Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TREATING MATRIX
METALLOPROTEINASE 9 (MMP9)-MEDIATED CONDITIONS
FIELD OF THE INVENTION
The present invention relates generally to metalloproteinases and
metalloproteinase-
mediated conditions, and more particularly to matrix metalloproteinases
(MMPs), MMP-
mediated conditions, and to MMP inhibitors and treatment of MMP-mediated
conditions.
Particularly preferred aspects relate to modulation (e.g., inhibition) of MMPs
(e.g., MMP9), 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.
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
Metalloproteinases are a superfamily of proteinases (enzymes) classified into
families
and subfamilies as described, for example, in N. M. Hooper FEBS Letters 354:1-
6, 1994.
Examples of metalloproteinases include the matrix metalloproteinases (MMPs)
such as the
collagenases (MMP1, MMP8, MMP13), the gelatinases (MMP2, MMP9), the
stromelysins
(MMP3, MMP10, MMP II), matrilysin (MMP7), metalloelastase (MMP12), enamelysin
(MMP19), the MT-MMPs (MMP14, MMP15, MMP16, MMP17); the reprolysin or
adamalysin
or MDC family which includes the secretases and sheddases such as TNF
converting enzymes
(ADAM 10 and TACE); the astacin family which include enzymes such as
procollagen
processing proteinase (PCP); and other metalloproteinases such as aggrecanase,
the endothelin
converting enzyme family and the angiotensin converting enzyme family.
Collectively, the metalloproteinases are known to cleave a broad range of
matrix
substrates such as collagen, proteoglycan and fibronectin. Metalloproteinases
are implicated in
the processing, or secretion, of biological important cell mediators, such as
tumour necrosis
factor (TNF); and the post translational proteolysis processing, or shedding,
of biologically
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important membrane proteins, such as the low affinity IgE receptor CD23 (see,
e.g., N. M.
Hooper et al., Biochem. J. 321:265-279, 1997).
Not surprisingly, therefore, metalloproteinases are believed to be important
in many
physiological disease processes that involve tissue remodeling (e.g.,
embryonic development,
bone formation, uterine remodelling during menstruation, disrupting the blood-
brain barrier etc.).
Moreover, inhibition of the activity of one or more metalloproteinases may
well be of benefit in
these diseases or conditions, for example: various inflammatory and allergic
diseases such as,
inflammation of the joint (especially rheumatoid arthritis, osteoarthritis and
gout), inflammation
of the gastro-intestinal tract (especially inflammatory bowel disease,
ulcerative colitis and
gastritis), inflammation of the skin (especially psoriasis, eczema,
dermatitis); in tumour
metastasis or invasion; in disease associated with uncontrolled degradation of
the extracellular
matrix such as osteoarthritis; in bone resorptive disease (such as
osteoporosis and Paget's
disease); in diseases associated with aberrant angiogenesis; the enhanced
collagen remodelling
associated with diabetes, periodontal disease (such as gingivitis), corneal
ulceration, ulceration
of the skin, post-operative conditions (such as colonic anastomosis) and
dermal wound healing;
demyelinating diseases of the central and peripheral nervous systems (such as
multiple
sclerosis); Alzieimer's disease; extracellular matrix remodelling observed in
cardiovascular
diseases such as restenosis and atherosclerosis; asthma; rhinitis; and chronic
obstructive
pulmonary diseases (COPD).
MMP12, also known as macrophage elastase or metalloelastase, was initially
cloned in
the mouse (Shapiro et al., Journal of Biological Chemistry 267: 4664, 1992)
and has also been
cloned in man by the same group in 1995. MMP12 is preferentially expressed in
activated
macrophages, and has been shown to be secreted from alveolar macrophages from
smokers
(Shapiro et al, 1993, Journal of Biological Chemistry, 268: 23824) as well as
in foam cells in
atherosclerotic lesions (Matsumoto et al, Am. J. Pathol. 153: 109, 1998). A
mouse model of
COPD is based on challenge of mice with cigarette smoke for six months, two
cigarettes a day
six days a week. Wild-type mice developed pulmonary emphysema after this
treatment. When
MMP12 knock-out mice were tested in this model they developed no significant
emphysema,
strongly indicating that MMP12 is a key enzyme in the COPD pathogenesis. The
role of MMPs
such as MMP12 in COPD (emphysema and bronchitis) is discussed in Anderson and
Shinagawa,
1999, Current Opinion in Anti-inflammatory and Immunomodulatory
Investigational Drugs
1(1): 29-38. It was recently discovered that smoking increases macrophage
infiltration and
macrophage-derived MMP- 12 expression in human carotid artery plaques
(Matetzky S, Fishbein
M C et al., Circulation 102:(18), 36-39 Suppl. S, Oct. 31, 2000).
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MMP9-(Gelatinase B; 92 kDa-TypeIV Collagenase; 92 kDa Gelatinase) is a
secreted
protein which was first purified, then cloned and sequenced, in 1989 (S. M.
Wilhelm et al., J.
Biol. Chem. 264 (29): 17213-17221, 1989; published erratum in J. Biol. Chem.
265 (36): 22570,
1990) (for review of detailed information and references on this protease see
T. H. Vu & Z.
Werb (1998) (In: Matrix Metalloproteinases, 1998, edited by W. C. Parks & R.
P. Mecham, pp.
115-148, Academic Press. ISBN 0-12-545090-7).
The expression of MMP9 is restricted normally to a few cell types, including
trophoblasts, osteoclasts, neutrophils and macrophages (Vu & Werb, supra).
However, the
expression can be induced in these same cells and in other cell types by
several mediators,
including exposure of the cells to growth factors or cytokines. These are the
same mediators
often implicated in initiating an inflammatory response. As with other
secreted MMPs, MMP9 is
released as an inactive Pro-enzyme, which is subsequently cleaved to form the
enzymatically
active enzyme. The proteases required for this activation in vivo are not
known. The balance of
active MMP9 versus inactive enzyme is further regulated in vivo by interaction
with TIMP-1
(Tissue Inhibitor of Metalloproteinases-1), a naturally-occurring protein.
TIMP-1 binds to the
C-terminal region of MMP9, leading to inhibition of the catalytic domain of
MMP9. The
balance of induced expression of ProMMP9, cleavage of Pro- to active MMP9 and
the presence
of TIMP-1 combine to determine the amount of catalytically active MMP9 which
is present at a
local site. Proteolytically active MMP9 attacks substrates which include
gelatin, elastin, and
native Type IV and Type V collagens; it has no activity against native Type I
collagen,
proteoglycans or laminins.
