U.S. patent application number 14/775907 was filed with the patent office on 2016-02-04 for liquids rich in noble gas and methods of their preparation and use.
The applicant listed for this patent is THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Yong-Jian GENG, Shao-Ling HUANG, Hyunggun KIM, Melvin KLEGERMAN, David MCPHERSON, Tao PENG, Xing YIN.
Application Number | 20160030470 14/775907 |
Document ID | / |
Family ID | 51538472 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160030470 |
Kind Code |
A1 |
HUANG; Shao-Ling ; et
al. |
February 4, 2016 |
LIQUIDS RICH IN NOBLE GAS AND METHODS OF THEIR PREPARATION AND
USE
Abstract
Provided herein is a novel composition for oral administration
and delivery of Noble gas, such as xenon or argon. Methods of
treating and preventing neuronal or cardiovascular damage with such
compositions are also provided. The present invention relates
generally to the fields of molecular biology, medicine and
nutraceuticals. More particularly, it concerns methods for oral
delivery of inert gas compositions, such as Xenon or Argon, for the
treatment and prevention of disease.
Inventors: |
HUANG; Shao-Ling; (Houston,
TX) ; MCPHERSON; David; (Houston, TX) ; GENG;
Yong-Jian; (Houston, TX) ; YIN; Xing;
(Houston, TX) ; KIM; Hyunggun; (Houston, TX)
; KLEGERMAN; Melvin; (Houston, TX) ; PENG;
Tao; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Family ID: |
51538472 |
Appl. No.: |
14/775907 |
Filed: |
March 17, 2014 |
PCT Filed: |
March 17, 2014 |
PCT NO: |
PCT/US14/30210 |
371 Date: |
September 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61788808 |
Mar 15, 2013 |
|
|
|
61889901 |
Oct 11, 2013 |
|
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Current U.S.
Class: |
424/439 ;
206/438; 424/600 |
Current CPC
Class: |
A23L 33/16 20160801;
A61J 1/1468 20150501; A61K 47/02 20130101; A23V 2002/00 20130101;
A61P 25/28 20180101; A61K 47/24 20130101; A61K 47/42 20130101; A61K
9/0095 20130101; A61K 9/107 20130101; A61P 9/04 20180101; A23D
7/0053 20130101; A61K 33/00 20130101; A61P 9/00 20180101; A23L
33/25 20160801; A61K 47/44 20130101; A61P 7/02 20180101; A61K 47/14
20130101; A23L 33/115 20160801; A61K 47/28 20130101; A61P 9/10
20180101; A61P 9/12 20180101; A61P 3/02 20180101; A61K 47/6951
20170801; A61K 33/00 20130101; A61K 2300/00 20130101; A23V 2002/00
20130101; A23V 2250/00 20130101; A23V 2250/10 20130101; A23V
2250/18 20130101 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61K 47/44 20060101 A61K047/44; A61K 47/28 20060101
A61K047/28; A61K 47/24 20060101 A61K047/24; A61J 1/14 20060101
A61J001/14; A61K 9/107 20060101 A61K009/107; A61K 9/00 20060101
A61K009/00; A23L 1/304 20060101 A23L001/304; A23L 1/30 20060101
A23L001/30; A23L 1/308 20060101 A23L001/308; A61K 47/48 20060101
A61K047/48; A61K 47/42 20060101 A61K047/42 |
Goverment Interests
[0002] The invention was made with government support under Grant
Nos. NS067454, HL074002, and HL059586 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A nutraceutical composition comprising: (i) a substantially
aqueous component comprising a dissolved Noble gas, a portion of
the Noble gas being encapsulated with a water-soluble polymer;
and/or (ii) an edible oil component comprising a dissolved Noble
gas.
2. The composition of claim 1, wherein the Noble gas is Helium,
Neon, Argon, Krypton or Xenon.
3. The composition of claim 1, wherein the Noble gas is Xenon.
4. The composition of claim 1, wherein the composition comprises a
mixture of two or more Noble gases.
5. The composition of claim 1, wherein the composition comprises an
emulsion.
6. The composition of claim 1, wherein water-soluble polymer is
cyclodextrin.
7. The composition of claim 6, wherein the cyclodextrin is gamma-
or beta-cyclodextrin.
8. The composition of claim 6, wherein the cyclodextrin is
beta-cyclodextrin.
9. The composition of claim 6, wherein the composition comprises
about 0.1 to about 1.0 mg/ml of a cyclodextrin molecule.
10. The composition of claim 1, wherein the oil comprises
polyunsaturated fatty acids (PUFA).
11. The composition of claim 1, wherein the oil comprises at least
1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% PUFAs.
12. The composition of claim 1, wherein the oil component is
saturated with xenon or argon gas.
13. The composition of claim 1, wherein the edible oil component
comprises soluble xenon gas.
14. The composition of claim 1, wherein the edible oil is
essentially free of oxygen.
15. The composition of claim 1, wherein the oil comprises canola
oil, flaxseed oil, rapeseed oil, soybean oil, walnut oil, fish oil,
safflower oil, chia seed oil, sunflower seed oil, sesame seed oil,
seaweed oil, corn oil, cotton seed oil, peanut oil, palm oil,
avocado oil, coconut oil, or olive oil.
16. The composition of claim 1, wherein the oil comprises omega-3
fatty acids.
17. The composition of claim 1, further defined as an emulsion
comprising: (a) 25% to 50% by volume oil, said oil being saturated
with a Noble gas; and (b) 30% to 75% by volume aqueous
solution.
18. The composition of claim 1, further defined as an emulsion
comprising: (a) 15% to 75% by volume oil, said oil being saturated
with a Noble gas; and (b) 25% to 85% by volume aqueous
solution.
19. The composition of claim 1, wherein the aqueous solution
comprises water, fruit juice, vegetable juice or an alcohol.
20. The composition of claim 1, further comprising phospholipid,
detergent, or protein components.
21. The composition of claim 20, wherein the detergent is a plant
surfactant, a synthetic detergent or a bile acid.
22. The composition of claim 20, wherein the detergent is
lithocholic acid, deoxycholic acid, taurocholic acid, glycocholic
acid, chenodeoxycholic acid, or cholic acid.
23. The composition of claim 20, wherein the phospholipid is egg
phosphocholine (egg PC), soybean PC, DPPC or DOPC.
24. The composition of claim 20, wherein the protein is milk
protein, whey protein, soy protein isolate, or bovine serum
albumin.
25. The composition of claim 1, further comprising between about 1
and 50 g of xenon per ml of oil.
26. The composition of claim 1, comprising: (a) 15% to 75% by
volume olive oil, said oil being saturated with xenon or argon gas;
(b) 25% to 85% by volume aqueous solution; and one or more of a
phospholipid, a detergent or a protein.
27. The composition of claim 1, comprising: (a) 25% to 50% by
volume olive oil, said oil being saturated with xenon or argon gas;
(b) 50% to 75% by volume aqueous solution; (c) 10-30 mg/ml
phosphocholine; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml lithocholic
acid.
28. The composition of claim 1, comprising: (a) 15% to 75% by
volume olive oil, said oil being saturated with xenon or argon gas;
(b) 25% to 85% by volume aqueous solution; (c) 10-30 mg/ml
phosphocholine; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml lithocholic
acid.
29. The composition of claim 1, further defined as a herbal,
vitamin or energy-providing nutraceutical beverage.
30. The composition of claim 1, further comprising a preservative,
flavoring agent, dye, vitamin, anti-oxidant, or plant extract.
31. The composition of any one of claims 1-30, said composition
comprised in a gas impermeable container.
32. The composition of claim 31, wherein the container comprises
about 1 ml to 2 liters of the composition.
33. The composition of claim 32, wherein the container comprises
about 1 mg to 20 g, 1 mg to 10 g, 1 mg to 1 g, 1 to 100 mg, 1 to 50
mg, 1 to 25 mg or 1 to 10 mg of Xe.
34. The composition of claim 31, wherein the container comprises a
one-way value to release the composition for oral consumption
without exposing the entire content to the atmosphere.
35. The composition of claim 32, wherein the container is
pressurized.
36. The composition of claim 35, wherein the container is
pressurized with a Noble gas.
37. The composition of claim 35, further comprising CO.sub.2.
38. A single serving nutraceutical comprising a composition of any
one of claims 1-30, in a gas impermeable container.
39. The single serving beverage of claim 38, wherein the container
is a bottle.
40. The single serving beverage of claim 39, wherein the container
has 1 ml to 1.0 1 ml of the composition.
41. The single serving beverage of claim 38, wherein the Noble gas
comprises Xe.
42. The single serving beverage of claim 41, having about 1 mg to
20 g, 1 mg to 10 g, 1 mg to 1 g, 1 to 100 mg, 1 to 50 mg, 1 to 25
mg or 1 to 10 mg of Xe.
43. A single serving nutraceutical composition comprising about 25
to 500 ml of a substantially aqueous component and about 0.1 mg to
20 g of dissolved Noble gas, wherein a portion of the Noble gas in
encapsulated to enhance aqueous solubility.
44. A method of improving the health or well-being of a subject
comprising providing a liquid or semi-liquid composition formulated
for oral consumption, comprising a dissolved Noble gas.
45. The method of claim 44, wherein the liquid composition is
saturated or supersaturated with a Noble gas.
46. The method of claim 45, wherein the Noble gas comprises Xe.
47. The method of claim 44, wherein the liquid composition is a
composition in accordance with any one of claims 1-42.
48. A method of providing neurological or cardiovascular protection
is a subject comprising orally administering an effective amount of
a composition comprising dissolved Noble gas.
49. The method of claim 48, wherein the subject has or is risk for
atherosclerosis, Alzheimer's disease, thrombotic stroke, hemorrhage
stroke, heart failure or cardiac hypertrophy.
50. The method of claim 48, wherein the subject is a human.
51. The method of claim 50, wherein the Noble gas comprises Xe.
52. The method of claim 51, comprising administering about 0.1 mg
to 20 g/day of Xe to the subject.
53. The method of claim 52, comprising administering about 500 mg
to 10 g, 500 mg to 5 g, 500 mg to 2 g, 1 to 100 mg, 1 to 50 mg, 1
to 25 mg or 1 to 10 mg per day of Xe to the subject.
54. The method of claim 48, wherein the composition is administered
weekly, daily, twice a day, three times a day, every six hours,
every three hours or hourly.
55. The method of claim 54, wherein the composition is administered
over the period of a week, two weeks, a month or a year.
56. The method of claim 48, further defined as a method for
treating or preventing a neurological disease or neurological
injury.
57. The method claim 56, wherein the neurological disease is
Alzheimer's disease.
58. The method of claim 57, further defined as a method reducing
beta-amyloid levels in the subject.
59. The method of claim 57, further defined as a method for
treating or preventing the progression of Alzheimer's disease is a
subject.
60. The method of claim 57, wherein the subject is at risk for
developing Alzheimer's disease.
61. The method of claim 57, wherein the subject has or is diagnosed
with a genetic predisposition for Alzheimer's disease.
62. The method of claim 56, wherein the neurological injury is
thrombotic or ischemic stroke.
63. A method for reducing a marker of inflammation in a subject
comprising orally administering an effective amount of a
composition dissolved Noble gas.
64. The method of any one of claims 48-63, wherein the composition
comprises Noble gas that has been encapsulated to enhance aqueous
solubility.
65. The method of any one of claims 48-63, wherein the composition
comprises: (i) a substantially aqueous component comprising a
dissolved Noble gas, a portion of the Noble gas being encapsulated
with a water-soluble polymer; and/or (ii) an oil component
comprising a dissolved Noble gas.
66. The method of claim 65, wherein the Noble gas is Helium, Neon,
Argon, Krypton or Xenon.
67. The method of claim 65, wherein the Noble gas is Xenon.
68. The method of claim 65, wherein the composition comprises a
mixture of two or more Noble gases.
69. The method of claim 65, wherein the composition comprises an
emulsion.
70. The method of claim 65, wherein water-soluble polymer is
cyclodextrin.
71. The method of claim 70, wherein the cyclodextrin is gamma- or
beta-cyclodextrin.
72. The method of claim 70, wherein the cyclodextrin is
beta-cyclodextrin.
73. The method of claim 70, wherein the composition comprises about
0.1 to about 1.0 mg/ml of a cyclodextrin.
74. The method of claim 65, wherein the oil comprises
polyunsaturated fatty acids (PUFA).
75. The method of claim 65, wherein the oil comprises at least 1%,
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% PUFAs.
76. The method of claim 65, wherein the oil component is saturated
with xenon or argon gas.
77. The method of claim 65, wherein the edible oil component
comprises soluble xenon gas.
78. The method of claim 65, wherein the edible oil is essentially
free of oxygen.
79. The method of claim 65, wherein the oil comprises canola oil,
flaxseed oil, rapeseed oil, soybean oil, walnut oil, fish oil,
safflower oil, chia seed oil, sunflower seed oil, sesame seed oil,
seaweed oil, corn oil, cotton seed oil, peanut oil, palm oil,
avocado oil, coconut oil, or olive oil.
80. The method of claim 65, wherein the oil comprises omega-3 fatty
acids, omega-6 fatty acids or omega-9 fatty acids.
81. The method of claim 65, further defined as an emulsion
comprising: (a) 25% to 50% by volume oil, said oil being saturated
with a Noble gas; and (b) 30% to 75% by volume aqueous
solution.
82. The method of claim 65, further defined as an emulsion
comprising: (a) 15% to 75% by volume oil, said oil being saturated
with a Noble gas; and (b) 25% to 85% by volume aqueous
solution.
83. The method of claim 65, wherein the aqueous solution comprises
spring water, fruit juice or vegetable juice.
84. The method of claim 65, further comprising phospholipid,
detergent, or protein components.
85. The method of claim 84, wherein the detergent is a plant
surfactant, a synthetic detergent or a bile acid.
86. The method of claim 84, wherein the detergent is lithocholic
acid, deoxycholic acid, taurocholic acid, glycocholic acid,
chenodeoxycholic acid, or cholic acid.
87. The method of claim 84, wherein the phospholipid is egg
phosphocholine (egg PC), soybean PC, DPPC or DOPC.
88. The method of claim 84, wherein the protein is milk protein,
whey protein, soy protein isolate, or bovine serum albumin.
89. The method of claim 65, further comprising between about 1 and
50 mg of xenon per ml of oil.
90. The method of claim 65, wherein the composition comprises: (a)
15% to 75% by volume olive oil, said oil being saturated with xenon
or argon gas; (b) 25% to 85% by volume aqueous solution; and one or
more of a phospholipid, a detergent or a protein.
91. The method of claim 65, wherein the composition comprises: (a)
25% to 50% by volume olive oil, said oil being saturated with xenon
or argon gas; (b) 50% to 75% by volume aqueous solution; (c) 10-30
mg/ml phosphocholine; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml
lithocholic acid.
92. The method of claim 65, wherein the composition comprises: (a)
15% to 75% by volume olive oil, said oil being saturated with xenon
or argon gas; (b) 25% to 85% by volume aqueous solution; (c) 10-30
mg/ml phosphocholine; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml
lithocholic acid.
93. A method of making an aqueous composition comprising a Noble
gas comprising: (a) incubating a Noble gas with a water soluble
encapsulating polymer; and (b) exposing the encapsulated Noble gas
to an aqueous to produce an aqueous composition comprising the
Noble gas. at a pressure of between about 2 atm and 10 atm, at a
temperature of between about 4.degree. C. and -180.degree. C. for
at least 4 hours to produce an encapsulated Noble gas solution at a
pressure of between about 2 atm and 10 atm, at a temperature of
between about 20.degree. C. and 1.degree. C. for at least 4
hours
94. The method of claim 93, wherein the incubating of step (a) is
performed at a pressure of between about 2 atm and 8 atm, 2 atm and
5 atm, 2 atm and 4 atm or at a pressure of about 3 atm.
95. The method of claim 93, wherein the incubating of step (a) is
performed at a temperature of between about 0.degree. C. and
-150.degree. C., -20.degree. C. and -150.degree. C., -20.degree. C.
and -100.degree. C., -40.degree. C. and -100.degree. C. or at a
temperature of about -80.degree. C.
96. The method of claim 93, wherein the incubating of step (a) is
for a period of at least 8 hours, 12 hours, 24 hours, 48 hours or
for between 8 hours and three days.
97. The method of claim 93, wherein the exposing of step (b) is
performed at a pressure of between about 2 atm and 8 atm, 2 atm and
5 atm, 2 atm and 4 atm or at a pressure of about 3 atm.
98. The method of claim 93, wherein the exposing of step (b) is
performed at a temperature of between about 15.degree. C. and
1.degree. C., 10.degree. C. and 1.degree. C., 8.degree. C. and
2.degree. C., 6.degree. C. and 2.degree. C. or at a temperature of
about 4.degree. C.
99. The method of claim 93, wherein the exposing of step (b) is for
a period of at least 8 hours, 12 hours, 24 hours, 48 hours or for
between 8 hours and three days.
100. The method of claim 93, wherein the aqueous solution comprises
a dissolved Noble gas.
101. The method of claim 99, wherein the aqueous solution is
saturated with a Noble gas.
102. The method of claim 100, wherein the aqueous solution is
saturated with a Noble gas by a method comprising: (i) obtaining a
degassed aqueous solution; and (ii) exposing the degassed aqueous
solution to a Noble gas at a pressure of between about 2 atm and 10
atm, at a temperature of between about 20.degree. C. and 1.degree.
