume
of 250 l. Therefore, albuterol sulfate was diluted (in the control article
and the 4 test articles)
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to concentrations of 0, 25, 50 and 100 g/ml. Thirty minutes prior to dosing,
solutions of
albuterol sulfate of 4 different concentrations (0, 25, 50 and 100 g/ml) was
made up in a 1 Ox
stock (500 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 pg/ml) was
prepared on the day
of dosing.
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 l/animal of test
article or control was administered. 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.
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 pg/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.
Once prior to test article administration (Day -1) and again at 2, 6, 10, 14,
18, 22 and 26
hours post-dose, 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 500ug/mL were aerosoloized for 30 seconds, animals were
exposed to the
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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).
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. Data considered unphysiological
was excluded
from further analysis. 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).
Results. The results from this experiment showed that 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.
Surprisingly,
however, administration of albuterol (representative data for the 25 g
albuterollanimal groups
are shown) formulated in the inventive electrokinetically generated fluids (at
all oxygen level
values tested; ambient, 20 ppm, 40 ppm and 60 ppm) 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.
Applicants also showed 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.
According to particular aspects, 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 2
(Effects of inventive electrokinetically-altered fluids on cytokine expression
were determined)
Overview. The inventive electrokinetically-altered fluids lowered the
production of pro-
inflammatory cytokines (IL-1B, TNF-a, IL-6, and GM-CSF), chemokines (IL-8, MIP-
la,
RANTES, and Eotaxin), inflammatory enzymes (iNOS, COX-2, and MMP-9), allergen
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responses (MHC class II, CD23, B7-1, and B7-2), and Th2 cytokines (IL-4, IL-
13, and IL-5)
when compared to control fluid and increased anti-inflammatory cytokines
(e.g., IL1R-a,
TIMPs) when compared to control fluid. The results disclosed in this Example
are also
disclosed in Applicants' WO 2009/055729.
In particular aspects, human mixed lymphocytes were stimulated with T3 antigen
or
PHA in Revalesio oxygen-enriched fluid, or control fluid, and changes in IL-
13, 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-
y, GM-CSF,
MIP-lB, MCP-1, G-CSF, FGFb, VEGF, TNF-a, RANTES, Leptin, TNF-B, TFG-B, and NGF
were evaluated. As was shown, pro-inflammatory cytokines (IL-1B, TNF-a, IL-6,
and GM-
CSF), chemokines (IL-8, MIP-la, 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-a, TIMPs) tested were
increased in test fluid
versus control fluid.
Additionally, 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.
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
(Al (OH)3) (200 mg/mL) once each on days 1, 2, and 3. The study was a
randomized 2 x 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.
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.
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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.
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 IA-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.
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 PAXgeneTM Blood
RNA Tube and
Qiagen PAXgeneTM 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 being 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-zolTM, homogenized, and sent to the lab for further
processing.
BAL analysis. Lung lavage was collected and centrifuged for 10 minutes at 4 C
at 600-
800 g to pellet the cells. The supernatants were transferred to fresh tubes
and frozen at -80 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 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.
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.
Blood analysis. 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-zolTM or PAXgeneTM. Next, the
clotted blood
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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
C.
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 L of 5N acetic acid per 0.2 mL of whole blood or plasma. Tubes were shaken
and stored at
-80 C. Utilizing PAXgeneTm, tubes were incubated for approximately two hours
at room
temperature. Tubes were then placed on their side and stored in the -20 C
freezer for 24 hours,
and then transferred to -80 C for long term storage.
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.
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 or PolytronTm homogenizer. Samples were stored at -
80 C.
Results from Blood Samples. 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
Revalesio treatment;
followed by saline challenged saline treatment; and saline challenged
Revalesio treatment. To
facilitate review, both the RDC1676-01 groups are highlighted with gray shaded
backdrops,
whereas the control saline treatment groups have unshaded backdrops.
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 RDC 1676-00
group.
Applicants determined that the level of RANTES (IL-8 super family) produced
after
treatment with the inventive electrokinetically-altered fluids was less than
that produced by the
saline only exposed groups. Applicants demonstrated that the inventive
electrokinetically-
altered fluids caused MCP-1 to be produce at lower levels when compared to
that which was
produced by the saline only exposed groups. Applicants determined that the
level of TNF alpha
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produced after treatment with the inventive electrokinetically-altered fluids
was less than that
produced by the saline only exposed groups.