There has been a growing body of data implicating roles for MMP9 in various
physiological and pathological processes. Physiological roles include the
invasion of embryonic
trophoblasts through the uterine epithelium in the early stages of embryonic
implantation; some
role in the growth and development of bones; and migration of inflammatory
cells from the
vasculature into tissues.
MMP9 release, measured using enzyme immunoassay, was significantly enhanced in
fluids and in AM supernantants from untreated asthmatics compared with those
from other
populations (Am. J. Resp. Cell & Mol. Biol., 5:583-591, 1997). Also, increased
MMP9
expression has been observed in certain other pathological conditions, thereby
implicating
MMP9 in disease processes such as COPD, arthritis, tumour metastasis,
Alzheimer's disease,
multiple sclerosis, and plaque rupture in atherosclerosis leading to acute
coronary conditions
such as myocardial infarction (see also W007087637A3, incorporated herein by
reference).
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Recently, it has been demonstrated that the levels of MMP-9 are significantly
increased
in patients with stable asthma 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, JAllergy Clin Immunol 2003;111:1278-1284).
SUMMARY OF EXEMPLARY EMBODIMENTS
Particular aspects provide a method for treating an MMP9-mediated condition or
disease,
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 an MMP9-mediated condition or disease. 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
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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 MMP9-mediated condition or disease comprises an
obstructive
airways disease, including but not limited to asthma and chronic obstructive
pulmonary disease.
In particular aspects, the MMP9-mediated condition or disease comprises at
least one of
rheumatoid arthritis, osteoarthritis, atherosclerosis, cancer, and multiple
sclerosis. In certain
embodiments, the MMP9-mediated condition or disease comprises at least one
disease or
disorder of the peripheral or central nervous system characterized by
persistent or sustained
expression and/or activity of MMP9, selected from the group consisting of
Alzheimer's disease,
stroke/cerebral ischemia, head trauma, spinal cord injury, multiple sclerosis,
amyotrophic lateral
sclerosis, Huntington's disease, Parkinson's disease, migraine, cerebral
amyloid angiopathy,
AIDS, age-related cognitive decline; mild cognitive impairment and prion
diseases in a mammal.
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 comprises administration of an
additional inhibitor of at
least one MMP, In certain aspects, the at least one MMP is selected from the
group consisting
of MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-
14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1, MMP-2, MMP-3,
MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-
16, MMP-17, MMP-18, MMP-19 and MMP-20. 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
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fusion proteins, including TSLPR-immunoglobulin Fc molecules or polypeptides
that encode
components of more than one receptor chain.
In certain aspects, the at least one additional therapeutic agent is selected
from the group
consisting of: standard non-steroidal anti-inflammatory drugs (NSAID'S),
piroxicam, diclofenac;
a propionic acid, naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen;
a fenamate,
mefenamic acid, indomethacin, sulindac, apazone; a pyrazolone, phenylbutazone;
a salicylate,
aspirin; an analgesic or intra-articular therapy, a corticosteroid; a
hyaluronic acid, hyalgan,
synvisc; an immune suppressant, cyclosporine, interferon; a TNF-.alpha.
inhibitor, EnbrelTm;
low dose methotrexate, lefunimide, hydroxychloroquine, d-penicilamine,
auranofin, parenteral
gold and oral gold.
In particular embodiments, the at least one additional therapeutic agent is
selected from
the CNS agent group consisting of: an antidepressant, sertraline, fluoxetine,
paroxetine; an anti-
Parkinsonian drug; deprenyl, L-dopa, requip, miratex; a MAOB inhibitor,
selegine, rasagiline; a
COMP inhibitor, tolcapone, Tasmar; an A-2 inhibitor, a dopamine reuptake
inhibitor, an NMDA
antagonist, a nicotine agonist, a dopamine agonist, an inhibitor of neuronal
nitric oxide synthase,
an anti-Alzheimer's drug; an acetylcholinesterase inhibitor, metrifonate,
donepezil, Aricept,
Exelon, ENA 713 or rivastigmine; tetrahydroaminoacridine, Tacrine, Cognex, or
THA; a COX-1
or COX-2 inhibitor, celecoxib, Celebrex, rofecoxib, Vioxx; propentofylline, an
anti-stroke
medication, an NR2B selective antagonist, a glycine site antagonist, and a
neutrophil inhibitory
factor (NIF).
In certain aspects, the at least one additional therapeutic agent is selected
from the group
consisting of: an estrogen; a selective estrogen modulator, estrogen,
raloxifene, tamoxifene,
droloxifene, lasofoxifene; an agent that results in reduction of A.beta.1-40/1-
42, an amyloid
aggregation inhibitor, a secretase inhibitor; an osteoporosis agent,
droloxifene, fosomax;
immunosuppressant agents, FK-506, rapamycin; an anticancer agent, endostatin,
angiostatin; a
cytotoxic drug, adriamycin, daunomycin, cis-platinum, etoposide, taxol,
taxotere; an alkaloid,
vincristine; an antimetabolite, methotrexate; a cardiovascular agent, calcium
channel blockers; a
lipid lowering agent, a statin; a fibrate, a beta-blocker, an ACE inhibitor,
an angiotensin-2
receptor antagonist , and a platelet aggregation inhibitor.
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
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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,
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
obstructive airways
disease, chronic obstructive pulmonary disease, asthma, rheumatoid arthritis,
osteoarthritis,
atherosclerosis, cancer, multiple sclerosis, Alzheimer's disease,
stroke/cerebral ischemia, head
trauma, spinal cord injury, amyotrophic lateral sclerosis, Huntington's
disease, Parkinson's
disease, migraine, cerebral amyloid angiopathy, AIDS, age-related cognitive
decline; mild
cognitive impairment and prion diseases.
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 adherins and desmasomes. 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.
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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,
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-alterd fluid is at least 8 ppm, at
least 15, ppm, at least 20
ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at
least 60 ppm
oxygen at atmospheric pressure.
In particular aspects, treating comprises administration by at least one of
topical,
inhalation, intranasal, and intravenous.
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
effect.
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.
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
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voltage protocols (A. stepping from zero mV; B. stepping from -60 mV; C
stepping from -120
mV) and the two time-points (15 minutes (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 CsC1 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.
DETAILED DESCRIPTION
Provided are methods for treating an MMP9-mediated condition or disease,
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 an MMP9-mediated condition or disease. 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 modulation or down-regulation of MMP-9 expression
and/or activity
have utility for treating MMP9-mediated diseases or conditions as disclosed
herein (e.g.,
obstructive airways disease, chronic obstructive pulmonary disease, asthma,
rheumatoid arthritis,
osteoarthritis, atherosclerosis, cancer, multiple sclerosis, Alzheimer's
disease, stroke/cerebral
ischemia, head trauma, spinal cord injury, amyotrophic lateral sclerosis,
Huntington's disease,
Parkinson's disease, migraine, cerebral amyloid angiopathy, AIDS, age-related
cognitive
decline; mild cognitive impairment and prion diseases).