C. for at least 4 hours to produce an aqueous solution saturated
with the Noble gas.
103. The method of claim 93, wherein the Noble gas is argon or
xenon.
104. The method of claim 103, wherein the Noble gas is xenon.
105. The method of claim 93, wherein the Noble gas comprises a
mixture of two or more Noble gases.
106. The method of claim 93, wherein water-soluble encapsulating
polymer is cyclodextrin.
107. The method of claim 106, wherein the cyclodextrin is gamma- or
beta-cyclodextrin.
108. The method of claim 106, wherein the cyclodextrin is
beta-cyclodextrin.
109. The method of claim 108, wherein the beta-cyclodextrin is
hydroxypropyl-beta-cyclodextrin.
110. The method of claim 93, further defined as a method for making
a pharmaceutical or nutraceutical composition.
111. The method of claim 93, wherein the aqueous solution comprises
a preservative, flavoring agent, dye, vitamin, anti-oxidant, or
plant extract.
112. The method of claim 93, further comprising the step of (c)
packaging the aqueous composition comprising the Noble gas in a gas
impermeable container.
113. The method of claim 112, wherein the container is
pressurized.
114. The method of claim 113, wherein the container is pressurized
with a Noble gas.
115. The method of claim 112, wherein the container is a pill, a
capsule, a foild or polymer packet or a bottle.
116. An aqueous composition comprising a Noble gas produced by a
method according to any one of claims 93-115.
117. A method of making a composition for oral administration of a
Noble gas comprising: (a) solubilizing a Noble gas in an edible oil
by mixing the oil and gas at a pressure of between about 2 atm and
6 atm, at a temperature of between about 0.degree. C. and
25.degree. C. to produce an edible oil comprising soluble Noble
gas.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Nos. 61/788,808, filed Mar. 15, 2013, and
61/889,901, filed Oct. 11, 2013, both of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
molecular biology, medicine and nutraceuticals. More particularly,
it concerns methods for oral delivery of inert gas compositions,
such as Xenon or Argon, for the treatment and prevention of
disease.
[0005] 2. Description of Related Art
[0006] Both Xenon (Xe) and Argon (Ar) are, pleiotypic
cytoprotective gases, which have unique advantages that include
rapid diffusion across biological barriers such as the blood-brain
barrier (BBB) and complete passage across cell membranes due to its
low blood-gas partition coefficient. In animal models, Xe given as
a continuous gas inhalation has demonstrated potent neuroprotective
and myocardical protective effects. Xe protects against oxygen and
glucose deprivation (OGD) and protects against hypoxia/ischemia by
alteration of molecules involved in neuronal ischemic tolerance. Xe
helps to induce transcription of several pro-survival genes
including brain-derived neurotrophic factor (BDNF) and pro-survival
proteins such as Bcl.sub.2 which promote cell tolerance to ischemic
injury. Xe interacts with the human immune system by modulating
inflammatory cytokines such as TNF-.alpha. and IL-6 in monocytes.
Xe helps to sustain release of hypoxia inducible factor 1 alpha
(HIF-1.alpha.) and other proteins. All these pathways are
implicated in organ protection.
[0007] Current methods for gas delivery involve inhalation and
administration of a gas donor. However, Xe or Ar inhalation cannot
be practically given in many situations as the required high Xe or
Ar concentration for inhalation limits the fraction of inspired
oxygen necessary for cell survival. In addition, there is the
difficulty of developing a continuous inhalation strategy for
patients as daily use. Improved methods of delivery of Xe or Ar are
greatly needed.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention relates generally to
treatment methods, pharmaceuticals and nutraceuticals (a
portmanteau of the words "nutrition" and "pharmaceutical", which is
a food or food product with health and medical benefits, including
the prevention and treatment of disease). More particularly, it
concerns methods for primarily oral delivery of inert gas
compositions to the gastrointestinal tract (GI), comprising a Noble
gas such as Helium, Neon, Argon, Krypton and/or Xenon. Such gases
can be delivered as part of a method for the prevention and/or
treatment of diseases in the heart and brain, including but not
limited to atherosclerosis-associated ischemic heart disease,
stroke and other neuron degenerative condition, such as dementia
(e.g., Alzheimer disease). In certain aspects, compositions
comprising Noble gases provide reduction in one or more marker of
inflammation or general improvement in well-being.
[0009] In a first embodiment there is provided a liquid (or
semi-liquid, e.g., pastes or gels) formulation having an enhanced
concentration of encapsulated Noble gas (e.g., the gas may be
encapsulated to achieve a specified concentration). The Noble gas
can be selected from Helium, Neon, Argon, Krypton, Xenon or a
mixture thereof. As used herein the encapsulated Noble gas can be
provided as a gas dissolved in a lipid and emulsified, encapsulated
in a liposome formulation and/or encapsulated in a water soluble
molecule (e.g., cyclodextrin). In general the encapsulation allows
the composition to achieve a higher gas content than can be
achieved in an equal amount of water (absent such encapsulation)
and the same temperature and pressure. In preferred aspects, the
composition is formulated for oral delivery
[0010] In a certain embodiments there is provided a nutraceutical
(such as a beverage) composition comprising a substantially aqueous
component and a dissolved Noble gas, wherein a portion of the Noble
gas in encapsulated to enhance aqueous solubility. In some aspects,
the encapsulated Noble gas may be encapsulated in a lipid, such as
in a liposome or in a lipid phase that is emulsified in the
composition. In still further aspects, the Noble gas (e.g., Xe Ar,
Kr, Ne or He) is encapsulated in a water-soluble molecule, such as
a water soluble polymer. In still further aspects, the Noble gas is
encapsulated in .alpha.-, .beta.-, or .gamma.-cyclodextrin or a
mixture thereof. Further molecules that may be used for Noble gas
encapsulation are detailed below.
[0011] In yet a further embodiment the invention provides single
serving nutraceutical composition including but not limited to
beverages, gels, pastes, tablets, and capsules. For example, the
composition may comprise about 1, 2, 3, 4, 5 or 10 to about 15, 20,
25, 30, 35, 40, 45 or 50 ml of the composition. In further aspects,
the composition may comprise about 10, 15, 25, 30, 35, 40, 45 or 50
to about 100, 150, 200, 250, 300, 350, 400, 450 or 500 ml of a
composition (e.g., a substantially aqueous composition). In some
aspects, a composition of the embodiments comprises 0.01 to 15 g or
about 0.1 to 200 mg of dissolved Noble gas (or a mixture of two or
more such gases), wherein a portion of the Noble gas is
encapsulated to enhance aqueous solubility. As detailed above the
Noble gas may be encapsulated or solubilized in a (emulsified)
lipid component, in a liposome or in a water-soluble molecule. For
example, in some aspects, the Noble gas (e.g., Xe) is encapsulated
in a water-soluble polymer, such as cyclodextrin. In certain
aspects, the amount of dissolved Noble gas (e.g., Xe) in the
formulation is about 0.1 mg to 10 g, 0.1 to 1,000 mg, 0.1 to 500
mg, 0.5 to 100 mg, 1 to 100 mg, 1 to 50 mg, 1 to 25 mg or 1 to 10
mg. Likewise, the volume of liquid component in the formulation can
be adjusted to provide an optimal amount of the Noble gas in a
single serving. For example, the single serving beverage may
comprise a total volume of between about 1 to 10 ml, 10 to 25 ml,
25 to 50 ml, 50 to 500 ml, 50 to 300 ml, 100 to 300 ml or 200 to
400 ml. As detailed further herein, in certain preferred
embodiments, such a single serving nutraceutical is provided in a
gas-impermeable sealed container, such as a bottle, can or foil or
polymer package. Likewise, in some aspects, a package of
single-serving nutraceutical beverages is provided comprising 4, 6,
8, 12, 24 or more single serving compositions such as beverages,
each comprised in a separate sealed container.
[0012] As detailed supra, certain aspects of the embodiments
concern compositions comprising Noble gases (e.g., Xe), at least a
portion of which is encapsulated in cyclodextrin (CD) (e.g.,
.beta.-cyclodextrin). In some cases, cyclodextrin for use according
to the embodiments will include hydrophilic groups that further
enhance the aqueous solubility. For example, in certain aspects,
the CD is a hydroxypropyl-CD, such as
hydroxypropyl-beta-cyclodextrin. A composition may comprise, for
instance, about 0.01 to about 5.0 mg/ml; about 0.05 to about 2.0
mg/ml; about 0.1 to about 1.5 mg/ml or about 0.1 to about 1.0 mg/ml
of a CD. In some specific aspects, the composition comprises 0.1 to
about 1.0 mg/ml of hydroxypropyl .beta.-CD. In still further
aspects, a composition comprises about 0.1 to about 5.0 ml; about
0.1 to about 4.0; about 0.1 to about 3.8; about 0.1 to about 2.0;
about 0.1 to about 1.0 ml; or 0.5 to about 1.0 ml of Noble gas
(e.g., Xe) per 0.5 mg of cyclodextrin in the composition (e.g., at
standard temperature-pressure). In particular, a composition may
comprise about 0.1 to about 5.0 ml; about 0.1 to about 4.0; about
0.1 to about 3.8; about 0.1 to about 2.0; about 0.1 to about 1.0
ml; or 0.5 to about 1.0 ml of Xe per 0.5 mg of .beta.-cyclodextrin
(e.g., hydroxypropyl .beta.-CD). In a very specific aspect, a
composition comprises up to about 3.8 ml of Xe to 0.5 mg of a CD
molecule (e.g., per ml of water).
[0013] Additional cyclodextrin molecules that can be used according
to the embodiments (in either oil or water compositions depending
on the desired solubility include, without limitation,
methyl-.beta.-cyclodextrin, randomly
methylated-.beta.-cyclodextrin, dimethyl-.beta.-cyclodextrin,
randomly dimethylated-.beta.-cyclodextrin,
trimethyl-.beta.-cyclodextrin, acetylated
dimethyl-.beta.-cyclodextrin, 2-Hydroxyethyl-.beta.-cyclodextrin,
2-Hydroxypropyl-.beta.-cyclodextrin,
3-Hydroxypropyl-.beta.-cyclodextrin,
hydroxybutenyl-.beta.-cyclodextrin,
2,3-Dihydroxypropyl-.beta.-cyclodextrin,
2-Hydroxypropyl-.gamma.-cyclodextrin, glucosyl-.beta.-cyclodextrin,
maltosyl-.beta.-cyclodextrin,
glucuronyl-glucosyl-.beta.-cyclodextrin, alkylated
.beta.-cyclodextrin, 2,6-Di-O-ethyl-.beta.-cyclodextrin,
2,3,6-Tri-O-ethyl-.beta.-cyclodextrin, acylated
.beta.-Cyclodextrin, 2,3,6-Tri-O-acyl(C2-C18)-.beta.-cyclodextrin,
2,3,6-Tri-O-butanoyl-.beta.-cyclodextrin,
2,3,6-Tri-O-valeryl-.beta.-cyclodextrin,
2,3,6-Tri-O-octyl-.beta.-cyclodextrin,
O-Carboxymethyl-O-ethyl-.beta.-cyclodextrin, .beta.-Cyclodextrin
sulfate, sulfobutyl ether group-.beta.-cyclodextrin, or sulfobutyl
ether group-.beta.-cyclodextrin.
[0014] The methodologies demonstrated herein can provide very high
concentrations of Noble gas in a liquid format. However, in some
cases, liquids or semi-liquids (including e.g., a beverage) of the
embodiments may comprise a relatively moderate amount of a Noble
gas such as Xe. For example, such a beverage may comprise a
dissolved Xe concentration (when in a sealed container) of about
1.0 to about 5,000 .mu.g/ml, about 10 to about 1,000 .mu.g/ml,
about 100 to about 800 .mu.g/ml, about 1.0 to about 100 .mu.g/ml,
5.0 to about 50 .mu.g/ml, about 10 to about 100 .mu.g/ml or about
10 to about 50 .mu.g/ml. For example, Xe dissolved in water at
standard temperature and pressure can have a concentration of about
600 .mu.g/ml. In a further aspect, a beverage is provided having a
high concentration of Xe, such as about 1 mg/ml to about 100 mg/ml,
1 mg/ml to about 50 mg/ml, about 1 mg/ml to about 30 mg/ml, or
about 10 mg/ml to about 50 mg/ml. In some aspects, a beverage is
provided comprising a Xe level of about 10 mg/ml to about 15, 20,
25, 30, 35, 40, 45 or 50 mg/ml Xe (e.g., such as a beverage
comprising pressurized and/or encapsulated Xe). For example, a
formulation comprising cyclodextrin (hydroxypropyl .beta.-CD)
encapsulated Xe can have a Xe concentration of about 22.4 mg/ml
when formulated at 3 atm and 4.degree. C., then brought to standard
ambient temperature and pressure (SATP)).
[0015] In some aspects, the methodologies and compositions herein
concern Argon gas provided in a liquid or semi-liquid format (e.g.,
as a beverage). For example, such a composition may comprise a
dissolved Ar concentration (when in a sealed container) of about
1.0 to about 1,000 .mu.g/ml, about 10 to about 500 .mu.g/ml, about
20 to about 500 .mu.g/ml, about 30 to about 250 .mu.g/ml, about 40
to about 200 .mu.g/ml, about 50 to about 100 .mu.g/ml or about 40
to about 75 .mu.g/ml. For example, Ar dissolved in water at
standard temperature and pressure can have a concentration of about
55 .mu.g/ml. In a further aspect, a beverage is provided having a
high concentration of Ar (such as Ar encapsulated in a polymer or
an oil), such as about 0.01 .mu.g/ml to about 1 mg/ml, 0.1 .mu.g/ml
to about 1 mg/ml, about 1 .mu.g/ml to about 500 .mu.g/ml, about 10
.mu.g/ml to about 500 .mu.g/ml, about 100 .mu.g/ml to about 500
.mu.g/ml, or about 200 .mu.g/ml to about 500 .mu.g/ml. For example,
a formulation comprising an oil encapsulated Ar at SATP may have an
Ar concentration of about 165 .mu.g/ml.
[0016] In still yet a further aspect, a composition of the
embodiments is characterized in having a Noble gas content (at
standard ambient temperature and pressure; SATP) of at least about
0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3
mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM or 2.0 mM. In
some aspects, the Noble gas is in a concentration of at least 4.5,
4.6., 4.7, 4.8, 4.9 or 5.0 mM. Thus, in some aspects, the
composition comprises a Noble gas content of between about 5.0 and
50 mM, 5.0 and 25 mM, 5.0 and 20.0 mM, 5.0 and 15 mM or 5.0 and 10
mM at SATP. In still further aspects, the composition comprises a
Xenon content of greater than about 5.0 mM, such as between about
5.0 and 50 mM, 5.0 and 25 mM, 5.0 and 20.0 mM, 5.0 and 15 mM or 5.0
and 10 mM at SATP. In still further aspects, a composition
comprises Ar at a concentration of greater than 0.5 mM, 0.6 mM, 0.7
mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM,
1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM or 2.0 mM. For example, the
composition may comprise between about 1.0 and 10 mM, 1.5 and 10
mM, 1.5 and 10 mM, 2.0 and 10 mM or 2.0 and 5 mM Ar at SATP.
[0017] In still further aspects, a composition of the embodiments
is defined by the Noble gas content as compared to the aqueous
component (e.g., water) content. For example, a composition may
comprise a Noble gas to aqueous component ratio of between about
1:20 and 4:1; about 1:10 and 4:1; about 1:9 and 4:1; about 1:2 and
4:1; 1:1 and 4:1; about 1.5:1 and 4:1; about 1:20 and 1:1; about
1:10 and 1:1; or about 2:1 and 3:1 (volume:volume). In some
aspects, the Noble gas to aqueous component ratio is greater than
1:10; 1:9; or 1:5. In certain aspects, the Noble gas to aqueous
component ratio is greater than 1:1 or greater than 2:1. Thus, in
some specific aspects, a composition comprises a Xe to water ratio
of between about 1:2 and 4:1; 1:1 and 4:1 or 1.5:1 and 4:1 or 2:1
and 3:1 (volume:volume), such as a ratio greater than about
2:1.
[0018] In certain embodiments compositions such as a beverage
composition of the embodiments further comprise additional
components such as preservatives, flavoring agents, dyes, vitamins,
anti-oxidants, plant or microbial extracts, salts (electrolytes,
glycerol, sodium, potassium and chloride), alcohols, lipids, oils,
or a mixture thereof. Thus, in some aspects, a beverage or other
form of the embodiments is further defined as an herbal, vitamin or
energy-providing nutraceutical composition.
[0019] In a further embodiment the invention provides, a
pharmaceutical or nutraceutical composition comprising lipids such
as an edible oil component comprising a soluble gas, such as Noble
gas (e.g., xenon or argon gas). Examples, of oils for such a
composition include, without limitation, flaxseed oil, rapeseed
oil, soybean oil, walnut oil, fish oil, safflower oil, sunflower
oil, corn oil, cotton seed oil, peanut oil, palm oil or olive oil.
In one aspect, the oil may comprise polyunsaturated fatty acids
(PUFA), such as an oil comprising at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or more PUFAs. In still further aspects,
the oil comprises omega-3 fatty acids, which may aid in biological
uptake of the gases of the embodiments. In still further aspects,
the oil component is saturated or super saturated with xenon or
argon gas.