In addition, Applicants demonstrated that the level of MIP- 1 alpha produced
after
treatment with the inventive electrokinetically-altered fluids was less than
that produced by the
saline only exposed groups. Applicants demonstrated that the inventive
electrokinetically-
altered fluids caused IL-1 alpha to be produce at lower levels when compared
to that which was
produced by the saline only exposed groups. Applicants observed that the level
of Vcam
produced after treatment with the inventive electrokinetically-altered fluids
was less than that
produced by the saline only exposed groups. Applicants observed that the level
of IL-1 beta
produced after treatment with the inventive electrokinetically-altered fluids
was less than that
produced by the saline only exposed groups. Applicants demonstrated that the
inventive
electrokinetically-altered fluids caused Eotaxin and MCP-3 to be produce at
lower levels when
compared to that which was produced by the saline only exposed groups.
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 3
(Effects of the inventive electrokinetically-altered fluids to modulate T-cell
proliferation were
determined)
Overview. The inventive electrokinetically-altered fluids improved regulatory
T-cell
function as shown by relatively decreased proliferation. The results disclosed
in this Example
are also disclosed in Applicants' WO 2009/055729.
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.
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Methods. Briefly, FITC-conjugated anti-CD25 (ACT-1) antibody used in sorting
was
purchased from DakoCytomation (Chicago, IL). 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 IgGl (isotype control), and XCL1 antibodies. All
antibodies were
used according to manufacturer's instructions.
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+CD25hiCD 12710/nTreg and CD4+CD25- responder T cells.
Suppression assays were performed in round-bottom 96 well microtiter plates.
3.75 x
103 CD4+CD25neg responder T cells, 3.75 x 103 autologous T reg, 3.75 x 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 C
in RPMI 1640
medium supplemented with 10% fetal bovine serum. Sixteen hours before the end
of the
incubation, 1.0 mCi of 3H-thymidine was added to each well. Plates were
harvested using a
Tomtec cell harvester and 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.
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 PhosFlowTm buffer and permeabilized in
denaturing
Permbuffer I11 . Cells were then stained with antibodies against each
particular selected
molecule.
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. Regulatory T cell proliferation was studied by stimulating cells with
diesel
exhaust particulate matter (PM, from EPA). Applicants determined that the
cells stimulated
with PM (no Rev, no Solas) resulted in a decrease in secreted IL-10, while
cells exposed to PM
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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. Note that treatment with Rev alone
resulted in higher IL-
levels relative to Saline and Media controls. Similar results were obtained
with GITR,
Granzyme A, XCL1, pStat5, and Foxp3, respectively.
Applicants also obtained AA PBMC data, obtained from an allergic asthma (AA)
profile
of peripheral blood mononuclear cells (PBMC) evaluating tryptase. The AA PBMC
data was
10 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 Solas).
Treatment with
Rev alone resulted in lower tryptase levels relative to Saline and Media
controls.
In summary, Applicants observed a decreased proliferation in the presence of
PM and
Rev relative to PM in control fluid (no Rev, no Solas), indicating that the
inventive
electrokinetically generated fluid Rev improved regulatory T-cell function as
shown by
relatively decreased proliferation in the assay. Moreover, the evidence
indicates that beta
blockade, GPCR blockade and Ca channel blockade affects the activity of Rev on
Treg function.
EXAMPLE 4
(Synergistic effects between the inventive electrokinetically-altered fluids
and Budesonide were
determined)
Overview. The inventive electrokinetically-altered fluids provided for
synergistic anti-
inflammatory effects with Budesonide in vivo in an art-recognized animal model
for allergic
asthma. The results disclosed in this Example are also disclosed in
Applicants' WO
2009/055729.
Applicants initially performed experiments 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
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induced by ovalbumin sensitization in this model resemble those observed in
man (Elwood et
al., JAllergy 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:
Brown Norway rats of strain Bn/Crl were obtained from Charles River Kingston,
weighing approximately 275 50g 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.
Sensitization. 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/100mg Aluminum Hydroxide per 1 ml of 0.9% Sodium
Chloride,
followed by repeat injection on day 3.
Treatment. 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 20L, 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.
Ovalbumin challenge. 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 2L/min).
Sample collection. 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:
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Eosinophil Count: As expected, treatment with Budesonide ("NS + Budesonide 750
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.
