U.S. patent application number 14/430506 was filed with the patent office on 2015-09-03 for treatment compositions.
The applicant listed for this patent is REOXCYN DISCOVERIES GROUP, INC.. Invention is credited to James Pack, Daniel Robinson, Gary Samuelson.
Application Number | 20150246071 14/430506 |
Document ID | / |
Family ID | 50341821 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246071 |
Kind Code |
A1 |
Robinson; Daniel ; et
al. |
September 3, 2015 |
TREATMENT COMPOSITIONS
Abstract
Compositions for and methods of treating diseases related to
oxidative stress are described.
Inventors: |
Robinson; Daniel; (Salt Lake
City, UT) ; Pack; James; (Salt Lake City, UT)
; Samuelson; Gary; (Salt Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REOXCYN DISCOVERIES GROUP, INC. |
Salt Lake City |
UT |
US |
|
|
Family ID: |
50341821 |
Appl. No.: |
14/430506 |
Filed: |
February 28, 2013 |
PCT Filed: |
February 28, 2013 |
PCT NO: |
PCT/US2013/028420 |
371 Date: |
March 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61707141 |
Sep 28, 2012 |
|
|
|
61706670 |
Sep 27, 2012 |
|
|
|
61704401 |
Sep 21, 2012 |
|
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Current U.S.
Class: |
424/666 ;
424/680; 424/722 |
Current CPC
Class: |
A61K 33/14 20130101;
A61K 31/192 20130101; A61K 31/4439 20130101; A61K 33/00 20130101;
A61K 33/00 20130101; C02F 2201/46115 20130101; A61K 31/216
20130101; A61K 31/557 20130101; A61K 33/14 20130101; A61P 43/00
20180101; C02F 1/46104 20130101; A61P 3/10 20180101; C02F 2201/4611
20130101; C02F 2201/46155 20130101; C02F 1/467 20130101; C25B 9/08
20130101; C02F 1/46109 20130101; A61K 31/20 20130101; A61K 31/421
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 45/06
20130101; A61K 31/422 20130101; A61P 35/00 20180101; C02F 1/4618
20130101; C02F 1/4698 20130101 |
International
Class: |
A61K 33/14 20060101
A61K033/14; A61K 45/06 20060101 A61K045/06; A61K 31/20 20060101
A61K031/20; A61K 31/421 20060101 A61K031/421; A61K 31/4439 20060101
A61K031/4439; A61K 31/216 20060101 A61K031/216; A61K 31/192
20060101 A61K031/192; A61K 31/422 20060101 A61K031/422; A61K 33/00
20060101 A61K033/00; A61K 31/557 20060101 A61K031/557 |
Claims
1. A composition comprising at least one redox signaling agent
(RXN) and a PPAR agonist.
2. The composition of claim 1, wherein the PPAR agonist comprises a
Free Fatty Acid (FFA).
3. The composition of claim 1, wherein the PPAR agonist comprises
an eicosanoid.
4. The composition of claim 1, wherein the PPAR agonist comprises a
thazolidinedione.
5. The composition of claim 1, wherein the PPAR agonist comprises a
fibrate.
6. The composition of claim 1, wherein the PPAR agonist comprises a
dual agonist.
7. The composition of claim 6, wherein the dual agonist comprises
aleglitazar.
8. The composition of claim 6, wherein the dual agonist comprises
muraglitazar.
9. The composition of claim 6, wherein the dual agonist comprises
tesaglitazar.
10. The use of a composition comprising RXN and a PPAR agonist in
the treatment of a PPAR-mediated disease.
11. The use of claim 10, wherein the PPAR-mediated disease is
diabetes mellitus.
12. The use of claim 11, wherein the diabetes mellitus is type 1
diabetes.
13. The use of claim 11, wherein the diabetes mellitus is type 2
diabetes.
14. The use of claim 10, wherein the PPAR-mediated disease is
obesity.
15. The use of claim 10, wherein the PPAR-mediated disease is
cancer.
16. The use of a composition comprising at least one RXN in the
treatment of cancer, comprising a first therapy which activates a
PPAR pathway and at least one other agent wherein the at least one
other agent does not activate a PPAR pathway.
17. The use of claim 16, wherein the at least one other agent
comprises radiation.
18. The use of claim 16, wherein the at least one other agent
comprises a chemotherapeutic agent.
19. The use of a composition comprising at least one RXN in the
treatment of cancer, comprising a first therapy which mobilizes
Free Fatty Acids (FFAS) and at least one other agent wherein the at
least one other agent does not mobilize FFAs.
20. The use of claim 19, wherein the at least one other agent
comprises radiation.
21. A method of treating an oxidative stress related disorder
comprising: administering a composition including at least one
species selected from O.sub.2, H.sub.2, Cl.sub.2, OCl.sup.-, HOCl,
NaOCl, HClO.sub.2, ClO.sub.2, HClO.sub.3, HClO.sub.4,
H.sub.2O.sub.2, Na.sup.+, Cl.sup.-, H.sup.+, H.sup.-, OH.sup.-,
O.sub.3, O.sub.4*.sup.-, .sup.1O, OH*.sup.-, HOCl--O.sub.2*.sup.-,
HOCl--O.sub.3, O.sub.2*.sup.-, HO.sub.2*, NaCl, HCl, NaOH, water
clusters, or a combination thereof to a patient experiencing
oxidative stress; and treating the oxidative stress related
disorder.
22. A method of treating a reduced mitochondrial DNA disorder
comprising: administering a composition including at least one
species selected from O.sub.2, H.sub.2, Cl.sub.2, OCl.sup.-, HOCl,
NaOCl, HClO.sub.2, ClO.sub.2, HClO.sub.3, HClO.sub.4,
H.sub.2O.sub.2, Na.sup.+, Cl.sup.-, H.sup.+, H.sup.-, OH.sup.-,
O.sub.3, O.sub.4*.sup.-, .sup.1O, OH*.sup.-, HOCl--O.sub.2*.sup.-,
HOCl--O.sub.3, O.sub.2*.sup.-, HO.sub.2*, NaCl, HCl, NaOH, water
clusters, or a combination thereof to a patient experiencing the
reduced mitochondrial DNA disorder; increasing mitochondrial DNA
density; and treating the reduced mitochondrial DNA disorder.
23. The method of claim 22, wherein the reduced mitochondrial DNA
disorder is sacropenia, diabetes, Alzheimer's disease, Parkinson's
disease, neurological disease, muscle loss due to aging, obesity,
or cardiovascular disorders.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 61/704,401, filed Sep. 21, 2012; 61/706,670, filed
Sep. 27, 2012; and 61/707,141, filed Sep. 28, 2012. Each of these
provisional applications is incorporated by reference in its
entirety.
TECHNICAL FIELD
[0002] Compositions useful for the treatment of diseases related to
oxidative stress and reduced mitochondrial DNA are disclosed.
BACKGROUND
[0003] Virtually all biochemistry takes place in salt water, from
the transcription of genes to the production of structures,
proteins and enzymes. Considering this, it is not surprising that
all life, from the most primitive prokaryotic bacteria to the most
advanced eukaryotic mammalian cells (even plants), has developed
the ability to oxidize and reduce salt water, thereby producing two
classes of reactive molecules, namely the "reduced species" (RS)
and "reactive oxygen species" (ROS). Both of these chemical species
are produced from salt water and are prevalent in mammalian
cells.
[0004] Mammalian cells utilize RS/ROS in a variety of biological
processes. RS/ROS are produced in large amounts in the cell during
the metabolism of sugar, specifically by the oxidative
phosphorylation process in the mitochondria.
SUMMARY
[0005] Embodiments include a composition comprising a redox
signaling agent (RXN) and a Peroxisome Proliferator-Activated
Receptor (PPAR) agonist. In an embodiment the PPAR agonist
comprises a Free Fatty Acid (FFA). In an embodiment the PPAR
agonist comprises an eicosanoid, or a thazolidinedione, or a
fibrate. In an embodiment the PPAR agonist comprises a dual agonist
such as aleglitazar, muraglitazar, or tesaglitazar. Embodiments
include the use of a composition comprising RXN and a PPAR agonist
in the treatment of a PPAR-mediated disease such as diabetes
mellitus, including type 1 diabetes, type 2 diabetes, gestational
diabetes, obesity, or cancer. Embodiments useful in the treatment
of cancer can comprise a first therapy which activates a PPAR
pathway and at least one other agent wherein the at least one other
agent does not activate a PPAR pathway, such as radiation or a
chemotherapeutic agent. Embodiments useful in the treatment of
cancer can comprise a first therapy which mobilizes Free Fatty
Acids (FFAS) and at least one other agent wherein the at least one
other agent does not mobilize FFAs, such as radiation.
[0006] Embodiments include a method of treating an oxidative stress
related disorder, such method including administering a composition
including at least one species selected from O.sub.2, H.sub.2,
Cl.sub.2, OCl.sup.-, HOCl, NaOCl, HClO.sub.2, ClO.sub.2,
HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2, Na.sup.+, Cl.sup.-,
H.sup.+, H.sup.-, OH.sup.-, O.sub.3, O.sub.4*.sup.-, .sup.1O,
OH*.sup.-, HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-,
HO.sub.2*, NaCl, HCl, NaOH, water clusters, or a combination
thereof to a patient experiencing oxidative stress; and treating
the oxidative stress related disorder.
[0007] Embodiments include a method of treating a reduced
mitochondrial DNA disorder comprising administering a composition
including at least one species selected from O.sub.2, H.sub.2,
Cl.sub.2, OCl.sup.-, HOCl, NaOCl, HClO.sub.2, ClO.sub.2,
HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2, Na.sup.+, Cl.sup.-,
H.sup.+, H.sup.-, OH.sup.-, O.sub.3, O.sub.4.sup.*-, .sup.1O,
OH*.sup.-, HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-,
HO.sub.2*, NaCl, HCl, NaOH, water clusters, or a combination
thereof to a patient experiencing the reduced mitochondrial DNA
disorder; increasing mitochondrial DNA density; and treating the
reduced mitochondrial DNA disorder. Such disorders include
sacropenia, diabetes, Alzheimer's disease, Parkinson's disease,
neurological disease, muscle loss due to aging, obesity, or
cardiovascular disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a flow chart of a process as described herein.
[0009] FIG. 2 illustrates an example diagram of the generation of
various molecules at the electrodes. The molecules written between
the electrodes depict the initial reactants and those on the
outside of the electrodes depict the molecules/ions produced at the
electrodes and their electrode potentials.
[0010] FIG. 3 illustrates a plan view of a process and system for
producing a composition according to the present description.
[0011] FIG. 4 illustrates an example system for preparing water for
further processing into a composition described herein.
[0012] FIG. 5 illustrates a Cl35 spectrum of a NaCl, NaClO solution
at a pH of 12.48, and a composition described herein (the
composition is labeled "ASEA").
[0013] FIG. 6 illustrates a .sup.1H NMR spectrum of a composition
of the present disclosure.
[0014] FIG. 7 illustrates a 31P NMR spectrum of DIPPMPO combined
with a composition described herein.
[0015] FIG. 8 illustrates a mass spectrum showing a parent peak and
fragmentation pattern for DIPPMPO with m/z peaks at 264, 222, and
180.
[0016] FIG. 9 illustrates oxygen/nitrogen ratios for a composition
described herein compared to water and NaClO (the composition is
labeled "ASEA").
[0017] FIG. 10 illustrates chlorine/nitrogen ratios for a
composition described herein compared to water and NaClO (the
composition is labeled "ASEA").
[0018] FIG. 11 illustrates ozone/nitrogen ratios for a composition
described herein compared to water and NaClO (the composition is
labeled "ASEA").
[0019] FIG. 12 illustrates the carbon dioxide to nitrogen ratio of
a composition as described herein compared to water and NaClO (the
composition is labeled "ASEA").
[0020] FIG. 13 illustrates an EPR splitting pattern for a free
electron.
[0021] FIG. 14 illustrates a flow chart of the mouse study
described in Example 3 (the composition-treatment group is labeled
"ASEA").
[0022] FIG. 15 is a flow chart showing a total overview of the
mouse preparation and study (the composition used is referred-to as
"ASEA").
[0023] FIG. 16 illustrates mice grouped into placebo and ASEA (a
composition described herein) treatment versus run time and versus
glycogen depletion.
[0024] FIG. 17A illustrates the fold change relate to ASEA (a
composition described herein) treatment group of different mouse
groups. FIG. 17B illustrates the fold change difference between
ASEA sedentary (non-running) and ASEA running groups.
[0025] FIG. 18 illustrates different mouse groups versus the amount
of liver Superoxide Dismutase (SOD) produced (the composition
embodiment used is referred-to as "ASEA").
[0026] FIGS. 19A and 19B illustrate different mouse groups
(sedentary/running; treatment/placebo) versus oxidized glutathione
(the composition embodiment used is referred-to as "ASEA").
[0027] FIG. 20 illustrates different mouse groups versus fold
change for IL-6 and TNF-alpha (the composition embodiment used is
referred-to as "ASEA").
[0028] FIG. 21 illustrates a comparison of A-B ratios between
conditions 24 hours post ingestion (the composition embodiment used
is referred-to as "ASEA").
[0029] FIG. 22 illustrates a flow chart of the human running
performance study protocol (the composition embodiment used in the
protocol is referred-to as "ASEA").
[0030] FIG. 23 illustrates a flow chart of a 12-week, randomized
trial performed accord to the protocol of Example 7 (the
composition embodiment used in the protocol is referred-to as
"ASEA").
[0031] FIG. 24 illustrates a graph of VCO.sub.2 versus VO.sub.2
resulting from the study in Example 8 (the composition embodiment
used in the protocol is referred-to as "ASEA").
[0032] FIG. 25 illustrates cell images for each culture results of
HMVEC-L Cells p65 subunit NF-kB screen for toxicity (the
composition embodiment used in the protocol is referred-to as
"ASEA").
[0033] FIG. 26 illustrates results for P-Jun screen for toxicity
(the composition embodiment used in the protocol is referred-to as
"ASEA").
[0034] FIG. 27 illustrates a graph showing the reduction of
oxidants over an 11 minute interval (RFU units on vertical
scale).
[0035] FIG. 28 illustrates a graph showing antioxidant activity
over an 11 minute interval (the composition embodiment used in the
protocol is referred-to as "ASEA").
[0036] FIG. 29 illustrates nuclear staining patterns for results of
HMVEC-L Nuclear Accumulation of NRF2 (the composition embodiment
used in the protocol is referred-to as "ASEA").
[0037] FIG. 30 illustrates serum-starved cell cultures exposed to
low-concentration ASEA (a composition disclosed herein).
[0038] FIG. 31 illustrates a western blot validation of NRF2
nuclear accumulation following ASEA treatment.
[0039] FIG. 32 illustrates results for proliferation of murine and
HMVEC-L cells and LDH activity following ASEA treatment.
[0040] FIG. 33 illustrates further results for proliferation of
murine and HMVEC-L cells and LDH activity following ASEA
treatment.
[0041] FIG. 34 illustrates results of HMVEC-L viability exposed
high-concentration ASEA and to escalating amounts of Cachexin
stressor (the composition embodiment used in the protocol is
referred-to as "ASEA").
[0042] FIG. 35 illustrates results of concentration-dependent
response of HMVEC-L cells to Cachexin insult (the composition
embodiment used in the protocol is referred-to as "ASEA").
DETAILED DESCRIPTION
[0043] Described herein are therapeutic compositions that generally
include at least one redox signaling agent (RXN). RXNs can include,
but are not limited to superoxides: O.sub.2*.sup.-, HO.sub.2*;
hypochlorites: OCl.sup.-, HOCl, NaOCl; hypochlorates: HClO.sub.2,
ClO.sub.2, HClO.sub.3, HClO.sub.4; oxygen derivatives: O.sub.2,
O.sub.3, O.sub.4*.sup.-, .sup.1O; hydrogen derivatives: H.sub.2,
H.sup.-; hydrogen peroxide: H.sub.2O.sub.2; hydroxyl free radicals:
OH*.sup.-; ionic compounds: Na.sup.+, Cl.sup.-, H.sup.+, OH.sup.-,
NaCl, HCl, NaOH; chlorine: Cl.sub.2; and water clusters:
n*H.sub.2O-induced dipolar layers around ions. A composition
comprising RXNs as described herein can mobilize free fatty acids
(FFAs) and increase mitochondrial density. FFAs may activate
Peroxisome Proliferator-Activated Receptors (PPARs).