[0020] In a further embodiment, an oil-gas composition may be
further comprised in an emulsion. For example, an emulsion can
comprise (a) 25% to 50%, 60%, 70%, 80%, 90%, 95% or more by volume
oil, (the oil comprising soluble xenon or argon gas) and (b) 1%,
2%, 3%, 5%, 10%, 20%, or 30% to about 75% or 85% by volume aqueous
solution. In some aspects, the aqueous solution comprises water
(e.g., spring water), fruit juice, vegetable juice or other
nutritious beverage. In some aspects, the composition may further
comprise phospholipid, detergent, or protein components. In some
aspects, the composition further comprises phospholipids,
detergents, flavorings, dyes, emulsifiers, co-emulsifiers and/or
protein components. For example, the detergent can be a plant
surfactant, a synthetic detergent or a bile acid. In certain
aspects, the detergent is lithocholic acid, deoxycholic acid,
taurocholic acid, glycocholic acid, chenodeoxycholic acid, or
cholic acid. Examples of phospholipids for use according to the
embodiments include, without limitation, egg phosphocholine (egg
PC), soybean PC, DPPC or DOPC. Proteins that may be included are,
for example, milk protein, whey isolate protein, soy protein
isolate, potassium caseinate, egg albumin, (Brown) rice protein,
hydrolyzed beef protein isolate, pea protein isolate, hemp protein,
or bovine serum albumin.
[0021] As detailed above, in certain aspects, oil compositions are
provided that comprise both soluble and insoluble, trapped, or free
gases and that may be in a liquid or semi-liquid form. Gases that
may be included in such compositions include without limitation
Noble gases (e.g., He, Ar, Kr, Ne or Xe), CO.sub.2, nitrous oxide,
isoflurane and servoflurane. In preferred embodiments, a lipid oil
composition comprises soluble xenon or argon gas (and, in some
cases, also comprise insoluble or free Xe or Ar or other Noble
gas). In some aspects, a composition comprises low oxygen or
nitrogen content, or is essentially free of these gases. In some
aspects, the oil composition is semi-saturated or saturated with a
gas. In still further aspects, a lipid oil can be supersaturated
with a gas (e.g., such that the gas is bubbling out of the oil when
exposed to the atmosphere). In some aspects, a lipid oil
composition of the embodiments may comprise between about 10 and
500 mg of xenon per ml of oil (e.g., between about 10, 20, 30, 40,
50, 60, 70, 80, 90 or 100 mg and about 150, 200, 250, 300, 350,
400, 450 or 500 mg per ml of oil). For example, at SATP an oil may
comprise about 0.1 to about 50; about 0.1 to about 20 or about 1 to
about 15 mg of Xe per ml of oil.
[0022] In certain specific aspects, a composition of the
embodiments may comprise (a) 25% to 50% by volume oil (e.g., olive
oil or other desired lipid), (the oil comprising soluble xenon or
argon gas); (b) 50% to 75% by volume water solution; (c) 10-30
mg/ml of a phospholipid (e.g., egg phosphocholine); (d) 10-50 mg/ml
of a protein (e.g., BSA); and (e) 1-5 mg/ml of a detergent (e.g.,
lithocholic acid). In some aspects, the composition may further
comprise a preservative, flavoring agent, vitamin, anti-oxidant, or
plant extract.
[0023] In some aspects, compositions comprising Noble gases may be
comprised in a gas impermeable container of any size or shape. In
some cases, the container may be pressurized (e.g., pressurized
with a Noble gas, such as xenon or argon gas). In some aspects,
such a container may comprise a single serving or unit dosage of a
composition (e.g., in a paste, a gel, a pill, a tablet or capsule).
In other cases, a container can comprise multiple doses (e.g., a
multiple dose bottle or a compartmentalized container). In this
later case to may be preferable that the container be pressurized
and comprise a one-way value to release the composition without
exposing the entire content to the atmosphere. In still further
aspects, such a container may comprise excess gas (e.g., argon or
xenon) that maintain the pressure in the vessel when doses of the
composition are released. Such a system is described, for example,
in U.S. Pat. Publn. No. 20030177784, which is incorporated herein
by reference. In some cases, the container comprises foil or
similar impermeable material such as a polymer, such that the
container may be pressured by effectively dispense liquids or
semi-liquids readily.
[0024] In a further embodiment, the present disclosure provides a
unit dosage of a composition of the embodiments comprised in a gas
impermeable container. In certain aspects, the container may be a
paste, gel, pill, tablet or a capsule. In another aspect, the
container may be a bottle. For example, a container can enclose 1-5
ml; 5-25 ml; 25-100 ml; 125-300 ml; 355-500 ml; 500 ml to 1 liter
or more of the composition, such as an emulsion of the
embodiments.
[0025] In some further embodiments there is provided a method of
improving the health or well-being of a subject comprising
administering to the subject (or providing the subject with) a
composition in accordance the embodiments. For example, the subject
can be provided with an amount to of a Noble gas composition of the
embodiments that is sufficient to reduce the level of at least one
marker of inflammation or cardiovascular risk (e.g., blood
pressure). For example, in some cases a Noble gas composition is
administered in as an oral liquid or semi-liquid formulation to
provide a daily dose equivalent to between about 0.1 to 200 mg/day
of Xe. In another example, a daily Xe dose may be between about 1
to 100 mg, 1 to 50 mg, 1 to 25 mg or 1 to 10 mg per day of Xe. In
another example, a Noble gas composition is administered as an oral
formulation to provide a daily dose equivalent to between about 0.1
to 5 g/day of Xe. For instance, the daily dosage of Xe can be
between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0
and 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 grams per day of Xe.
In a further embodiment, a method comprises administering an oral
Xe composition in a dose of between about 0.1 and 10; 0.1 and 5;
0.5 and 5; 1.0 and 3.0; or 2.0 and 3.0 mg (of Xe)/Kg/day. In still
further aspects, a method of the embodiment comprises administering
(or providing to a subject) a dose of Noble gas (e.g., Xe) to
achieve an initial maximal blood concentration of between 10 .mu.M
and 500 .mu.M, between 10 .mu.M and 100 .mu.M, between 10 .mu.M and
50 .mu.M or an initial maximal blood concentration of at least 50
or 100 .mu.M. For example, in some aspects, such a dose of
composition of the embodiments is administered daily, every two
days, or weekly.
[0026] In a further embodiment, the present disclosure provides a
method of providing neurological or cardiovascular protection is a
subject comprising orally administering an effective amount of a
composition in accordance with any one of the embodiments. In
certain aspects, the subject (e.g., a human subject) has or is at
risk for Alzheimer's disease, thrombotic stroke, ischemic stroke or
cardiac hypertrophy. In some aspects, the method further comprises
administering about 25-300 ml/day (70-1,350 mg/day, e.g., 100-1,200
mg/day, 200-1000 mg/day, 500-1000 mg/day or 800-1,200 mg/day) to
the subject. The composition may be administered weekly, daily,
twice a day, three times a day, every six hours, every three hours
or hourly. Likewise, a composition may be administered over the
period of a week, two weeks, a month or a year. In some aspects,
the method is a method for treating or preventing a neurological
disease or neurological injury, such as Alzheimer's disease or
thrombotic or ischemic stroke. In further aspects, methods are
provided for treating or preventing cardiac hypertrophy, or
providing protection from myocardial ischemia.
[0027] In still a further embodiment, the present disclosure
provides a method of reducing beta-amyloid levels in a subject
comprising administering an effective amount of xenon or argon to
the subject. In certain aspects, the xenon or argon is administered
orally in a composition in accordance with any one of the
embodiments. In still other aspects, the xenon or argon or other
Noble gasare administered via inhalation or injection (e.g.,
comprised liposomes).
[0028] In a related embodiment, the present disclosure provides a
method of treating or preventing the progression of Alzheimer's
disease in a subject comprising administering an effective amount
of xenon or argon or other Noble gas to the subject. In certain
aspects, the xenon or argon is administered orally. In other
aspects, the xenon or argon or other Noble gas (e.g., He, Ne or Kr)
are administered via inhalation or injection In some aspects, the
subject is a subject who is at risk for developing Alzheimer's
disease, such as a subject who has or is diagnosed with a genetic
predisposition for Alzheimer's disease.
[0029] In yet a further embodiment, the present disclosure provides
a method of making a composition for oral administration of xenon
or argon comprising (a) solubilizing xenon or argon gas in an
edible oil by mixing the oil and gas to produce an edible oil
comprising soluble xenon or argon gas. In some aspects, the method
further comprises (b) emulsifying the edible oil comprising the
soluble gas in an aqueous solution (e.g., a solution comprising a
detergent or other emulsifier) to produce an emulsion comprising
soluble xenon or argon gas. In some aspects, oil and gas are mixed
at a pressure of between about 1 atm and 6 atm, 2 atm and 6 atm or
2 atm and 4 atm, at a temperature of between about 0.degree. C. and
37.degree. C. or 0.degree. C. and 25.degree. C. In certain aspects,
solubilizing xenon or argon gas in an edible oil comprises
saturating or supersaturating the oil with xenon or argon gas. In
some aspects, the method further comprises bottling or capturing
the oil or emulsion in a gas impermeable container (e.g., a bottle,
capsule or pill). For example, the container may be pressurized,
such as container pressurized at 2-6 atm. In certain aspects, steps
(a)-(b) of the method may be performed under a xenon or argon or
other Noble gas atmosphere (e.g., He, Ne or Kr).
[0030] In yet a further embodiment there is provided a method of
making a substantially aqueous composition comprising a Noble gas
comprising: (a) incubating a Noble gas (or a mixture of Noble
gases) with a water soluble encapsulating molecule (e.g., a water
soluble polymer); and (b) exposing the encapsulated Noble gas to an
aqueous solution to produce an aqueous composition comprising the
encapsulated Noble gas. In some aspects, step is (a) is performed
at a pressure of between about 2 atm and 10 atm (e.g., a pressure
between about 2 atm and 8 atm, 2 atm and 5 atm, 2 atm and 4 atm or
at a pressure of about 3 atm), at a temperature of between, e.g.,
about 25.degree. C. or 4.degree. C. and -180.degree. C. (e.g., for
a period of at least 1, 2, 3, 4 or more hours) to produce an
encapsulated Noble gas. In certain aspects the incubating of step
(a) is performed at a temperature of between about 0.degree. C. and
-150.degree. C., -20.degree. C. and -150.degree. C., -20.degree. C.
and -100.degree. C., -40.degree. C. and -100.degree. C. or at a
temperature of about -80.degree. C. and for a period of at least 8
hours, 12 hours, 24 hours, 48 hours or for between 8 hours and
three days. In some cases, step (b) comprises exposing the
encapsulated Noble gas to an aqueous solution at a pressure of
between about 2 atm and 10 atm (e.g., a pressure between about 2
atm and 8 atm, 2 atm and 5 atm, 2 atm and 4 atm or at a pressure of
about 3 atm), e.g., at a temperature of between about 20.degree. C.
and 1.degree. C. For example, step (b) can occur over a period of
at least 1, 2, 3, 4 or more hours. In still further aspects, the
exposing of step (b) is performed at a pressure of between about 2
atm and 8 atm, 2 atm and 5 atm, 2 atm and 4 atm or at a pressure of
about 3 atm. In some aspects, the exposing of step (b) is performed
at a temperature of between about 15.degree. C. and 1.degree. C.,
10.degree. C. and 1.degree. C., 8.degree. C. and 2.degree. C.,
6.degree. C. and 2.degree. C. or at a temperature of about
4.degree. C. In still further aspects, the exposing of step (b) is
for a period of at least 8 hours, 12 hours, 24 hours, 48 hours or
for between 8 hours and three days. In still further aspects, the
exposing of step (b) comprises exposing the encapsulated Noble gas
to an aqueous solution that comprises of is a saturated with a
Noble gas or a mixture of Noble gases. For example, the solution
can be saturated with a Noble gas by a method comprising: (i)
obtaining a degassed aqueous solution; and (ii) exposing the
degassed aqueous solution to a Noble gas at a pressure of between
about 2 atm and 10 atm, at a temperature of between about
20.degree. C. and 1.degree. C. for at least 4 hours to produce an
aqueous solution saturated with the Noble gas.
[0031] In yet a further embodiment, the present disclosure provides
a composition for use in providing neurological or cardiovascular
protection in a subject, the composition comprising edible oil
saturated with a Noble gas, such as xenon or argon gas.
[0032] In a particular aspect of the embodiments there is provided
a method of delivery of a Noble in a substantially aqueous
composition comprising supplying a beer, cider, soda or other
carbonated beverage from a tap operably coupled to a Noble gas
canister (e.g., a Xe gas canister), such that the Noble gas is used
to maintain tap pressure and it thereby dissolved into the beverage
(e.g., beer) being dispensed from the tap.
[0033] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0034] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0035] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0036] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0038] FIG. 1. Echocardiographic Measurements to Assess Cardiac
Hypotrophy and Function of C57BL/6J wild type (WT) and
apolipoprotein-E (ApoE) knockout (KO) mice in Response to Xe-rich
Solution Pre-treatment. FIG. 1A. Intra-ventricular septum in
diastole (IVS:d) (mm) FIG. 1B. Left ventricular posterior wall
diameter in diastole (LVPW:d) (mm) FIG. 1C. Left ventricular (LV)
volume in diastole (LV Vol;d) (.mu.L). *p<0.05, ** p<0.01,
KO/KO6w compared to WT/WT6w, respectively; # p<0.05, KO
control/vehicle compared to KO 6w; .sctn.p<0.05,
.sctn..sctn.p<0.01, .sctn..sctn..sctn.p<0.001, KO xenon
compared to KO vehicle. WT=Wild type mice; KO=Apo E knockout
mice.
[0039] FIG. 2. Echocardiographic Measurements on % Ejection
fraction (% EF) (FIG. 2A). % Fractional shortening (% FS) (FIG.
2B), and Cardiac output (ml/min)(FIG. 2C). *p<0.05, **
p<0.01, KO/KO6w compared to WT/WT6w, respectively; # p<0.05,
KO control/vehicle compared to KO 6w; .sctn.p<0.05,
.sctn..sctn.p<0.01, .sctn..sctn..sctn.p<0.001, KO xenon
compared to KO vehicle.
[0040] FIG. 3. Alterations in Cardiac Mass Morphology in Wild Type
(WT) and Apo-E Knockout (KO) Mice. FIG. 3A. LV mass corrected (mg).
FIG. 3B. Ratio of heart weight (HW) to body weight (BW) (mg/g).
WT-6w (n=5): WT mice fed with regular diet for 6.sup.th week. KO-6w
(n=4): the KO mice fed with regular diet for 6.sup.th week. KO-6w
control (n=5): the KO mice fed with high fat diet and administered
by PBS gavage for 6.sup.th week. KO-6w vehicle (n=7): the KO fed
with high fat diet and vehicles for 6.sup.th week. KO-6w Xenon
(n=6): the KO fed with high fat diet and administered with
Xenon-rich-solution for 6.sup.th week. *p<0.05, KO/KO6w compared
to WT/WT6w, respectively; .sctn.p<0.05, KO6w xenon compared to
KO6w vehicle.
[0041] FIG. 4. Myocardiographic Alterations in Cardiac Function in
Wild Type and Apo-E Knockout Mice in response to Xe-rich Solution
Pre-treatment. WT (n=9): wild type mice fed with regular diet at
baseline; KO (n=25): Apo E-KO mice fed with regular diet at
baseline. WT-6w (n=5): WT mice fed with regular diet for 6.sup.th
week. KO-6w (n=5): the KO mice fed with regular diet for 6.sup.th
week. KO-6w control (n=4): the KO mice fed with high fat diet and
administered by PBS gavage for 6.sup.th week. KO-6w vehicle (n=7):
the KO fed with high fat diet and vehicles for 6.sup.th week. KO-6w
Xenon (n=6): the KO fed with high fat diet and administered with
Xenon-rich-solution for 6.sup.th week.
[0042] FIG. 5. Levels of Brain-derived Neurotrophic Factor (BDNF)
in the Heart (FIG. 5A) and Brain (FIG. 5B) in Response to
Xe-Rich-Solution Per-treatment. WT-6w (n=4): WT mice fed with
regular diet for 6.sup.th week. KO-6w (n=5): the KO mice fed with
regular diet for 6.sup.th week. KO-6w vehicle (n=7): the KO fed
with high fat diet and administered by the solution gavage at
6.sup.th week. KO-6w Xenon (n=6): the KO fed with high fat diet and
administered by Xenon gavage at 6.sup.th week. *p<0.05, **
p<0.01, *** p<0.001, KO6w/vehicle/Xenon compared to WT6w,
respectively; # p<0.05, KO6w vehicle compared to KO 6w;
.sctn.p<0.05, KO6w Xenon compared to the vehicle.
[0043] FIG. 6. Levels of Beta-Amyloid in the Blood (FIG. 6A) and
Brain (FIG. 6B) in Response to Xe-Rich-Solution Per-treatment.
WT-6w (n=5): WT mice fed with regular diet for 6.sup.th week. KO-6w
(n=4): the KO mice fed with regular diet for 6.sup.th week. KO-6w
control (n=5): the KO mice fed with high fat diet and administered
by PBS gavage for 6th week. KO-6w vehicle (n=7): the KO fed with
high fat diet and vehicles for 6.sup.th week. KO-6w Xenon (n=6):
the KO fed with high fat diet and administered by
Xenon-rich-solution for 6.sup.th week. *p<0.05, KO6w vehicle
compared to WT6w; .sctn..sctn.p<0.01, KO6w Xenon compared to the
vehicle.