Additionally, while
treatment with the inventive fluid "RDC1676-03" alone did not significantly
reduce the
eosinophil count, it nonetheless displayed a substantial synergy with
Budesonide in reducing the
eosinophil count ("RDC1676-03 + Budesonide 750 pg/Kg). Similarly, the
Eosinophil % also
reflected a similar trend. While RDC1676-03 or Budesonide 750 ug/kg alone did
not have a
significant effect on Eosinophil % count in the challenged animals, the two in
combination
reduced the Eosinophil % significantly.
Therefore, Applicants determined, according to particular aspects, that the
inventive
electrokinetically generated fluids (e.g., RDC1676-03) 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:
Applicants also demonstrated 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).
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.
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. 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
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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.
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:
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.
Applicants observed 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).
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.
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. Applicants observed such effects on
Interferon
gamma and IL 10, respectively. 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 ug/kg
or 750 ug/kg
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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).
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.
Applicants observed that 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 ug/kg or 750 ug/kg. Once again, there was a clear synergistic
effect of
Budesonide 750 ug/kg and Rev 60. A similar downward trend was observed for a
number of
other pro-inflammatory cytokines, such as KC or IL8, MCP3, ILlb, 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 5
(Effects of the inventive electrokinetically-altered fluids on intercellular
tight junctions were
determined)
Overview. The inventive electrokinetically-altered fluids were shown to
modulate
intercellular tight junctions. The results disclosed in this Example are also
disclosed in
Applicants' WO 2009/055729.
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).
Example Overview. 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
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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:
Intratracheal drug delivery. 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.
Animal groups and dosing. 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 g sCT
in 200 L per animal).
Assays. In particular aspects, blood samples (e.g., 200 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 = -70 C until assayed for sCT using
an ELISA.
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 or PolytronTm homogenizer. Samples were stored at -
80 C.
Results:
Enhancement of tight junctions. Applicants observed 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
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tight junctions. RDC1676-00 signifies sterile saline processed according to
the presently
disclosed methods, but without oxygenation.
Additionally, according to particular aspects, tight junction related proteins
were
upregulated in lung tissue. Applicants showed upregulation of the junction
adhesion molecules
JAM 2 and 3, GJAl, 3, 4 and 5 (junctional adherens), OCLN (occludin), claudins
(e.g., CLDN
3, 5, 7, 8, 9, 10), TJP1 (tight junction protein 1), respectively.
EXAMPLE 6
(Effects of the inventive electrokinetically-altered fluids on whole-cell
conductance were
determined)
Overview. The inventive electrokinetically-altered fluids decreased the whole-
cell
conductance as demonstrated by patch clamp analysis conducted on bronchial
epithilial cells
(BEC). Patch clamp analysis conducted on bronchial epithilial cells (BEC)
perfused with
inventive electrokinetically-altered fluid (RNS-60) revealed that exposure to
RNS-60 resulted in
a decrease in whole cell conductance. In addition, stimulation with a cAMP
stimulating
"cocktail", which dramatically increased the whole-cell conductance, also
increased the drug-
sensitive portion of the whole-cell conductance, which was ten-times higher
than that observed
under basal conditions. The results disclosed in this Example are also
disclosed in Applicants'
WO 2009/055729.
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.
Overview. Applicants showed that 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, Applicants showed in the context of T-
regulatory cells
stimulated with particulate matter (PM), that there was a decreased
proliferation of T-regulatory
cells in the presence of PM and Rev relative to PM in control fluid (no Rev,
no Solas),
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
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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 indicate that beta
blockade, GPCR blockade
and Ca channel blockade affects the activity of the electrokinetically
generated fluids on Treg
and PBMC function. Furthermore, Applicants demonstrated, according to
additional aspects,
upon expose to the inventive fluids, tight junction related proteins were
upregulated in lung
tissue. Applicants showed upregulation of the junction adhesion molecules JAM
2 and 3,
GJA1,3,4 and 5 (junctional adherens), OCLN (occludin), claudins (e.g., CLDN 3,
5, 7, 8, 9, 10),
TJP1 (tight junction protein 1), respectively. Patch clamp studies were
performed to further
investigate and confirm said utilities.
Materials and Methods:
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").
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: 135mM NaCl, 5mM KC1, 1.2mM CaC12, 0.8mM MgC12, and 10mM HEPES
(pH
adjusted to 7.4 with N-methyl D-Glucamine). Basal currents were measured after
which RNS-
60 was perfused onto the cells.