[0044] Embodiments can further comprise a PPAR agonist. PPARs can
be nuclear receptors. The PPAR family comprises three isoforms,
designated alpha, gamma and delta (also called beta), each encoded
by a different gene. These receptors, which form part of the
superfamily of nuclear receptors and of transcription factors, may
play a major role in regulation of lipid and carbohydrate
metabolism. PPARs may play important roles in the regulation of
cellular differentiation, development, and metabolism
(carbohydrate, lipid, protein), and tumorigenesis of higher
organisms.
[0045] PPAR-alpha may control lipid metabolism (hepatic and
muscular) and glucose homeostasis, and may influence intracellular
metabolism of lipids and sugars by direct control of transcription
of the genes coding for proteins involved in lipid homeostasis.
PPAR-alpha may also exert anti-inflammatory and antiproliferative
effects and may prevent the proatherogenic effects of accumulation
of cholesterol in macrophages by stimulating the outflow of
cholesterol. PPAR-gamma is a key regulator of adipogenesis. It may
also be involved in the lipid metabolism of mature adipocytes, in
glucose homeostasis, in insulin resistance, in inflammation, in
accumulation of cholesterol at the macrophage level and in cellular
proliferation. PPAR-gamma consequently may play a role in the
pathogenesis of obesity, insulin resistance and diabetes.
PPAR-delta may be involved in controlling lipid and carbohydrate
metabolism, in the energy balance, in neurodegeneration, in
obesity, in the formation of foam cells and in inflammation.
[0046] Methods of making therapeutic compositions are described
comprising: electrolyzing salinated water having a salt
concentration of about 10 g NaCl/gal, such as 10.75 g NaCl/gal
using a set of electrodes with an amperage of about 50-60 amps,
such as 56 amps to form a life enhancing composition, wherein the
water is chilled below room temperature and the water is circulated
during electrolyzing.
[0047] A method of producing the disclosed compositions can include
one or more of the steps of (1) preparation of an ultra-pure
homogeneous solution of sodium chloride in water, (2) temperature
control and flow regulation through a set of inert catalytic
electrodes and (3) a modulated electrolytic process that results in
the formation of such stable molecular moieties and complexes. In
one embodiment, such a process includes all these steps.
[0048] The saline generally should be free from contaminants, both
organic and inorganic, and homogeneous down to the molecular level.
In particular, metal ions can interfere with the electro-catalytic
surface reactions, and thus it may be helpful for metals to be
avoided. In one embodiment, a brine solution is used to salinate
the water. The brine solution can have a NaCl concentration of
about 540 g NaCl/gal, such as 537.5 g NaCl/gal. In one embodiment,
the composition can include at least one species such as O.sub.2,
H.sub.2, Cl.sub.2, OCl.sup.-, HOCl, NaOCl, HClO.sub.2, ClO.sub.2,
HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2, Na.sup.+, Cl.sup.-,
H.sup.+, H.sup.-, OH.sup.-, O.sub.3, O.sub.4*.sup.-, .sup.1O,
OH*.sup.-, HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-,
HO.sub.2*, NaCl, HCl, NaOH, water clusters, or a combination
thereof.
[0049] In one embodiment, the composition can include at least one
species such as H.sub.2, Cl.sub.2, OCl.sup.-, HOCl, NaOCl,
HClO.sub.2, ClO.sub.2, HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2,
O.sub.3, O.sub.4*.sup.-, .sup.1O.sub.2, OH*.sup.-,
HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-, HO.sub.2*,
water clusters, or a combination thereof.
[0050] In one embodiment, the composition can include at least one
species such as HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2, O.sub.3,
O.sub.4.sup.*-, .sup.1O.sub.2, OH*.sup.-, HOCl--O.sub.2*.sup.-,
HOCl--O.sub.3, O.sub.2*.sup.-, HO.sub.2*, water clusters, or a
combination thereof.
[0051] In one embodiment, the composition can include O.sub.2. In
one embodiment, the composition can include H.sub.2. In one
embodiment, the composition can include Cl.sub.2. In one
embodiment, the composition can include OCl.sup.-. In one
embodiment, the composition can include HOCl. In one embodiment,
the composition can include NaOCl. In one embodiment, the
composition can include HClO.sub.2. In one embodiment, the
composition can include ClO.sub.2. In one embodiment, the
composition can include HClO.sub.3. In one embodiment, the
composition can include HClO.sub.4. In one embodiment, the
composition can include H.sub.2O.sub.2. In one embodiment, the
composition can include Na.sup.+. In one embodiment, the
composition can include Cl.sup.-. In one embodiment, the
composition can include H.sup.+. In one embodiment, the composition
can include H. In one embodiment, the composition can include
OH.sup.-. In one embodiment, the composition can include O.sub.3.
In one embodiment, the composition can include O.sub.4*.sup.-. In
one embodiment, the composition can include .sup.1O.sub.2. In one
embodiment, the composition can include OH*.sup.-. In one
embodiment, the composition can include HOCl--O.sub.2.sup.*-. In
one embodiment, the composition can include HOCl--O.sub.3,
O.sub.2*.sup.-. In one embodiment, the composition can include
HO.sub.2*. In one embodiment, the composition can include NaCl. In
one embodiment, the composition can include HCl. In one embodiment,
the composition can include NaOH. In one embodiment, the
composition can include water clusters. Embodiments can include
combinations thereof.
[0052] With this in mind, a step in such a process is shown in FIG.
1. 100 is an optional reverse osmosis procedure 102. Water can be
supplied from a variety of sources, including but not limited to
municipal water, filtered water, nanopure water, or the like.
[0053] The reverse osmosis process can vary, but can provide water
having a total dissolved solids content of less than about 10 ppm,
about 9 ppm, about 8 ppm, about 7 ppm, about 6 ppm, about 5 ppm,
about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, or the
like.
[0054] The reverse osmosis process can be performed at a
temperature of about 5.degree. C., about 10.degree. C., about
15.degree. C., about 20.degree. C., about 25.degree. C., about
30.degree. C., about 35.degree. C., or the like. The reverse
osmosis step can be repeated as needed to achieve a particular
total dissolved solids level. Whether the optional reverse osmosis
step is utilized, an optional distillation step 104 can be
performed.
[0055] The distillation process can vary, but can provide water
having a total dissolved solids content of less than about 5 ppm,
about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, about 0.9 ppm,
about 0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5 ppm, about
0.4 ppm, about 0.3 ppm, about 0.2 ppm, about 0.1 ppm, or the like.
The temperature of the distillation process can be performed at a
temperature of about 5.degree. C., about 10.degree. C., about
15.degree. C., about 20.degree. C., about 25.degree. C., about
30.degree. C., about 35.degree. C., or the like.
[0056] The distillation step can be repeated as needed to achieve a
particular total dissolved solids level. After water has been
subjected to reverse osmosis, distillation, both, or neither, the
level of total dissolved solids in the water can be less than about
5 ppm, about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, about
0.9 ppm, about 0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5
ppm, about 0.4 ppm, about 0.3 ppm, about 0.2 ppm, about 0.1 ppm, or
the like.
[0057] The reverse osmosis, distillation, both, or neither, can be
preceded by a carbon filtration step.
[0058] Purified water can be used directly with the systems and
methods described herein.
[0059] In one embodiment, contaminants can be removed from a
commercial source of water by the following procedure: water flows
through an activated carbon filter to remove the aromatic and
volatile contaminants and then undergoes Reverse Osmosis (RO)
filtration to remove dissolved solids and most organic and
inorganic contaminants. The resulting filtered RO water can contain
less than about 8 ppm of dissolved solids. Most of the remaining
contaminants can be removed through a distillation process,
resulting in dissolved solid measurements less than 1 ppm. In
addition to removing contaminants, distillation may also serve to
condition the water with the correct structure and Oxidation
Reduction Potential (ORP) to facilitate the oxidative and reductive
reaction potentials on the platinum electrodes in the subsequent
electro-catalytic process.
[0060] After water has been subjected to reverse osmosis,
distillation, both or neither, a salt is added to the water in a
salting step 106. The salt can be unrefined, refined, caked,
de-caked, or the like. In one embodiment, the salt is sodium
chloride (NaCl). In some embodiments, the salt can include an
additive. Salt additives can include, but are not limited to
potassium iodide, sodium iodidie, sodium iodate, dextrose, sodium
fluoride, sodium ferrocyanide, tricalcium phosphate, calcium
carbonate, magnesium carbonate, fatty acids, magnesium oxide,
silicone dioxide, calcium silicate, sodium aluminosilicate, calcium
aluminosilicate, ferrous fumarate, iron, or folic acid. Any of
these additives can be added at this point or at any point during
the described process. For example, the above additives can be
added just prior to bottling.
[0061] Salt can be added to water in the form of a brine solution.
To mix the brine solution, a physical mixing apparatus can be used
or a circulation or recirculation can be used. In one embodiment,
pure pharmaceutical grade sodium chloride is dissolved in the
prepared distilled water to form a 15 wt % sub-saturated brine
solution and continuously re-circulated and filtered until the salt
has completely dissolved and all particles >0.1 microns are
removed. This step can take several days. The filtered, dissolved
brine solution is then injected into tanks of distilled water in
about a 1:352 ratio (salt:water) in order to form a 0.3% saline
solution. In one embodiment, a ratio 10.75 g of salt per 1 gallon
of water can be used to form the composition. In another
embodiment, 10.75 g of salt in about 3-4 g of water, such as 3.7875
g of water can be used to form the composition. This solution then
can be allowed to re-circulate and diffuse until homogeneity at the
molecular scale has been achieved.
[0062] In one embodiment, the homogenous saline solution is chilled
to about 4.8.+-.0.5.degree. C. Temperature regulation during the
entire electro-catalytic process can be employed as thermal energy
generated from the electrolysis process itself may cause heating.
In one embodiment, process temperatures at the electrodes can be
constantly cooled and maintained at about 4.8.degree. C. throughout
electrolysis.
[0063] Brine can then be added to the previously treated water or
to fresh untreated water to achieve a NaCl concentration of between
about 1 g NaCl/gal water and about 25 g NaCl/gal water, between
about 8 g NaCl/gal water and about 12 g NaCl/gal water, or between
about 4 g NaCl/gal water and about 16 g NaCl/gal water. Once brine
is added to water at an appropriate amount, the solution can be
thoroughly mixed. The temperature of the liquid during mixing can
be at room temperature or controlled to a desired temperature or
temperature range.
[0064] To mix the solution, a physical mixing apparatus can be used
or a circulation or recirculation can be used. The salt solution
can then be chilled in a chilling step 108.
[0065] For large amounts of composition, various chilling and
cooling methods can be employed. For example cryogenic cooling
using liquid nitrogen cooling lines can be used. Likewise, the
solution can be run through propylene glycol heat exchangers to
achieve the desired temperature. The chilling time can vary
depending on the amount of liquid, the starting temperature and the
desired chilled temperature.
[0066] Products from the anodic reactions can be effectively
transported to the cathode to provide the reactants necessary to
form the stable complexes on the cathode surfaces. Maintaining a
high degree of homogeneity in the fluids circulated between the
catalytic surfaces can also be helpful. A constant flow of about
2-8 mL/cm.sup.2*sec can be used, with typical mesh electrode
distances 2 cm apart in large tanks. This flow can be maintained,
in part, by the convective flow of gasses released from the
electrodes during electrolysis.
[0067] The mixed solution, chilled or not, can then undergo
electrochemical processing through the use of at least one
electrode in an electrolyzing step 110. Each electrode can be or
include a conductive metal. Metals can include, but are not limited
to copper, aluminum, titanium, rhodium, platinum, silver, gold,
iron, a combination thereof or an alloy such as steel or brass. The
electrode can be coated or plated with a different metal such as,
but not limited to aluminum, gold, platinum or silver. In an
embodiment, each electrode is formed of titanium and plated with
platinum. The platinum surfaces on the electrodes by themselves can
be optimal to catalyze the required reactions. Rough, double
layered platinum plating can assure that local "reaction centers"
(sharply pointed extrusions) are active and that the reactants not
make contact with the underlying electrode titanium substrate.
[0068] In one embodiment, rough platinum-plated mesh electrodes in
a vertical, coaxial, cylindrical geometry can be optimal, with, for
example, not more than 2.5 cm, not more than 5 cm, not more than 10
cm, not more than 20 cm, or not more than 50 cm separation between
the anode and cathode. The amperage run through each electrode can
be between about 2 amps and about 15 amps, between about 4 amps and
about 14 amps, at least about 2 amps, at least about 4 amps, at
least about 6 amps, or any range created using any of these values.
In one embodiment, 7 amps is used with each electrode.
[0069] The amperage can be run through the electrodes for a
sufficient time to electrolyze the saline solution. The solution
can be chilled during the electrochemical process. The solution can
also be mixed during the electrochemical process. This mixing can
be performed to ensure substantially complete electrolysis.
[0070] Electric fields between the electrodes can cause movement of
ions. Negative ions can move toward the anode and positive ions
toward the cathode. This can enable exchange of reactants and
products between the electrodes. In some embodiments, no barriers
are needed between the electrodes.
[0071] In the mitochondria, fluctuations of the mitochondrial
potential, specifically pulsing of the potentials have been seen to
take place. Similarly, pulsing potentials in the power supply of
the production units of compositions disclosed herein can also be
utilized. Lack of filter capacitors in the rectified power supply
can cause the voltages to drop to zero 120 times per second,
resulting in a hard spike when the alternating current in the house
power lines changes polarity. This hard spike, under Fourier
transform, can emit a large bandwidth of frequencies. In essence,
the voltage is varying from high potential to zero 120 times a
second.
[0072] After amperage has been run through the solution for a
sufficient time, an electrolyzed solution is created. The solution
can be stored and or tested for particular properties in
storage/testing step 112.
[0073] The end products of this electrolytic process can react
within the saline solution to produce many different chemical
entities. The compositions described herein can include one or more
of these chemical entities, known as redox signaling agents or
RXNs.
[0074] The chlorine concentration of the electrolyzed solution can
be between about 5 ppm and about 34 ppm, between about 10 ppm and
about 34 ppm, or between about 15 ppm and about 34 ppm. In one
embodiment, the chlorine concentration is about 32 ppm.
[0075] The saline concentration in the electrolyzed solution can
be, for example, between about 0.10% w/v and about 0.20% w/v,
between about 0.11% w/v and about 0.19% w/v, between about 0.12%
w/v and about 0.18% w/v, between about 0.13% w/v and about 0.17%
w/v, or between about 0.14% w/v and about 0.16% w/v.
[0076] The composition generally can include electrolytic and/or
catalytic products of pure saline that mimic redox signaling
molecular compositions of the native salt water compounds found in
and around human cells. The composition can be fine tuned to mimic
or mirror molecular compositions of different biological media. The
composition can have reactive species other than chlorine present.
As described, species present in the compositions described herein
can include, but are not limited to O.sub.2, H.sub.2, Cl.sub.2,
OCl.sup.-, HOCl, NaOCl, HClO.sub.2, ClO.sub.2, HClO.sub.3,
HClO.sub.4, H.sub.2O.sub.2, Na.sup.+, Cl.sup.-, H.sup.+, H.sup.-,
OH.sup.-, O.sub.3, O.sub.4*.sup.-, .sup.1O.sub.2, OH*.sup.-,
HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-, HO.sub.2*,
NaCl, HCl, NaOH, and water clusters: n*H.sub.2O-induced dipolar
layers around ions, and the like.
[0077] Compositions disclosed herein can also include PPAR
agonists, such as, for example, FFAs, fibrates (fenofibrate,
bezafibrate, ciprofibrate, gemfibrozil), thiazolidinediones
(rosiglitazone and pioglitazone), L-165041, GW501516, KD3010,
eicosanoids, prostaglandins (E1--alprostadil, I2--prostacyclin,
PGJ2), prostacyclins (epoprostenol, treprostinil, FLOLAN.RTM.,
veletri, remodulin, ventavis (iloprost), the thromboxanes
(thromboxane A2, thromboxane B2), leukotrienes (LTC4, LTD4, LTE4
and LTF4), perfluorooctanoic acid, perfluorononanoic acid,
Berberine, RS5444, and the like. The PPAR agonist can be "dual",
"balanced" or "pan" PPAR ligands ("glitazars"), including, for
example, aleglitazar, muraglitazar, tesaglitazar, AM3102, CAY10506,
CP 775146, DRF 2519, (+)-etomoxir sodium salt hydrate, GSK 3787,
GW0742, GW 1929, GW 7647, GW1929 hydrate, W501516, L-165041,
methyl-8-hydroxy-8-(2-pentyl-oxyphenyl)-oct-5-ynoate, NPC 15199,
nTZDpa, PAz-PC, pioglitazone, rosiglitazone (potassium salt),
rosiglitazone-d3 maleate, S26948, WY 14643, and the like.