[0044] FIG. 7. Xenon-rich-Solution to Increase Brain Tolerance to
Ischemic Injury. FIG. 7A. Infarct size. FIG. 7B. Percent infarct
volume. FIG. 7C. Limb placement. FIG. 7D. Grid walking.
[0045] FIG. 8. Example of the exemplary mouse experimental
protocol.
[0046] FIG. 9. Example of the rat experimental protocol.
[0047] FIG. 10. Initial Xenon caging experiments. (A) A schematic
showing the structure of CD used for caging of Xe. (B) A schematic
showing the physical properties of .alpha.-, .beta.- and .gamma.-CD
as compared to a Xe atom. (C) Graph shows the results of studies to
determine the effects of pressure on Xe encapsulation in CD. (D)
Graph shows the effects of temperature on the on Xe encapsulation
in CD.
[0048] FIG. 11. Upper panel shows an exemplary protocol for
production of Xe enhanced water using CD caging. Lower panel is a
graph showing the volume of dissolved Xe per 5 ml water that was
achieved using the indicated methods.
[0049] FIG. 12. Echocardiographic Measurements of Mice Treated with
Xe Water. Graphs show the results of echocardiographic measurement
of (1) WT mice fed with regular diet for 6th week (WT-6w, n=5); (2)
Apo E knockout mice fed with high fat diet and normal water control
(KO6w control, n=13); (3) vehicle control mice feed with water
containing cyclodextrin only (KO6w vehicle, n=5); or (4) Apo E
knockout mice fed with Xe-rich-water (KO6w Xenon, n=5) after 6
weeks of treatment. Graphs show the results of measurements of
intra-ventricular septum (IVS) volume, percent left ventricular
(LV) ejection fraction (EF), left ventricular posterior wall
thickness (LVPW), percent LV fractional shortening (FS), LV volume
and cardiac output (CO).
[0050] FIG. 13. Effects of Xe on ischemic stress. Graph in the left
panels show the results of studies to measure CKMB Creatine Kinase
(CKMB) levels in the control mice versus Xe-treated animals. Graph
in the right panel shows the results of studies to measure troponin
expression levels in the control mice versus Xe-treated animals
(Mean.+-.SE, n=5, .sup..sctn.p<0.01).
[0051] FIG. 14. Xe-rich-water decreases expression of
.beta.-amyloid in brain and blood. Graphs show the amount of
.beta.-amyloid that was found plasma (left panel) or brain (right
panel) after 6 weeks of treatment. WT6w (n=10) indicates mice fed
with regular diet at 6th week; KO-6w (n=5) indicates ApoE knock
mice fed with regular diet at 6th week; KO-6w vehicle (n=7)
indicates ApoE knock mice fed with high fat diet and water
containing cyclodextrin at 6th week; KO-6w Xenon (n=6) indicates
ApoE knock mice fed with high fat diet and Xe-rich-Water at 6th
week. *p<0.05, KO6w vehicle compared to WT6w;
.sctn..sctn.p<0.01, KO6w Xenon compared to the vehicle.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0052] Nobel gases such as Xenon (Xe) and Argon (Ar) are attractive
since they may improve health and well-being at low dosages and are
also potential therapeutics if given at higher dosages. However,
there are a wide range of difficulties in attempting to administer
such gases to humans. In particular, the amount of gas that can be
administered via inhalation is a very serious limitation. Likewise,
because these gases are chemically neutral and non-polar
formulation into other delivery vehicles has proved to a very
difficult challenge.
[0053] Disclosed herein are solutions rich in a Noble gas, such as
Xe or Ar for oral delivery to humans and in some cases other
animals of interest. In some aspects, these solutions use lipids
including but not limited to oils such as edible oils (e.g.,
omega-3 rich oils) from known foods as a carrier media to provide
increased solubility of such Noble gases. Alternatively or
additionally aqueous solutions can incorporate a Noble gas
encapsulated in polymer (e.g., cyclodextrin includes:
.alpha.-cyclodextrin: 6-membered sugar ring molecule,
.beta.-cyclodextrin: 7-membered sugar ring molecule, and
.gamma.-cyclodextrin: 8-membered sugar ring molecule and various
derivatives). Derivatives of cyclodextrins include but are not
limited to Methyl-.beta.-cyclodextrin, randomly
methylated-.beta.-cyclodextrin, dimethyl-.beta.-cyclodextrin,
randomly dimethylated-.beta.-cyclodextrin,
Trimethyl-.alpha.-cyclodextrin; Acetylated
dimethyl-.beta.-cyclodextrin: 2-Hydroxyethyl-.beta.-cyclodextrin,
2-Hydroxypropyl-.beta.-cyclodextrin,
3-Hydroxypropyl-.beta.-cyclodextrin;
Hydroxybutenyl-.beta.-cyclodextrin:
2,3-Dihydroxypropyl-.beta.-cyclodextrin,
2-Hydroxypropyl-.gamma.-cyclodextrin; glucosyl-.beta.-cyclodextrin;
maltosyl-.beta.-cyclodextrin;
glucuronyl-glucosyl-.beta.-cyclodextrin; 2, hydrophobic CD that can
be combined with lipid/oil: alkylated .beta.-cyclodextrin,
2,6-Di-O-ethyl-.beta.-cyclodextrin,
2,3,6-Tri-O-ethyl-.beta.-cyclodextrin; acylated
.beta.-Cyclodextrin:
2,3,6-Tri-O-acyl(C.sub.2-C.sub.18)-.beta.-cyclodextrin,
2,3,6-Tri-O-butanoyl-3-cyclodextrin,
2,3,6-Tri-O-valeryl-.beta.-cyclodextrin,
2,3,6-Tri-O-octyl-.beta.-cyclodextrin,
O-Carboxymethyl-O-ethyl-.beta.-cyclodextrin, .beta.-Cyclodextrin
sulfate; sulfobutyl ether group-.beta.-cyclodextrin; and sulfobutyl
ether group-.beta.-cyclodextrin. Studies herein demonstrate that
both types of solutions are able to provide a significant level of
Noble gas in an aqueous-based system. Upon oral delivery these
solutions have preventive effects in the brain and heart tissues.
For example, Xe solutions are shown to increase tolerance of
tissues to ischemic damage and provide cardio-protective effects.
In model systems for heart disease the compositions are able not
only to have a direct positive effect marker of cardiac function
(see, e.g., FIG. 12) but are also able to lower overall blood
pressure in mice deficient for Apo-E (results shown in Table 2).
Moreover, these solutions also demonstrate biologically significant
(therapeutic) effects in model systems for Alzheimer's disease. In
particular, Xe based compositions are shown effective in reducing
.beta.-amyloid load in both the blood and brain tissues of treated
animals (FIG. 14). Thus, compositions are provided comprising
effective amounts of dissolved or trapped Ar or Xe that can be used
to provide cardiovascular and neuroprotective effects to a
subject.
[0054] The Noble gas compositions and therapeutic methods disclosed
herein offer new avenues for the increasing wellbeing as well as
for the treatment and prevention of a wide range of chronic
diseases. Importantly, the compositions provided herein have been
shown to provide potent cardio-protective and blood pressure lowing
effects at specific dosages that could be useful in treatment of
patients with heart disease or who have a high risk of stroke.
Likewise, the provided Noble gas compositions are shown able to
reduce amyloid load in body tissues and therefore offer a unique
therapy to treat and prevent the onset of Alzheimer's disease.
Given the convenient aqueous formulations that have now been
achieved, effective amounts of non-toxic Noble gases can now be
easily delivered via oral formulations. Given the stability of the
formulations a variety of doses could be easily distributed without
complex packaging, dosing systems or even refrigeration that
improve wellbeing by increasing or improving certain physiological
parameters (e.g. reducing inflammation, reducing stress, increased,
relaxation, reducing blood pressure, clearing the mind) at certain
dosages to therapeutic/preventives at other dosages (e.g. improved
cardiac and neurological function). Accordingly, a range of
compositions intended for primarily oral delivery including but not
limited to beverages could be used for gas delivery to provide an
effective and convenient nutraceutical or therapeutic that is
easily incorporated into standard preventative therapies such as
diet modification and exercise. Moreover, because of the ease of
delivery and the lack of toxicity formulations, provided here could
likely be administered with little or no supervision from medical
professionals.
I. PHARMACEUTICAL AND NUTRACEUTICAL FORMULATIONS
[0055] Pharmaceutical and nutraceutical compositions provided
herein comprise an effective amount of a tissue or cell protective
gas, such as Xe or Ar, and, optionally additional agents such as
further gases, dissolved or dispersed in an acceptable carrier can
be included. In some aspects, such an acceptable carrier includes
components formulated to increase or control the content of soluble
gas to desired levels, such as lipids including edible oils or
caging molecule as detailed above. The Phrase "containing" means
the dissolving, emulsifying, suspending, trapping and other like
means of obtaining a solution with Nobel gas for primarily oral
delivery. The phrase "acceptable carrier" refer to molecular
entities and compositions that do not produce an adverse, allergic
or other untoward reaction when administered to (e.g., ingested by)
an animal, such as, for example, a human, as appropriate. The
preparation of a pharmaceutical or nutraceutical composition that
contains a Noble gas is detailed herein. Further addition of active
or inactive ingredients to such a composition will be known to
those of skill in the art in light of the present disclosure, and
as exemplified by Remington's Pharmaceutical Sciences, 18th Ed.
Mack Printing Company, 1990, incorporated herein by reference.
Moreover, for animal (e.g., human) administration, it will be
understood that preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0056] An "acceptable carrier" may include any and all solvents,
dispersion media, coatings, surfactants, antioxidants,
preservatives (e.g., antibacterial agents, antifungal agents),
isotonic agents, absorption delaying agents, salts, preservatives,
drugs, drug stabilizers, gels, binders, excipients, disintegration
agents, lubricants, sweetening agents, flavoring agents, dyes, such
like materials and combinations thereof, as would be known to one
of ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.
1289-1329, incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated. In general the carriers of the present embodiments
all comprise an oil-based component that comprises a dissolved
Noble gas, such as Ar or Xe.
[0057] In certain embodiments, the pharmaceutical composition may
comprise different types of carriers depending on whether it is to
be administered in solid, liquid or aerosol form, and whether it
need to be sterile for such routes of administration as injection.
In certain embodiments, pharmaceutical compositions provided herein
can be administered intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally,
intratracheally, intranasally, intravitreally, intravaginally,
intrarectally, topically, intratumorally, intramuscularly,
intraperitoneally, subcutaneously, subconjunctival,
intravesicularlly, mucosally, intrapericardially, intraumbilically,
intraocularally, orally, topically, locally, inhalation (e.g.
aerosol inhalation), injection, infusion, continuous infusion,
localized perfusion bathing target cells directly, via a catheter,
via a lavage, in cremes, in lipid compositions (e.g., liposomes),
or by other method or any combination of the forgoing as would be
known to one of ordinary skill in the art (see, for example,
Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing
Company, 1990, incorporated herein by reference).
[0058] In other embodiments, one may use eye drops, nasal solutions
or sprays, aerosols or inhalants in the present embodiments. Such
compositions are generally designed to be compatible with the
target tissue type. In a non-limiting example, nasal solutions are
usually aqueous solutions designed to be administered to the nasal
passages in drops or sprays. Nasal solutions are prepared so that
they are similar in many respects to nasal secretions, so that
normal ciliary action is maintained. Thus, in preferred embodiments
the aqueous nasal solutions usually are isotonic or slightly
buffered to maintain a pH of about 5.5 to about 6.5. In addition,
antimicrobial preservatives, similar to those used in ophthalmic
preparations, drugs, or appropriate drug stabilizers, if required,
may be included in the formulation. For example, various commercial
nasal preparations are known and include drugs such as antibiotics
or antihistamines.
[0059] Additional formulations which are suitable for other modes
of administration include suppositories. Suppositories are solid
dosage forms of various weights and shapes, usually medicated, for
insertion into the rectum, vagina or urethra. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0060] Sterile injectable solutions can also be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains
the basic dispersion medium and/or the other ingredients. In the
case of sterile powders for the preparation of sterile injectable
solutions, suspensions or emulsion, the preferred methods of
preparation are vacuum-drying or freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered liquid medium
thereof. The liquid medium should be suitably buffered if necessary
and the liquid diluent first rendered isotonic prior to injection
with sufficient saline or glucose. The preparation of highly
concentrated compositions for direct injection is also
contemplated, where the use of DMSO as solvent is envisioned to
result in extremely rapid penetration, delivering high
concentrations of the active agents to a small area. In particular
embodiments, prolonged absorption of an injectable composition can
be brought about by the use in the compositions of agents delaying
absorption, such as, for example, aluminum monostearate, gelatin or
combinations thereof.
[0061] The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. Where
clinical application of liposomes (e.g., liposomes comprising
gases) is undertaken, solutions of therapeutic compositions can be
prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms. The therapeutic compositions of the present
invention are advantageously administered in the form of injectable
compositions either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid prior to
injection may also be prepared. These preparations also may be
emulsified. A typical composition for such purpose comprises a
pharmaceutically acceptable carrier. For instance, the composition
may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human
serum albumin per milliliter of phosphate buffered saline. Other
pharmaceutically acceptable carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, buffers and
the like.
[0062] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well-known parameters. Additional formulations are suitable for
oral administration. Oral formulations include such typical
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate and the like. The compositions generally will
take the form of solutions or suspensions.
[0063] The therapeutic compositions of the present embodiments may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. In this case, intravenous injection or infusion may be
preferred. Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other
excipients.
Oral Formulations
[0064] In certain preferred embodiments, a composition of the
embodiments is administered orally and is formulated to facilitate
such oral administration (e.g., as a beverage formulation). Thus,
in some embodiments a composition (such as an emulsion of
oil-encapsulated gas or polymer encapsulated gas) may comprise, for
example, solutions, suspensions, emulsions, tablets, pills,
capsules (e.g., hard or soft shelled gelatin capsules), sustained
release formulations, buccal compositions, troches, elixirs,
suspensions, syrups, or combinations thereof. Oral compositions may
be incorporated directly with a food or drink product (e.g., along
with a fruit juice or alcohol). Preferred carriers for oral
administration comprise inert diluents, assimilable edible carriers
or combinations thereof. In other aspects, the oral composition may
be prepared as a syrup or elixir. A syrup or elixir, and may
comprise, for example, at least one active agent, a sweetening
agent, a preservative, a flavoring agent, a dye, a preservative, or
combinations thereof.
[0065] In further aspects, a composition comprising dissolved Noble
gas, such as Xe or Ar, can be formulated into a capsule or tablet
for oral administration. In some aspects, the capsule is
substantially impermeable to gas, and preferably the capsule is
formulated to dissolve in the gastrointestinal tract of a
subject.
[0066] In certain preferred embodiments an oral composition may
comprise one or more binders, excipients, disintegration agents,
lubricants, flavoring agents, and combinations thereof. In certain
embodiments, a composition may comprise one or more of the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc.; or combinations thereof the foregoing. When
the dosage unit form is a capsule, it may contain, in addition to
materials of the above type, carriers such as a liquid carrier.
Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both.
[0067] A composition may comprise various antioxidants to retard
oxidation of one or more component. Additionally, the prevention of
the action of microorganisms can be brought about by preservatives
such as various antibacterial and antifungal agents, including but
not limited to parabens (e.g., methylparabens, propylparabens),
chlorobutanol, phenol, sorbic acid, thimerosal or combinations
thereof.
[0068] In embodiments where the composition is in a liquid form, a
carrier can comprise a solvent or dispersion medium comprising but
not limited to, water, ethanol, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, etc.), lipids (e.g.,
triglycerides, vegetable oils, liposomes) and combinations thereof.
The proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin; by the maintenance of the required
particle size by dispersion in carriers such as, for example liquid
polyol or lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof such methods. In
many cases, it will be preferable to include isotonic agents, such
as, for example, sugars, sodium chloride or combinations
thereof.
Additional Components for Pharmaceutical and Nutraceutical
Formulations
[0069] Oral Noble gas formulations of the embodiments may comprise
additional components as detailed herein below. It is contemplated
that such additional components may be included, for example, as at
least or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 113,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89 or 90% of the total composition on a
weight:weight or volume:volume basis. In certain aspects, an
additional component comprises less than about 20%, 10%, 5% or less
of the total composition on a weight:weight or volume:volume
basis.
[0070] In some embodiments micronutrients can included, such as
(without limitation) L-carnitine, choline, coenzyme Q10,
alpha-lipoic acid, omega-3-fatty acids, pepsin, phytase, trypsin,
lipases, proteases, cellulases, and a combination comprising at
least one of the foregoing micronutrients.
[0071] Antioxidants can include materials that scavenge free
radicals. In some embodiments, exemplary antioxidants can include
citric acid, rosemary oil, vitamin A, vitamin E, vitamin E
phosphate, tocopherols, di-alpha-tocopheryl phosphate,
tocotrienols, alpha lipoic acid, dihydrolipoic acid, xanthophylls,
beta cryptoxanthin, lycopene, lutein, zeaxanthin, astaxanthin,
beta-carotene, carotenes, mixed carotenoids, polyphenols,
flavonoids, and a combination comprising at least one of the
foregoing antioxidants.
[0072] Exemplary nutrients can also include amino acids such as
L-tryptophan, L-lysine, L-leucine, L-methionine,
2-aminoethanesulfonic acid (taurine), and L-carnitine; creatine;
glucuronolactone; inositol; and a combination comprising at least
one of the foregoing nutrients.