More specifically, patch pipettes were pulled from borosilicate glass (Garner
Glass Co,
Claremont, CA) 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, NY). The pipettes were filled with an intracellular solution
containing (in
mM): 135 KC1, 10 NaCl, 5 EGTA, 10 Hepes, pH was adjusted to 7.4 with NMDG (N-
Methyl-
D-Glucamine).
The cultured Calu-3 cells were placed in a chamber containing the following
extracellular solution (in mM): 135 NaCl, 5 KC1, 1.2 CaC12, 0.5 MgC12 and 10
Hepes (free
acid), pH was adjusted to 7.4 with NMDG.
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Cells were viewed using the 40X 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
extracellular solutes and pH as for the above control fluid, and whole-cell
currents at different
holding potentials were recorded with the same protocols.
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, CA). 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 I/V relationship is the whole cell conductance.
Drugs and Chemicals. Whenever indicated, cells were stimulated with a cAMP
stimulatory cocktail containing 8-Br-cAMP (500 mM), IBMX (isobutyl-l-
methylxanthie, 200
mM) and forskolin (10 mM). The cAMP analog 8-Br-cAMP (Sigma Chem. Co.) was
used from
a 25 mM stock in H2O 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:
Applicants determined whole-cell currents under basal (no cAMP) conditions,
with a
protocol stepping from zero mV holding potential to +/-100 mV. Representative
tracings
(control, followed by the whole-cell tracings while perfusing the test
solution) were made on an
average of n=12 cells. Composite `delta' tracings, obtained by subtraction of
the test average
values, from those under control conditions were obtained. The whole-cell
conductance,
obtained from the current-to-voltage relationships was 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) was also linear, and the reversal
potential was near zero mV.
There was a decrease in the whole cell conductance under hyperpolarizing
conditions.
In addition, Applicant determined whole-cell currents under basal conditions,
with a
protocol stepping from -40 mV holding potential to 100 mV. Representative
tracings (control,
followed by the whole-cell tracings while perfusing the test solution) were
made on an average
of n=12 cells. Composite delta tracings were obtained by subtraction of the
test average values,
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from those under control conditions. The whole-cell conductance obtained from
the current-to-
voltage relationships was highly linear under both conditions, and reflected 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) was also linear, and the reversal potential was near zero mV. Values
were comparatively
similar to those obtained with the zero mV protocol.
Applicants determined whole-cell currents under basal conditions, with a
protocol
stepping from -60 mV holding potential to 100 mV. Representative tracings
(control, followed
by the whole-cell tracings while perfusing the test solution) were made on an
average of n=12
cells. Composite `delta' tracings were obtained by subtraction of the test
average values, from
those under control conditions. The whole-cell conductance obtained from the
current -to-
voltage relationships was highly linear under both conditions, and reflected 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
was near zero mV. Values were comparatively similar to those obtained with the
zero mV
protocol.
Applicants also determined whole-cell currents under basal conditions, with a
protocol
stepping from -120 mV holding potential to 100 mV. Representative tracings
(control,
followed by the whole-cell tracings while perfusing the test solution) were
made on an average
of n=12 cells. Composite `delta' tracings were obtained by subtraction of the
test average
values, from those under control conditions. The whole-cell conductance
obtained from the
current -to-voltage relationships was highly linear under both conditions, and
reflected 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 was near zero mV. Values were comparatively similar to those
obtained with the zero
mV protocol.
In addition, Applicants determined 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. Representative tracings
(control, followed
by the whole-cell tracings after cAMP stimulation, followed by perfusion with
the drug-
containing solution) were made on an average of n=12 cells. Composite `delta'
tracings
(corresponding to voltage protocols at zero mV, and at -40 mV) were obtained
by subtraction of
the test average values in drug + cAMP, from those under control conditions
(cAMP alone).
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The whole-cell conductance obtained from the current-to-voltage relationships
was highly linear
under all conditions, and reflected a change in conductance due to the test
conditions.
Applications demonstrated whole-cell currents under cAMP-stimulated
conditions,
obtained with protocols stepping from various holding potentials to 100 mV.
Representative
tracings (control, followed by the whole-cell tracings after cAMP stimulation,
followed by
perfusion with the drug-containing solution) were made on a average of n=5
cells. Composite
`delta' tracings (voltage protocols at -60 mV, and-120 mV) were obtained by
subtraction of the
test average values in drug + cAMP, from those under control conditions (cAMP
alone). The
whole-cell conductance, obtained from the current-to-voltage relationships,
was highly linear
under all conditions, and reflected a change in conductance due to the test
conditions.