[0078] In embodiments, the PPAR agonist can be packaged separately
from the RXN composition comprising at least one RXN. For example,
in an embodiment the PPAR agonist can be added to the RXN
composition just prior to administration to a patient. In
embodiments the PPAR agonist and the RXN can be administered in
separate compositions. In embodiments the PPAR agonist can be
contained within microspheres suspended within a RXN composition.
In embodiments the PPAR agonist/RXN composition can comprise a
gelcap.
[0079] Administration of PPAR agonist and/or RXN compositions can
be achieved via any suitable method, including, for example,
parenterally, by injection, epicutaneous, inhalational, enema, eye
drops, ear drops, through mucous membranes in the body, by mouth
(orally), gastric feeding tube, duodenal feeding tube, gastrostomy,
rectally, intravenous, intra-arterial, intraosseous infusion,
intra-muscular, intracerebral, intracerebro-ventricular,
subcutaneous, or the like.
[0080] Compositions of the invention can be formulated into any
suitable aspect, such as, for example, aerosols. liquids, elixirs,
syrups, tinctures, creams, ointments, lotions, thin films, solids,
gelcaps, a microsphere suspension, a soft gelatin capsule, and the
like.
[0081] When administered as a liquid composition, it can be taken
once, twice, three times, four times or more a day. Each
administration can be about 1 oz, about 2 oz, about 3 oz, about 4
oz, about 5 oz, about 6 oz, about 7 oz, about 8 oz, about 9 oz,
about 10 oz, about 11 oz, about 12 oz, about 16 oz, about 20 oz,
about 24 oz, about 28 oz, about 32 oz, about 34 oz, about 36 oz,
about 38 oz, about 40 oz, about 46 oz, between about 1 oz and about
32 oz, between about 1 oz and about 16 oz, between about 1 oz and
about 8 oz, at least about 2 oz, at least about 4 oz, or at least
about 8 oz. In one embodiment, the composition can be administered
at a rate of about 4 oz twice a day.
[0082] In other embodiments, the administration can be acute or
long term. For example, the composition can be administered for a
day, a week, a month, a year or longer.
[0083] The compositions described herein when administered can be
used to treat a condition or a disease. For example, when
administered alongside exercise or not, the compositions described
herein can increase the density of mitochondrial DNA. For example,
an increase in mitochondrial DNA of about 1%, about 5%, about 10%,
about 15%, about 20%, about 21%, about 22%, about 23%, about 24%,
about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,
about 32%, about 34%, about 36%, about 38%, about 40%, about 45%,
between about 1% and about 40%, between about 1% and about 10%,
between about 20% and about 30%, at least about 5%, at least about
10%, or at least about 20% when compared to an individual who has
not taken the composition. An increase in mitochondrial DNA can
result in a lower level of free radicals in the blood which can in
turn lead to a reduced amount of oxidative stress.
[0084] Compositions disclosed herein can be useful in treating
diseases related to mitochondrial DNA. As such, the compositions
described can treat conditions or diseases such as, but not limited
to sacropenia, Parkinson's disease, neuro-related age disease,
obesity, aging, life stresses such as those caused by fear,
neurodegenerative diseases, cognitive disorders, obesity, reduced
metabolic rate, metabolic syndrome, diabetes mellitus,
cardiovascular disease, hyperlipidemia, neurodegenerative disease,
cognitive disorder, mood disorder, stress, and anxiety disorder;
for weight management, or to increase muscle performance or mental
performance, AIDS, dementia complex, Alzheimer's disease,
amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander
disease, Alper's disease, ataxia telangiectasia, Batten disease,
bovine spongiform encephalopathy (BSE), Canavan disease,
corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with
Lewy bodies, fatal familial insomnia, frontotemporal lobar
degeneration, Huntington's disease, Kennedy's disease, Krabbe
disease, Lyme disease, Machado-Joseph disease, multiple sclerosis,
multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease,
Pick's disease, primary lateral sclerosis, progressive supranuclear
palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic
sclerosis, spinocerebellar ataxia, subacute combined degeneration
of spinal cord, tabes dorsalis, Tay-Sachs disease, toxic
encephalopathy, transmissible spongiform encephalopathy, and wobbly
hedgehog syndrome, cognitive function abnormalities, perception
abnormalities, attention disorders, speech comprehension disorders,
reading comprehension disorders, creation of imagery disorders,
learning disorders, reasoning disorders, mood disorders,
depression, postpartum depression, dysthymia, bipolar disorder,
generalized anxiety disorder, panic disorder, panic disorder with
agoraphobia, agoraphobia, social anxiety disorder,
obsessive-compulsive disorder, post-traumatic stress disorder,
musculoskeletal disorder, lack of strength, lack of endurance,
cancer, atherosclerotic lesions, atherosclerosis, oxidative stress,
atherogenesis, hypertension, hypercholesterolemia, and degenerative
diseases.
[0085] Compositions disclosed herein can be useful in treating
diseases involving PPAR pathways, including, for example, metabolic
syndrome, cardiovascular disease, diabetes, obesity, glucose
intolerance, hyperinsulinemia, hypercholesterolemia,
hypertriglyceridemia, and hypertension, inflammation, vascular
function, and vascular remodeling, cancer, inflammation,
neurodegenerative diseases, diseases relating to mitochondrial
biogenesis, ageing, and the like.
[0086] In embodiments, compositions of the invention can comprise a
component of a therapy acting through multiple mechanisms. For
example, a dual acting therapy can comprise a first therapy which
activates a PPAR pathway and at least one other agent that does not
activate a PPAR pathway. In embodiments the first agent can be at
least one RXN. In embodiments the at least one other agent is a
mAb, radiation, surgery, angiogenesis inhibitor, transplantation, a
cancer vaccine, gene therapy, laser treatment, photodynamic
therapy, an alkylating agent, an antimetabolite, an anti-tumor
antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a
corticosteroid, hormone therapy, immunotherapy, and the like.
[0087] Embodiments can provide a substrate in vitro for superoxides
and/or fatty acid oxidation (FAO) enzymes comprising RXNs.
Embodiments can include a method of treating cancer comprising a
combination therapy wherein one therapy comprises compositions
comprising at least one RXN which increases FFAs, and at least one
other agent wherein the at least one other agent does not increase
FFAs.
[0088] In other embodiments, methods of treating an oxidative
stress related disorders are described comprising: administering a
composition including at least one species selected from O.sub.2,
H.sub.2, Cl.sub.2, OCl.sup.-, HOCl, NaOCl, HClO.sub.2, ClO.sub.2,
HClO.sub.3, HClO.sub.4, H.sub.2O.sub.2, Na.sup.+, Cl.sup.-,
H.sup.+, H.sup.-, OH.sup.-, O.sub.3, O.sub.4*.sup.-, .sup.1O,
OH*.sup.-, HOCl--O.sub.2*.sup.-, HOCl--O.sub.3, O.sub.2*.sup.-,
HO.sub.2*, NaCl, HCl, NaOH, water clusters, or a combination
thereof to a patient experiencing oxidative stress; and treating
the oxidative stress related disorder. In some embodiments, the
administration occurs twice a day or once a day. Each
administration can include between about 1 oz and about 16 oz per
day. In other embodiments, the oxidative stress related disorder is
diabetes, cardiovascular disease, or obesity.
Example 1
[0089] FIG. 3 illustrates a plan view of a process and system for
producing a life enhancing composition according to the present
description. One skilled in the art understands that changes can be
made to the system to alter the life enhancing composition, and
these changes are within the scope of the present description.
[0090] Incoming water 202 can be subjected to reverse osmosis
system 204 at a temperature of about 15-20.degree. C. to achieve
purified water 206 with about 8 ppm of total dissolved solids.
Purified water 206, is then fed at a temperature of about
15-20.degree. C. into distiller 208 and processed to achieve
distilled water 210 with about 0.5 ppm of total dissolved solids.
Distilled water 210 can then be stored in tank 212.
[0091] FIG. 4 illustrates an example system for preparing water for
further processing into a therapeutic beverage. System 300 can
include a water source 302 which can feed directly into a carbon
filter 304. After oils, alcohols, and other volatile chemical
residuals and particulates are removed by carbon filter 304, the
water can be directed to resin beds within a water softener 306
which can remove dissolved minerals. Then, as described above, the
water can pass through reverse osmosis system 204 and distiller
208.
[0092] As needed, distilled water 210 can be gravity fed from tank
212 into saline storage tank cluster 214 using line 216. Saline
storage tank cluster 214 in one embodiment can include twelve tanks
218. Each tank 218 can be filled to about 1,300 gallons with
distilled water 210. A handheld meter can be used to test distilled
water 210 for salinity.
[0093] Saline storage tank cluster 214 is then salted using a brine
system 220. Brine system 220 can include two brine tanks 222. Each
tank can have a capacity of about 500 gallons. Brine tanks 222 are
filled to 475 gallons with distilled water 210 using line 224 and
then NaCl is added to the brine tanks 222 at a ratio of about 537.5
g/gal of liquid. At this point, the water is circulated 226 in the
brine tanks 222 at a rate of about 2,000 gal/hr for about 4
days.
[0094] Prior to addition of brine to tanks 218, the salinity of the
water in tanks 218 can be tested using a handheld conductivity
meter such as an YSI ECOSENSE.RTM. ecp300 (YSI Inc., Yellow
Springs, Ohio). Any corrections based on the salinity measurements
can be made at this point. Brine solution 228 is then added to
tanks 218 to achieve a salt concentration of about 10.75 g/gal. The
salted water is circulated 230 in tanks 218 at a rate of about
2,000 gal/hr for no less than about 72 hours. This circulation is
performed at room temperature. A handheld probe can again be used
to test salinity of the salinated solution. In one embodiment, the
salinity is about 2.8 ppth.
[0095] In one method for filling and mixing the salt water in the
brine holding tanks, the amount of liquid remaining in the tanks is
measured. The amount of liquid remaining in a tank is measured by
recording the height that the liquid level is from the floor that
sustains the tank, in centimeters, and referencing the number of
gallons this height represents. This can be done from the outside
of the tank if the tank is semi-transparent. The initial liquid
height in both tied tanks can also be measured. Then, after
ensuring that the output valve is closed, distilled water can be
pumped in. The amount of distilled water that is being pumped into
a holding tank can then be calculated by measuring the rise in
liquid level: subtracting the initial height from the filled height
and then multiplying this difference by a known factor.
[0096] The amount of salt to be added to the tank is then
calculated by multiplying 11 grams of salt for every Gallon of
distilled water that has been added to the tank. The salt can be
carefully weighed out and dumped into the tank.
[0097] The tank is then agitated by turning on the recirculation
pump and then opening the top and bottom valves on the tank. Liquid
is pumped from the bottom of the tank to the top. The tank can be
agitated for three days before it may be ready to be processed.
[0098] After agitating the tank for more than 6 hours, the salinity
is checked with a salinity meter by taking a sample from the tank
and testing it. Salt or water can be added to adjust the salinity
within the tanks. If either more water or more salt is added then
the tanks are agitated for 6 more hours and tested again. After
about three days of agitation, the tank is ready to be
processed.
[0099] Salinated water 232 is then transferred to cold saline tanks
234. In one embodiment, four 250 gal tanks are used. The amount of
salinated water 232 moved is about 1,000 gal. A chiller 236 such as
a 16 ton chiller is used to cool heat exchangers 238 to about
0-5.degree. C. The salinated water is circulated 240 through the
heat exchangers which are circulated with propylene glycol until
the temperature of the salinated water is about 4.5-5.8.degree. C.
Chilling the 1,000 gal of salinated water generally takes about 6-8
hr.
[0100] Cold salinated water 242 is then transferred to processing
tanks 244. In one embodiment, eight tanks are used and each can
have a capacity of about 180 gal. Each processing tank 244 is
filled to about 125 gal for a total of 1,000 gal. Heat exchangers
246 are again used to chill the cold salinated water 242 added to
processing tanks 244. Each processing tank can include a cylinder
of chilling tubes and propylene glycol can be circulated. The heat
exchangers can be powered by a 4-5 ton chiller 248. The temperature
of cold salinated water 242 can remain at 4.5-5.8.degree. C. during
processing.
[0101] Prior to transferring aged salt water to processing tanks,
the aged salt water can be agitated for about 30 minutes to
sufficiently mix the aged salt water. Then, the recirculation
valves can then be closed, the appropriate inlet valve on the
production tank is opened, and the tank filled so that the salt
water covers the cooling coils and comes up to the fill mark
(approximately 125 gallons).
[0102] Once the aged salt water has reached production temperature,
the pump is turned off but the chiller left on. The tank should be
adequately agitated or re-circulated during the whole duration of
electrochemical processing and the temperature should remain
constant throughout.
[0103] Each processing tank 244 includes electrode 250. Electrodes
250 can be 3 inches tall circular structures formed of titanium and
plated with platinum. Electrochemical processing of the cold
salinated water can be run for 8 hr. A power supply 252 is used to
power the eight electrodes (one in each processing tank 244) to 7
amps each for a total of 56 amps. The cold salinated water is
circulated 254 during electrochemical processing at a rate of about
1,000 gal/hr.
[0104] An independent current meter can be used to set the current
to around 7.0 Amps. Attention can be paid to ensure that the
voltage does not exceed 12V and does not go lower than 9V. Normal
operation can be about 10V.
[0105] A run timer can be set for a prescribed time (about 4.5 to 5
hours). Each production tank can have its own timer and/or power
supply. Electrodes should be turned off after the timer has
expired.
[0106] The production tanks can be checked periodically. The
temperature and/or electrical current can be kept substantially
constant. At the beginning, the electrodes can be visible from the
top, emitting visible bubbles. After about 3 hours, small bubbles
of un-dissolved oxygen can start building up in the tank as oxygen
saturation occurs, obscuring the view of the electrodes. A slight
chlorine smell can be normal.
[0107] After the 8 hour electrochemical processing is complete,
life enhancing water 256 has been created with a pH of about
6.8-8.2, 32 ppm of chlorine, 100% OCl.sup.- and 100% O.sup.-2. The
composition 256 is transferred to storage tanks 258.
Example 2
Characterization of a Beverage Produced as Described in Example
1
[0108] A composition produced as described in Example 1 and
marketed under the trade name ASEA.RTM. was analyzed using a
variety of different characterization techniques. ICP/MS and
.sup.35Cl NMR were used to analyze and quantify chlorine content.
Headspace mass spectrometry analysis was used to analyze adsorbed
gas content in the beverage. .sup.1H NMR was used to verify the
organic matter content in the beverage. .sup.31P NMR and EPR
experiments utilizing spin trap molecules were used to explore the
beverage for free radicals.
[0109] The composition was received and stored at about 4.degree.
C. when not being used.
Chlorine NMR
[0110] Sodium hypochlorite solutions were prepared at different pH
values. 5% sodium hypochlorite solution had a pH of 12.48.
Concentrated nitric acid was added to 5% sodium hypochlorite
solution to create solutions that were at pH of 9.99, 6.99, 5.32,
and 3.28. These solutions were then analyzed by NMR spectroscopy.
The beverage had a measured pH of 8.01 and was analyzed directly by
NMR with no dilutions.
[0111] NMR spectroscopy experiments were performed using a 400 MHz
Bruker spectrometer equipped with a BBO probe. .sup.35Cl NMR
experiments were performed at a frequency of 39.2 MHz using single
pulse experiments. A recycle delay of 10 seconds was used, and 128
scans were acquired per sample. A solution of NaCl in water was
used as an external chemical shift reference. All experiments were
performed at room temperature.
[0112] .sup.35Cl NMR spectra were collected for NaCl solution,
NaClO solutions adjusted to different pH values, and the
composition. FIG. 5 illustrates a Cl35 spectrum of NaCl, NaClO
solution at a pH of 12.48, and the composition. The chemical shift
scale was referenced by setting the Cl.sup.- peak to 0 ppm. NaClO
solutions above a pH=7 had identical spectra with a peak at
approximately 5.1 ppm. Below pH of 7.0, the ClO.sup.- peak
disappeared and was replaced by much broader, less easily
identifiable peaks. The composition was presented with one peak at
approximately 4.7 ppm, from ClO.sup.- in the composition. This peak
was integrated to estimate the concentration of ClO.sup.- in the
composition, which was determined to be 2.99 ppt or 0.17 M of
ClO.sup.- in the composition.