[0073] Phytochemicals ("phytonutrients") are plant derived
compounds which may provide a beneficial effect on the health or
well-being of the consumer. Phytochemicals include plant derived
antioxidants, phenolic compounds including monophenols and
polyphenols, and the like. Exemplary phytochemicals include lutein,
lycopene, carotene, anthocyanin, capsaicinoids, flavonoids,
hydroxycinnamic acids, isoflavones, isothiocyanates, monoterpenes,
chalcones, coumestans, dihydroflavonols, flavanoids, flavanols,
quercetin, flavanones, flavones, flavan-3-ols (catechins,
epicatechin, epigallocatechin, epigallocatechingallate, and the
like), flavonals (anthocyanins, cyanidine, and the like); phenolic
acids; phytosterols, saponins, terpenes (carotenoids), and a
combination comprising at least one of the foregoing
phytochemicals.
[0074] The phytochemicals can be provided in substantially pure or
isolated form or in the form of natural plant extracts. Suitable
plant extracts which contain one or more phytochemicals include
fruit skin extracts (grape, apple, crab apple, and the like), green
tea extracts, white tea extracts, green coffee extract, and a
combination comprising at least one of the foregoing extracts.
[0075] Various herbals, aromatic plants or plant parts or extracts
thereof, can also be included in the compositions for a variety of
reasons such as for flavor or for their potential health benefits.
Exemplary herbals include Echinacea, Goldenseal, Calendula,
Rosemary, Thyme, Kava Kava, Aloe, Blood Root, Grapefruit Seed
Extract, Black Cohosh, Ginseng, Guarana, Cranberry, Ginko Biloba,
St. John's Wort, Evening Primrose Oil, Yohimbe Bark, Green Tea, Ma
Huang, Maca, Bilberry, extracts thereof, and a combination
comprising at least one of the foregoing herbals. Additional plant
extracts for inclusion in a composition of the embodiments include,
but are not limited to, extracts or components from Acai,
Spirulina, Chlorella, Wheat Grass, Black Soy Bean, Turmeric, Chia
Seeds, Coconut Oil, Cocoa, Lingon Berries, Eggs, Beat Juice,
Mustard Greens, Sweet Potatoes, Red Wine, Avocados, Blue Berries,
Black Berries, Almonds, Green Tea, Lentils, Black Beans and Aloe
Vera. For example, in some aspects, a composition of the
embodiments includes a protein source selected from the group
consisting of whey protein concentrate, potassium caseinate, egg
albumin, soy isolate, and whey isolate, (Brown) rice protein,
hydrolyzed beef protein isolate, Pea Protein Isolate, and hemp
protein.
[0076] In still further aspects, a composition of the embodiment
can include a diuretic, such a watermelon extract or dandelion leaf
extract (e.g., 4:1).
[0077] In some embodiments, the composition can have a Brix
measurement as measured by a Brix refractometer at 20.degree. C. of
about 8.0 to about 9.5.degree. Brix, specifically about 8.5 to
about 8.9.degree. Brix. In another embodiment, the composition can
have a Brix measurement as measured by a Brix densitometer at
20.degree. C. of about 7.5 to about 9.1.degree. Brix, specifically
about 7.9 to about 8.3.degree. Brix.
Electrolytes
[0078] The inclusion of electrolytes in the various aspects of the
compositions of the invention is contemplated. Exemplary
electrolytes include salts of a metal of the groups I and II of the
periodic table, preferably the inorganic and organic salts of
sodium, potassium, calcium and/or magnesium. Examples of such salts
include, but are not limited to, are sodium acetate, acidic sodium
citrate, acidic sodium phosphate, sodium amino salicylate, sodium
bicarbonate, sodium bromide, sodium chloride, sodium citrate,
sodium lactate, sodium phosphate, sodium salicylate, sodium
sulphate (anhydrous), sodium sulphate (Glauber's salt), potassium
acetate, potassium bicarbonate, potassium bromide, potassium
chloride, potassium citrate, potassium-D-gluconate, mono- and
dibasic potassium phosphate, calcium acetate, calcium chloride,
calcium citrate, calcium-D-gluconate, calcium lactate, calcium
laevulinate, dibasic calcium phosphate, magnesium chloride and
magnesium sulfate. In one aspect, the electrolytes are sodium
chloride, monopotassium phosphate and magnesium sulfate and, when
present in an 8 oz. volume, are included in amounts of about 50 mg
to about 500 mg, from about 10 mg to about 200 mg and from about 10
mg to about 200 mg, respectively. In other aspects, sodium
chloride, when present in an 8 oz. volume, is included in an amount
ranging from about 50 mg to about 60 mg, about 70 mg, about 80 mg,
about 90 mg or about 100 mg and magnesium sulfate and monopotossium
phosphate, when present in an 8 oz. volume, are included in amounts
of about 10 mg to about 20 mg, about 30 mg, about 40 mg, about 50
mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100
mg, or about 200 mg. Compositions of the embodiments can likewise
include glycerol.
[0079] Additional electrolytes and liquid formulation for
composition are provides in U.S. Pat. Nos. 4,981,687, 5,089,477,
5,147,650, 5,236,712, and 5,238,684, each of which are incorporated
herein by reference.
Vitamins and Minerals
[0080] It is contemplated to include vitamin and/or minerals into
various aspects of the compositions of the embodiments. Vitamins
for inclusion include, but are not limited to, Vitamins and
Co-Vitamins such as Vitamin A (beta-carotene), Choline, Vitamin B1
(thiamin), Vitamin B2 (riboflavin, vitamin G), Vitamin B3 (niacin,
vitamin P, vitamin PP), Vitamin B5 (pantothenic acid), Vitamin B6
(pyridoxine, pyridoxamine, or pyridoxal), Vitamin B7 (biotin,
vitamin H), Vitamin B9 (folic acid, folate, vitamin M), Vitamin B12
(cobalamin), Vitamin C (ascorbic acid), Vitamin D (ergocalciferol,
or cholecalciferol), Vitamin E (tocopherol) and Vitamin K
(naphthoquinoids). Minerals for possible inclusion include, without
limitation, Calcium (Ca), Chloride (Cl--), Chromium (Cr), Cobalt
(Co) (as part of Vitamin B12), Copper (Cu), Iodine (I), Iron (Fe),
Magnesium (Mg), Manganese (Mn), Molybdenum (Mo), Phosphorus (P),
Potassium (K), Selenium (Se), Sodium (Na) and Zinc (Zn).
[0081] Vitamin A, for example, helps in the formation and
maintenance of healthy teeth, skeletal and soft tissue, mucous
membranes, and skin. It is also known as retinol because it
generates the pigments that are necessary for the working of the
retina. It promotes good vision, especially in dim light.
Beta-carotene is a precursor to vitamin A that has antioxidant
properties, helping the body deal with unstable chemicals called
free radicals.
[0082] Thiamine (B-1) helps the body cells convert carbohydrates
into energy. It is also essential for the functioning of the heart
and for healthy nerve cells, including those in the brain.
Riboflavin (B-2) works with the other B vitamins and is important
for body growth and red blood cell production. Similar to thiamine,
it helps in releasing energy from carbohydrates. Niacin (B-3) is a
B vitamin that helps maintain healthy skin and nerves. It is also
important for the conversion of food to energy and may have
cholesterol-lowering effects. Vitamin B-6 is also known as
pyridoxine and aids in the formation of red blood cells and in the
maintenance of normal brain function. It also assists in the
synthesizing of antibodies in the immune system. Vitamin B-12, like
the other B vitamins, is important for metabolism, participating
in, for example, the formation of red blood cells. Pantothenic acid
is essential for the metabolism of food. It is also essential in
the synthesis of hormones and cholesterol. Biotin is essential for
the metabolism of proteins and carbohydrates, and in the synthesis
of hormones and cholesterol. Folate (folic acid) works with vitamin
B-12 in the production of red blood cells and is necessary for the
synthesis of DNA.
[0083] Vitamin C, also called ascorbic acid, promotes healthy teeth
and gums, helps in the absorption of iron, and helps maintain
normal connective tissue. It also promotes wound healing and is an
antioxidant.
[0084] Vitamin D promotes the body's absorption of calcium, which
is essential for the normal development and maintenance of healthy
teeth and bones. It also helps maintain adequate blood levels of
calcium and phosphorus, which are minerals necessary for many
functions.
[0085] Vitamin E is also known as tocopherol and is an antioxidant.
It is also important in the formation of red blood cells and the
use of vitamin K.
[0086] Therefore, it is desirable to incorporate various vitamin
types into the various aspects of the compositions of the
invention. In one embodiment, vitamin B1 (thiamin) when present in
an 8 oz. volume, is included in an amount ranging from about 0.1 mg
to about 5 mg; vitamin B2 (riboflavin), when present in an 8 oz.
volume, is included in an amount ranging from about 0.1 mg to about
5 mg; vitamin B3 (niacin), when present in an 8 oz. volume, is
included in an amount ranging from about 1 mg to about 50 mg;
vitamin B5 (pantothenoic acid), when present in an 8 oz. volume, is
included in an amount ranging from about 1 mg to about 50 mg;
vitamin B6, when present in an 8 oz. volume, is included in an
amount ranging from about 0.1 mg to about 5 mg; and vitamin B12,
when present in an 8 oz. volume, is included in an amount ranging
from about 1 .mu.g to about 50 .mu.g. In a further embodiment,
vitamins B1, B2 and B6, when present in an 8 oz. volume, are
included in amounts of about 0.1 mg to about 2 mg, about 3 mg,
about 4 mg, or about 5 mg; vitamins B3 and B5, when present in an 8
oz. volume, are included in amounts of 1 mg, to about 3 mg, about 4
mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg,
about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg;
and vitamin B12, when present in an 8 oz. volume, is included in
amounts of 1 .mu.g to about 10 .mu.g, about 20 .mu.g, about 30
.mu.g, about 40 .mu.g, or about 50 .mu.g.
[0087] In yet another embodiment, a composition of the invention
provided further comprising vitamin A, when present in an 8 oz.
volume, is included in an amount ranging from about 50 IU to about
1000 IU. In one aspect, vitamin A, when present in an 8 oz. volume,
is included in amounts of about 50 IU to about 100 IU, about 200
IU, about 300 IU, about 400 IU, about 500 IU, about 600 IU, about
700 IU, about 800 IU, about 900 IU or about 1000 IU.
[0088] In another embodiment, a composition of the embodiments is
provided further comprising, vitamin C, when present in an 8 oz.
volume, is included in an amount ranging from about 10 mg to about
100 mg. In some aspects, vitamin C, when present in an 8 oz.
volume, is included in amounts of 10 mg to about 20 mg, about 30
mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80
mg, about 90 mg, or about 100 mg.
[0089] In yet another embodiment, a composition of the invention is
provided further comprising vitamin E, when present in an 8 oz.
volume, is included in an amount ranging from about 1 IU to about
50 IU. In aspect, vitamin E, when present in an 8 oz. volume, is
included in amounts of about 1 IU to about 10 IU, about 20 IU,
about 30 IU, about 40 IU, or about 50 IU.
Amino Acids
[0090] In a further embodiment, the aforementioned composition is
provided further comprising one or more amino acids selected from
the group consisting of alanine, arginine, creatine, cysteine,
glysine, histidine, glutamine, lysine, methionine, ornithine,
leucine, isoleucine, serine, tyrosine, aspartagine, aspartic acid,
threonine, proline, tryptophan, valine, phenylalanine, and
selenocysteine. For example, creatine can be supplied in its
various forms such as creatine monohydrate, creatine magnesium
chelate or creatine nitrate.
[0091] For example, glutamine, when present in an 8 oz. volume, is
included in an amount ranging from about 5 mg to about 100 mg or in
amounts of about 5 mg to about 20 mg, about 30 mg, about 40 mg,
about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg or
about 100 mg.
[0092] Furthermore, the inclusion of cysteine in a composition of
the invention is contemplated. For example, cysteine, when present
in an 8 oz. volume, is included in an amount ranging from about 10
mg to about 100 mg.
Carbohydrates
[0093] As mentioned supra, in some aspects including a carbohydrate
source in the composition of the invention is contemplated.
Exemplary carbohydrates include, but are not limited to,
monosaccharides, a disaccharides, oligosaccharides and a glucose
polymers. Modified carbohydrates, such as sucrolose, are also
contemplated. In another aspect, carbohydrate of the formulation is
derived from citric acid.
Flavoring Agents
[0094] One or more flavoring agents may be added to the
compositions of the invention in order to enhance their
palatability. Any natural or synthetic flavor agent can be used in
the present invention. For example, one or more botanical and/or
fruit flavors may be utilized herein. As used herein, such flavors
may be synthetic or natural flavors.
[0095] Exemplary fruit flavors include exotic and lactonic flavors
such as, for example, passion fruit flavors, mango flavors,
pineapple flavors, cupuacu flavors, guava flavors, cocoa flavors,
papaya flavors, peach flavors, and apricot flavors. Besides these
flavors, a variety of other fruit flavors can be utilized such as,
for example, apple flavors, citrus flavors, grape flavors,
raspberry flavors, cranberry flavors, cherry flavors, grapefruit
flavors, and the like. These fruit flavors can be derived from
natural sources such as fruit juices and flavor oils, or may
alternatively be synthetically prepared.
[0096] Exemplary botanical flavors include, for example, tea (e.g.,
black and green tea), aloe vera, guarana, ginseng, ginkgo,
hawthorn, hibiscus, rose hips, chamomile, peppermint, fennel,
ginger, licorice, lotus seed, schizandra, saw palmetto,
sarsaparilla, safflower, St. John's Wort, curcuma, cardimom,
nutmeg, cassia bark, buchu, cinnamon, jasmine, haw, chrysanthemum,
water chestnut, sugar cane, lychee, bamboo shoots, vanilla, coffee,
and the like.
[0097] The flavor agent can also comprise a blend of various
flavors. If desired, the flavor in the flavoring agent may be
formed into emulsion droplets which are then dispersed in the
beverage composition or concentrate. Because these droplets usually
have a specific gravity less than that of water and would therefore
form a separate phase, weighting agents (which can also act as
clouding agents) can be used to keep the emulsion droplets
dispersed in the beverage composition or concentrate. Examples of
such weighting agents are brominated vegetable oils (BVO) and resin
esters, in particular the ester gums. See L. F. Green, Developments
in Soft Drinks Technology, Vol. 1, Applied Science Publishers Ltd.,
pp. 87-93 (1978) (Incorporated herein by reference) for a further
description of the use of weighting and clouding agents in liquid
beverages. Typically the flavoring agents are conventionally
available as concentrates or extracts or in the form of
synthetically produced flavoring esters, alcohols, aldehydes,
terpenes, sesquiterpenes, and the like.
[0098] The amount of flavor agent used will vary, depending on the
agents used and the intensity desired in the finished product. The
amount can be readily determined by one skilled in the art.
Generally, if utilized, the flavor agent should be present at a
level of from about 0.0001% to about 0.5%.
Flavanols
[0099] Flavanols are natural substances present in a variety of
plants (e.g., fruits, vegetables, and flowers). The flavanols which
may be utilized in the present invention can be extracted from, for
example, fruit, vegetables, green tea or other natural sources by
any suitable method well known to those skilled in the art.
Flavanols may be extracted from either a single plant or mixtures
of plants. Plants containing flavanols are known to those skilled
in the art.
[0100] The amount of flavanols in the various aspect of the
compositions of the invention can vary. However, wherein one or
more flavanols are utilized, preferably from about 0.001% to about
5% by weight of the composition.
Sensate Formulations
[0101] In some aspects, compositions include "sensates", trigeminal
nerve stimulants which can alter the taste of e.g., a beverage
composition and decrease the perception of off-notes. Sensates
include "warming agents", compounds which provide a sensation of
warmth; "cooling agents", compounds which provide a cooling
sensation; and "tingling agents", compounds which provide a
tingling, stinging or numbing sensation. The sensate may be a
warming, a cooling, a tingling agent, or any combination comprising
at least one of the foregoing sensates.
[0102] Warming agents may be selected from a wide variety of
compounds known to provide the sensory signal of warming to the
individual user. These compounds offer the perceived sensation of
warmth, particularly in the oral cavity, and often enhance the
perception of flavors, sweeteners and other organoleptic
components. Useful warming agents include those having at least one
allyl vinyl component, which may bind to oral receptors. Examples
of suitable warming agents include vanillyl alcohol n-butyl ether
(TK-1000, supplied by Takasago Perfumery Company Ltd., Tokyo,
Japan); vanillyl alcohol n-propyl ether; vanillyl alcohol isopropyl
ether; vanillyl alcohol isobutyl ether; vanillyl alcohol n-amino
ether; vanillyl alcohol isoamylether; vanillyl alcohol n-hexyl
ether; vanillyl alcohol methyl ether; vanillyl alcohol ethylether;
gingerol; shogaol; paradol; zingerone; capsaicin; dihydrocapsaicin;
nordihydrocapsaicin; homocapsaicin; homodihydrocapsaicin; ethanol;
isopropyl alcohol; iso-amyl alcohol; benzyl alcohol; glycerine;
chloroform; eugenol; cinnamon oil; cinnamic aldehyde; phosphate
derivatives thereof, and the like, or a combination comprising at
least one of the foregoing warming sensates.