Methods for making the electrokinetically generated fluid used herein have
been
previously described (see, e.g., US-2008-0219088, PCT/US2007/082578), which
are
incorporated herein by reference in their entirety).
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Particular aspects provide methods for treating an MMP (e.g., MMP9)-mediated
disease
or condition, comprising administering, to a subject in need thereof, a
therapeutically effective
amount of a composition comprising at least one electrokinetically generated
fluid (including
gas-enriched (e.g., oxygen enriched) electrokinetically generated fluids) as
disclosed herein.
Additional aspects provide methods for treating an obstructive airways
disease,
comprising administering a therapeutic composition comprising at least one
electrokinetically
generated fluid (including gas-enriched (e.g., oxygen enriched)
electrokinetically generated
fluids) as disclosed herein. In particular embodiments, the obstructive
airways disease
comprises at least one of asthma and chronic obstructive pulmonary disease.
Particular aspects
comprise treating at least one of rheumatoid arthritis, osteoarthritis,
atherosclerosis, cancer, and
multiple sclerosis.
Additional aspects provide novel methods and compositions having substantial
utility for
treatment of COPD, arthritis, tumor metastasis, Alzheimer's disease, multiple
sclerosis, and
plaque rupture in atherosclerosis leading to acute coronary conditions such as
myocardial
infarction. The novel methods have utility for treating a disease or disorder
of the peripheral or
central nervous system characterized by persistent or sustained expression
and/or activity of
MMP9, including but not limited to Alzheimer's disease, stroke/cerebral
ischemia, head trauma,
spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis,
Huntington's disease,
Parkinson's disease, migraine, cerebral amyloid angiopathy, AIDS, age-related
cognitive
decline; mild cognitive impairment and prion diseases in a mammal, which
comprises
administering to said mammal a therapeutically effective amount of a MMP9
inhibitor as
described herein.
Particular aspects of the present invention provide novel methods and
compositions
having substantial utility for treatment of cognitive impairment (e.g.,
dementia, cognitive decline
in aged individuals, Alzheimer's disease, etc.). Preferably, inhibitors of MMP-
9 as described
herein are used.
Additional aspects provide methods treating an MMP9-mediated disease or
condition,
comprising administering, to a subject in need thereof, a therapeutically
effective amount of a
composition comprising at least one electrokinetically generated fluid
(including gas-enriched
(e.g., oxygen enriched) electrokinetically generated fluids) as disclosed
herein, in combination
with administration of an additional inhibitor of at least one other MMP
(e.g., an inhibitor of
MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14,
MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1, MMP-2, MMP-3,
MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-
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16, MMP-17, MMP-18, MMP-19 and MMP-20, etc.). In particular embodiments, the
at least
one additional matrix metalloproteinase (MMP) inhibitor is suitable to inhibit
at least one MMP
having the characteristic elevated expression or activity. In additional
embodiments, the at least
one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least
two MMPs having
the characteristic elevated expression or activity. In certain embodiments,
the at least one matrix
metalloproteinase (MMP) inhibitor is MMP-specific or substantially specific to
a particular
MMP or inhibits a limited number of MMPs (e.g., from one to two MMPs, from one
to three
MMPs, or from about one to about four MMPs). In certain embodiments the at
least one matrix
metalloproteinase (MMP) inhibitor is a broad spectrum MMP inhibitor inhibiting
at least three,
or at least 4 MMPs (e.g., having the characteristic elevated expression or
activity).
Preferred exemplary embodiments:
Particular aspects provide a method for treating an MMP9-mediated condition or
disease,
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 an MMP9-mediated condition or disease. 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.
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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 MMP9-mediated condition or disease comprises an
obstructive
airways disease, including but not limited to asthma and chronic obstructive
pulmonary disease.
In particular aspects, the MMP9-mediated condition or disease comprises at
least one of
rheumatoid arthritis, osteoarthritis, atherosclerosis, cancer, and multiple
sclerosis. In certain
embodiments, the MMP9-mediated condition or disease comprises at least one
disease or
disorder of the peripheral or central nervous system characterized by
persistent or sustained
expression and/or activity of MMP9, selected from the group consisting of
Alzheimer's disease,
stroke/cerebral ischemia, head trauma, spinal cord injury, multiple sclerosis,
amyotrophic lateral
sclerosis, Huntington's disease, Parkinson's disease, migraine, cerebral
amyloid angiopathy,
AIDS, age-related cognitive decline; mild cognitive impairment and prion
diseases in a mammal.
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 comprises administration of an
additional inhibitor of at
least one MMP. In certain aspects, the at least one MMP is selected from the
group consisting
of MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-
14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1, MMP-2, MMP-3,
MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-
16, MMP-17, MMP-18, MMP-19 and MMP-20. 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 certain aspects, the at least one additional therapeutic agent is selected
from the group
consisting of: standard non-steroidal anti-inflammatory drugs (NSAID'S),
piroxicam, diclofenac;
a propionic acid, naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen;
a fenamate,
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mefenamic acid, indomethacin, sulindac, apazone; a pyrazolone, phenylbutazone;
a salicylate,
aspirin; an analgesic or intra-articular therapy, a corticosteroid; a
hyaluronic acid, hyalgan,
synvisc; an immune suppressant, cyclosporine, interferon; a TNF-.alpha.
inhibitor, EnbrelTm;
low dose methotrexate, lefunimide, hydroxychloroquine, d-penicilamine,
auranofin, parenteral
gold and oral gold.
In particular embodiments, the at least one additional therapeutic agent is
selected from
the CNS agent group consisting of: an antidepressant, sertraline, fluoxetine,
paroxetine; an anti-
Parkinsonian drug; deprenyl, L-dopa, requip, miratex; a MAOB inhibitor,
selegine, rasagiline; a
COMP inhibitor, tolcapone, Tasmar; an A-2 inhibitor, a dopamine reuptake
inhibitor, an NMDA
antagonist, a nicotine agonist, a dopamine agonist, an inhibitor of neuronal
nitric oxide synthase,
an anti-Alzheimer's drug; an acetylcholinesterase inhibitor, metrifonate,
donepezil, Aricept,
Exelon, ENA 713 or rivastigmine; tetrahydroaminoacridine, Tacrine, Cognex, or
THA; a COX-1
or COX-2 inhibitor, celecoxib, Celebrex, rofecoxib, Vioxx; propentofylline, an
anti-stroke
medication, an NR2B selective antagonist, a glycine site antagonist, and a
neutrophil inhibitory
factor (NIF).
