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(12) United States Patent (10) Patent No.: US 9.446,072 B2 Samuelson Et Al

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(12) United States Patent (10) Patent No.: US 9.446,072 B2 Samuelson Et Al USOO9446O72B2 (12) United States Patent (10) Patent No.: US 9.446,072 B2 Samuelson et al. (45) Date of Patent: *Sep. 20, 2016 (54) METHODS OF AMELIORATING OXIDATIVE A633/40 (2006.01) STRESS BY INCREASING THE EFFICIENCY A6II 45/06 (2006.01) OF GPX AND SOD ACTIVITY A633/20 (2006.01) (52) U.S. Cl. (71) Applicant: Reoxeyn Discoveries Group, INC. CPC ............... A61K 33/40 (2013.01); A61K 33/00 Salt Lake City, UT (US) (2013.01); A61K 33/14 (2013.01); A61K 33/20 (72) Inventors: Gary Samuelson, Sandy, UT (US); (2013.01); ) A6 IK 45/06 ((2013.01 ) Verdis Norton, Sandy, UT (US) (58) Field of Classification Search s s None (73) Assignee: Reoxcyn Discoveries Group, Inc., Salt See application file for complete search history. Lake City, UT (US) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S. PATENT DOCUMENTS U.S.C. 154(b) by 108 days. 2006/0076248 A1* 4/2006 Kindred ........................ 205,743 This patent is Subject to a terminal dis claimer. OTHER PUBLICATIONS (21) Appl. No.: 13/857,225 Hanaoka1313).* (Journal of Applied Electrochemistry 2001, 31, 1307 (22) Filed: Apr. 5, 2013 * cited by examiner (65) Prior Publication Data US 2013/0236563 A1 Sep 12, 2013 Primary Examiner — Abigail Fisher O. I Z. Assistant Examiner — Jessica Kassa Related U.S. Application Data (74) Attorney, Agent, or Firm — David R. Conklin; Kirton McConkie (63) Continuation-in-part of application No. 12/592.402, filed on Nov. 24, 2009, now Pat. No. 8,455,010, which is a continuation-in-part of application No. (57) ABSTRACT 12/383,212, filed on Mar. 20, 2009, now Pat. No. Methods of ameliorating cellular oxidative stress are dis 8.367,120, which is a continuation-in-part of closed. The methods include contacting at least one cell with application No. 12/290,398, filed on Oct. 30, 2008, a composition comprising a mixture of reduced species (RS) now abandoned. and reactive oxygen species (ROS). The mixture of reduced (60) Provisional application No. 61/001010, filed on Oct. Spec1es (RS) and reactive oxygen species (ROS) 1OS a 30, 2007 target mixture where the target mixture is the reduced s species (RS) and reactive oxygen species (ROS) found in a (51) Int. Cl. known biological system. A6 IK33/00 (2006.01) A6 IK 33/14 (2006.01) 17 Claims, 27 Drawing Sheets U.S. Patent Sep. 20, 2016 Sheet 1 of 27 US 9,446,072 B2 Reverse Osmiosis 102 Distillation 104 Salting 106 Chilling 108 Electrolyzing 110 Storage/Testing 112 Bottling 114 FIG, 1 U.S. Patent Sep. 20, 2016 Sheet 3 of 27 US 9.446,072 B2 220 212 226 ? 222 208 O ?et GO ? 214 / S /K () V WWQA V II:232 3. O 218 234 240 > 242 (D 250 * LGBC(EGCGS-24sKY-> <>< t tlets 254 256 258 3. FIG 3 U.S. Patent Sep. 20, 2016 Sheet 4 of 27 US 9,446,072 B2 Input 304 208. FIG 4 U.S. Patent Sep. 20, 2016 Sheet 5 Of 27 US 9,446,072 B2 FIG 5 U.S. Patent Sep. 20, 2016 Sheet 6 of 27 US 9,446,072 B2 & S S U.S. Patent Sep. 20, 2016 Sheet 7 Of 27 US 9,446,072 B2 r d ? yar tex s o d we n N T 3 s S S s S U.S. Patent US 9.446,072 B2 U.S. Patent Sep. 20, 2016 Sheet 9 Of 27 US 9,446,072 B2 FIG 9 0.7000000 Oxygen/Mitrogen 0, 6000000 O5000000 0,4000000 03000000 O, 2000000 0, 1000000 0.0000000 Water ASEA WaCIO Chlorine/Mitrogen 00000180 00000160 00000140 00000120 00000100 00000080 00000060 00000040 00000020 00000000 Water ASEA WaCIO FIG, 10 U.S. Patent Sep. 20, 2016 Sheet 10 of 27 US 9,446,072 B2 FIG 11 Ozone/Nitrogen 0.0002000 0.0001800 00001600 0.0001400 0.0001200 00001000 00000800 00000600 00000400 00000200 00000000 Carbon Dioxide/Mitrogen 0, 0080000 0.0070000 0.0060000 0.0050000 0.0040000 00030000 0.0020000 0, 0010000 0.0000000 