[0103] A variety of well-known cooling agents may be employed in
instant compositions. Exemplary cooling agents include menthol,
xylitol, erythritol, menthane, menthone, menthyl acetate, menthyl
salicylate, N,2,3-trimethyl-2-isopropyl butanamide (WS-23),
N-ethyl-p-menthane-3-carboxamide (WS-3), menthyl succinate,
3,1-menthoxypropane 1,2-diol and glutarate esters, among others,
and the like, or a combination comprising at least one of the
foregoing cooling sensates.
[0104] Tingling agents may be employed in the beverage compositions
to provide a tingling, stinging or numbing sensation to the user.
Exemplary tingling agents include Jambu Oleoresin or para cress
(Spilanthes sp.), in which the active ingredient is Spilanthol;
Japanese pepper extract (Zanthoxylum peperitum), including the
ingredients known as Saanshool-I, Saanshool-II and Sanshoamide;
black pepper extract (piper nigrum), including the active
ingredients chavicine and piperine; Echinacea extract; Northern
Prickly Ash extract; red pepper oleoresin; and the like, or a
combination comprising at least one of the foregoing tingling
sensates.
[0105] A sensate may be present in a composition, such as a
composition (e.g., a beverage composition), in an amount of about
0.01 to about 10 weight percent, specifically about 0.1 to about
5.0, and yet more specifically about 1.0 to about 3.0 weight
percent based on the total weight of the beverage composition.
Stimulants
[0106] In some aspects, a composition of the embodiments includes a
stimulant or an agent that provides a feeling of enhanced energy
level. For example, a composition can include Caffeine (anhydrous),
Green Tea Extract (Camellia sinensis) (leaf, e.g., 45% EGCG),
Hoodia gordonii, Advantra Z.RTM. (Citrus aurantium, e.g., 60%
synephrine alkaloids), L-Taurine, Panax Ginseng Powder,
Glucuronolactone, Adenosine, Octopamine, L-Carnitine, Yohimbine,
Vinpocetine, NADH, Evodiamine Cinnulin PF.RTM. Cinnamon Bark
Extract (Cinnamonum burmannii), Banaba Leaf Extract, or
Zychrome.RTM. [Chromium (as Chromium Dinicocysteinate).
Coloring Agent
[0107] Small amounts of one or more coloring agents may be utilized
in the compositions of the present invention. FD&C dyes (e.g.,
yellow #5, blue #2, red #40) and/or FD&C lakes are preferably
used. By adding the lakes to the other powdered ingredients, all
the particles, in particular the colored iron compound, are
completely and uniformly colored and a uniformly colored beverage
mix is attained. Preferred lake dyes which may be used in the
present invention are the FDA-approved Lake, such as Lake red #40,
yellow #6, blue #1, and the like. Additionally, a mixture of
FD&C dyes or a FD&C lake dye in combination with other
conventional food and food colorants may be used. Riboflavin and
b-carotene may also be used. Additionally, other natural coloring
agents may be utilized including, for example, fruit, vegetable,
and/or plant extracts such as grape, black currant, aronia, carrot,
beetroot, red cabbage, and hibiscus.
[0108] The amount of coloring agent used will vary, depending on
the agents used and the intensity desired in the finished product.
The amount can be readily determined by one skilled in the art.
Generally, if utilized, the coloring agent should be present at a
level of from about 0.0001% to about 0.5%, preferably from about
0.001% to about 0.1%, and most preferably from about 0.004% to
about 0.1%, by weight of the composition.
Preservatives
[0109] Preservatives may or may not be needed for use in the
present compositions. Techniques such as aseptic and/or clean-fill
processing may be utilized to avoid preservatives. One or more
preservatives may, however, optionally be added to the present
compositions. Preferred preservatives include, for example,
sorbate, benzoate, and polyphosphate preservatives (for example,
sodium hexametapolyphosphate).
[0110] Preferably, wherein a preservative is utilized herein, one
or more sorbate or benzoate preservatives (or mixtures thereof) are
utilized. Sorbate and benzoate preservatives suitable for use in
the present invention include sorbic acid, benzoic acid, and salts
thereof, including (but not limited to) calcium sorbate, sodium
sorbate, potassium sorbate, calcium benzoate, sodium benzoate,
potassium benzoate, and mixtures thereof.
[0111] Wherein a composition comprises a preservative, the
preservative is preferably included at levels from about 0.0005% to
about 0.5%, more preferably from about 0.001% to about 0.4% of the
preservative, still more preferably from about 0.001% to about
0.1%, even more preferably from about 0.001% to about 0.05%, and
most preferably from about 0.003% to about 0.03% of the
preservative, by weight of the composition. Wherein the composition
comprises a mixture of one or more preservatives, the total
concentration of such preservatives is preferably maintained within
these ranges.
Acidulants
[0112] If desired, the present compositions may optionally comprise
one or more acidulants. An amount of an acidulant may be used to
maintain the pH of the composition. Compositions of the present
invention, in various aspects, have a pH of from about 2 to about
9, from about 2.5 to about 8.5, from about 3 to about 8, from about
03.5 to about 7.5, from about 4 to about 7, from about 4.5 to about
6.5, or from about 5 to about 6.
[0113] Acidity of a composition can be adjusted to and maintained
within the requisite range by known and conventional methods, e.g.,
the use of one or more of the aforementioned acidulants. Typically,
acidity within the above recited ranges is a balance between
maximum acidity for microbial inhibition and optimum acidity for
the desired beverage flavor.
[0114] Organic as well as inorganic edible acids may be used to
adjust the pH of the beverage, and may be added additional to the
acid serving as part of the second component herein. The acids can
be present in their undissociated form or, alternatively, as their
respective salts, for example, potassium or sodium hydrogen
phosphate, potassium or sodium dihydrogen phosphate salts. The
preferred acids are edible organic acids which include citric acid,
malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic
acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid or
mixtures thereof. The most preferred acids are citric and malic
acids.
[0115] The acidulant can also serve as an antioxidant to stabilize
beverage components. Examples of commonly used antioxidant include
but are not limited to ascorbic acid, EDTA
(ethylenediaminetetraacetic acid), and salts thereof.
[0116] The amount of acidulant used will vary, depending on the
agent used and the pH desired in the finished product. The amount
can be readily determined by one skilled in the art. Generally, if
utilized, the acidulant should be present at a level of from about
0.0001% to about 0.5% by weight of composition.
Antioxidants
[0117] A composition of the embodiment may, in some aspects,
further comprise an antioxidant. For example, the antioxidant may
be natural or synthetic. Suitable antioxidants include, but are not
limited to, ascorbic acid and its salts, ascorbyl palmitate,
ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl
isothiocyanate, o-, m- or p-amino benzoic acid (o is anthranilic
acid, p is PABA), butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), caffeic acid, canthaxantin, alpha-carotene,
beta-carotene, beta-carotene, beta-apo-carotenoic acid, carnosol,
carvacrol, cetyl gallate, chlorogenic acid, citric acid and its
salts, clove extract, coffee bean extract, p-coumaric acid,
3,4-dihydroxybenzoic acid, N,N'-diphenyl-p-phenylenediamine (DPPD),
dilauryl thiodipropionate, distearyl thiodipropionate,
2,6-di-tert-butylphenol, dodecyl gallate, edetic acid, ellagic
acid, erythorbic acid, sodium erythorbate, esculetin, esculin,
6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethyl gallate, ethyl
maltol, ethylenediaminetetraacetic acid (EDTA), eucalyptus extract,
eugenol, ferulic acid, flavonoids (e.g., catechin, epicatechin,
epicatechin gallate, epigallocatechin (EGC), epigallocatechin
gallate (EGCG), polyphenol epigallocatechin-3-gallate), flavones
(e.g., apigenin, chrysin, luteolin), flavonols (e.g., datiscetin,
myricetin, daemfero), flavanones, fraxetin, fumaric acid, gallic
acid, gentian extract, gluconic acid, glycine, gum guaiacum,
hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic
acid, hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid,
hydroxytryrosol, hydroxyurea, lactic acid and its salts, lecithin,
lecithin citrate; R-alpha-lipoic acid, lutein, lycopene, malic
acid, maltol, 5-methoxy tryptamine, methyl gallate, monoglyceride
citrate; monoisopropyl citrate; morin, beta-naphthoflavone,
nordihydroguaiaretic acid (NDGA), octyl gallate, oxalic acid,
palmityl citrate, phenothiazine, phosphatidylcholine, phosphoric
acid, phosphates, phytic acid, phytylubichromel, pimento extract,
propyl gallate, polyphosphates, quercetin, trans-resveratrol rice
bran extract, rosemary extract, rosmarinic acid, sage extract,
sesamol, silymarin, sinapic acid, succinic acid, stearyl citrate,
syringic acid, tartaric acid, thymol, tocopherols (i.e., alpha-,
beta-, gamma- and delta-tocopherol), tocotrienols (i.e., alpha-,
beta-, gamma- and delta-tocotrienols), tyrosol, vanilic acid,
2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., lonox 100),
2,4-(tris-3',5'-bi-tert-butyl-4'-hydroxybenzyl)-mesitylene (i.e.,
lonox 330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary
butyl hydroquinone (TBHQ), thiodipropionic acid, trihydroxy
butyrophenone, tryptamine, tyramine, uric acid, vitamin K and
derivates, resveratrol, CoQ-10 (coenzyme Q10), vitamin C, vitamin
E, beta-carotene, other related carotenoids, selenium, manganese,
glutathione, lipoic acid, flavonoids, phenols, polyphenols,
phytoestrogens, N-Acetyl Cysteine, wheat germ oil, zeaxanthin, or
combinations thereof. Preferred antioxidants include tocopherols,
ascorbyl palmitate, ascorbic acid, and rosemary extract. The
concentration of the additional antioxidant or combination of
antioxidants may range from about 0.001% to about 5% by weight, and
preferably from about 0.01% to about 1% by weight.
Water
[0118] The compositions of the invention may comprise from 0% to
about 99.999% water, by weight of the composition. The compositions
may comprise at least about 4% water, at least about 20% water, at
least about 40% water, at least about 50% water, at least about 75%
water, and at least about 80% water. The water included at these
levels includes all added water and any water present in
combination components, for example, fruit juice.
[0119] In various embodiments, the composition is provided in an 1
oz volume, 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 12 oz,
about 14 oz, about 16 oz, about 18 oz, about 20 oz, about 22 oz,
about 24 oz, about 30 oz, or about 40 oz volume or in that volume
of water. In one aspect, a water component of the formulation is
demineralized water.
Alcohol
[0120] In some aspects, a beverage composition of the embodiments
includes ethanol, such as between about 1% to 60% alcohol (ABV), or
about 1 to 40% alcohol (ABV), or about 1% to 20% alcohol (ABV), or
about 1% to 10% alcohol (ABV) (alcohol by volume, ABV). For
example, the composition may include distilled spirits, e.g. vodka,
rum, whiskey, gin, burbon, rye, or other sweetened or unsweetened
distilled liquors. In some aspects, the beverage may be composed of
substantial amount of beer, wine, cider or malt liquor.
Sea Minerals
[0121] In some aspects, compositions of the embodiments further
comprise sea minerals. Sea minerals are nature's perfect balance of
macrominerals, microminerals, and ultra trace minerals. They are
present in the most readily assimilated and bio-available form
known. Sea mineral levels are almost identical to the mineral
levels found in human blood serum and are in pH balance very
similar to human blood. Sea minerals are free of toxic heavy
metals, such as arsenic, cadmium, mercury, lead, radon, ruthenium,
and uranium.
[0122] Sea salt, for example, is primarily composed of the
following ions, listed in order of descending abundance by weight:
Chloride (Cl.sup.-) 55.03% Sodium (Na.sup.+) 30.59% Sulfate
(SO.sub.4.sup.2-) 7.68% Magnesium (Mg.sup.2+) 3.68% Calcium
(Ca.sup.2+) 1.18% Potassium (K.sup.+) 1.11% Bicarbonate
(HCO.sub.3.sup.-) 0.41% Bromide (Br) 0.19% Borate (BO.sub.3.sup.3-)
0.08% Strontium (Sr.sup.2+) 0.04% other ions 0.01%. Sea salt allows
liquids to freely cross body membranes, e.g.: the glomeruli of the
kidney or blood vessel walls. Sea salt is necessary for the proper
breakdown of plant carbohydrates into usable and assimilable
nutrients.
Lipid Components
[0123] As further detailed below compositions of the embodiments
may further comprise a lipid component alone or as part of an oil
(such as a lipid component that comprises dissolved Noble gas).
Lipids for inclusion in compositions of the embodiments include,
but are not limited to .omega.-3 fatty acids such as
.alpha.-linolenic acid (ALA, 18:3), eicosapentaenoic acid or EPA
(20:5n-3), docosahexaenoic acid or DHA (22:6n-3); .omega.-6 fatty
acids such as linoleic acid or (LA, 18:2), an omega-6 fatty acid,
gamma-linolenic acid or GLA (18:3n-6), dihomo-gamma-linolenic acid
or DGLA (20:3n-6) or arachidonic acid or AA (20:4n-6) or .omega.-9
fatty acids. For example, polyunsaturated oils can be derived from
Walnuts, Canola Oil, Sunflower Seeds, Sesame Seeds, Chia Seeds,
Peanuts, Peanut Butter, Olive Oil, Seaweed, Sardines, Soybeans,
Tuna, Wild Salmon or a Whole Grain Wheat, any of which may be used
in a composition of the embodiments.
II. DOSING FOR NOBLE GAS FORMULATIONS
[0124] The amount of a Noble gas incorporated into the compositions
of the embodiments depend upon the specific formulation used and
its intended use. An effective amount of the composition is
determined based on the intended goal, such to provide neuro- or
cardiovascular protectective effect or to provide and improve
feeling of well-being to a subject (e.g., reduce inflammation,
stress and/or blood pressure). The term "unit dose" or "dosage"
refers to physically discrete units suitable for use in a subject,
each unit containing a predetermined-quantity of the composition
calculated to produce the desired effect. The quantity of a
composition to be administered will also depend, both on the
frequency of administration and unit dose, depends on the
protection desired.
[0125] In certain embodiments, the actual dosage amount of a
composition provided to a subject can be determined by physical and
physiological factors such as body weight, health condition,
previous or concurrent therapeutic interventions, diet, and on the
route of administration.
[0126] An effective dose range of a nutraceutical or therapeutic
can be extrapolated, for example, from effective doses determined
in animal studies. In general a human equivalent dose (HED) in
mg/kg can be calculated in accordance with the following formula
(see, e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008,
which is incorporated herein by reference):
HED (mg/kg)=Animal dose (mg/kg).times.(Animal K.sub.m/Human
K.sub.m)
[0127] Use of the K.sub.m factors in conversion results in more
accurate HED values, which are based on body surface area (BSA)
rather than only on body mass. K.sub.m values for humans and
various animals are well known. For example, the K.sub.m for an
average 60 kg human (with a BSA of 1.6 m.sup.2) is 37, whereas a 20
kg child (BSA 0.8 m.sup.2) would have a K.sub.m of 25. K.sub.m for
some relevant animal models are also well known, including: mice
K.sub.m of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster
K.sub.m of 5 (given a weight of 0.08 kg and BSA of 0.02); rat
K.sub.m of 6 (given a weight of 0.15 kg and BSA of 0.025) and
monkey K.sub.m of 12 (given a weight of 3 kg and BSA of 0.24).
[0128] For example, in a mouse system it is possible to administer
between about 200 .mu.l to 5 ml of orally ingested aqueous solution
saturated with Xe per day (i.e., about 0.12-3.0 mg/day or about 7.2
to about 180 mg/Kg/day for a mouse). Thus, for a human subject that
would translate to a dosage of about 500 .mu.g/kg/day to about 12.2
mg/kg/day or for a human of average mass (60 kg) that would be
about 30 to about 732 mg/day.
[0129] As noted above, the precise amounts of an active gas
component depend on the particular formulation. Nonetheless, a
calculated HED dose can provide a general guide for dosing that may
provide beneficial effects. For the instant embodiments, it is
envisioned that the amount of gas, such as Xenon, to be provided in
a unit dosage would be from about 0.1 to about 200 mg, considering
application of 1-2 doses a day to a an average subject. For
example, one .about.6 oz. cold bottle of aqueous Xe drink (e.g.,
comprising cyclodextrin encapsulated Xe) could comprise 4 grams of
Xe, while 2 ml of Xe in water at room temperature and 1 atm
pressure would comprise 1.2 mg of Xe. In general, oil formulations
of Xe can comprise about 20 times more Xe than water (without an
encapsulation system. For example, at room temperature and 1 atm,
solutions of about 12 mg Xe/ml could be achieved in an oil, such as
olive oil.
III. NOBLE GAS ENCAPSULATION
[0130] Oil Components
[0131] Certain aspects of the embodiments concern oils that
comprise dissolved gases, such as, Ar or Xe. In some aspects the
oil is a flaxseed oil, rapeseed oil, soybean oil, walnut oil, fish
oil, safflower oil, sunflower oil, avocado oil, coconut oil, corn
oil, cotton seed oil, peanut oil, palm oil, olive oil, chia oil,
echium oil, krill oil or vegetable oil. In further aspects, the oil
is a mixture of two or more oils. It will be understood by the
skilled artisan that the oil is preferable an edible, substantially
non-toxic oil. Thus, in some aspects, the oil is a non-petroleum
based oil, such as an animal or vegetable-derived oil. Preferably
the oil comprises a high concentration of omega-3 omega-6- and/or
omega-9-fatty fatty acids (e.g., eicosapentaenoic acid,
docosahexaenoic acid, stearidonic acid, and/or linolenic acid). In
still further aspects, an oil is selected for its concentration of
polyunsaturated fatty acids (PUFA), such as oil having at least
about 5%, 10%, 20% or more PUFA content.