Applicants also demonstrated 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). 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 was
further observed
in the subtracted currents, where the contribution of the whole cell
conductance at zero mV was
further subtracted for each protocol (n=5).
Summary of Example. 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.).
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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.
Taken together with Applicants' other data, 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, and 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 components) including
but not limited
to GPCRs and/or g-proteins, and TSLP. 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 7
(Effects of inventive electrokinetically-altered fluids on whole-cell
conductance were
determined)
Overview. 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. Applicants performed patch clamp studies to further confirm the
utilities, as described
herein, of the inventive electrokinetically generated saline fluids (RNS-60
and Solas), including
the utility to modulate whole-cell currents. Two sets of experiments were
conducted.
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
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time. The contribution of the non-linear whole cell conductance, as previously
observed, was
voltage sensitive, although present at all voltage protocols.
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. See above 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.
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 hour. 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. Figures 3 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.
Figures 4 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.
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
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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. See above 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.
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 KC1). The external saline
was then replaced
with a solution where NaCl was replaced by CsCI 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: Figures 5 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 CsCI (square symbols) instead of NaCl as the external solution,
increased whole cell
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conductance with a linear behavior when compared to the control (diamond
symbols); and 2)
CaC12 at both 20 mM CaC12 (circle symbols) and 40 mM CaC12 (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)
CaC12 at 40 mM (triangle symbols) increased whole cell conductance in a non-
linear manner.
Figures 6 A-D show the graphs resulting from the subtraction of the CsCl
current data
(shown in Figure 5) from the 20 mM CaC12 (diamond symbols) and 40 mM CaC12
(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.
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.
The accumulated evidence suggests activation by Revalesio saline of ion
channels,
which make different contributions to the basal cell conductance.
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.
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EXAMPLE 8
(Effects of inventive electrokinetically-altered fluids on whole-cell
conductance were
investigated, and a dose response curve was generated)
Overview. In this experiment Applicants assessed the effect of dilutions of
the
electrokinetically-altered fluid (e.g., RNS-60) on epithelial cell membrane
polarity and ion
channel activity.
Methods. See above for general patch clamp methods. In the following
experiment,
patch clamp studies were performed to further confirm the utility of the
inventive
electrokinetically generated saline fluids (RNS-60) to modulate whole-cell
currents. In
particular, the experiment assessed the effect of dilutions of the inventive
electrokinetically
generated saline fluid. The solutions were made by diluting the inventive
electrokinetically
generated saline fluid in normal saline at concentrations of: 100% (Rev), 75%
(3:4), 50% (1:1),
25% (4:3), and 0% (Sal).
Results. Figures 7 A and B show the results of a series of patch clamp
experiments that
assessed the effects of diluted electrokinetically generated fluid (e.g., RNS-
60) on epithelial cell
membrane polarity and ion channel activity. Panel A demonstrates the volts
versus current of
whole cell conductance for each diluted sample as indicated on the graph (Rev,
3:4, 1:1, 4:3, and
Sal). Panel B demonstrates the dilution amount versus the change in current
comparing the
dilution to normal saline. The results indicate that the mechanism of action
of the RNS-60
solution occurs in a linear dose responsive manner.
EXAMPLE 9
(Treatment of primary bronchial epithelial cells (BEC) with the inventive
electrokinetically
generated fluids, as well as with non-electrokinetic control pressure pot
fluid, resulted in
reduced expression and/or activity of two key proteins of the airway
inflammatory pathways,
MMP9 and TSLP)
Overview. Applicants have now shown (using Bio-Layer Interferometry biosensor,
Octet Rapid Extended Detection (RED) (forteBioTm)), that in the presence of
electrokinetically
generated fluids (e.g., Rev; gas-enriched electrokinetically generated fluid)
of the instant
disclosure compared to normal saline, Bradykinin binding to the B2 receptor
was concentration
dependent, and binding affinity was increased. Additionally, in the context of
T-regulatory cells
stimulated with diesel exhaust particulate matter (PM, standard commercial
source), Applicants'
data showed a decreased proliferation of T-regulatory cells in the presence of
PM and Rev
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relative to PM in control fluid (no Rev, no Solis), 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, Diptheria
toxin (DT390, a
truncated diphtheria toxin molecule; 1:50 dilution of std. commercial
concentration) resulted in
beta blockade, GPCR blockade and Ca channel blockade of the effects the
activity of the
electrokinetically generated fluids on Treg and PBMC function. Furthermore,
Applicants' has
shown that upon exposure to the inventive fluids, tight junction related
proteins (e.g., JAM 2 and
3, GJA1, 3, 4 and 5 (junctional adherens), OCLN (occludin), claudins (e.g.,
CLDN 3, 5, 7, 8, 9,
10), TJP1 (tight junction protein 1)) were upregulated in lung tissue.