Proton NMR
[0113] An ASEA sample was prepared by adding 550 .mu.L of ASEA and
50 .mu.L of D.sub.2O (Cambridge Isotope Laboratories) to an NMR
tube and vortexing the sample for 10 seconds. .sup.1H NMR
experiments were performed on a 700 MHz Bruker spectrometer
equipped with a QNP cryogenically cooled probe. Experiments used a
single pulse with pre-saturation on the water resonance experiment.
A total of 1024 scans were taken. All experiments were performed at
room temperature.
[0114] A .sup.1H NMR spectrum of the composition was determined and
is presented in FIG. 6. Only peaks associated with water were able
to be distinguished from this spectrum. This spectrum show that
very little if any organic material can be detected in the
composition using this method.
Phosphorous NMR and Mass Spectrometry
[0115] DIPPMPO (5-(Diisopropoxyphosphoryl)-5-1-pyrroline-N-oxide)
(VWR) samples were prepared by measuring about 5 mg of DIPPMPO into
a 2 mL centrifuge tube. This tube then had 550 .mu.L of either the
composition or water added to it, followed by 50 .mu.L of D.sub.2O.
A solution was also prepared with the composition but without
DIPPMPO. These solutions were vortexed and transferred to NMR tubes
for analysis. Samples for mass spectrometry analysis were prepared
by dissolving about 5 mg of DIPPMPO in 600 .mu.L of the composition
and vortexing, then diluting the sample by adding 100 .mu.L of
sample and 900 .mu.L of water to a vial and vortexing.
[0116] NMR experiments were performed using a 700 MHz Bruker
spectrometer equipped with a QNP cryogenically cooled probe.
Experiments performed were a single 30.degree. pulse at a .sup.31P
frequency of 283.4 MHz. A recycle delay of 2.5 seconds and 16384
scans were used. Phosphoric acid was used as an external standard.
All experiments were performed at room temperature.
[0117] Mass spectrometry experiments were performed by directly
injecting the ASEA/DIPPMPO sample into a Waters/Synapt Time of
Flight mass spectrometer. The sample was directly injected into the
mass spectrometer, bypassing the LC, and monitored in both positive
and negative ion mode.
[0118] .sup.31P NMR spectra were collected for DIPPMPO in water,
the composition alone, and the composition with DIPPMPO added to
it. An external reference of phosphoric acid was used as a chemical
shift reference. FIG. 7 illustrates a .sup.31P NMR spectrum of
DIPPMPO combined with the composition. The peak at 21.8 ppm was
determined to be DIPPMPO and is seen in both the spectrum of
DIPPMPO with the composition (FIG. 7) and without the composition
(not pictured). The peak at 24.9 ppm is most probably DIPPMPO/OH.
as determined in other DIPPMPO studies. This peak may be seen in
DIPPMPO mixtures both with and without the composition, but is
detected at a much greater concentration in the solution with the
composition. In the DIPPMPO mixture with the composition, there is
another peak at 17.9 ppm. This peak may be from another radical
species in the composition such as OOH. or possibly a different
radical complex. The approximate concentrations of spin trap
complexes in the composition/DIPPMPO solution are as follows:
TABLE-US-00001 Solution Concentration DIPPMPO 36.6 mM
DIPPMPO/OH.cndot. 241 .mu.M DIPPMPO/radical 94 .mu.M
[0119] Mass spectral data was collected in an attempt to determine
the composition of the unidentified radical species. The mass
spectrum shows a parent peak and fragmentation pattern for DIPPMPO
with m/z peaks at 264, 222, and 180, as seen in FIG. 8. FIG. 8 also
shows peaks for the DIPPMPO/Na adduct and subsequent fragments at
286, 244, and 202 m/z. Finally, FIG. 8 demonstrates peaks for one
DIPPMPO/radical complex with m/z of 329. The negative ion mode mass
spectrum also had a corresponding peak at m/z of 327. There are
additional peaks at 349, 367, and 302 at a lower intensity as
presented in FIG. 8. None of these peaks could be positively
confirmed. However, there are possible structures that would result
in these mass patterns. One possibility for the peak generated at
329 could be a structure formed from a radical combining with
DIPPMPO. Possibilities of this radical species include a
nitroxyl-peroxide radical (HNO--HOO.) that may have formed in the
beverage as a result of reaction with nitrogen from the air.
Another peak at 349 could also be a result of a DIPPMPO/radical
combination. Here, a possibility for the radical may be
hypochlorite-peroxide (HOCl.sup.-HOO.). However, the small
intensity of this peak and small intensity of the corresponding
peak of 347 in the negative ion mode mass spectrum indicate this
could be a very low concentration impurity and not a compound
present in the ASEA composition.
ICP/MS Analysis
[0120] Samples were analyzed on an Agilent 7500 series
inductively-coupled plasma mass spectrometer (ICP-MS) in order to
confirm the hypochlorite concentration that was determined by NMR.
A stock solution of 5% sodium hypochlorite was used to prepare a
series of dilutions consisting of 300 ppb, 150 ppb, 75 ppb, 37.5
ppb, 18.75 ppb, 9.375 ppb, 4.6875 ppb, 2.34375 ppb, and 1.171875
ppb in deionized Milli-Q water. These standards were used to
establish a standard curve.
[0121] Based on NMR hypochlorite concentration data, a series of
dilutions was prepared consisting of 164.9835 ppb, 82.49175 ppb,
41.245875 ppb, 20.622937 ppb, 10.311468 ppb, and 5.155734 ppb.
These theoretical values were then compared with the values
determined by ICP-MS analysis. The instrument parameters were as
follows:
TABLE-US-00002 Elements analyzed .sup.35Cl, .sup.37Cl # of points
per mass 20 # of repetitions 5 Total acquisition time 68.8 s Uptake
speed 0.50 rps Uptake time 33 s Stabilization time 40 s Tune No Gas
Nebulizer flow rate 1 mL/min Torch power 1500 W
[0122] The results of the ICP-MS analysis are as follows:
TABLE-US-00003 Measured Concentration Concentration by NMR Dilution
(ppb) (ppb) 1 81 82 2 28 41 3 24 21 4 13 10 5 8 5
[0123] Dilutions were compared graphically to the ICP-MS signals
and fit to a linear equation (R.sup.2=0.9522). Assuming linear
behavior of the ICP-MS signal, the concentration of hypochlorite in
the beverage was measured to be 3.02 ppt. Concentration values were
determined by calculating the concentration of dilutions of the
initial beverage and estimating the initial beverage hypochlorite
concentration to be 3 ppt (as determined from .sup.35Cl NMR
analysis). The ICP-MS data correlate well with the .sup.35Cl NMR
data, confirming a hypochlorite concentration of roughly 1/3% (3
ppt). It should be noted that ICP-MS analysis is capable of
measuring total chlorine atom concentration in solution, but not
specific chlorine species. The NMR data indicate that chlorine
predominantly exists as ClO.sup.- in the beverage.
Gas Phase Quadrupole MS
Sample Prep
[0124] Three sample groups were prepared in triplicate for the
analysis: 1) Milli-Q deionized water 2) the composition, and 3) 5%
sodium hypochlorite standard solution. The vials used were 20 mL
headspace vials with magnetic crimp caps (GERSTEL). A small stir
bar was placed in each vial (VWR) along with 10 mL of sample. The
vials were capped, and then placed in a Branson model 5510
sonicator for one hour at 60.degree. C.
[0125] The sonicator was set to degas which allowed for any
dissolved gasses to be released from the sample into the headspace.
After degassing, the samples were placed on a CTC PAL autosampler
equipped with a heated agitator and headspace syringe. The agitator
was set to 750 rpm and 95.degree. C. and the syringe was set to
75.degree. C. Each vial was placed in the agitator for 20 min prior
to injection into the instrument. A headspace volume of 2.5 mL was
collected from the vial and injected into the instrument.
Instrument Parameters
[0126] The instrument used was an Agilent 7890A GC system coupled
to an Agilent 5975C El/Cl single quadrupole mass selective detector
(MSD) set up for electron ionization. The GC oven was set to
40.degree. C. with the front inlet and the transfer lines being set
to 150.degree. C. and 155.degree. C. respectively. The carrier gas
used was helium and it was set to a pressure of 15 PSI.
[0127] The MSD was set to single ion mode (SIM) in order to detect
the following analytes:
TABLE-US-00004 Analyte Mass Water 18 Nitrogen 28 Oxygen 32 Argon 40
Carbon Dioxide 44 Chlorine 70 Ozone 48
[0128] The ionization source temperature was set to 230.degree. C.
and the quadrupole temperature was set to 150.degree. C. The
electron energy was set to 15 V.
[0129] Mass spectrometry data was obtained from analysis of the gas
phase headspace of the water, the composition, and hypochlorite
solution. The raw area counts obtained from the mass spectrometer
were normalized to the area counts of nitrogen in order to
eliminate any systematic instrument variation. Both nitrogen and
water were used as standards because they were present in equal
volumes in the vial with nitrogen occupying the headspace and water
being the solvent. It was assumed that the overall volume of water
and nitrogen would be the same for each sample after degassing. In
order for this assumption to be correct, the ratio of nitrogen to
water should be the same for each sample. A cutoff value for the
percent relative standard deviation (% RSD) of 5% was used. Across
all nine samples, a % RSD of 4.2 was observed. Of note, sample
NaClO.sup.-3 appears to be an outlier, thus, when removed, the %
RSD drops to 3.4%.
[0130] FIGS. 9-11 illustrate oxygen/nitrogen, chlorine/nitrogen,
and ozone/nitrogen ratios. It appears that there were less of these
gases released from the composition than from either water or
nitrogen. It should be noted that the signals for both ozone and
chlorine were very weak. Thus, there is a possibility that these
signals may be due to instrument noise and not from the target
analytes.
[0131] FIG. 12 illustrates the carbon dioxide to nitrogen ratio. It
appears that there may have been more carbon dioxide released from
the composition than oxygen. However, it is possible that this may
be due to background contamination from the atmosphere.
[0132] Based on the above, more oxygen was released from both water
and sodium hypochlorite than the composition.
EPR
[0133] Two different composition samples were prepared for EPR
analysis. The composition with nothing added was one sample. The
other sample was prepared by adding 31 mg of DIPPMPO to 20 mL of
the composition (5.9 mM), vortexing, and placing the sample in a
4.degree. C. refrigerator overnight. Both samples were placed in a
small capillary tube which was then inserted into a normal 5 mm EPR
tube for analysis.
[0134] EPR experiments were performed on a Bruker EMX 10/12 EPR
spectrometer. EPR experiments were performed at 9.8 GHz with a
centerfield position of 3500 Gauss and a sweepwidth of 100 Gauss. A
20 mW energy pulse was used with modulation frequency of 100 kHz
and modulation amplitude of 1 G. Experiments used 100 scans. All
experiments were performed at room temperature.
[0135] EPR analysis was performed on the composition with and
without DIPPMPO mixed into the solution. FIG. 9 shows the EPR
spectrum generated from DIPPMPO mixed with the composition. The
composition alone showed no EPR signal after 100 scans (not
presented). FIG. 13 illustrates an EPR splitting pattern for a free
electron. This electron appears to be split by three different
nuclei. The data indicate that this is a characteristic splitting
pattern of OH. radical interacting with DMPO (similar to DIPPMPO).
This pattern can be described by .sup.14N splitting the peak into
three equal peaks and .sup.1H three bonds away splitting that
pattern into two equal triplets. If these splittings are the same,
it leads to a quartet splitting where the two middle peaks are
twice as large as the outer peaks. This pattern may be seen in FIG.
13 twice, with the larger peaks at 3457 and 3471 for one quartet
and 3504 and 3518 for the other quartet. In this case, the .sup.14N
splitting and the .sup.1H splitting are both roughly 14 G, similar
to an OH. radical attaching to DMPO. The two quartet patterns in
FIG. 13 are created by an additional splitting of 47 G. This
splitting is most likely from coupling to .sup.31P, and similar
patterns have been seen previously. The EPR spectrum in FIG. 13
indicates that there is a DIPPMPO/OH. radical species in the
solution.
Example 3
Delivery of Composition to Exercising Mice
[0136] Studies have shown that supplementation with green tea
extract for 8-10 weeks in mice resulted in increased treadmill time
to exhaustion compared to control mice. Higher muscle glycogen and
increased fatty acid beta-oxidation were measured in exercised mice
treated with green tea extract. Based on these studies, further
exploration into other supplements that can increase physical
properties such as time to exhaustion, VO.sub.2max, and the like
may be useful.
[0137] The effect of composition (ASEA) ingestion on treadmill
endurance capacity, fuel substrate utilization, tissue
inflammation, and tissue oxidative stress in mice was studied. If
ASEA causes increased fatty acid mobilization then endurance
capacity can be improved in mice taking ASEA (compared to placebo).
Sparing of muscle glycogen can be seen when taking ASEA. Mice were
given the equivalent of about half the human ASEA dose.
[0138] Six-month old male specific pathogen-free C57BL/6 laboratory
mice (n=60) were purchased from Jackson Laboratory. Mice were
randomly assigned to each of four treatment groups (n=15 each) as
illustrated in FIG. 14. A total overview of the mouse preparation
and study is illustrated in FIG. 15.
[0139] This particular strain and model of mouse has been used in
previous studies involving both exercise and nutritional
intervention studies. Thus, the use of this strain allowed
comparison to data from other studies. Mice can be a suitable
substitute for humans for this type of study because mice are
genetically similar to humans and thus data obtained in this study
will be translatable to human intervention studies.
[0140] All animal procedures took place in the Center for
Laboratory Animal Sciences (CLAS) at the North Carolina Research
Campus and protocols were reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC).
[0141] ASEA or placebo (same ingredients as ASEA composition
without the proprietary signaling molecules added) was administered
to the mice via gavage once per day for 1-week. The average body
mass of all the mice at the start of the study and the volume of
ASEA used for the gavaging were determined, but the volume did not
exceed 0.3 mL per mouse. Guidelines for gavage are as the follows:
"the volume should not exceed 1-2% of body weight (=0.2-0.4 mL for
a 20 g mouse)". Thus, a volume of 0.3 mL for a 6 month old 30 g
mouse is well below this volume suggestion.
[0142] The composition was not palatable and the mice did not drink
it willingly. Gavage was an acceptable alternative to ensure the
mice did not become dehydrated simply because they would not drink
the study composition. The gavaging was performed by the animal
husbandry staff at CLAS. In one embodiment, mice can be given an
amount of the composition that is equivalent to a daily human dose
as described herein.
[0143] Following the 1-week (7 days) treatment period mice were
euthanized and tissues harvested for further analysis of outcome
measures. The four groups of mice were phased into the 1-week
protocol each day. For example, if Group 1 started the protocol on
a given day, Group 2 would begin the protocol on the following day,
Group 3 would be begin the following day, and Group 4 the day after
that. Mice from Group 1 would then be euthanized following the
final treadmill test (7th day of treatment), Group 2, Group 3, and
Group 4 each on subsequent days. Thus, total time for the mouse
protocol was 11 days. There was overlap of orientation treadmill
days, with maximal treadmill testing and euthanasia days. As
stated, prior to euthanasia, mice from Group 1 and Group 3
underwent an endurance treadmill test to exhaustion using the
protocol summarized in the following Table.
TABLE-US-00005 Time (min) Speed (m/min) Notes 1 0 Adjustment to
treadmill 5 10 "warm up" period 2 12 2 14 2 16 2 18 2 20 2 22
Speeds between 20-24 m/min correspond to roughly 80% VO.sub.2max
for mice 2 to end 24 Mice stay at this speed until they reach
exhaustion (e.g., sit on shock grid for 5 full seconds)
[0144] During the three day period preceding the maximal endurance
test, mice were oriented (trained) to the treadmill for 15 min/day.
Speeds for the training days were about 10 m/min, 15 m/min, and 18
m/min respectively. Then, on the final day of treatment mice
underwent a maximal endurance capacity test on the treadmill.
[0145] Mice from Group 2 and Group 4 were not submitted to an
endurance capacity test and were euthanized at the end of 1-week
treatment. Tissues harvested from these mice were collected to
assess the chronic effects of the test composition in absence of an
exercise intervention. All blood/plasma and tissues were
snap-frozen in liquid nitrogen and stored at -80.degree. C. until
assayed.
[0146] For the treadmill orientation and endurance protocols, mice
were run on a multi-lane rodent treadmill (Columbus Instruments,
Columbus Ohio) equipped with a shock grid at the back. Once each
mouse was placed in a treadmill lane, a 1 minute resting period was
initiated. At this point, the mouse was able to adjust to the
inside of the treadmill chamber. Following the 1 minute rest
period, the treadmill belt was started at a speed of about 10
m/min, and the protocol described in the above Table was
followed.