[0132] In certain aspects, oil compositions or emulsions of the
embodiments comprise one or more phospholipid component.
Phospholipids include, for example, phosphatidylcholines,
phosphatidylglycerols, phosphatidylethanolamines
glycerophospholipids and certain sphingolipids. Thus, phospholipids
for use herein include, but are not limited to,
dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl
phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoyl
phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoyl
phosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoyl
phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin
("DSSP"), distearoylphophatidylethanolamine ("DSPE"),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine
("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain
phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"),
dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine
("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl
phosphatidylcholine ("POPC"), palmitoyloeoyl
phosphatidylethanolamine ("POPE"), lysophosphatidylcholine,
lysophosphatidylethanolamine, and
dilinoleoylphosphatidylcholine.
[0133] In addition to solublization of Noble gases in lipid
components (e.g., for emulsification) it is also contemplated that
such gases can be provided in an aqueous formulation encapsulated
in a liposome. Such liposome encapsulation of gas has been
previously demonstrated, see, e.g., U.S. Pat. No. 7,976,743,
incorporated herein by reference.
[0134] Water Soluble Molecules
[0135] As detailed further herein, in certain aspects, Noble gas
solubility in an aqueous component is enhanced by encapsulating the
gas (gases) in a water soluble molecule such as polymer. In general
the molecule used for encapsulation will be a molecule that can
form a pocket with increased hydrophobicity that is configured so
encompass (at portion of) a Noble gas atom. Such an encapsulting
molecule thereby shields the hydrophobic atom from the surrounding
polar environment of the aqueous component thereby effectively
increasing the content of Noble gas that can be solubilized in the
aqueous component.
[0136] For example, as shown herein cyclodextrin and its
derivatives are well-adapted for encapsulating Noble gas. In the
case of the large Xe atom, .beta.-cyclodextrin was used to
encapsulate Xe (see, e.g., FIG. 10B). Theoretically, one skilled in
the art could increase the concentration of cyclodextrin or
Hydroxypropyl-beta-cyclodextrin (hp-beta-CD) or other derivatives
to increase the amount of included Xe (i.e. molecular caged Xe). An
acceptably safe dose for cyclodextrin can be about 1,000 mg/kg/day
for chronic oral administration. Solubility of hp-beta-CD, for
example, is 330 mg/ml. That means one can increase cyclodextrin
concentration significantly to about 0.5 mg/ml by use of soluble
derivatives (See Example 3 and 4). This will be able to bring
dissolved gas concentration to at least 500 mg/ml. Additional
molecules that are contemplated for gas encapsulation include,
without limitation, carcer and or hemicarcerands (see, Saleh 2007),
macroglobulin, cucurbituril (see, US 20030140787, incorporated
herein by reference), calixarenes (Adams et al., 2008),
pillararenes (Cao et al., 2009), prophyrins, metallacrowns, crown
ethers, cyclotriveratrylenes, crypotophenes, foldamers, additional
cyclodextrin polymers, silsesquioxanes (Skelton et al., 2013),
tenas porous polymer, HayeSep.RTM. Porous Polymer and Porapak.TM.
Porous Polymer (reach of the foregoing citations incorporated
herein by reference). Selection of the particular polymer for
encapsulation will depend not only on the Noble gas to be
encapsulated, but also the particular type of formulation to be
made (e.g., an oral formulation).
IV. EXAMPLES
[0137] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Methods for Studies of Example 2
[0138] Preparation of Xe-Rich-Solution.
[0139] Xenon-rich-solution was composed of olive oil (or can be
made with other oils such as flaxseed oil, rapeseed oil, soybean
oil, walnut oil, fish oil etc), egg phosphocholine (Avanti, polar
lipid. Alabama, USA), BSA (or other protein such as milk), and
lithocholic acid (Sigma-Aldrich, St. Louis, Mo., USA). The
solution, comprising 25% oil component, was emulsified using a
sonication method and stabled by fabrication of the emulsion with
surfactants such as phospholipids (egg PC, soybean PC, DPPC, DOPC
etc), proteins and lithocholic acid. Xenon (Matheson Tri-Gas.RTM.,
Houston, Tex., USA) was saturated into oil by pressurization at low
temperature.
[0140] Murine Experiment Setting.
[0141] All animal studies were approved by the Animal Welfare
Committee at The University of Texas Health Science Center at
Houston. C57BL/6J wild type (WT) and apolipoprotein-E (Apo E)
knockout (KO) mice were purchased from Jackson Laboratory (Bar
Harbor, Me., USA), see, e.g., Meir et al., 2004, incorporated
herein by reference. The control WT mice used were C57BL/6J, which
share the same genetic background with the Apo E KO mice. Six- to
eight-month-old male WT and KO mice were fed control or high fat
diet (Harlan Laboratories, USA) and administered control or Xe-rich
solution (200 .mu.l, once a day) and -water drink for 6 weeks,
since the Apo E KO mice type fed a high fat diet develop
atherosclerotic lesions. See FIG. 8.
[0142] Echocardiographic Measurements and Electrocardiographic
Images (In Vivo).
[0143] Baseline measurements by echocardiography were obtained
before feeding with the high fat diet. Cardiac morphology and
function were assessed by serial M-mode echocardiography using a
Vevo 770 Imaging System (VisualSonics Inc., Ontario, Canada)
equipped with a 30 MHz microprobe. M-mode ventricular measurements
were taken at 6-weeks after the feeding. Electrocardiographic (ECG)
data was obtained. Echo data (HR, heart rate; LVID, left
ventricular internal dimension; IVS, intra-ventricular septum;
LVPW, left ventricular posterior wall; FS, fractional shortening;
SV, stroke volume; EF, ejection fraction; CO, cardiac output; LV
Vol, LV volume; LV mass corrected) were analyzed with the analysis
software (VisualSonics Inc., Ontario, Canada).
[0144] Protein Assays.
[0145] Fresh frozen heart and brain tissues were slightly thawed on
crushed ice to allow dissection of the hearts and brains. Tissue
samples were homogenized by sonicating for 2-3 bursts of 20 seconds
on ice using a minimal volume of radioimmunoprecipitation assay
(RIPA) buffer (Cell Signaling Technology, Inc., MA, USA) containing
protease inhibitors (complete protease inhibitor cocktail, Sigma)
and centrifuged for 10 min at 4.degree. C. at 14,000.times.g. The
supernatant was removed. The protein concentration was determined
using a Bradford Protein Assay (Bio-Rad, CA, USA).
[0146] Brain-Derived Neurotrophic Factor (BDNF) and .beta.-Amyloid
Measurements.
[0147] Except for the heart and brain extracts, the samples (such
as plasma) were thawed and clarified at 12,000 rpm for 10 min at
4.degree. C. prior to ELISA assays for .beta.-amyloid per the
manufacturer's instructions. BDNF and .beta.-amyloid peptide
(A.beta.1-40) content were determined by using BDNF Sandwich ELISA
Kit (Millipore Corporation, MA, USA) and a Mouse/Rat Amyloid .beta.
(1-40) High Specific ELISA Assay Kit (IBLAmerican, Minneapolis,
Minn., USA). Following instructions, the samples were added in the
pre-coated 96-well microtiter plates for incubation overnight at
4.degree. C. After washing, the antibodies were added and
incubated. Fluorescence was measured by using SpectroMax Microplate
reader (Bio-Tek Instruments) at 450 nm. All samples were analyzed
in duplicate.
[0148] Rat Experiment Setting.
[0149] Male Sprague-Dawley rats (260-280 g, Harlan Laboratories
Inc., Indianapolis, Ind.) were randomly divided into two groups
(n=8 in each group). One group was given water via a gastric tube
and the other given the xenon-rich solution. After two weeks, the
rats underwent middle cerebral occlusion for 2 hours in a double
blind manner. Behavioral function was evaluated and then after
scarifice, infarct volumes were evaluated at 24 hours after brain
injury under a double blind manner. See FIG. 9.
[0150] Rat Model of Middle Cerebral Artery Occlusion (MCAO).
[0151] Cerebral ischemia was induced by occluding the right middle
cerebral artery (MCA) for 2 hours using the intraluminal suture
method. In brief, the right common carotid artery (CCA) was exposed
under an operating microscope. The external carotid artery was
ligated close to its distal end. The internal carotid artery (ICA)
was isolated and separated from adjacent tissues. A 4-0
monofilament nylon suture (Ethicon, Somerville, N.J., USA) coated
with poly L-lysine (0.1% [wt/vol]) and heparin (1000 U/mL) was
inserted into the MCA lumen located 18 to 20 mm from the external
carotid artery/common carotid artery bifurcation for 2 hours to
provoke ischemia. As soon as the suture was removed, external
carotid artery was ligated, allowing blood reperfusion through
common carotid artery into MCA. In all experiments, body
temperature was monitored and maintained at 37.degree. C. during
ischemia and over the first hour of reperfusion with the use of a
feed-forward temperature controller equipped with a heating lamp
and heating pad (Harvard Apparatus, Holliston, Mass., USA). A
polyethylene catheter was introduced into the right femoral artery
for pressure recording. Cerebral blood flow was monitored with the
use of a PR407-1 straight-needle laser Doppler flowmeter probe
(Perimed, Jarfalla, Stockholm, Sweden) connected to a standard
laser Doppler monitor (PF5010 LDPM unit and PF5001 main unit;
Perimed, Jarfalla, Stockholm, Sweden). Interruption of blood flow
was recorded in the region of ischemic penumbra (2 mm lateral and 2
mm posterior to the bregma).
[0152] Neurologic Assessment.
[0153] Neurologic assessment was conducted at 24 hours after brain
injury. All behavioral tests were conducted in a quiet and low-lit
room by an observer blinded with respect to the treatment groups.
Animals were tested for motor function and neurologic outcomes by
recording limb placement, beam walking, and grid walking
abilities.
[0154] Infarct Volume Measurement.
[0155] After neurologic assessment at 24 hours after surgery,
animals were sacrificed and the brains harvested. Using a
Jacobowitz brain slicer, 2-mm thick coronal sections were stained
with 2% TTC. Infarct size was normalized with respect to the whole
brain volume and presented as normalized infarct volume (%).
[0156] Statistical Analysis.
[0157] Data were processed using Microsoft Excel and GraphPad Prism
5.0. All values are expressed as the mean.+-.S.E.M. Comparisons
between each of two groups was determined using the unpaired
2-tailed Student's t test. Analysis of multiple groups was
performed using one-way ANOVA, followed by a Tukey's post hoc
multiple comparison test. P values of less than 0.05 were
considered significant.
Example 2
Xe Administration Studies
[0158] Resistance to Cardiac Hypertrophy in Response to Xenon (Xe)
Exposure.
[0159] To examine the effect of Xe activity on heart disease an
apolipoprotein E knockout (apoE-/-; or "KO" as used herein) mouse
model was employed. This is a well-established model for
atheriosclerosis, as the animals will develop atherosclerotic
lesions even on a normal chow diet, while a high fat diet
significantly accelerates this process (Meir et al., 2004).
Accordingly, the model has previously been successfully employed to
evaluate the effects of both natural compounds and pharmaceuticals
on atheriosclerosis and cardiovascular disease.
[0160] Animals were divided into 5 groups (see experiment setting).
Echocardiography was used to assess cardiac dimensions and function
at baseline, and at 6-weeks following the Xe exposure (FIGS. 1 and
2; Table 1). Cardiac size of WT and KO hearts were determined with
LV mass (corrected) and normalized to body weight (mg/g) following
6-weeks of the Xe exposure (FIG. 3A).
[0161] As expected, LV mass increased in Apo E KO mice fed
with/without high fat diet at 6-weeks and baseline versus WT and WT
after 6 weeks, respectively. The increase in LV mass was blocked in
KO mice treated with Xe (KO6w Xe) as compared to the KO6w vehicle
and KO6w control groups. The presence of a hypertrophic heart was
confirmed in that heart weight normalized to body weight was
significantly increased in the KO6w control and vehicle groups.
Decreased heart-to-body weight was observed in the KO6w Xe mice in
response to the Xe exposure at 6 weeks, compared to the KO6w
vehicle (FIG. 3B).
[0162] Intra-ventricular septum (IVS), LV posterior wall thickness
(PW), LV volume (V), and LV internal dimensions (ID) were measured
by echocardiography at baseline and following 6 weeks of Xe
exposure a diastole and systole, in WT and ApoE-KO hearts (FIG. 1
and Table 1). The in wall thickness increase was significantly
blocked in KO6w Xe mice in response to Xe exposure at 6 weeks, as
compared to the KO6w vehicle mice, as well as versus KO6w/control
mice (FIGS. 1A and 1B). Heart rate (HR) increased in
KO/KO6w/control/vehicle animals compared to WT and WT6w,
respectively. Again, this increase was blocked in KO6w given Xe
(representative M-mode data are shown in FIG. 4A). Taken together,
these results indicate that Xe activity suppresses progression of
cardiac hypertrophy.
[0163] Improved Cardiac Function and Myocardial Ischemia in
Response to Xe Exposure.
[0164] Cardiac function was assessed at baseline and 6 weeks
following Xe exposure (FIG. 2). At baseline and 6 weeks, LV
fractional shortening (FS), LV ejection fraction (EF), and cardiac
output (CO) were decreased in KO mice fed with/without high fat
diet, as compared to WT and WT6w, respectively. On the other hand,
KO6w Xe hearts, in response to Xe exposure, significantly blocked
these decreases at 6 week time point as compared to KO6w vehicle
(FIGS. 2A-C; respectively).
[0165] ECG data show changes in T wave, ST segment, and QRS complex
in KO/KO6w/control/vehicle hearts compared to WT/WT6w,
respectively, consistent with myocardial ischemia. However, these
changes did not occur in KO6w treated with Xe at the 6-weeks time
point. These data indicate an improvement in myocardial ischemia in
response to Xe exposure and suggest that Xe-treated hearts have no
hypertrophic changes and less myocardial ischemia. These changes
also suggest a protective role of xenon-rich solution in heart
diseases (FIG. 4B and suggest that Xe activity improves cardiac
function and protects from myocardial ischemia.
[0166] Increased Expression of BDNF in Heart and Brain Pretreated
with Xe.
[0167] Xe preconditioning has the roles of neuroprotection in
stroke (Peng et al, CNS Neurosci Ther; 2013 October; 19(10):773-84)
and in brain damage from neonatal asphyxia (Ma et al, Journal of
Cerebral Blood Flow & Metabolism (2006),199-208) in rats via
the regulation of genes and synthesis of BDNF. Previous studies
have shown that BDNF is expressed in heart and may be involved in
the molecular mechanisms of heart disease (Okada et al., 2012). To
assess the possible role of BDNF in Xe activity on the
cardiovascular system, relative BDNF expression levels in heart and
brain were measured by ELISA in the presence of absence of XE
treatment (FIG. 5). As shown in FIG. 5A, relative BDNF levels were
increased in KO6w/control/vehicle, as compared to WT6w. Further
increased BDNF levels were observed in KO6w vehicle versus KO6w. In
response to Xe exposure BDNF levels were further increased in the
heart (KO6w Xe). Similar results were also observed in brain (FIG.
5B). These data indicate that in the heart and brain, BDNF may be
involved in Xe-mediated changes (Pagel et al., 2010).
[0168] Levels of .beta.-Amyloid in Plasma and Brain Pretreated with
Xenon-Rich Solution.
[0169] It has previously been reported that Xe may be involved in
lipid homeostasis, however the exact role and effects of Xe on such
homeostasis was unclear (Golden et al., 2010; Jung et al., 2011).
.beta.-amyloid is a main component of brain deposits associated
with Alzheimer's disease and also is related to lipid homeostasis
(Shankar et al., 2008; Selkoe et al., 2001). To investigate whether
Xe activity could regulate .beta.-amyloid levels Apo E-KO mice fed
with high fat diets were examined. In particular, .beta.-amyloid
levels in the plasma and brains of treated and untreated animals
were examined by ELISA (FIG. 6). As shown in FIG. 6A, relative
plasma .beta.-amyloid levels were significantly increased in KO6w
animals treated with the vehicle (and increase, although not
significantly in KO6w) as compared to WT6w animals. This increase
was markedly attenuated in KO6w mice given Xe-rich solution.
Similar results were also observed in brain tissue (FIG. 6B).
Importantly, these data demonstrate that administration of a
Xenon-rich solution has the potential for treatment of cerebral
deposition disease. Accordingly, such solution may be used to
attenuate the effects or delay the progression of amyloid disease,
such as Alzheimer's disease.
[0170] Xenon-Rich Solution to Increase Brain Tolerance to Ischemic
Injury.
[0171] For these studies rats were divided into two groups, one
group was given gastric delivery with water and another group was
given gastric delivery with xenon-rich-solution. After two weeks,
the rats underwent middle cerebral occlusion for 2 hours. Infarct
volumes were evaluated at 24 hours after the brain injury. Rats
given the xenon-rich solution developed smaller infarct size than
controls (FIGS. 7A and 7B). Likewise, behavioral assessments
including limb placement (FIG. 7C) and grid walking (FIG. 7D),
which are indicative of neurological function were completed. The
group pre-treated with an oral Xe-rich solution showed better
ability to perform the behavioral tasks. These data indicate that
administration of a xenon-rich-solution increases the brain
tolerance to ischemic insults and can result in significant
abatement of neurological effects even following significant
ischemic injury.