Furthermore, as shown in
patch clamp studies, 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.
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:
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 -80% confluence. The cells
were then treated for
6 hours with normal saline, control fluid Solas, non-electrokinetic control
pressure pot fluid, or
the test fluid Revera 60 at a 1:10 dilution (100ul in lml of airway epithelial
growth medium)
along with the diesel exhaust particulate matter (DEP or PM) before being
lifted for FACS
analysis. Both MMP9 and TSLP receptor antibodies were obtained from BD
Biosciences and
used as per manufacturer's specifications.
Results:
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In Figures 1 and 2, DEP represents cells exposed to diesel exhaust particulate
matter
(PM, standard commercial source) alone, "NS" represents cells exposed to
normal saline alone,
"DEP+NS" 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. "PP60" represents cells
exposed to the
non-electrokinetic control pressure pot fluid, and "DEP + PP60" refers to
cells treated with
particulate matter in the presence of the non-electrokinetic control pressure
pot fluid (i.e., having
60 ppm dissolved oxygen).
Figure 1 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 DEP induced TSLP receptor expression, while Normal Saline failed
to produce
similar level of reduction in DEP induced TSLP receptor expression
(approximately 20%
reduction). Additionally, the non-electrokinetic control pressure pot fluid
PP60 resulted in
approximately 50% reduction in DEP induced TSLP receptor expression.
The effect of the inventive Revera 60, Solas, and also of the PP60 solutions
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, YJ,
Thymic stromal
lymphopoietin: Master switch for allergic inflammation, J Exp Med 203:269-273,
2006; Al-
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)).
Likewise, Figure 2 shows the effect of Revera 60, Solas, non-electrokinetic
control
pressure pot fluid (PP60), 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.
Additionally, the
non-electrokinetic control pressure pot fluid PP60 resulted in approximately
30% reduction in
DEP-induced cell surface attached MMP9 levels. 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
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WO 2010/048425 PCT/US2009/061710
and even higher in patients with 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).
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.
According to yet additional aspects, non-electrokinetic control pressure pot
fluid (i.e.,
having 60 ppm dissolved oxygen) have 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. Without being bound by
mechanism,
Applicants' collective data indicates that the action of the non-
electrokinetic control pressure pot
fluid in this system is mediated by a mechanism that is distinct from that of
Applicants'
electrokinetically-generated fluids. This is not only because the effects are
relatively smaller,
but also because non-electrokinetic control pressure pot fluid has not
displayed activity in other
assays displaying activity with Applicants' electrokinetically generated
fluids. Nonetheless,
Applicants' discovery of the herein disclosed activity of non-electrokinetic
control pressure pot
fluid in this system represents a novel use for such pressure pot fluid in the
context of asthma
and related conditions as disclosed herein.
According to particular aspects, therefore, the inventive methods comprising
administration of Applicants' electrokinetically generated fluids provide for
modulation (down-
regulation of TSLP expression and/or activity) are applicable to the treatment
of at least one
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disease or condition selected from the TSLP-mediated group consisting of
disorders of the
immune system, allergic inflammation, allergic airway inflammation, DC-
mediated
inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma,
obstructive airways
disease, chronic obstructive pulmonary disease, and food allergies,
inflammatory arthritis,
rheumatoid arthritis and psoriasis.
The results disclosed herein are entirely consistent with the art-recognized
role of TSLP
as a master switch of allergic inflammation at the epithelial cell-DC
interface (Yong-Jun et al., J.
Exp. Med., 203:269-273, 2006), and are further consistent with the phenotypes
of mice lacking
the TSLPR (e.g., fail to develop asthma in response to inhaled antigens; Zhou
et al., supra and
Al-Shami et al., J. Exp. Med., 202:829-839, 2005), and with results obtained
from pretreating
OVA-DCs with anti-TSLPR (e.g., resulting in a significant reduction of
eosinophils and
lymphocyte infiltration as well as IL-4 and IL-5 levels.
The presently disclosed subject matter further illuminates the role that TSLPR
plays in
DC-primed allergic disease, and provides for novel compositions and methods
comprising
administration of Applicants' electrokinetic ally generated fluids.