[0147] Mice were allowed to run until they were no longer able to
keep up with the belt and the hind limbs stayed on the shock grid
for more than about 5 seconds. When the mouse was no longer running
(as assessed by sitting on the shock grid with all 4 paws off of
the belt for more than 5 seconds), the mouse was removed from the
shock grid immediately and placed back into the home cage. The mice
were then monitored for recovery for a period of at least about 20
minutes following the orientation bouts.
[0148] The maximal endurance test occurred only once per mouse, and
mice were euthanized immediately following the test. The test ended
when the mouse could not run off the shock grid onto the treadmill
at any point during the test or if signs of exhaustion (signs of
above normal heart rate and ventilation) were evident.
[0149] The signs of exhaustion used included a mouse sitting on the
shock grid for more than 5 seconds, rapid breathing, and/or
increased heart rate. It has been our experience that mice that are
not fatigued do not show these signs and will continue to run
within 5 seconds of stopping. These procedures follow national
recommendations (American Physiological Society's, Resource Book
for the Design of Animal Exercise Protocols, 2006) based on
research in the area. If at any point during the test a mouse got
its foot caught between the shock grid and the treadmill the test
was immediately terminated. If the mouse was injured and needed
treatment, proper procedures were followed and vivarium staff was
notified. If the mouse was deemed not injured, it was allowed to
recover and placed back in its home cage and re-tested the
following day. Once the mouse completed the protocol the mouse was
placed back into its home cage. Generally, mice are usually back up
and jumping around the cage within 30 seconds of re-exposure to the
home cage following an endurance test. However, mice were still
monitored several times during the 20-60 minutes following the
procedure and notes were taken of any abnormalities such as apathy
or decreased food consumption.
[0150] Some form of motivation was needed to make the mice run on
the treadmill, particularly in the orientation sessions. A variety
of forms of motivation can be used. The three most common
techniques are, use of shock grid, use of air puffs, and manually
tapping a mouse's tail. Use of air puffs have the potential to be
ineffective and possibly confounding to data analysis. Given the
standard rodent treadmill that is used in this type of testing that
encloses the treadmill, manually tapping the tail was not ideal.
Thus, shock grids were the best method of motivation for exercise
on the treadmill.
[0151] The shock grid was positioned at the back of the treadmill.
The shock grid delivered pulsed shock at an average current of 1.0
milliamperes at 150 volts (the shock grid was adjustable within a
range of 0-3.4 mA). The shock grid was regularly checked with an
ampmeter to ensure proper functioning. The shock levels used were
22 times less than that accepted in the literature. Also, the
amperage of the system was 167-500 times less than lethal levels
for mice, and the total power of the system was 60 times less than
lethal levels for mice. No new data or guidelines existed to
suggest that the use of a shock grid with our proposed settings was
anything but appropriate.
[0152] Based on a similar study the effect size was calculated to
be 1.647. Using p=0.0125 for significance during a priori power
analysis. Using G-Power the following calculation was made and a
least significant number of animals is assumed to 12/group. 15
animals per group were used (with estimated power of 0.95) to
account for any loss of power if any animals do not make it through
the protocol.
Analysis: A Priori: Compute Required Sample Size
TABLE-US-00006 [0153] Input Tail(s) Two Effect size d 1.6470588
.alpha. err prob 0.0125 Power (1-.beta. err prob) 0.95 Allocation
ratio N2/N1 1 Output Noncentrality parameter .delta. 4.5106563
Critical t 2.6694793 Df 28 Sample size 15 Actual power
0.9604227
[0154] Based on the results from the mouse study, results are
illustrated in FIGS. 16-20. FIG. 16 (A and B) illustrates that mice
who were administered ASEA had an increased run time to exhaustion.
As such, ASEA can be used to increase time to exhaustion in
athletes when exercising.
[0155] FIGS. 17A and 17B illustrate the fold change relate to ASEA
of different mouse groups; P=0.042. This measurement tracks 12sRNA
(mitochondrial DNA copy number). One week ASEA consumption in
sedentary mice did not increase muscle mitochondria density. An
interaction between one long endurance exercise bout to exhaustion
was observed with ASEA vs. ASEA sedentary (P<0.05). Fold change
increased when ASEA was delivered along with exercise, but fell
when exercise was not present. This supports that ASEA helped
decrease the level of oxidative stress in the muscle.
[0156] FIG. 18 illustrates that SOD produced in the liver decreases
in mice when administered ASEA and subjected to exercise. U is the
amount of enyzme needed to inhibit 50% dismutation of the
superoxide radical. An acute bout of exercise activates CuZnSOD
activity, but most studies reported no change in its mRNA and
enzyme protein levels, suggesting that the increased activity was
due to increased O.sup.2- concentration. This result can indicate
that ASEA linked to exercise can reduce oxidative stress.
[0157] FIGS. 19A and 19B illustrate that oxidized glutothione
decreases in mice when administered ASEA and subjected to exercise.
This result can indicate that ASEA linked to exercise can reduce
oxidative stress.
[0158] FIG. 20 illustrates that exercise increased mRNA (gene
expression) for IL-6 and TNF-alpha, indicating the typical
pro-inflammatory response. ASEA tended to reduce gene expression
for these inflammatory cytokines.
Example 4
Human Biking Exercise Study
[0159] A study was performed to estimate the increase in metabolism
of individuals using the present systems and methods wherein the
subjects drank an ASEA composition(s). The study was performed at
the Metabolomics Laboratory, North Carolina Research Campus, David
H. Murdock Research Institute and Appalachian State University. A
goal of the study was to measure the influence of ASEA on small
molecules (metabolites) that can shift in response to
supplementation. The shift in metabolites, depending on the
nutritional product, may represent effects on inflammation,
oxidative stress, and physiologic stress.
[0160] Twenty-two subjects participated in the study. Each subject
was tested for baseline values of VO.sub.2max and body composition.
Then, ten participants were given an ASEA composition once a day
for seven days and ten subjects were given a placebo once a day for
seven days.
[0161] On the day of the first phase of the study, blood and urine
were collected from all twenty-two participants and then each of
the twenty-two participants biked 75 km. Blood and urine were
collected just prior to finishing the 75 km biking and one hour
thereafter. Results are tabulated below.
[0162] A washout period of three weeks then lapsed throughout which
participants did not entertain an ASEA composition. After the three
weeks, the participants crossed-over and were given the opposite
composition for 7 days. The same routine was again performed (blood
urine, 75 km biking, blood urine, blood urine one hour post). Data
is tabulated below.
[0163] Athletes ingesting ASEA for seven days started the 75 km
cycling trial with high blood free fatty acids leading to increased
fat oxidation and a sparing of amino acids (and potentially muscle
glycogen).
Example 5
Human Running Performance
[0164] A study to determine if a composition of the invention
versus placebo ingestion during a 2-week period improves run time
to exhaustion when athletes run on treadmills with the speed
adjusted to 70% VO.sub.2max. A flow chart of the study protocol is
illustrated in FIG. 22.
[0165] Blood and skeletal muscle biopsy samples are collected and
analyzed for shifts in metabolites and glycogen utilization,
respectively, to study underlying mechanisms.
[0166] Metabolites and glycogen utilization are altered when a
composition of the invention is used alongside exercise.
Example 6
Efficacy of Ingesting ASEA on Disease Risk Factor Change in
Overweight/Obese Women
[0167] A 12-Week, randomized trial is performed accord to the
protocol in FIG. 23. The study evaluates the effectiveness of 4 fl
oz/day ASEA compared to placebo over a 12-week period in helping
adult women improve disease risk factors associated with arterial
stiffness, inflammation, cholesterol status, blood pressure,
oxidative stress and capacity, fasting serum glucose, and metabolic
hormones.
[0168] Ingestion of ASEA over a 12 week period decreases arterial
stiffness, decreases inflammation, improves cholesterol status,
decreases blood pressure, decreases oxidative stress and capacity,
decreases fasting serum glucose, and alters metabolic hormones.
Example 7
Effect of an Immune-Supporting Supplement, ASEA, on Athletic
Performance
[0169] Described is a pilot study used to measure the possible
effects of an immune-supporting supplement on athletic performance
as measured by a standard VO.sub.2max and Ventilatory Threshold
(VT) athletic endurance test.
[0170] The objectives of the pilot study were to (1) confirm the
general observation that an immune-supporting supplement has an
effect on athletic performance and (2) determine the specific
physiological parameters: Heart Rates (HR), volume of O.sub.2
inspired (VO.sub.2), volume of CO.sub.2 expired (VCO.sub.2), volume
of expired gas (VE), Respiration Rate (RR), Respiratory Exchange
Ratio (RER), Aerobic Threshold (AeT), Anaerobic Threshold (AT),
VO.sub.2max and Ventilatory Threshold (VT) that are affected by
oral ingestion of this supplement during both the aerobic and
anaerobic phases of exercise.
[0171] The immune-supporting supplement, a composition of the
invention, contains a balanced mixture of Redox Signaling molecules
that purportedly increases the efficiency of the communication
channels between cells, enabling faster response of the immune
system and cellular healing activities. Enzymes in the body also
break down these Redox Signaling molecules into salt water and
nascent oxygen. There are two proposed mechanisms involving Redox
Signaling that can affect athletic performance, (1) increased
efficiencies in cellular absorption or use of oxygen, prolonging
aerobic metabolism, and (2) more efficient processing of lactate
energy stores and tissue repair mechanisms, prolonging anaerobic
metabolism.
[0172] During physical activity, the increased power requirements
from muscle tissues require increased metabolism of available
energy stores. Sustainable aerobic metabolism of sugars can supply
this energy demand as long as there is an adequate supply of oxygen
and sugars in the blood. As energy demands exceed the ability of
the respiratory and cardiovascular system to deliver sufficient
oxygen to the muscle tissue, methods involving the anaerobic
metabolism of carbohydrates, creatines, pyruvates, etc. start to
become prevalent.
[0173] Anaerobic metabolism supplies the excessive demand for
energy but is accompanied by the production of CO.sub.2 and
lactates. Prolonged or excessive anaerobic metabolism depletes the
available energy stores faster than they can be renewed; the
buildup of CO.sub.2 and lactates can also interfere with aerobic
metabolism and thus, when the energy stores are spent, exhaustion
will result.
[0174] Because anaerobic metabolism is marked by an excess in
CO.sub.2 and lactate production, it can be monitored by measuring
the excess CO.sub.2 exhaled during exercise or the buildup of
lactates in the blood. The Ventilatory Threshold (VT) is the point
where the excess CO.sub.2 is first detected in the expired breath;
it is related to the point at which anaerobic metabolism is
starting to become prevalent.
[0175] In this pilot study, VT was determined graphically from the
VCO.sub.2 vs. VO.sub.2 graph. VCO.sub.2 is the volume of CO.sub.2
expired per minute and VO.sub.2 is the volume of O.sub.2 inspired
per minute. VO.sub.2max is simply the maximum volume of O.sub.2
inspired per minute possible for any given individual. VO.sub.2max
is measured in mL/kg/min (milliliters of O.sub.2 per kilogram of
body weight per minute). VO.sub.2max is measured at the peak of the
VO.sub.2 curve. The Aerobic Threshold (AeT) was determined by the
software and indicates when fat-burning metabolic activities start
to be dominated by aerobic metabolism. The Anaerobic Threshold (AT)
was also software-determined and marks the point where the
anaerobic metabolism starts to completely dominate.
[0176] Recruitment Methods: A standard VO.sub.2max test was run on
18 athletes who responded to recruitment flyers posted in athletic
clubs and to invitations extended to a local competitive Triathlon
team. The participants were selected based on answers from
qualification questionnaire which affirmed that they:
[0177] 1. Perform a rigorous physical workout at least five hours
per week on average.
[0178] 2. Have no medical conditions that might prevent
participation
[0179] 3. Agree to follow diet and hydration instructions.
[0180] 4. Will perform only normal daily routines during the
study.
[0181] 5. Have no history of heart problems in the family.
[0182] The final selections were athletes of a caliber much higher
that the expectations reflected in the recruitment flyers, a
majority being athletes involved in regular athletic competitions.
All of the participants had never taken the supplement prior to the
study.
[0183] The participants did not receive any monetary compensation,
but did receive a case of product and results from the VO.sub.2max
tests.
[0184] The VO.sub.2max testing was done at an athletic club by
accredited professionals holding degrees in exercise physiology and
with more than 10 years daily experience in administering VO.sub.2
tests. The participants were given a choice of performing the test
on either a treadmill or a stationary cycle. A CARDIOCOACH.RTM.
metabolic cart measured heart rate (HR), inspired and expired gases
(VO.sub.2, VCO.sub.2, VE) and recorded weight, height, age, and
body mass indexes (BMI). Power settings on the treadmill or cycle
were recorded every minute.
[0185] Each participant was scheduled to take two VO.sub.2max
tests, (1) a baseline test and (2) a final test. The baseline test
was performed before any supplement ingestion. The participants
drank 4 oz. of the supplement per day between the baseline test and
the final test (7 to 10 days later) and drank 8 oz. of the
supplement ten minutes before starting the final test. For the
baseline test, the power settings on the cycle or treadmill were
determined by the test administrator. The power settings for the
final test were matched exactly to the power settings of the
baseline test for each participant. Participants were encouraged to
strictly maintain their regular diet and exercise routine and to
come to each test well hydrated (at least 8 oz. of water in the
last 2 hours before each test).
[0186] Each participant was fitted with a breathing mask and heart
monitor. Each VO.sub.2max test consisted of a 10 minute warm up
period where participants walked or cycled at a low power setting
determined by the administrator. This was followed by a ramp up
period, where the administrators increased the power settings every
minute, according to their evaluation of the physical condition of
the participant, and termination when the administrators started
seeing the indications of a maximum VO.sub.2 reading when RER
(VCO.sub.2/VO.sub.2)>1.0 or at the administrator's discretion.
The administrators had ample experience in obtaining consistent
VO.sub.2max results on this equipment, estimated at about 6% test
to test variation over the last 5 years.
[0187] The raw data (HR, VO.sub.2, VCO.sub.2, VE, Power Settings)
were collected from the CARDIOCOACH.RTM. software for analysis.
Data points were automatically averaged over 15 to 25 second breath
intervals by the software, VO.sub.2max is also determined by the
software with an averaged VO.sub.2 peak method. VT was determined
graphically from the slope of the VCO.sub.2 vs. VO.sub.2 graph.
[0188] Linear regression methods were used to determine the slope,
change in VCO.sub.2 over change in VO.sub.2. In theory, when
aerobic metabolism switches to anaerobic metabolism, the volume of
CO.sub.2 expelled (VCO.sub.2) is increased in proportion to the
Volume of O.sub.2 inhaled (VO.sub.2). This is reflected as an
increase of slope on the VCO.sub.2 vs. VO.sub.2 graph, seen as a
clear kink on the graph around the VT point. Linear regression was
used to determine the slope both before VT and after. Slopes were
determined by linear regression on the linear region of data points
before and after VT point, excluding points surrounding the VT and
near VO.sub.2max. The intersection of the before and after lines
was used to determine the reported VT point (FIG. 24).
[0189] Methods for determining the VT point on any individual
participant were kept consistent from the baseline test to the
final test. Average HR was averaged over the linear range of HR
increase during the power ramp, excluding points a few minutes into
the beginning and before the end of the data set. In every case,
the same data analysis methods were used for the final test as were
used for the baseline test for each participant.
[0190] Compliance to protocol was very high by both participants
and administrators, based on answers to compliance questions. One
data set was discarded for low VCO.sub.2 values, probably due to a
loose mask. The ventilatory data for this one participant was
rejected, leaving 17 valid data ventilatory data sets. The Heart
Rates (HR), however, were compared for all 18 participants.
TABLE-US-00007 Total Average Partic- Average Male/ Weight Cycle/
Average Data Sets ipants Age Female (Kg) Treadmill BMI Selected 18
41 .+-. 9 16/2 76 .+-. 11 7/11 24.4 .+-. 3.4 17
[0191] The average VO.sub.2max reading over all participants (N=17)
was measured at the relatively high value of 62.5 mL/kg/min,
indicative of the quality of athletes in the sample. Only four
participants had VO.sub.2max readings below 55 mL/kg/min; these
four were not involved in competitive training programs.