[0172] Summary.
[0173] Studies presented here demonstrate the beneficial effects of
oral Xe delivery for the neurological and cardiovascular systems.
In particular, it has been shown that Xe intake was protective in a
model system of cardiovascular disease, where an wide range of
disease markers could be improved by Xe treatment. Likewise, it has
been shown that oral Xe is neuroprotective. Not only was the orally
delivered Xe able to significantly protect from ischemic injury,
but Xe was also shown to reduce beta-amyloid load in treated
animals, indicating that it may be useful in treatment or
prevention of degenerative neurological disease. Importantly, the
data also show that even in an oral delivery system (e.g., such a
lipid system described herein) can deliver a sufficient amount of
Xe to provide measurable benefit treated animals.
TABLE-US-00001 TABLE 1 Echocardiographic Measurements LVID; d IVS;
s LVID; s LVPW; s LV Vol; s Heart Rate (mm) (mm) (mm) (mm) (.mu.L)
(BPM) WT 4.14 .+-. 0.18 1.26 .+-. 0.09 2.86 .+-. 0.12 1.11 .+-.
0.08 30.55 .+-. 5.34 360 .+-. 11 wild type mice fed with regular
diet at baseline KO 3.6 .+-. 0.11* 1.28 .+-. 0.07 2.65 .+-. 0.11
1.13 .+-. 0.04 27.99 .+-. 3.46 439 .+-. 9* Apo E-KO mice fed with
regular diet at baseline WT6w 4.01 .+-. 0.05 1.18 .+-. 0.07 2.66
.+-. 0.08 1.18 .+-. 0.07 29.67 .+-. 3.38 377 .+-. 15 WT mice fed
with regular diet for 6 weeks KO6w 3.4 .+-. 0.22* 1.32 .+-. 0.10
2.42 .+-. 0.13 1.2 .+-. 0.07 26.85 .+-. 2.83 428 .+-. 19* KO mice
fed with regular diet for 6 weeks KO6w 3.4 .+-. 0.16* 1.34 .+-.
0.11 2.47 .+-. 0.09 1.41 .+-. 0.05 25.45 .+-. 4.19 459 .+-. 15*
control KO mice fed with high fat diet and administered by PBS
gavage for 6 weeks KO6w 3.4 .+-. 0.14* 1.36 .+-. 0.06 2.57 .+-.
0.10 1.3 .+-. 0.05 26.26 .+-. 3.71 457 .+-. 14 vehicle KO fed with
high fat diet and vehicles for 6th week KO6w Xenon 4.0 .+-.
0.07.sup..sctn..sctn. 1.29 .+-. 0.05 2.65 .+-. 0.10 1.26 .+-. 0.07
26.01 .+-. 3.82 402 .+-. 13.sup..sctn. KO fed with high fat diet
and administered with Xenon- rich-solution for 6th week *p <
0.05, **p < 0.01, KO/K06w compared to WT/WT6w, respectively; # p
< 0.05, KO control/vehicle compared to KO 6w; .sup..sctn.p <
0.05, .sup..sctn..sctn.p < 0.01, .sup..sctn..sctn..sctn. p <
0.001, KO xenon compared to KO vehicle. Vehicle is caged molecular
water with cyclodextrin without Xenon loaded.
Example 3
Material and Methods for Example 4
Preparation of Caged Molecular Enclosed Xenon
[0174] Xenon was enclosed into a soluble caged molecules (e.g.,
cyclodextrin). To remove possible residue molecules from cage, the
caged molecular was baked at 40-80.degree. C. under vacuum for
overnight. To enclose xenon into caged molecules, xenon was
incubated with caged molecular in a sealed vial under 2-10 atm
pressure at 4 to -180.degree. C. for overnight to 3 days.
Preparation of Pure Xenon Supersaturated Water
[0175] Pure water was degassed under 20-80 mbar vacuum at room
temperature for overnight. Xenon (99.999% Medical grade, Matheson
Tri-Gas.RTM., Houston, Tex., USA) was re-dissolved into degased
water by pressure water with 2-10 atm xenon gas at 4.degree. C. for
overnight to 3 days.
Preparation of Xe-Rich-Water
[0176] Xe-rich-water is composed of Xe directly dissolved in water
and caging with hydroxypropyl-beta-cyclodextrin (hp-beta-CD) in
water. To prepare a Xe-rich-water, Xenon supersaturated water (10
ml) was injected into the vial containing 5 mg caged
molecular-xenon complexes (0.5 mg/ml). The resulting mixture was
incubated under 2-10 atm pressure at 4.degree. C. for overnight to
3 days.
Measurement of Xe Dissolved in Xenon-Rich-Water
[0177] To measure the amount of Xe dissolved in a Xe-rich-water.
The solution was warmed to room temperature and the pressure over
Xe-rich-water sample was released. The solution was then warmed up
to 80.degree. C. in a vial with a silicone-rubber seal (Thermo
Scientific SepraSeal.RTM. (Thermo Scientific, Hudson, N.H., USA)
sealed top for 2 hours. After cooling down to room temperature, a
syringe with a 17 gauge needle was inserted into a vial through the
silicone-rubber seal. The released Xe gas present in the top spacer
formed pressure, which pushed Xe gas into the syringe. The amount
of Xe released into syringe was then measured.
Animals
[0178] All animal studies were approved by the Animal Welfare
Committee at The University of Texas Health Science Center at
Houston. Wild type (WT) and Apo E knockout (KO) transgenic mice
were purchased from Jackson Laboratory (Bar Harbor, Me., USA). Wild
type control mice used were C57BL/6J to compare with the Apo E KO
mice with the same genetic background. Eight- to eleven-month old
male and female WT and KO mice were fed with a high fat diet
(Harlan Laboratories, USA) and administered with caged molecular
water with CD, but without Xe (vehicle) or Xe-rich-water, which
included caged molecular water with CD loaded with Xe (0.2 to 10 ml
per day) for 6 weeks.
Echocardiographic Measurements and Electrocardiographic Images (In
Vivo)
[0179] Baseline measurements by echocardiography were obtained
before feeding with the high fat diet. Cardiac morphology and
function were assessed by serial M-mode echocardiography using a
Vevo 770 Imaging System (VisualSonics Inc., Ontario, Canada)
equipped with a 30 MHz microprobe. M-mode ventricular measurements
were taken at 6-weeks after the feeding. Electrocardiographic (ECG)
data was obtained. Echo data (HR, heart rate; LVID, left
ventricular internal dimension; IVS, intra-ventricular septum;
LVPW, left ventricular posterior wall; FS, fractional shortening;
SV, stroke volume; EF, ejection fraction; CO, cardiac output; LV
Vol, LV volume; LV mass corrected) were analyzed with the analysis
software (VisualSonics Inc., Ontario, Canada).
Blood Pressure Measurement
[0180] Mice blood pressure was monitored non-invasively utilizing a
tail-cuff placed on mice tail to occlude the blood flow.
Protein Assays
[0181] Fresh frozen heart and brain tissues were slightly thawed on
crushed ice to allow dissection of the hearts and brains. Tissue
samples were homogenized by sonicating for 2-3 bursts of 20 seconds
on ice using a minimal volume of radioimmunoprecipitation assay
(RIPA) buffer (Cell Signaling Technology, Inc. MA. USA)) containing
protease inhibitors (complete protease inhibitor cocktail, Sigma)
and centrifuged for 10 min at 4.degree. C. at 14,000.times.g. The
supernatant was removed. The protein concentration was determined
by using a Bradford Protein Assay (Bio-Rad, CA, USA)
.beta.-Amyloid Measurements
[0182] .beta.-amyloid peptide (A.beta.1-40) content in both brain
and blood were determined using a Mouse/Rat Amyloid .beta. (1-40)
High Specific ELISA Assay Kit (IBLAmerican, Minneapolis, Minn.,
USA). Following instructions, the samples were added in the
pre-coated 96-well microtiter plates for incubation overnight at
4.degree. C. After washing, the antibodies were added and
incubated. Absorbance was measured by using SpectroMax Microplate
reader (Bio-Tek Instruments) at 450 nm. All samples were analyzed
in duplicate.
Western Blot Analysis Cardiac Troponin Expression in Heart
Tissue
[0183] Western blot analysis was carried out as previously
described (Yin, X, Molecular Pharmacology) using cardiac troponin I
(cTnI) (Cell Signaling Technology, Inc., Danvers, Mass., USA). For
immunoblot analyses, samples were resolved by SDS-PAGE (4-12%)
gradient gels, and transferred to Polyvinilidene Fluoride (PVDF)
membranes. Blots were then incubated overnight at 4.degree. C. with
primary antibodies and washed three times with TBS containing 0.1%
Tween 20 (TBST), and then probed with secondary antibodies (LI-COR
Biosciences, Lincoln, Nebr., USA) following the manufacturer's
instructions. Densitometric analyses of the immunoblots were
performed with an Odyssey Infared Imager (LI-COR Biosciences).
Statistical Analysis
[0184] Data were processed using Microsoft Excel and GraphPad Prism
5.0. All values are expressed as the mean.+-.S.E.M. Comparisons
between two groups were determined using unpaired 2-tailed
Student's t test. Analysis was performed using one-way ANOVA,
followed by a Tukey's post hoc multiple comparison test when
multiple groups were compared. P values of less than 0.05 were
considered significant.
Example 4
Results of Further Studies with Xe Enhanced Water
Xenon Dissolved in Xe-Rich-Water
[0185] Cyclodextrin (CD) is a multifunctional caged molecule
employed the in food, pharmaceutical, and chemical industries.
Cyclodextrins provide a hydrophobic interior and a hydrophilic
exterior. Studies described here were undertaken to determine if
these properties could be used, to increase the solubility of Noble
gases, such as xenon.
[0186] Data from initial studies demonstrated that the inclusion of
Xenon into cyclodextrin (hp-beta-CD) is highly related to pressure
(FIG. 10C) and temperature (FIG. 10D). Increased pressure resulted
in increased amounts of Xe encapsulation as shown in FIG. 10C.
Likewise, encapsulation of gaseous Xe at low temperature was more
efficient (FIG. 10D). For example, the studies showed that, at 3
atm, -80.degree. C., a total of 5 ml of xenon could be encapsulated
in a hp-beta-CD cage (0.5 mg/ml of hp-beta-CD was used).
[0187] Xenon solubility in water is, likewise, highly related to
the pressure and temperature of the solution. By incubation of
degassed water with pure xenon gas at 4.degree. C., 3 atm for 4
hours to overnight, a total 6.5 ml xenon gas was dissolved into 5
ml water. To make Xe-rich-water, Xe-saturated water was incubated
with Xe-CD under 3 atm pressure at 4.degree. C. A pressure of 3 atm
was used here since, typically, a standard beverage container can
withstand 80-90 psi (5.4-6.1 atm) pressure (i.e., a typical can of
a soft drink such as Coca-Cola.TM. classic products have an
internal pressure of 55 psi (3.7 atm) at 75.degree. F.). As showed
in FIG. 11, in the presence of water as a media, 19 ml Xenon gas
was included into caged molecules and dissolved in water (with a
starting volume of 5 ml of water). Thus, the formulation achieved a
total Xe content of 22.4 mg Xe per ml of the CD-water solution (at
CD concentration of 0.5 mg/ml hp-beta-CD).
Xenon-Rich-Water Increases the Tolerance of Heart to Ischemic
Stress
[0188] To examine the effect of Xe activity on prevention of heart
disease mice were divided into 4 groups, (1) wild type fed with
normal food and water control (WT); (2) Apo E knockout mice fed
with high fat diet and normal water control; (3) Apo E knockout
mice fed with high fat diet and vehicle control (feed with water
containing cyclodextrin but no xenon), and (4) Apo E knockout mice
fed with high fat diet and Xe-rich-water (Xenon loaded into
cyclodextrin, i.e. molecular caged xenon). Echocardiography was
used to assess cardiac dimensions and function at baseline, and at
6-weeks after feeding.
[0189] Intra-ventricular septum (IVS), left ventricular posterior
wall thickness (LVPW), left ventricular (LV) volume, and LV
internal dimensions (ID) were measured at diastole and systole by
echocardiography at baseline and following 6-weeks of treatment
(FIG. 12). High fat diet caused the ventricular wall thickness to
be significantly increased, as is typical for ApoE KO animals.
However, this pathological change did not occur in ApoE KO mice
receiving 6-weeks of Xe-rich-water treatment, as compared to mice
which received only water with cyclodextrin (FIG. 12). Heart rate
(HR) also increased in ApoE KO mice fed a high fat diet and treated
with vehicle. Again, treatment with Xe-rich-water prevented this
increase. These results suggest, as indicated in the studies above,
that oral Xe-rich-water consumption suppresses progression of
cardiac hypertrophy. Furthermore, levels of Xe in the encapsulated
water formulations were high enough to achieve beneficial
effect.
[0190] Cardiac function was also assessed at baseline and 6-weeks
of administration of Xe-rich-water (FIG. 12). At baseline and
6-weeks, LV fractional shortening (FS), (EF), and cardiac output
(CO) were decreased in ApoE KO mice fed with a high fat diet, as
compared to WT and WT6w treated mice. In ApoE KO mice that received
administration of Xe-rich-water (KO6w Xe) hearts from the mice
significantly protected from these decreases as compared to KO6w
vehicle at the 6-week time point, (FIG. 12A-C; respectively).
[0191] ECG data show changes in T wave, ST segment, and QRS complex
in KO/KO6w/control/vehicle hearts compared to WT/WT6w respectively,
compatible with myocardial ischemia. These changes did not occur in
the hearts of ApoE KO mice that received administration of
Xe-rich-water (KO6wXe hearts) 6-weeks. This indicates that
administration of Xe-rich-water reduces myocardial ischemia.
[0192] Troponin and CKMB (Creatine Kinase) are two markers of heart
ischemia. Further studies measured the plasma CKMB level and
troponin expression in heart tissue. These studies showed that the
level of the two markers were increased in controls and was
decreased in ApoE KO mice that received administration of
Xe-rich-water (FIG. 13). These data further confirmed that the
consumption of Xe-rich water increased the tolerance of the heart
to ischemic stress.
Xenon-Rich-Water Stabilize Blood Pressure
[0193] Further analysis of Xe-treated mice indicated that oral
administration of the Xe-rich drinking water on a daily basis for 6
weeks significantly decreases both systolic and diastolic blood
pressure (Table 2), while increasing the heart contractility.
TABLE-US-00002 TABLE 2 Xe-rich-water stabilize blood pressure ApoE
ApoE ApoE Baseline 6W-control 6W-vehicle 6W-Xenon (n = 19) (n = 3)
(n = 5) (n = 5) Systolic BP 98 .+-. 7 122 .+-. 4* 121 .+-. 5* 103
.+-. 3.sup..sctn. (mmHg) Diastolic BP 74 .+-. 5 94 .+-. 3* 91 .+-.
2* 77 .+-. 3.sup..sctn. (mmHg) *p < 0.05, KO/KO6W compared to
Apo E fed without high fat diet (baseline); .sup..sctn.p < 0.05,
KO Xenon compared to KO vehicle
Xenon-Rich-Water Reduces Beta-Amyloid in Both Brain Tissue and
Blood
[0194] Studies were also undertaken to determine the effects of
Xe-water administration on .beta.-amyloid in brain and blood (see
FIG. 14). For these studies the well characterized ApoE-KO mouse
model system for Alzheimer's disease was used. These mice exhibit
increased levels of serum and brain .beta.-amyloid levels as
compared to control mice. However, administration of Xe-water to
the mice over a period of six weeks resulted in decreased levels of
both serum and brain .beta.-amyloid (achieving levels similar to
control animals).
[0195] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0196] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0197] U.S. Pat. No. 4,981,687 [0198] U.S. Pat. No. 5,089,477
[0199] U.S. Pat. No. 5,147,650 [0200] U.S. Pat. No. 5,236,712
[0201] U.S. Pat. No. 5,238,684 [0202] U.S. Pat. No. 7,976,743
[0203] U.S. Pat. Publn. No. 20030177784 [0204] Adams et al., J.
Phys. Chem. A, 112(30):6829-6839, 2008. [0205] Baumert et al., Acta
Anaesthesiol. Scan., 49:743-749, 2005. [0206] Cao et al.,
Angewandte Chemie International Edition. 48(51):9721-9723, 2009.
[0207] Golden et al., PLoS One, 5:e10099, 2010. [0208] Hein et al.,
Acta Anaesthesiol. Scan., 54:470-478, 2010. [0209] Huang, Sl,
circulation [0210] Jung et al., Eur. J. Appl. Physiol.,
111:303-311, 2011. [0211] Meir et al., "Atherosclerosis in the
Apolipoprotein E-Deficient Mouse--A Decade of Progress,"
Arterioscler. Thromb. Vasc. Biol., 24:1006-1014, 2004. [0212] Okada
et al., Arteriosclerosis, Thrombosis, and Vascular Biol.,
32:1902-1909, 2012. [0213] Pagel, J. Cardiothorac. Vasc. Anesth.,
24:143-163, 2010. [0214] Saleh "Novel Phenomena In Encapsulating
Hydrocarbon Gases," in Novel Phenomena In Encapsulating Hydrocarbon
Gases, 2007. [0215] Selkoe, Neuron, 32:177-180, 2001. [0216]
Skelton et al., Phys Chem Chem Phys. 15(12):4341-54, 2013. [0217]
Shankar et al, Nat. Med., 14:837-842, 2008.
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