In certain aspects, the at least one additional therapeutic agent is selected
from the group
consisting of: an estrogen; a selective estrogen modulator, estrogen,
raloxifene, tamoxifene,
droloxifene, lasofoxifene; an agent that results in reduction of A.beta.1-40/1-
42, an amyloid
aggregation inhibitor, a secretase inhibitor; an osteoporosis agent,
droloxifene, fosomax;
immunosuppressant agents, FK-506, rapamycin; an anticancer agent, endostatin,
angiostatin; a
cytotoxic drug, adriamycin, daunomycin, cis-platinum, etoposide, taxol,
taxotere; an alkaloid,
vincristine; an antimetabolite, methotrexate; a cardiovascular agent, calcium
channel blockers; a
lipid lowering agent, a statin; a fibrate, a beta-blocker, an ACE inhibitor,
an angiotensin-2
receptor antagonist , and a platelet aggregation inhibitor.
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
obstructive airways
disease, chronic obstructive pulmonary disease, asthma, rheumatoid arthritis,
osteoarthritis,
atherosclerosis, cancer, multiple sclerosis, Alzheimer's disease,
stroke/cerebral ischemia, head
trauma, spinal cord injury, amyotrophic lateral sclerosis, Huntington's
disease, Parkinson's
disease, migraine, cerebral amyloid angiopathy, AIDS, age-related cognitive
decline; mild
cognitive impairment and prion diseases.
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 adherins and desmasomes. 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,
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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,
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-alterd fluid is at least 8 ppm, at
least 15, ppm, at least 20
ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at
least 60 ppm
oxygen at atmospheric pressure.
In particular aspects, treating comprises administration by at least one of
topical,
inhalation, intranasal, and intravenous.
Electrokineticall-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).
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 nanostructures
substantially having an
average diameter of less than about 100 nanometers and stably configured in
the ionic aqueous
fluid in an amount sufficient to provide, upon contact of a living cell by the
fluid, modulation of
at least one of cellular membrane potential and cellular membrane
conductivity.
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,
CA 02741341 2011-04-20
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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."
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
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(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.
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
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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
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
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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 S/cm, at least 40 S/cm, at
least 80 S/cm, at least
100 S/cm, at least 150 S/cm, at least 200 S/cm, at least 300 S/cm, or at
least 500 S/cm, at
least 1 mS/cm, at least 5, mS/cm, 10 mS/cm, at least 40 mS/cm, at least 80
mS/cm, at least 100
mS/cm, at least 150 mS/cm, at least 200 mS/cm, at least 300 mS/cm, or at least
500 mS/cm. In
particular aspects, any salt may be used in preparing the inventive
electrokinetically-altered
fluids, provided that they allow for formation of biologically active salt-
stabilized nanostructures
(e.g., salt-stabilized oxygen-containing nanostructures) as disclosed herein.
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 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
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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.
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),,. 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
nano structures):
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'
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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
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, NaC1) 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
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WO 2010/048455 PCT/US2009/061744
(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),
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., CY 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 paticles 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 strutures 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.
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WO 2010/048455 PCT/US2009/061744
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
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,
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CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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.
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 nanobubles (e.g., hydrophobid nanobubbles) of about 20 nm x
1.5 nm, comprise
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CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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=0.082^057111.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%,
greater than about 10%, greater than about 15%, or greater than about 20%. In
additional
aspects, the substantial size of the charge-stabilized oxygen-containing
nanostructures, or arrays
thereof, having a charge-stabilized configuration in the ionic aqueous fluid
is a size selected
from the group consisting of less than: 100 nm; 90 nm; 80 nm; 70 nm; 60 nm; 50
nm; 40 nm; 30
nm; 20 nm; 10 nm; 5 nm; 4 nm; 3 nm; 2 nm; and 1 nm. Preferably, this size is
less than about
50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or
less than about
10 nm.
In certain aspects, the inventive electrokinetic fluids comprise solvated
electrons. In
further aspects, the inventive electrokinetic fluids comprises charge-
stabilized nanostructures
and/or charge-stabilized oxygen-containing nanostructures, and/or arrays
thereof, which
comprise at least one of: solvated electron(s); and unique charge
distributions (polar, symmetric,
asymmetric charge distribution). In certain aspects, the charge-stabilized
nanostructures and/or
charge- stabilized oxygen-containing nanostructures, and/or arrays thereof,
have paramagnetic
properties.
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
CA 02741341 2011-04-20
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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.
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)
BI 32-36
B2 169-200
B3 20-180*
B4 40-60
*wound depth minimal, majority >150, occasional 20 s
MMP-9 Mediated Conditions
MMP-9 is believed to be involved in many types of diseases, disorders, and/or
conditions
including, but not limited to, several types of cancers (e.g. breast cancer,
gastric cancer,
endometrial carcinoma, glioblastomas, and primary central nervous system
lymphoma
(PCNSL)), cardiovascular diseases (e.g. atherosclerosis and restenosis),
neuropsychiatric
disorders (e.g. schizophrenia and bipolar illness), pulmonary diseases (e.g.
asthma and chronic
bronchitis), neuroinflammatory degenerative diseases (e.g. Alzheimer's
disease, Parkinson's
disease, Huntington's disease, amyotrophic lateral sclerosis (ALS, also known
as Lou Gehrig's
disease), and diabetic retinopathy), autoimmune diseases (e.g. multiple
sclerosis, lupus
erythematosus, and rheumatoid arthritis), and nervous system-related disorders
and conditions
(e.g. stroke/cerebral ischemia, head trauma, spinal cord injury, migraine,
cerebral amyloid
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WO 2010/048455 PCT/US2009/061744
angiopathy, HIV-associated dementia (AIDS), age-related cognitive decline;
mild cognitive
impairment, and prion diseases in a mammal).