[0192] The data shows that two significant changes in physiological
parameters could be attributed to ingestion of the composition, as
determined by a statistical paired t-test analysis. The average
time taken to arrive at VO.sub.2max was increased by 10% with very
high confidence (P=0.006) and the average time taken to arrive at
Ventilatory Threshold (VT) was increased by 12% with a marginal
level of confidence (P=0.08).
[0193] Given that the power ramp-up-points between the baseline and
final test for each participant were identical, an increase in the
amount of time to obtain VO.sub.2max and VT on the final test also
indicates a higher average power outputs at such thresholds.
Calibrated power output measurements were not available. However,
the test administrator for the final test, upon reaching the
maximum power recorded for the baseline test, regularly surpassed
this maximum power before the participant reached VO.sub.2max on
the final test.
[0194] All other physiological parameters (VO.sub.2max, VT, AeT,
AT, Start HR, HR at AeT, HR at AT, HR at VO.sub.2max, and overall
average HR) were not significantly changed by supplement ingestion.
The high level of consistency between the baseline and final test
for these parameters, however, supports the repeatability of the
tests. The test to test repeatability has an estimated standard
deviation of less than 5% for all parameters.
TABLE-US-00008 Averages (N = 17) Baseline Final Change % Change
P-Value VO.sub.2max 62.5 63.6 +1.1 +2% -- (mL/kg/min) VT 36.4 38.7
+2.3 +6% 0.34 (mL/kg/min) Aerobic 43.6 43.8 +0.2 +0% -- Thresh.
(AeT) Anaerobic 55.5 56.5 +1.0 +2% -- Thresh. (AT) Pre VT Slope
1.030 1.030 0.0 0% -- of VCO.sub.2/VO.sub.2 Post VT 1.997 1.944
-0.053 -2.7%.sup. -- slope of VCO.sub.2/VO.sub.2 Start Heart 87.4
85.9 -1.5 -1% -- Rate (bpm) Heart Rate at 147 145 -2 -2% -- AeT
Heart Rate at 165 165 0 0% -- AT Heart Rate at 174 175 +1 +1% --
VO.sub.2max Heart Rate 137 134 -3 -2% -- Overall Time to VT 306 344
38 +12% 0.08 (secs) Time to 639 703 64 +10% 0.006
VO.sub.2max(s)
[0195] Of the 17 participants in the study, 70% of them experienced
a significant increase in time to VO.sub.2max, 18% of the
participants showing more than a 25% increase, 41% showing more
than a 10% increase, 18% of the participants exhibiting no
significant change and 12% showing a mild decrease (under 10%).
[0196] There was a moderate but significant correlation between the
increases in "time to VO.sub.2max" and "time to VT" (correlation
coefficient 0.35), meaning that an increase in time to reach
VO.sub.2max was moderately but not always proportional to the
increase in the time it took to get to VT. There is a strong
correlation between increase in time to VO.sub.2max and decrease in
the average overall heart rate (correlation coefficient -0.67),
meaning that an increase in time to VO.sub.2max would most often be
accompanied by a decrease in average overall heart rate.
[0197] Ingestion of the test supplement, a composition of the
invention, for 7-10 days prior to and immediately before a
VO.sub.2max test, was shown to significantly increase the time it
took for 70% of the participants to reach VO.sub.2max under
equivalent carefully regulated power ramp-up conditions. Time to VT
likewise was significantly extended.
[0198] The extension of time to reach VT, under similar increasing
demands for energy, is a direct indication that the aerobic phase
of metabolism is being extended and/or the anaerobic phases somehow
are being delayed as the demand for energy increases.
[0199] The lack of any other changes in the physiological
parameters (VO.sub.2max, VT, AeT, AT and associated heart rates)
suggests that cardiovascular capacity, lung capacity and blood
oxygen capacity and regulation were not affected. This assumption
is reasonable, given that the short duration of this study excluded
the possibility of training effects.
[0200] One feasible explanation for the results lies in the
enhancement of aerobic efficiencies, meaning that more aerobic
energy can be extracted at the same physiological state, or that
the clearance of lactates or CO.sub.2 becomes more efficient, again
allowing greater aerobic efficiency. Note that "time to AeT" and
"time to AT" were not compiled in this study, however changes in
these parameters would be expected and might offer clues to
determine the underlying mechanisms.
[0201] The results of this pilot test indicate that there is a
strong case for athletic performance enhancement and further
investigation is warranted. A placebo-based double-blind test,
measuring the more subtle effects in ventilation and heart rates
along with increases in blood lactate levels during a controlled,
calibrated power ramp would provide defensible evidence for this
effect and better support for some specific underlying mechanisms
of action.
Example 8
In Vitro Bioactivity Study
[0202] Described are a variety of results from in vitro
experiments, performed at national research institutions,
investigating the bioactivity of a composition disclosed herein,
when placed in direct physical contact with living cells. Specific
investigations include in vitro toxicity and antioxidant
efficiencies of the master antioxidants glutathione peroxidase
(GPx) and Superoxide Dismutase (SOD) inside living cells and the
translocation of two well-studied transcription factors (NF-kB,
NRF2) known to regulate toxic response and antioxidant production
in human cells. Some preliminary work on concentration dependence
was also done as well as cell proliferation, counts associated with
induced oxidative stress in human cells.
[0203] The objectives of the investigations were (1) to determine
if any signs of toxicity (NF-kB activation) are manifest when
varying concentrations of a certain redox signaling compound, ASEA,
are placed in physical contact with living cells, (2) to determine
if such direct contact affects the antioxidant efficacy of
glutathione peroxidase (GPx) and superoxide dismutase (SOD) and (3)
to determine if such contact activates translocational
transcription (NRF2) associated with increased expression of
antioxidants in living human endothelial cells and to verify the
expression of such transcription factors by Western Blot analysis,
(4) to determine the effect of this redox signaling compound on
proliferation cell counts of human cells and associated markers
(LDH) for cell viability and health, (5) to determine the effects
of this redox signaling compound on cells that were stressed with
cytokines (Cachexin), radiation and serum starvation.
[0204] The immune-supporting composition contains a redox-balanced
mixture of RXN [both reactive oxygen species (ROS) and reduced
species (RS)] that are involved in a large variety of pathways and
receptor-site activity in human cells. For example, when cells are
damaged, for any reason (ex. toxins, DNA breaks or infections), the
native redox signaling messengers inside the cells can become
imbalanced, most often manifest by the accumulation of
intracellular oxidants and ROS (oxidative stress). The cell, so
affected, will activate defense and repair mechanisms aimed to
restore proper redox-signaling homeostasis and proper cellular
function. If repair efforts are unsuccessful and normal homeostatic
redox balance is not able to be restored, then within a few hours,
the excess oxidants and ROS in such cells will facilitate apoptotic
processes to internally digest and destroy the dysfunctional cell.
Healthy neighboring cells will then divide to replace it. A
complete field of science called "redox signaling" has been founded
to study such processes, with literally thousands of references
available.
[0205] It is the nature of certain redox signaling molecules, when
unbalanced or isolated, to elicit immediate recognizable toxic
responses in exposed living cells; hydrogen peroxide is one example
of such a redox signaling molecule. The first-line cellular
response to toxic substances involves the translocation of NF-kB
into the nucleus as a precursor to the inflammatory response and
other defense mechanisms. The movement of NF-kB into the nucleus
can be visibly tracked in a living cell under a fluorescence
microscope with the aid of fluorescent tag molecules. The
observation of nuclear translocation of NF-kB is a sure marker that
a toxic response has been initiated. Even low-level toxicity is
detectable with this catch-all method; low-level concentrations of
hydrogen peroxide, for example, produce an easily distinguishable
positive toxic response.
[0206] A separate transcription factor, NRF2, moves into the
nucleus in response to low-level oxidative stress and facilitates
the increased production of antioxidants. Again, by the use of
fluorescent tags, the nuclear translocation of NRF2 can be seen in
cells under a fluorescence microscope. NRF2 nuclear translocation
is a second-line-of-defense mechanism known to increase the
production of protective enzymes and antioxidants such as
glutathione peroxidase and superoxide dismutase. NRF2 translocation
will often accompany low-level NF-kB activation and NF-kB
activation (almost) always precedes NRF2 translocation. Substances
that exhibit low-level toxicity, such as trace homeopathic toxins,
have long been used to activate the NRF2 pathway in order to
stimulate these natural defend-repair-replace mechanisms.
[0207] Enzymatic efficacy of antioxidants, such as Glutathione
Peroxidase (GPx) and Superoxide Dismutase (SOD), can be determined
through standardized ELISA tests that measure the time-related
reduction of certain oxidants introduced into cell lysates after
the living cells have been exposed to the test substance for a
given period of time. The reagents of the ELISA test must be chosen
as not to interfere or interact with the test substance. Other
critical factors such as the time of exposure and concentration
dependence must be experimentally determined.
[0208] Western Blot methods also exist to experimentally determine
the quantities of GPx or SOD in cell lysates. These
well-established molecular separation techniques and can be used to
directly verify whether the quantity of such antioxidant enzymes
has been increased in the sample. Measured antioxidant efficiency,
however, remains the best indication of cellular antioxidant
defense.
[0209] Monitoring cellular proliferation, cell counts and chemical
indicators of cellular death are also commonly used to determine
cellular viability and gross response to stressors such as
radiation, cytokines and toxins. Cachexin, for example, is a potent
toxin, a cytokine, that elicits immediate toxic responses and
build-up of oxidative stress in exposed cells. Cells, so stressed,
exhibit a greater tendency to undergo apoptosis and die, thereby
releasing internal proteins (such as LDH) into the surrounding
serum.
[0210] Normally, when the introduction of such stressors and toxins
elicits oxidative stress conditions in the cell cultures, cell
counts will fall, cellular proliferation will subside, and serum
LDH levels will rise, indicating that cell death is occurring in
the culture. Hydrogen peroxide, radiation and serum starvation can
also elicit similar responses. Redox signaling messengers, as
outlined above, are intimately involved in cellular reception of
and response to such stressors; redox messengers are involved in
mediating antioxidant production and action to protect the cells,
repair mechanisms necessary to fix DNA and structural damage and
also in mediating the apoptotic process that results in cell
death.
[0211] Increasing the concentration of such redox messengers in the
serum may serve to augment the efficiency of these normal cellular
processes. The exact action of various redox signaling mixtures
must be determined experimentally. Independent unpublished studies,
involving Mass Spectroscopy, Florescent Spectroscopy and Electron
Spin Resonance, have unmistakably verified the existence of several
kinds redox signaling molecules in the composition described
herein. Well-established redox electrochemistry also validates the
existence of such redox signaling molecules. The stability of this
redox-balanced mixture is many orders of magnitude greater than
expected. The confirmed preservation of unstable moieties in this
supplement might be explained by the existence of certain stable
molecular complexes, some of them verified by mass spectroscopy
that can shield radical interactions. Intellectual property
agreements, however, prevent the disclosure of the details.
[0212] The following research was conducted on a best efforts basis
by a senior researcher at a national laboratory and is designed to
assess basic mode-of-action when a composition of the invention is
placed into direct contact with human cells:
[0213] 1. The initial dose range projected for in vitro studies was
extrapolated from a 10 mL of a composition of the invention/kg
equivalent oral dose from human trials.
[0214] 2. Glutathione peroxidase (GPx) and superoxide dismutase
(SOD) ELISAs were used to determine whether a composition of the
invention alters enzymatic activity in murine epidermal (JB6)
cells.
[0215] 3. LDH (non-specific cellular death) levels and cell
proliferation rates were determined for various cell types exposed
to a composition of the invention.
[0216] 4. Human microvascular endothelial lung cells (HMVEC-L) were
treated with a composition of the invention and cell lysates were
analyzed by GSH-Px and SOD ELISAs to determine whether antioxidant
enzyme activities are altered.
[0217] 5. HMVEC-L cells were treated with a phosphate buffered
saline solution (PBS) negative control, 5% and 20% concentrations
of a composition of the invention and a Cachexin positive control
to determine the nuclear translocation activity of the p65 subunit
of NF-kB (cytokine transcription) at 30, 60, 90 and 120 min
intervals. Fluorescent microscopy techniques were employed to image
cellular response.
[0218] 6. Step (4) was repeated except nuclear translocation
activity of P-Jun was determined as an extension/verification of
step 4.
[0219] 7. Two cultures of HMVEC-L cells, one with normal random
cell cycles and another with serum starvation were treated with low
<1% concentrations of a composition of the invention to
determine the nuclear activity of NRF2 (antioxidant transcription)
at 30, 60, 90 and 120 minute intervals compared to a negative (PBS)
control.
[0220] 8. A Western Blot analysis was done on extra-nuclear and
intra-nuclear fractions, separated by differential centrifugation,
of serum starved HMVEC-L cell cultures exposed to <1% of a
composition of the invention compared with a positive hydrogen
peroxide control to determine phosphorylation events (oxidant
action) in the extra-nuclear fraction and NRF2 (antioxidant
transcription) in the intra-nuclear fraction at 0, 30, 60, 90 and
120 min intervals.
[0221] 9. Normal random cell phases of HMVEC-L cells were exposed
to radiation and then treated with a composition of the invention.
Cell counts were taken to determine survival.
[0222] 10. The efficacy of Cachexin reception in confluent-phase
and normal-phase HMVEC-L cells was determined through changes in
extracellular and intracellular LDH activity in cells exposed to
various mixtures of Cachexin, PBS and a composition of the
invention solutions.
[0223] Experimental Methods used to Assess Toxic Response in
Primary Human Lung Microvascular Endothelial Cells (HMVEC-L):
HMVEC-L cells (catalog #CC-2527) were purchased from Lonza
(Walkersville, Md.) as cryopreserved cells (Lot #7F4273). Cells
were thawed and maintained according to manufacturer's directions.
Cell culture medium (proprietary formulation provided by Lonza)
contained epidermal growth factor, hydrocortisone, GA-1000, fetal
bovine serum, vasoactive endothelial growth factor, basic
fibroblast growth factor, insulin growth factor-1 and ascorbic
acid.
[0224] HMVEC-L Cell cultures in normal random cell cycles were
exposed to high-concentration ASEA in the serum medium,
concentrations of 5% and 20%, and analyzed in conjunction with
cultures exposed to phosphate buffered saline solution (PBS) as
non-toxic negative control and Cachexin (5 ng/mL) as a positive
control (highly toxic). At intervals of 0, 30, 60, 90, and 120
minutes, aliquots of cells from each culture were placed under a
fluorescent microscope, stained by fluorescent dyes designed to tag
the p65 subunit of NF-kB along with a DAPI fluorescent nuclear
stain that aids the computer software to find the nuclei. Computer
automated imaging techniques were used to determine the relative
degree of translocation NF-kB into the nucleus via fluorescent
analysis over several cells. As a reminder to the reader, P65 NF-kB
translocation is the first-phase non-specific cellular response to
toxicity. Thus the movement of the NF-kB into the nucleus, as seen
visually in the microscope images, is a sensitive indicator of
general toxic response.
[0225] Results of HMVEC-L Cells p65 subunit NF-kB screen for
toxicity: Typical cell images are shown below for each culture.
Translocation of p65 subunit of NF-kB into the nucleus was not seen
in any cell cultures exposed to high-concentration a composition of
the invention. Automated analysis confirmed this and indicated no
toxic response at 0, 30, 90 and 120 minutes. In contrast, Cachexin
exposed cells exhibited an immediate sustained toxic response (FIG.
25).
[0226] Cachexin is positive control and induces the translocation
of p65 subunit of NF-kB from cytosol into nucleus. DAPI staining
shows position of nuclei in these images (see white arrow). A
composition of the invention (5 and 20% final v/v) did not induce
nuclear translocation of NF-kB at 30, 60 and 120 min time
points.
[0227] Given this null indication of toxicity after exposure to
high concentrations of ASEA, another test was performed to confirm
behavior.
[0228] Additional Method to Assess Toxic Response of HMVEC-L Cells
(P-Jun): A similar methodology as that employed with NF-kB was
employed to determine the nuclear translocation of an
anti-phospho-Jun (AP-1 P-Jun) antibody index (P-Jun is another
toxicity-related redox-responsive transcription factor). HMVEC-L
cells were again exposed to high-concentration ASEA. All procedures
were similar to the NF-kB analysis except for the substitution of
P-Jun fluorescent indicators and automated measurements taken over
100 cells in order to increase sensitivity. An additional naive
(untouched) culture was also analyzed.