Indications
Wound healing. MMP-9 has been shown to have a role in scar-free wound healing
in
nude mice (Manuel and Gawronska-Kozak, Matrix Biology, 25:505-514, 2006). In
particular,
Manuel and Gawronska-Kozak showed that post-injured skin tissue had high
levels of MMP-9
mRNA and protein when compared to wildtype mice during the remodeling phase of
wound
healing. Another study found that MMP-9 was highly expressed in human oral
mucosa post
wounding. Since wound healing requires keratinocyte migration and granulation
tissue
remodeling, the study thus indicated that in wound healing, MMP-9 is involved
in keratinocyte
migration and granulation tissue remodeling (Salo et al., Lab Invest. 70:176-
82, 1994). These
results indicate that MMP-9 plays an important role in wound healing. However,
a more recent
study demonstrated that prolonged MMP-9 production resulted in poor wound
healing in ulcers
from diabetic patients, due at least in part to prolonged inflammation (Liu et
al., Diabetes Care,
32:117-119, 2009). This result indicates that MMP-9, while necessary during
the remodeling
phase of wound healing, inhibits full healing of ulcers. Applicants show
herein, using a BEC
model, that the inventive electrokinetically-altered fluids significantly
downregulated production
of MMP-9. According to certain embodiments, the inventive electrokinetically-
altered fluids
have substantial utility for treating wounds and similar conditions.
Cancers. A recent review describes the role of MMP-9 in different cancers
(Rybakowski.,
Cardiovascular Psychiatry and Neurology, Vol. 2009, Article ID 904836, 7
pages). More
specifically, the review discusses how a genetic mutation that increases
expression of MMP-9
mRNA is frequently associated with an increased risk of some kinds of cancer
and with more
severe progression of the tumor and/or greater dynamics of metastases. In
addition, several
studies have shown that this particular genetic mutation was associate with
malignancy, growth
of tumors and the severity of lymph node metastases in colorectal cancer,
breast cancer, gastric
cancer, and urinary bladder cancer. In addition, a recent study examining the
level of MMP-9 in
tumors from gastric carcinomas compared to normal gastric mucosa demonstrated
that the
carcinomas had a significantly increased level of MMP-9 protein and that
increase was
significantly associated with worse survival of the patients (Sier et al., J.
Thrombosis and
Haemostasis, 4:127-127, 2006). Another study demonstrated that MMP-9 mRNA and
MMP-9
activity were higher in gliomas than in normal brain and more strongly
correlated with severity
of tumor (Forsyth, et al., British J. Cancer, 79:1828-1835, 1999). These
studies indicate that
MMP-9 plays an important role in the pathogenesis of many types of cancers.
Applicants show
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WO 2010/048455 PCT/US2009/061744
herein, using a BEC model, that the inventive electrokinetically-altered
fluids significantly
downregulated production of MMP-9. According to certain embodiments, the
inventive
electrokinetically-altered fluids have substantial utility for treating
certain cancers (e.g.
colorectal cancer, breast cancer, gastric cancer, gliomas and urinary bladder
cancer) and similar
conditions and limiting lymph node metastases and alleviating complications
relating to
cancerous conditions.
Pulmonary diseases (e.g. asthma and chronic bronchitis). Recently, it has been
demonstrated that the levels of MMP-9 are significantly increased in patients
with stable asthma
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 hyper-responsiveness 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). In addition, a recent study demonstrated
that apical
treatment using MMP-9 resulted in 1) decreased immunostaining of proteins
found in tight
junctions and 2) increased infection efficiency by virus (e.g. viral access to
epithelial basolateral
surface), both of which indicate disruption of barrier function (Vermeer et
al., Am J Physiol
Lung Cell Mol Physiol, 296:L751-L762, 2009). These studies indicate that MMP-9
plays an
important role in the pathogenesis of pulmonary diseases (e.g. asthma and
chronic bronchitis).
Applicants show herein, using a BEC model, that the inventive
electrokinetically-altered fluids
significantly downregulated production of MMP-9.. According to certain
embodiments, the
inventive electrokinetically-altered fluids have substantial utility for
treating pulmonary diseases
(e.g. asthma and chronic bronchitis) and similar conditions.
Cardiovascular diseases. A recent review describes the role of MMP-9 in
cardiovascular diseases (e.g. CHD, atherosclerosis, and hypertension.)
(Rybakowski, 2009).
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WO 2010/048455 PCT/US2009/061744
More specifically, the review discusses how the genetic mutation that
increases expression of
MMP-9 mRNA is related to an increased progression and mortality of coronary
heart disease
(CHD), increased atherosclerosis, and increased progression of hypertension.
In further studies
correlations between higher MMP-9 levels and coronary artery ectasia, higher
MMP-9 levels
and hypertrophic cardiomyopathy, and higher MMP-9 levels and hypertensive
patients were
found. These studies indicate that MMP-9 plays an important role in the
pathogenesis of
cardiovascular diseases (e.g. CHD, atherosclerosis, and hypertension.).
Applicants show herein,
using a BEC model, that the inventive electrokinetically-altered fluids
significantly
downregulated production of MMP-9.. According to certain embodiments, the
inventive
electrokinetically-altered fluids have substantial utility for treating
cardiovascular diseases (e.g.
CHD, atherosclerosis, and hypertension.) and similar conditions.
Neuropsychiatric disorders. A recent review describes the role of MMP-9 in
neuropsychiatric disorders (e.g. schizophrenia and bipolar mood disorder)
(Rybakowski., 2009).
More specifically, the review discusses how patients with bipolar mood
disorder had a
significant prevalence of the genetic mutation that increases expression of
MMP-9 mRNA when
compared to healthy control subjects. However, schizophrenia patients when
compared to
normal patients had a significantly lower association of the genetic mutation
that increases
expression of MMP-9 mRNA. These studies indicate that MMP-9 has a correlative
role in
neuropsychiatric disorders (e.g. schizophrenia and bipolar mood disorder).
Applicants show
herein, using a BEC model, that the inventive electrokinetically-altered
fluids significantly
downregulated production of MMP-9.. According to certain embodiments, the
inventive
electrokinetically-altered fluids have substantial utility for treating
neuropsychiatric disorders
(e.g. schizophrenia and bipolar mood disorder) and similar conditions.
Neuroinflammatory degenerative diseases. For several years MMP-9 has been
implicated in the pathogenesis of diseases like Alzheimer's disease and ALS.
Increased
expression of MMP-9 also has been reported in postmortem Alzheimer's disease
and ALS brain
tissue (Lorenzl et al., Neurochem. Int. 43:191-6, 2003). Lorenzl et al. also
reported increased
levels of MMP-9 circulating in plasma in Alzheimer's patients. Interestingly,
a recent study
demonstrated that MMP-9 can degrade tightly aggregated amyloid-beta fibrils
and may
contribute to ongoing clearance of plaques from amyloid-laden brains (Yan et
al., J Biol Chem.
281:24566-74, 2006 ). Another recent study showed increased expression of MMP-
9 in the
cortex and cerebellum of post-mortem Huntington's disease patient samples
compared to
controls (Silverstroni, et al., Clinical Neuroscience and Neuropathology,
20:1098-1103, 2009).
These studies indicate that MMP-9 has an important role in neuroinflammatory
degenerative
29
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
diseases (e.g. Alzheimer's disease, Huntington's disease and ALS). Applicants
show herein,
using a BEC model, that the inventive electrokinetically-altered fluids
significantly
downregulated production of MMP-9.. According to certain embodiments, the
inventive
electrokinetically-altered fluids have substantial utility for treating
neuroinflammatory
degenerative diseases (e.g. Alzheimer's disease, Huntington's disease and ALS)
and similar
conditions.
HIV-associated dementia (AIDS). MMP-9 is involved with degrading the
extracellular
matrix and leads to reduced fortitude of the blood-brain barrier, which may
lead to blood-brain
barrier dysfunction if MMP-9 is too active. According to certain embodiments,
the inventive
electrokinetically-altered fluids significantly downregulate production of MMP-
9 (see herein
below) and can thus increase the resilience of the blood-brain barrier thereby
reducing HIV
infection of the brain and subsequent dementia.
Autoimmune disorders. For several years, studies have demonstrated the
presence or
increased presence of MMP-9 in bodily fluids from many autoimmune disorders
including but
not limited to multiple sclerosis, SLE, and rheumatoid arthritis. A recent
review discussed and
summarized studies that examined the presence and amount of MMP-9 in serum,
cerebrospinal
fluid (CSF), and synovial fluid from patients afflicted with multiple
sclerosis, SLE, and
rheumatoid arthritis (Ram et al., J. Clinical Immunology, 26:299-307, 2006).
In general, each
study found an increase in MMP-9 in the respective body fluid when compared to
controls. In
another study, Ainiala, et al., discovered a correlation between increased
serum MMP-9 and
neuropsychiatric manifestations (e.g. patients having at least one
neuropsychiatric symptom, like
cognitive dysfunction), small-vessel cerebral vasculopathy, and an increased
risk of cerebral
ischemic events in patients with SLE (Ainiala, et al., Arthritis Rheum 50:858-
65, 2004).
For rheumatoid arthritis, a study has demonstrated substantial elevation of
MMP-9 levels
in patient sera and synovial fluid (Gruber et al., Clin Immunol Immunopathol,
78:161-71, 1996).
The study also discovered that the source of MMP-9 in rheumatoid arthritis is
from rheumatoid
arthritis synovium. The in situ reverse transcriptase PCR found that the MMP-9
transcript was
produced in several different types of rheumatoid synovial cells. In another
study, MMP-9
levels were found to be substantially higher in joint fluids from rheumatoid
arthritis when
compared to joint fluids from osteoarthritis (Seki, et al., Modern
Rheumatology, 7:197-209,
2009). In addition, the study demonstrated that active MMP-9 concentrations in
joint fluids
from rheumatoid arthritis patients was positively correlated with the MMP-9
positive cells in
rheumatoid arthritis synovium and to the score of diffuse infiltrates of
lymphocytes. These
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
results indicate that active MMP-9 actively participates in joint destruction
of rheumatoid
arthritis.
For multiple sclerosis, studies have shown that MMP-9 predominates in multiple
sclerosis acute lesions. Several studies have also demonstrated that the
levels of MMP-9 in sera
and CSF are different depending on which type of the disease the patient has.
In particular, one
study showed that short disease duration and relapsing-remitting multiple
sclerosis had much
higher levels of MMP-9 than primary progressive multiple sclerosis (Avolio, et
al., J
Neuroimmunol, 136:46-53, 2003). However, the primary progressive multiple
sclerosis had
much higher levels of MMP-9 than a patient with inactive multiple sclerosis. A
recent study
examined the effects of estriol, a pregnancy hormone having anti-inflammatory
properties, on
the production of MMP-9 from peripheral blood mononuclear cells (PBMCs)
collected from
female multiple sclerosis patients (Gold, et al., Lab Investigation 89:1076-
83, 2009). Gold, et
al., demonstrated that production of MMP-9 was significantly decreased in
PBMCs from those
patients having the relapsing-remitting type of multiple sclerosis.
Interestingly, this decrease of
MMP-9 correlated with a decrease in enhancing lesions on MRI and a decrease in
T cell and
macrophage infiltration into the central nervous system as shown in the EAE
mouse model.
These studies indicate that MMP-9 has an important role in autoimmune
disorders (e.g.
multiple sclerosis, SLE, and rheumatoid arthritis). Applicants show herein,
using a BEC model,
that the inventive electrokinetically-altered fluids significantly
downregulated production of
MMP-9.. According to certain embodiments, the inventive electrokinetically-
altered fluids have
substantial utility for treating autoimmune disorders (e.g. multiple
sclerosis, SLE, and
rheumatoid arthritis) and similar conditions.
Migraine. Recently a study has shown an increased production of MMP-9 in
migraine
patients during migraine attacks, indicating that a migraine attack has an
inflammation and/or
blood-brain barrier disruption component. These studies indicate that MMP-9
has a role in
migraine attacks. Applicants show herein, using a BEC model, that the
inventive
electrokinetically-altered fluids significantly downregulated production of
MMP-9.. According
to certain embodiments, the inventive electrokinetically-altered fluids have
substantial utility for
treating migraine attacks and similar conditions.
Stroke. MMP-9 involvement in remodeling extracellular matrix and more
specifically
degrading the blood-brain barrier has been long appreciated in the art. MMP-9
role in post-
stroke recovery has been examined (Clark, et al., Neuroscience Letters, 238:53-
56, 1997). Clark,
et al., demonstrated that MMP-9 activity is markedly elevated in the infarcted
tissue two days
post-infarction. Interestingly, those patients that died from the stroke had
elevated levels of
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active MMP-9 starting from 2 days and lasting until death, indicating that the
prolonged active
MMP-9 has a role in morality resulting from a stroke. These studies indicate
that MMP-9 has a
role in stroke recovery. Applicants show herein, using a BEC model, that the
inventive
electrokinetically-altered fluids significantly downregulated production of
MMP-9.. According
to certain embodiments, the inventive electrokinetically-altered fluids have
substantial utility for
treating patient recovering from a stroke and similar conditions.
MMP inhibitors:
A number of metalloproteinase inhibitors are known (see, for example, the
reviews of
MMP inhibitors by Beckett R. P. and Whittaker M., 1998, Exp. Opin. Ther.