[0229] Results for P-Jun screen for toxicity (FIG. 26): AP-1 index
determined using anti-phospho-Jun (P-Jun) antibody. AP-1 is nuclear
localized and upon activation, the phosphorylation status of P-Jun
is increased. Anti-P-Jun antibody binds to the phosphorylated form
reflected as an increase in fluorescence intensity (see Cachexin
control). A consistent trend reflecting an increase in P-Jun levels
was not observed for cells treated with 5% or 20% ASEA at 30, 60
and 120 min time points, while the Cachexin positive control
significantly increased nuclear P-Jun levels at 30 min.
[0230] Again no toxic response was observed; there was no
significant accumulation of P-Jun in the nuclei of cell cultures
exposed to high concentrations of a composition of the invention.
Automated analysis indicated no toxic response at 0, 30, 90 and 120
minutes, with a slight but non-significant increase for 20% a
composition of the invention at the 30 minute time point; at other
time points no increase was detected. In contrast, the Cachexin
exposed cells (positive control), as expected exhibited an
immediate sustained toxic response.
[0231] The results of the P-Jun analysis concurred with the
response seen in the NF-kB analysis. For both tests, there was no
significant difference between a composition of the invention
exposure and that of the negative PBS control for healthy
random-phase HMVEC-L cells. This confirmed lack of toxicity was
somewhat unexpected for this mixture of redox signaling molecules,
considering that some of them, if isolated from the mixture, are
known to elicit an immediate response.
[0232] Since nuclear translocation of NF-kB and P-Jun are typically
the first responders to serum toxicity and are known to initiate
the inflammatory response, especially in the ultra-sensitive human
endothelial cells, healthy human cells when directly exposed to a
composition of the invention, are not expected to exhibit defensive
behavior nor initiate inflammatory processes (such as the release
of inflammatory cytokines). It is not certain from this data
whether exposure would suppress or reverse the inflammatory
process.
[0233] Blood serum levels of such redox signaling molecules, for
all in vivo oral applications, would not exceed serum
concentrations of 1% and typically would be less than 0.1%. Serum
levels are expected to drop over time due to enzymatic breakdown of
the components. Independent in vivo pharmacokinetic studies
indicate that the active components in ASEA have approximately a 17
minute half-life in the blood and thus would be effectively cleared
from the blood within a few hours. Thus no toxic response is
expected due to exposure of healthy human cells at such levels. It
has been seen in these in vitro studies that direct exposure of
human cells to serum concentrations of up to 20% is still well
tolerated. The complete lack of toxicity, comparable to the PBS
control, is extremely rare and indicates that despite the
reactivity of this mixture, it is well tolerated by human tissues
and is native to or compatible with the extracellular
environments.
[0234] Experimental Methods Used to Determine Antioxidant Efficacy
of Glutathione Peroxidase (GPx): Cell cultures of standard murine
epidermal cells (JB6) were exposed to various small concentrations
of a composition of the invention (less than 1%) and PBS solution
for 24 hours. Cell lysates were prepared for measurements of GPx
enzymatic activity using a commercially available ELISA kit (GPx
activity kit, Cat #900-158) according to directions of the
manufacturer (Assay Designs, Ann Arbor, Mich.). Decrease of
oxidants due to GPx enzymatic activity was monitored over an 11
minute period of time after a chemical agent (cumene hydroperoxide)
initiated the reaction. The decrease of oxidants is an indication
of antioxidant efficacy. To determine GPx efficacy at various
concentrations of PBS or a composition of the invention, three
replications of oxidant residual in the samples were read every 2
min to generate the slope, indicating the decrease in relative
fluorescence units (RFU)(oxidant residual) per minute.
[0235] Results and Observations for GPx Antioxidant Efficacy Test:
After activation, the reduction of oxidants over time was closely
linear, as seen in the graphs below (RFU units on vertical scale).
A well-defined slope was established over the 11 minute interval
(FIG. 27). Antioxidant activity is measured by reduction of
oxidants over time (FIG. 28).
[0236] A significant increase in antioxidant activity was seen in
samples infused with ASEA compared to the PBS control (second
graph).
[0237] Concentration dependency, however, was not seen between the
5 ul, 10 ul and 20 ul infusions. This suggests that GPx antioxidant
activity might saturate at concentrations lower than that
represented by the 5 ul infusion. Such considerations will be
discussed later.
[0238] The table below summarizes the data shown on the preceding
graphs.
TABLE-US-00009 Sample Infusion Volume Slope for PBS Control Slope
for ASEA (<1% total volume) (% reduction/minute) (%
reduction/minute) 0 ul 0.1% 0.1% 5 ul 0.1% 3.6% 10 ul 0.2% 3.6% 20
ul 0.3% 3.7%
[0239] The raw data reflects more than a 10 fold increase in
antioxidant activity related to ASEA infusion. Taking into account
experimental uncertainties, it is 98% certain that the serum
infusion of small concentrations (<1%) of a composition of the
invention increased antioxidant efficiencies by at least 800%.
Further investigations should be done to confirm this increase and
explore concentration dependence for these low-level serum
concentrations.
[0240] Experimental Methods Used to Determine Antioxidant Efficacy
of Superoxide Dismutase (SOD): Human HMVEC-L cells were treated
with 10% phosphate buffered saline (PBS; vehicle control), 5% or
10% of a composition of the invention for 24 hr at which time cell
lysates were prepared for measurements of SOD activity using a
commercially available kit (SOD activity, cat #900-157) according
to manufacturer's (Assay Designs, Ann Arbor, Mich.) directions.
Cell culture medium was assayed for SOD activity in parallel.
Limited trials with smaller concentrations of a composition of the
invention <1% and murine epidermal cells were also
attempted.
[0241] Results of First-Attempt Methods to Determine SOD activity
for high serum a composition of the invention concentration:
Diluted lysates showed a marginal increase in enzymatic activity
associated with treatment with a composition of the invention.
Changes in enzymatic activity were marginal in the initial range of
5-10% a composition of the invention (final concentration, v/v).
The data represent the first attempt to measure SOD activity using
primary HMVEC-L cells treated with a composition of the invention.
It is feasible that the lack of SOD activity associated with 5-10%
a composition of the invention might be related to non-specific
inhibition at high dose. The primary concern is that we have little
understanding of the primary human HMVEC-L cell model and cannot
determine whether these cells are optimal for investigating
antioxidant defense regulation induced by a composition of the
invention. For example, ascorbic acid, known to break down certain
redox signaling complexes in A a composition of the invention, is
supplemented into the medium and it is feasible that some
modification of the medium formula (such as omission of ascorbic
acid for short periods of time defined empirically) could produce
more optimal conditions for detecting antioxidant defense regulated
by a composition of the invention. Initial efforts to serum-starve
these cells, as one approach to increase sensitivity and optimize
the model, were unsuccessful and resulted in extensive cell death
over 24 hours, indicating that the cells are dependent on the
growth factors supplemented in the cell culture medium to maintain
cell viability. If we interpret the initial a composition of the
invention concentrations (5-10%) to be high (based on inhibition of
medium enzymatic activity and cell proliferation), then it is
possible that the marginal increase in enzymatic activity
associated with cell lysates observed here may not accurately
reflect antioxidant defense regulation possibly occurring at lower
concentrations. The use of an in vitro model system with a well
defined and robust NRF2-regulated antioxidant defense response
would help address some of these uncertainties. In retrospect, we
have observed that a lower concentration of a composition of the
invention (1%) induces the nuclear translocation of the NRF2
transcription factor. In addition, the 24 hr time point was chosen
for the initial screen as a general time point for in vitro
investigations that would capture transcriptional regulation;
however, this time point was not optimal.
[0242] Results of Further Investigations into SOD enzymatic
activity at low composition of the invention concentrations
(<1%): It was found in another investigation that NRF2 nuclear
translocation (data and results are in the following sections),
took place at low doses of a composition of the invention (less
than 1%) and elicited peak SOD antioxidant activity at about 30 to
120 minutes after exposure. Thus when SOD antioxidant activity was
measured due to low-concentration composition of the invention
exposure at 30 to 120 minute time points, results similar to the
GPx enzymatic activity were seen both with murine epidermal (JB6)
cells and serum-starved HMVEC-L cells at a time point 90 to 120
minutes. A 500% increase in peak SOD enzymatic activity was
estimated over a short 120 minute term, with 95% confidence.
[0243] Experimental Methods Used to Determine Nuclear Translocation
of NRF2 in HMVEC-L Cells and Western Blot Verification: HMVEC-L
cells were again thawed and maintained according to manufacturer's
directions. The culture medium contained epidermal growth factor,
hydrocortisone, GA-1000, fetal bovine serum, vasoactive endothelial
growth factor, basic fibroblast growth factor, insulin growth
factor-1 and ascorbic acid in randomly cycling cultures. Ascorbic
acid was withheld from serum-starved cultures.
[0244] HMVEC-L Cell cultures in both normal random cell cycles and
in serum starvation were exposed to high-concentration (5-20%) and
low-concentration (1%) ASEA in the serum medium and analyzed in
conjunction with cultures exposed only to phosphate buffered saline
solution (PBS), as a negative control. At time points of 30, 60,
90, and 120 minutes, aliquots of cells from each of the cultures
were placed under a fluorescent microscope, stained by a
fluorescent dye designed to tag the NRF2 transcription factor along
with the DAPI fluorescent nuclear stain that aids the computer
software to find the nuclei. Computer automated imaging techniques
were used to determine the relative degree of nuclear accumulation
of NRF2 via fluorescent analysis over several cells. NRF2 regulates
the transcription of a number of phase II antioxidant defense
enzymes and raises the possibility that additional antioxidant
defense enzymes, such as glutathione transferase, may be expressed
through exposure to ASEA. Thus the accumulation of NRF2 into the
nucleus, as seen visually in the microscope images, is an indicator
of increased antioxidant expression in the cells.
[0245] Results of HMVEC-L Nuclear Accumulation of NRF2: Initial
screen of human endothelial cells suggests a subpopulation of cells
showed increased nuclear staining pattern (focal) following
treatment with high-concentration of a composition of the
invention. The positions of nuclei are indicated by DAPI stain in
lower panel. Foci appear brighter in a composition of the invention
stimulated cells which indicates higher level of NRF2 transcription
factor in the nucleus. H.sub.2O.sub.2 was used as positive control.
This effect was difficult to quantify based on nuclear staining
pattern. (FIG. 29).
[0246] Typical cell images are shown below for indicated cell
cultures exposed to low-concentrations of a composition of the
invention. Accumulation of NRF2 into the nucleus was clearly seen
in serum-starved cell cultures exposed to low-concentrations of a
composition of the invention. Automated analysis revealed strong
time-dependent nuclear accumulation of NRF2 in serum-starved cells,
relative to the negative control, at the 30 and 60 minute time
points (FIG. 30).
[0247] The nuclear staining profile was qualitatively different
from the cells maintained in optimal growth medium (randomly
cycling group). There was weak qualitative nuclear accumulation of
NRF2 induced by exposure to a composition of the invention in these
cells at 30, 60 and 120 minute time points, and yet the effect was
not nearly as pronounced as in the serum-starved cultures. However,
serum-starvation induced significant cell death complicating
interpretation of the data. The trends appeared weak and require
validation by Western Blot.
[0248] Experimental Methods for Western Blot Validation of NRF2
Nuclear Accumulation: HMVEC-L were treated with 1% of a composition
of the invention, nuclear extracts were separated through
centrifugal differentiation from the extra-nuclear cytosol at 30,
60 and 120 min and subjected to Western Blot analysis for NRF2. In
the Western blot experiment the extra-nuclear fraction was probed
for phosphorylated proteins using a combination of anti-phospho
serine, threonine and tyrosine antibodies. Virtually all cellular
processes are regulated by posttranslational modifications and
protein phosphorylation is a prevalent mechanism. Observable
changes in protein phosphorylation can lead to a mechanistic
understanding of the cellular processes perturbed by compositions
of the invention and provide a defined endpoint to better define
dose-dependent regulation of cell function by compositions of the
invention in vitro, as well as provide a potential candidate
molecular marker that may be used to provide in vitro-in vivo
correlates. Hydrogen peroxide (H.sub.2O.sub.2) was included as a
positive control for oxidant damage.
[0249] Results for Western Blot Validation of NRF2 Nuclear
Accumulation: NRF2 levels were increased in a time-dependent
fashion in nuclear extracts prepared from HMVEC-L cells treated
with 1% ASEA. H.sub.2O.sub.2 (30 min) did not increase nuclear NRF2
levels. In contrast, when protein phosphorylation was examined in
the extra-nuclear fraction (separated from nuclei by differential
centrifugation) we observed a single band by Western blot analysis
and this is likely due to the dilution of the extra-nuclear
fraction during the cell fractionation process (other
phosphorylated proteins are obviously present but are below
detection limits under these conditions) or specificity of the
anti-phospho-antibodies used was insufficient to detect a broad
range of phosphorylated proteins. However, we did observe a marked
increase in the phosphorylation of the protein detected following
H.sub.2O.sub.2 treatment, indicating that this phosphorylation
event is highly sensitive to redox regulation or activation of
protein kinase/deactivation of protein phosphatase activities
subsequent to oxidative damage. Treatment of cells with 1% of a
composition of the invention decreased phosphorylation levels
associated with this protein in a time-dependent fashion (FIG.
31).
[0250] Reductions in phosopho-protein regulation in extra-nuclear
fractions were seen along with strong time-dependent NRF2
accumulations in the nuclear fractions, indicating clear
time-dependent up-regulation of antioxidant expression.
[0251] At this point it is worth mentioning that NRF2 activity has
been clearly detected in conjunction with exposure to a
low-concentration of a composition of the invention without the
normal prior NF-kB activity. This suggests that phase II
antioxidant defense mechanisms have been stimulated without the
normal prior phase I toxic response. This behavior has no precedent
or is extremely rare. It appears from the data that compositions of
the invention are able to stimulate antioxidant expression without
ever eliciting a prior low-level phase I toxic response.
[0252] Experimental Methods to Determine Proliferation of Murine
(JB6) Cells and HMVEC-L Cells and LDH Activity with Exposure to
ASEA: HMVEC-L cells were treated with 5-20% ASEA for 72 hr and cell
number was determined using a Coulter Counter. Control (0
concentration group) was treated with 20% PBS. Serum LDH levels
were also measured as an indicator of cell culture viability at 0
to 20% concentration of the compositions of the invention/serum
concentrations. Recall that lower serum LDH concentrations indicate
less cell membrane failure. Similar experiments were performed for
murine (JB6) epidermal cells.
[0253] Results for Proliferation of Murine and HMVEC-L cells and
LDH activity (FIG. 41, 42): The initial in vitro screen indicates
that high-concentrations of compositions of the invention in serum
may inhibit cell proliferation (for both murine epidermal cells
[JB6] and primary human lung microvascular endothelial cells
[HMVEC-L]) in the concentration range of 5-20%. In this
concentration range we also observed direct inhibition of LDH
enzymatic activity. The data are somewhat contradictory as the
decreasing cell counts indicate cell death, yet lower serum LDH
levels indicate higher cellular membrane integrity. At the highest
concentration tested (20% v/v), cell proliferation was inhibited by
approximately 20%.
[0254] The mechanism behind reduced proliferation cannot be deduced
and could be related to interference with growth factor
responsiveness or other possible interpretations such as enhanced
programmed death (apoptotic response) for damaged cells. It is
noteworthy that high serum concentrations of composition of the
invention for in vitro enzymatic enhancement studies is not
optimal, it is possible that the initial screens underestimated or
even missed antioxidant defense (SOD) regulation by a composition
of the invention and thus indicate that low-concentration (<1%)
compositions of the invention and/or short exposure times should be
employed for such purpose.
[0255] Further studies were done that investigated the action of
stressed cells upon exposure to compositions of the invention; the
source of stress resulting from a variety of chemical and
environmental stressors. These investigations offer clues for the
possible mechanisms.
[0256] Experimental Methods to Determine cell viability of HMVEC-L
exposed to various mixtures of Cachexin stressor and
high-concentration compositions of the invention: HMVEC-L cultures
with normal random cell cycles (pS) and cultures approaching
confluence (A2), which are generally less sensitive to Cachexin,
were infused with escalating concentrations of Cachexin stressor
(0-5 ng/mL). These cultures had been pretreated with either a 10%
PBS control or 5-10% concentration of a composition of the
invention for 24 hours. Two indicators for cell viability were
employed. Serum LDH levels were obtained as an indication of
membrane integrity and Neutral Red dye was used as an indication of
lysosomal integrity. Recall that as cell membranes fail, LDH is
released into the serum medium. Lower quantities of LDH indicate
higher cell viability. The integrity of lysosomes, necessary for
viable cell function, are measured by absorption of Neutral Red dye
stain. Higher quantities of Neutral Red absorbance indicate higher
cell viability.
[0257] Results of HMVEC-L viability exposed high-concentration
composition of the invention and to escalating amounts of Cachexin
stressor (FIG. 34): Both confluent (A2) and normal (pS) HMVEC-L
cultures exhibited up to 30% improvement (relative to PBS controls)
in LDH levels related to exposure to compositions of the invention
after acute (up to 5 nm/mL) Cachexin insult. The LDH data suggest
that HMVEC-L cells stressed by Cachexin are less likely to die due
to cell membrane failure after being exposed to compositions of the
invention.
[0258] Behavior of lysosomal integrity in HMVEC-L cells as measured
by Neutral Red absorption exhibited behavior dependent on cell
culture phase. As expected, the confluent (A2) cells in the PBS
control were much less sensitive to Cachexin insult than cells in
the PBS control normal random phase (pS) culture; this is evidenced
in the 5 ng/mL Cachexin data: Lysosomal levels in A2 cells dropped
only 50% compared to 70% in the pS culture. Exposure of the normal
(pS) cultures to compositions of the invention made little
difference in lysosomal integrity under similar Cachexin insult,
yet exposure of confluent (A2) cell cultures to ASEA made them much
more sensitive to Cachexin insult, regressing to behavior similar
to that exhibited by the normal more sensitive (pS) cells.
[0259] This is the first evidence presented that suggests that
exposure of abnormal (Cachexin-insensitive) HMVEC-L cells to
compositions of the invention can make them more sensitive. The
data suggest that confluent (A2) cells stressed by Cachexin are
more likely to die when exposed to compositions of the invention,
these abnormal cells when exposed to ASEA exhibit closer to normal
behavior in the presence of Cachexin. This behavior was initially
unexpected as the hypothesis of the experiment was that
compositions of the invention would help cells protect themselves
against toxic insult. As it turns out, it appears that compositions
of the invention exposure only helps normal healthy cells to
protect themselves against oxidative insult and yet seems not to
help cells protect themselves against Cachexin. Exposure to
compositions of the invention may even help facilitate the death of
the stressed cells that are close to the end of their normal life
cycle. Incidentally, the normal role of Cachexin in the tissues is
to facilitate the death and replacement of damaged cells.
[0260] Experimental methods to determine the compositions of the
invention concentration-dependent response of A2 and pS phase
HMVEC-L cells to Cachexin insult: HMVEC-L cell cultures, prepared
in two phases, in the confluent end-of-life-cycle A2 phase (a phase
typically insensitive to Cachexin insult) and in the normal random
cycle pS phase were exposed for 24 hours to serum concentrations
(v/v of 2.5%, 5%, 10%, 15% and 20%) of either the PBS control or a
composition of the invention. Cachexin responsiveness was then
determined by monitoring LDH activity in both the intracellular
cytosol and in the surrounding growth media. Recall that increased
LDH activity in the growth media indicates cell membrane rupture
and death (LDH release) and the decrease of intracellular LDH
activity indicates loss of cellular integrity. Thus the cell
cultures that are responsive to Cachexin insult would experience an
increase in medium LDH activity and a decrease in intracellular LDH
activity.
[0261] LDH activity in untouched cell culture controls were
compared to that of cell cultures insulted with 5 ng/mL Cachexin
for each composition of the invention concentration considered. The
concentration dependence of compositions of the invention was then
graphed against LDH activity for each insulted culture and
control.
[0262] Results of concentration-dependent response of HMVEC-L cells
to Cachexin insult (FIG. 35): Relative to the PBS control, the
Cachexin response for the normal pS cells was much smaller than
expected. Only slight decreases in cell membrane integrity were
seen in the PBS control cultures and the intracellular LDH activity
remained the same. With exposure to compositions of the invention,
by itself, the normal pS cell cultures suffered a slight decrease
in overall cellular integrity and increase in cell death. It should
be noted that since the large expected response of the control pS
cells to Cachexin was not manifest, it is probable that the pS cell
cultures used in this investigation were nearing a confluent or
non-responsive state.
[0263] There was, however, a clear response when Cachexin insult
was added to the pS cell cultures exposed to various composition of
the invention concentrations, cultures demonstrated a clear loss of
intracellular LDH function and integrity. However, the accompanying
indication of cell death was not seen. This seems to indicate that
the "normal pS" cells were made more sensitive to Cachexin
reception by composition of the invention exposure, yet not brought
completely to the point of cell death.
[0264] The A2 cell culture response was very clear. Composition of
the invention exposure, even without Cachexin, seemed to cause loss
of intracellular LDH integrity, though it did not affect cell
death. However, when Cachexin insult was applied to such A2
cultures, composition of the invention exposure clearly amplified
the Cachexin reception rapidly decreasing cellular function and
there were also clear indications of concentration-dependent cell
death. There is strong evidence that exposure to compositions of
the invention increases Cachexin responsiveness in the A2 cell
cultures.
[0265] The results imply that exposure to compositions of the
invention significantly increases Cachexin responsiveness in A2 and
borderline pS HMVEC-L cell cultures. Of possible interest, exposure
to compositions of the invention alone might decrease integrity of
cellular LDH activity in A2 type cells; recall that zero toxic
response was detected in randomly cycling cells even under large
concentrations, so effects due to toxicity are not expected in
normal cells. It appears that exposure to compositions of the
invention may tend to accelerate the removal of non-responsive
confluent cells. This is evidently true when Cachexin is present.
These results might also bear on the observations that exposure to
compositions of the invention seemed to diminish cell proliferation
in high concentrations. No such trend was tried for
low-concentration exposure. Note that it is difficult to discount
the possibility that high-concentration effects might simply be
artifacts due to the interference of compositions of the invention
with the growth medium.
[0266] Experimental methods to determine effects of 5-10%
composition of the invention exposure to cells stressed by
radiation and serum starvation: Murine (JB6) cell cultures were
subjected to high-level radiation exposure (X-rays) and, in a
separate investigation, cultures were subject to serum starvation
of growth factors for 24 hours. The cells were then exposed to
5-10% ASEA exposure as means to determine the effect of composition
of the invention exposure on such stressed cells. Cell counts were
taken before and after composition of the invention exposure.
[0267] Results of effects of 5-10% composition of the invention
exposure on radiation and serum-starved murine cells: Quantitative
analysis was not compiled for these experiments. Qualitative
analysis, however, reveals results that might be of some interest.
For the radiation-damaged culture, immediate cell death was
observed for more than half of the culture upon exposure to
composition of the invention. No further cell-death was seen
thereafter. Upon inspection under a microscope, the remaining
living cells appeared normal and healthy. It appears that exposure
to a composition of the invention may have helped accelerate cell
death among the more seriously damaged cells and allowed for the
survival of healthy or repairable cells.
[0268] For serum-starved cell cultures similar observations were
made, except the cell death was not nearly as severe, amounting to
less than roughly a 20% loss. Surviving cells appeared to be very
robust and viable. Similar losses, however, were also seen in
serum-starved cultures that were not exposed to compositions of the
invention in later experiments.
[0269] A better understanding of the bioactivity of a certain
mixture of redox signaling molecules has been determined from in
vitro studies involving direct contact of compositions of the
invention with viable living HMVEC-L human cells and murine
epidermal JB6 cells. Five specific objectives were pursued to
determine:
[0270] 1) In vitro toxicity (based on NF-kB, P-Jun
translocation)
[0271] 2) Effects on antioxidant efficacy (for GPx and SOD)
[0272] 3) Effects on antioxidant transcriptional activity
(NRF2)
[0273] 4) Effects on cell proliferation and viability (cell
counts)
[0274] 5) Effects on stressed cells (Cachexin, radiation,
starvation)
[0275] No toxic response was observed for any healthy cell culture
in normal random phases (HMVEC-L or JB6) upon exposure to high
concentration compositions of the invention (up to 20%) of serum.
Two methods were used to determine toxic response, the
translocation and accumulation of NF-kB and P-Jun in the nuclei.
Both of these methods are known to be sensitive to low-levels of
toxicity, as verified by the positive control. A complete lack of
toxic indication and/or inflammatory cytokines was observed.
[0276] An 800% increase in GPx antioxidant efficacy in HMVEC-L
cells was seen after 24 hours exposure from low-concentration
composition of the invention (no concentration dependence seen). A
transitory increase of up to 500% was seen in SOD antioxidant
efficacy between 30 to 90 min. again after exposure to a
low-concentration of a composition of the invention (<1%). In
both cases, the low concentrations of compositions of the invention
were comparable to blood concentrations possible from oral dosing,
though data is not available to confirm this. Concentration
dependence at very low concentrations might be seen if such was
carefully investigated.
[0277] Exposure to high-concentration compositions of the
invention, in comparison, elicited only a small relative increase
in GPx antioxidant efficacy that was not concentration dependent.
An increase in SOD efficacy was not seen for either
high-concentration compositions of the invention or after long (24
hr) exposures. In subsequent investigations, this information will
be used to determine optimal concentrations and time points to
study concentration dependence (<0.1% and 0-120 minutes).
[0278] Studies examining the nuclear translocation of redox
responsive transcription factors suggest that compositions of the
invention at a lower concentration (less than 1%) induces a 20-30%
increase in the nuclear translocation of the NRF2 transcription
factor in HMVEC-L cells that appears to be transient (30-60 min).
We also observed that a composition of the invention induced a
parallel decrease in the phosphorylation of an extra-nuclear
protein whose phosphorylation status is clearly increased in
response to hydrogen peroxide treatment, consistent with an
antioxidant mode of action.
[0279] Serum-starving HMVEC-L cells, as an approach to increase
sensitivity, significantly increased the nuclear NRF2 signal
induced by composition of the invention (1%). However,
serum-starvation induced significant cell death complicating
interpretation of the data.
[0280] Cellular proliferation for both HMVEC-L and JB6 cell types
(determined from cell counts) was inhibited by high concentrations
(5-20% v/v) of exposure to compositions of the invention. The
HMVEC-L inhibition was clearly concentration dependent, with a 20%
loss of cell count at 20% ASEA concentration. In contrast to
decreased proliferation, serum LDH levels significantly decreased
with compositions of the invention concentration between 5-20%,
indicating increased cell membrane integrity. The results seem to
indicate that cellular proliferation is decreased while cell
membrane viability is increased at high concentrations. The
mechanism behind such behavior cannot be deduced from the data, yet
further evidence will be seen in the next section.
[0281] The response of HMVEC-L cells when stressed with Cachexin
depends upon cell phase. Normal randomly cycling HMVEC-L cells (pS)
exhibited typical behavior when stressed with Cachexin: exhibiting
decrease in cell viability accompanied by cell death. Confluent
end-of-life-cycle (A2) and borderline HMVEC-L cells, as expected,
were less sensitive to Cachexin insult, exhibiting less pronounced
decreases in cell viability and less cell death.
[0282] Exposure to compositions of the invention caused no
significant change in the response of the normal random cycling pS
cells to Cachexin (showing similar loss of cell viability and
cell-death). However, A2 cell cultures exposed to a composition of
the invention exhibited increased sensitivity to Cachexin,
restoring behavior similar to that of normal cells. This behavior
was reinforced as concentration dependence was examined. Borderline
A2 cells, exhibiting a relatively small Cachexin response, and A2
cells that are normally insensitive to Cachexin insult, exhibited a
much stronger response to Cachexin when exposed to compositions of
the invention, both in decrease in viability and increased cell
death.
[0283] It appears that exposure to compositions of the invention
causes increased rates of A2 cell death, enhancing the natural
reception of Cachexin in such end-of-life-cycle cells. Yet exposure
to composition of the invention is not expected to cause any change
in normal cell viability.
[0284] Cachexin is normally secreted to instigate cell death in
damaged or dysfunctional tissues, allowing surrounding healthy
cells to divide and fill in voids. Thus, increasing the sensitivity
to Cachexin in dysfunctional cells may help accelerate such a
process and is not always deleterious.
[0285] Acceleration of cell death was also seen in tissues that
were stressed with radiation and serum-starvation associated with
exposure to compositions of the invention.
[0286] The infusion of a certain balanced mixture of redox
signaling molecules using compositions of the invention into viable
HMVEC-L and JB6 cell cultures has been seen to elicit distinct
bioactivity. No indications of toxicity or the expression of
inflammatory cytokines were observed and yet there was increased
antioxidant and protective enzyme expression (as evidenced by
increased nuclear NRF2) and greatly increased efficacy for the two
master antioxidants, GPx and SOD. This behavior suggests that
infusion with compositions of the invention might tend to induce
and enhance oxidative defense mechanisms without inducing toxic or
inflammatory responses in such cells. Such action is unprecedented
or extremely rare. Normally, low-level toxicity induces slight
oxidative stress and inflammatory response which in turn induces
oxidative defense and cell repair mechanisms. It would be of
interest to determine concentration dependency of this effect with
ultra-low-concentration infusions of compositions of the
invention.
[0287] The induction of cell death in cultures of dysfunctional,
stressed or damaged cells by infusion of compositions of the
invention should also be explored. Natural healing processes
involve a repair or replace mechanism by which marginally damaged
cells are repaired, when possible, or undergo apoptosis, programmed
death, if they cannot be repaired and then are replaced through
mitosis of healthy neighboring cells. It is fairly evident that
infusion of composition of the invention, of itself, is not causing
direct stress to exposed cells, however, it might tend to increase
the efficiency of certain cytokine "death domain" messengers
(Cachexin) that are designed to induce cell death in dysfunctional
or damaged cells. The nuclear translocation of NRF2 can be
considered part of the phase II oxidative defense response which
includes expression of antioxidants, DNA repair molecules and other
known repair mechanisms.
[0288] Apoptosis is part of the replace mechanism when cells have
undergone unrepairable damage and must be removed and replaced.
Both antioxidant defense and apoptotic mechanisms are central to
normal tissue repair and regeneration. Redox signaling is involved
in several of the pathways, such as p53 gene expression, that can
determine whether a cell undergoes apoptosis or not. Chronic
oxidative stress tends to favor cell death. Certainly the presence
of Cachexin and other death domain messengers favor cell death. The
observation that infusion with compositions of the invention
enhances Cachexin reception might indicate that infusion with
compositions of the invention also might serve to enhance reception
of messengers in the signaling process that determines whether
defense, repair or replace mechanisms are activated.
Example 9
Treatment of Type 2 Diabetes
[0289] A 42 year old man diagnosed with type 2 diabetes is treated
by intravenously injecting a combination product comprising a PPAR
inhibitor (an eicosanoid) and a redox signaling agent (RXN)
composition of the invention. After about 1 week, the patient
maintains a more regular glucose profile with minimal glucose
excursions as compared to prior to treatment.
Example 10
Treatment of Type 1 Diabetes
[0290] A 22 year old woman diagnosed with type 1 diabetes is
treated by intravenously injecting a combination product comprising
a PPAR inhibitor (a thazolidinedione) and a redox signaling agent
(RXN) composition of the invention. After about 1 week, the patient
maintains a more regular glucose profile with minimal glucose
excursions as compared to prior to treatment.
Example 11
Treatment of Cancer
[0291] A 31 year old woman diagnosed with cancer is treated by
intravenously injecting a combination product comprising a PPAR
inhibitor (a fibrate) and a redox signaling agent (RXN) composition
of the invention. After about 1 month, the patient shows a
regression in her symptoms.
Example 12
Treatment of Type 2 Diabetes
[0292] A 72 year old man diagnosed with type 2 diabetes is treated
by orally administering a combination product comprising a PPAR
inhibitor (an eicosanoid) and a redox signaling agent (RXN)
composition of the invention. After about 1 week, the patient
maintains a more regular glucose profile with minimal glucose
excursions as compared to prior to treatment.
Example 13
Treatment of Type 1 Diabetes
[0293] A 61 year old woman diagnosed with type 1 diabetes is
treated by orally administering a combination therapy comprising a
PPAR inhibitor (a thazolidinedione) and a redox signaling agent
(RXN) composition of the invention. After about 1 week, the patient
maintains a more regular glucose profile with minimal glucose
excursions as compared to prior to treatment.
Example 14
Treatment of Cancer
[0294] A 31 year old woman diagnosed with oral cancer is treated by
orally administering a combination product comprising a PPAR
inhibitor (a fibrate) and a redox signaling agent (RXN) composition
of the invention. After about 1 month, the patient shows a
regression in her symptoms.
[0295] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the invention are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contains certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements.
[0296] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0297] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0298] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0299] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
* * * * *