Patents, 8(3):259-
282; and by Whittaker M. et al, 1999, Chemical Reviews 99(9):2735-2776). WO
02/074767
discloses hydantoin derivatives of formula that are useful as MMP inhibitors,
particularly as
potent MMP12 inhibitors. U.S. Patent Application Serial No. 11/721,590
(published as
20080032997) discloses a further group of hydantoin derivatives that are
inhibitors of
metalloproteinases and are of particular interest in inhibiting MMPs such as
MMP12 and MMP9.
Novel triazolone derivatives for inhibiting MMPs such as MMP12 and MMP9 are
disclosed in
U.S. Patent Application Serial No. 10/593543 (published as 20070219217).
Additional MMP12
and MMP9 inhibitors are disclosed in 11/509,490 (published as 20060287338)
(see also
10/831265 (published as 20040259896)).
Additional exemplary MMP inhibitors are summarize in Table 2 below:
//
//
//
//
//
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CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
0
0
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_ _ C? N
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o
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= o o p o
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CA 02741341 2011-04-20
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CA 02741341 2011-04-20
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53
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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
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
54
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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
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
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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
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,
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CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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,
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 pM 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-
57
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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
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
58
CA 02741341 2011-04-20
WO 2010/048455 PCT/US2009/061744
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
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;
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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
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
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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.
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
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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 MMP-9-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 I
(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
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 L of 50 g/mL albuterol
sulfate in one or
two diluents: Group A was deionized water that had passed through the
inventive device,
without the addition of oxygen, while Group B was inventive gas-enriched
water. Each group
was dosed intratracheally with solutions using a Penn Century Microsprayer. In
addition, the
animals were stratified across BUXCO plethysmograph units so that each
treatment group is
represented equally within nebulizers feeding the plethysmographs and the
recording units.
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
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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 (Cavia porcellus) were Hartley albino,
Crl:(HA)BR
from Charles River Canada Inc. (St. Constant, Quebec, Canada). Weight:
Approximately 325
50 g at the onset of treatment; number of groups was 32, with 7 male animals
per group (plus 24
spares form same batch of animals). Diet; all animals had free access to a
standard certified
pelleted commercial laboratory diet (PMI Certified Guinea Pig 5026; PMI
Nutrition
International Inc.) except during designated procedures. 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)
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 g/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 1/animal of test
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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
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).
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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 pg
albuterol/animal 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-113, 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)
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-
1B, 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-
1B, 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-113, 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-
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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.
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 1A-1B). The other 6 rats from the RDC
1676-00
group were challenged with saline as the control group delivered by way of
intratreacheal
instillation. The following day, the procedure was repeated with the RDC 1676-
01 group.
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
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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 be employed in order to preserve the integrity of the 4 lobes on
the right side of
the lung. The left "large" lobe was lavaged, while the 4 right lobes were tied
off and
immediately placedinot TRI-zo1TM, 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-zo1TM or PAXgeneTM. Next, the
clotted blood
sample was centrifuged for 10 minutes at 1200 g at room temperature. The serum
(supernatant)
was removed and placed into two fresh tubes, and the serum was stored at -80
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 pL 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
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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 TM 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 RDC1676-00 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
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
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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.
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 IgGi (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 1271o/nTreg and CD4+CD25- responder T cells.
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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 III . 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
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-
10 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
consistent with the above T-regulatory cell data, as cells stimulated with
particulate matter (PM)
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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
induced by ovalbumin sensitization in this model resemble those observed in
man (Elwood et al.,
J Allergy Clin Immuno 88:951-60, 1991; Sirois & Bissonnette, Clin Exp Immunol
126:9-15,
2001). The inhaled route was selected to maximize lung exposure to the test
material or the
control solution. The ovalbumin-sensitized animals were treated with
budesonide alone or in
combination with the test material RDC 1676-03 for 7 days prior to ovalbumin
challenge. 6 and
24 hours following the challenge, total blood count and levels of several pro
and anti-
inflammatory cytokines as well as various respiratory parameters were measured
to estimate any
beneficial effect of administering the test material on various inflammatory
parameters.
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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:
Eosinophil Count: As expected, treatment with Budesonide ("NS + Budesonide 750
pg/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
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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
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.
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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
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).
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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
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
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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 pg 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 TM 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
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
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JAM 2 and 3, GJA1, 3, 4 and 5 (junctional adherins), OCLN (occludin), claudins
(e.g., CLDN 3,
5, 7, 8, 9, 10), TJP1 (tight junction protein 1), respectively.
EXAMPLE 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
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.
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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 adherins), 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.
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
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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 UV 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,
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
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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 refleced 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).
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
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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.).
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.
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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
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
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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
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
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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 CsC1 (square symbols) instead of NaCl as the external solution,
increased whole cell
conductance with a linear behavior when compared to the control (diamond
symbols); and 2)
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
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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) (forteBio)), 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
relative to PM in control fluid (no Rev, no Solis), indicating that the
inventive electrokinetically
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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 adherins), 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 1ml 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:
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,
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"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
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
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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
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
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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' electrokinetically generated fluids.
EXAMPLE 10
(Effects of the electrokinetic fluids on wound healing were determined)
The effects of a gas-enriched fluid (enriched with oxygen) were tested for the
ability of
cultured human epidermal keratinocytes to seal a wound. The results disclosed
in this Example
are also disclosed in Applicants' published applications US 2008/0139674 and
WO
2008/115290.
Human epidermal keratinocytes were isolated from neonatal foreskins that were
obtained from routine circumcision and de-identified. Foreskins were washed
twice in PBS and
incubated in 2.4 U/mL Dispase II in order to separate the dermis from the
epidermis. The
epidermis was incubated with 0.25% trypsin/1 mM EDTA, neutralized with soy
bean trypsin
inhibitor, agitated, and passed through a 70 um sieve to separate the cells.
Next, the cell
suspension was centrifuged and resuspended in cell culture medium (M154)
supplemented with
0.07 mM CaC12, and human keratinocyte growth supplements (0.2% hydrocortisone,
0.2 ng/mL
human epidermal growth factor) and penicillin/streptomycin, amphoteracin
antibiotic cocktail.
The keratinocyte cell suspensions were plated onto uncoated 12-well culture
dishes and the
medium replaced after 24 hours, and every 48 hours after the initial seeding.
Upon reaching cellular confluence, linear scratches were made with a sterile
p1000
pipette tip, which resulted in a uniform cell-free wound. The monolayers were
washed several
times with Dulbecco's PBS in order to remove any cellular debris. The wound
monolayers
were then incubated in the following media: i) the complete growth media (as
described above
in this Example); ii) the complete growth media diluted 1:1 with a sheared
version of saline
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without oxygen (control fluid that was processed using the disclosed diffuser
device but without
adding a gas); and iii) the complete growth media diluted 1:1 with oxygen-
enriched saline.
Each study was done in triplicate.
Prior to incubation, the wells were filled with the respective media and
sealed by placing
a 25 x 25 mm glass coverslip on top of each well. At 6, 12, 24, and 48 hours
post-wounding,
oxygen measurements were made, and cultures were imagined.
Results. Six hours post-wounding, the edges of the wounds in the saline and
gas-
enriched media were more ruffled than those in the media control that was
processed with the
diffuser device disclosed herein, but without the addition of a gas. Twelve
hours post-
wounding the edges of the wounds in all three media appeared uneven, with
keratinocytes along
the borders migrating toward the center of the wounds. Quantification of
migrating
keratinocytes revealed approximately the same level of keratinocyte migration
in the saline and
gas-enriched media.
EXAMPLE 11
(Effect of the electrokinetic fluids on improved wound healing was
demonstrated)
A study was performed to determine the improved healing characteristics of
wounds that
were exposed to an oxygen-enriched saline solution that was processed
according to
embodiments disclosed herein. In this experiment, bandages were placed on
porcine dermal
excision biopsy wounds. The bandages soaked in oxygen-enriched saline solution
or a control
group of bandages soaked in a saline solution that was not oxygen-enriched.
Microscopically,
several factors were evaluated by the study including: 1) epidermalization; 2)
neovascularization; 3) epidermal differentiation; 4) mast cell migration; and
5) mitosis. The
results disclosed in this Example are also disclosed in Applicants' published
applications US
2008/0139674 and WO 2008/115290.
Results. Externally, the wounds appeared to heal at varying rates. The wounds
treated
with the oxygen-enriched saline solution showed an increase in wound healing
at days 4 through
11. However, both wounds seemed to complete healing at approximately the same
time. The
study showed that between days 3 and 11, the new epidermis in wounds treated
with the oxygen-
enriched saline solution migrated at two to four times as fast as the
epidermis of the wounds
treated with the normal saline solution. The study also showed that between 15
and 22 days, the
wound treated by the oxygen-enriched saline solution differentiated at a more
rapid rate as
evidenced by the earlier formation of more mature epidermal layers. At all
stages, the
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thickening that occurs in the epidermis associated with normal healing did not
occur within the
wounds treated by the oxygen-enriched saline solution.
Without wishing to be bound by any particular theory, it is believed that the
oxygen-
enriched saline solution may increase the localized level of NO within the
wounds. NO
modulates growth factors, collagen deposition, inflammation, mast cell
migration, epidermal
thickening, and neovascularization in wound healing. Furthermore, nitric oxide
is produced by
an inducible enzyme that is regulated by oxygen.
Thus, while not wishing to be bound to any particular theory, the inventive
gas-enriched
fluid may stimulate NO production, which is in accordance with the spectrum of
wound healing
effects seen in these experiments.
The epidermis of the healing pigs experienced earlier differentiation in the
oxygen-
enriched saline group at days 15 through 22. In the case of mast cell
migration, differences also
occurred in early and late migration for the oxygen-enriched solution. A
conclusive result for
the level of mitosis was unascertainable due to the difficulty in staining.
The results indicated that the wound treated with the oxygen-enriched saline
solution
showed much greater healing characteristics than the untreated wound. In
addition, the results
show a greater differentiated epidermis with more normal epidermal/dermal
contour.
Incorporation by Reference
All of the above U.S. patents, U.S. patent application publications, U.S.
patent
applications, foreign patents, foreign patent applications and non-patent
publications referred to
in this specification and/or listed in the Application Data Sheet, are
incorporated herein by
reference, in their entirety.
It should be understood that the drawings and detailed description herein are
to be
regarded in an illustrative rather than a restrictive manner, and are not
intended to limit the
invention to the particular forms and examples disclosed. On the contrary, the
invention
includes any further modifications, changes, rearrangements, substitutions,
alternatives, design
choices, and embodiments apparent to those of ordinary skill in the art,
without departing from
the spirit and scope of this invention, as defined by the following claims.
Thus, it is intended
that the following claims be interpreted to embrace all such further
modifications, changes,
rearrangements, substitutions, alternatives, design choices, and embodiments.
The foregoing described embodiments depict different components contained
within, or
connected with, different other components. It is to be understood that such
depicted
architectures are merely exemplary, and that in fact many other architectures
can be
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implemented which achieve the same functionality. In a conceptual sense, any
arrangement of
components to achieve the same functionality is effectively "associated" such
that the desired
functionality is achieved. Hence, any two components herein combined to
achieve a particular
functionality can be seen as "associated with" each other such that the
desired functionality is
achieved, irrespective of architectures or intermedial components. Likewise,
any two
components so associated can also be viewed as being "operably connected", or
"operably
coupled", to each other to achieve the desired functionality.
While particular embodiments of the present invention have been shown and
described,
it will be obvious to those skilled in the art that, based upon the teachings
herein, changes and
modifications may be made without departing from this invention and its
broader aspects and,
therefore, the appended claims are to encompass within their scope all such
changes and
modifications as are within the true spirit and scope of this invention.
Furthermore, it is to be
understood that the invention is solely defined by the appended claims. It
will be understood by
those within the art that, in general, terms used herein, and especially in
the appended claims
(e.g., bodies of the appended claims) are generally intended as "open" terms
(e.g., the term
"including" should be interpreted as "including but not limited to," the term
"having" should be
interpreted as "having at least," the term "includes" should be interpreted as
"includes but is not
limited to," etc.). It will be further understood by those within the art that
if a specific number
of an introduced claim recitation is intended, such an intent will be
explicitly recited in the
claim, and in the absence of such recitation no such intent is present. For
example, as an aid to
understanding, the following appended claims may contain usage of the
introductory phrases "at
least one" and "one or more" to introduce claim recitations. However, the use
of such phrases
should not be construed to imply that the introduction of a claim recitation
by the indefinite
articles "a" or "an" limits any particular claim containing such introduced
claim recitation to
inventions containing only one such recitation, even when the same claim
includes the
introductory phrases "one or more" or "at least one" and indefinite articles
such as "a" or "an"
(e.g., "a" and/or "an" should typically be interpreted to mean "at least one"
or "one or more");
the same holds true for the use of definite articles used to introduce claim
recitations. In
addition, even if a specific number of an introduced claim recitation is
explicitly recited, those
skilled in the art will recognize that such recitation should typically be
interpreted to mean at
least the recited number (e.g., the bare recitation of "two recitations,"
without other modifiers,
typically means at least two recitations, or two or more recitations).
Accordingly, the invention
is not limited except as by the appended claims.
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