FIG, 12 U.S. Patent Sep. 20, 2016 Sheet 11 of 27 US 9,446,072 B2 s s s U.S. Patent Sep. 20, 2016 Sheet 12 of 27 US 9,446,072 B2 FIG. I.4 10 Power Source FIG. I5 O U.S. Patent Sep. 20, 2016 Sheet 13 of 27 US 9.446,072 B2 FIG. I6 Wall Terminal Strip 10 - Potentiometer FIG. 1 7 — Current Transformer 104 102 Potentiometer -- 105 Transformer -- Rectifier -(- -- U.S. Patent Sep. 20, 2016 Sheet 14 of 27 US 9,446,072 B2 OneWay ANOVA, P=0.002; - PK0,015 WS, Sed-Pl Sed-Pl Sed ASEA Run-Pl Run-ASEA FIG, 18 U.S. Patent Sep. 20, 2016 Sheet 15 Of 27 US 9,446,072 B2 Sed-PI. Sed-ASEA Run-Pl Run-ASEA FIG, 19A Oxidized Glutathlone (GSSG) in muscle 6/jourd FIG. 19B U.S. Patent Sep. 20, 2016 Sheet 16 of 27 US 9,446,072 B2 FIG. 20 O ---------------was-nox-ca--- E : s ax 4s Y O U.S. Patent Sep. 20, 2016 Sheet 17 Of 27 US 9,446,072 B2 FIG 21 & Series - Expon. (Series1) y = 98.615e0031: U.S. Patent Sep. 20, 2016 Sheet 18 of 27 US 9,446,072 B2 FIG. 22 Bottle versus Pouch 12O 100 8 Bottle Decay 80 a Pouch Decay 60 40 -Expon.pon. ((Bottle Decay) 20 -Expon. (Pouch Decay) O y = 94.731e 0.0448 O 5 10 15 Month y = 99.495e003. U.S. Patent Sep. 20, 2016 Sheet 19 Of 27 US 9,446,072 B2 FIG. 23 Expt. 5f07 ROS Assay -- using data from 0-250 min 0.01 0.1 1 AAPH Concentration mM FIG. 24 Determination of Intraassay Wariation Using TWO Leves of AAPH 0.369E3 uM buffer control 200 400 600 800 1000 1200 1400 Time mins U.S. Patent Sep. 20, 2016 Sheet 20 Of 27 US 9,446,072 B2 NF-kBScreen pBS 5% 20% Positive Control ASEA ASEA Control DAPI FIG.25 U.S. Patent Sep. 20, 2016 Sheet 21 of 27 US 9,446,072 B2 |583#0z &### S. 9,'91-7 U.S. Patent Sep. 20, 2016 Sheet 22 of 27 US 9,446,072 B2 -- 0 ---A--- 5th/ASEA 1300 GPX -- 10 til ASEA ASEA -- 0 ------5ul PBS 1300 GPY - x - 10 ti? PBS PBS ---20th PBS U.S. Patent Sep. 20, 2016 Sheet 23 of 27 US 9.446,072 B2 20% PBS 5% ASEA h202 NRF2 DAP1 FIG, 29 % ASEA - goal-in-end 120 min NRF2 serum-starved DAP1 NRF2 fandomly cycling DAPi Nuclear NRF2staining profiles in andomly cycling and serum-starved HMVFC-1 Cells, F.IG, 30 U.S. Patent Sep. 20, 2016 Sheet 24 of 27 US 9,446,072 B2 Time (min) 1% ASEA 0 30 60 120 ho -- phospho-protein see 1% ASFA 0 30 60 120 H2O2 NRF2 is is ess - s's 10000 tin -- SS9s 9000\ regiationASEA actin see a isSS 800 is 43000 is 7000 S$41000 Sš 6000 0 30 60 90 20 S. S39000S. Time (min) SS 37000 regulation S35000 0 30 60 90 120 f Time (min) Cytos/exta-nuclear differential Vi?ion F.IG, 31 cytos/exta-nuclear ge U.S. Patent Sep. 20, 2016 Sheet 25 Of 27 US 9,446,072 B2 30000 28000 26000 24000 22000 FIG 32 20000 O 10 20 ASEA%(v/v) HMVEC-l counts indicate reduced proliferation for high-concentration ASFA after 72 hours 290 2.80 s 270 0. 5 10 15 20 Final Concentration 1% w/v) U.S. Patent Sep. 20, 2016 Sheet 26 of 27 US 9,446,072 B2 SSS S. S. SS t Sgs. S.s SS S SSS 'S (10 JuO %) BuelloSippy EjngN (10 JuO2Sgd %) Mihalpy HGT7 unpay U.S. Patent Sep. 20, 2016 Sheet 27 Of 27 US 9,446,072 B2 US 9,446,072 B2 1. 2 METHODS OF AMELORATING OXDATIVE the cell. This condition is known as oxidative stress and it STRESS BY INCREASING THE EFFICIENCY acts as a clear signal to the cell that something is wrong. The OF GPX AND SOD ACTIVITY cell reacts to this signal by producing the enzymes and repair molecules necessary to attempt repairs to the damage and it also can send messengers to activate the immune system to CROSS-REFERENCE TO RELATED identify and eliminate threats. If oxidative stress persists in APPLICATIONS the cell for more than a few hours, then the cells repair attempts are considered unsuccessful and the cell kills and This application is a Continuation-in-part of, and claims dismantles itself and is replaced by the natural cellular the benefit of, U.S. patent application Ser. No. 12/592.402, division of healthy neighboring cells. filed Nov. 24, 2009, which is a Continuation-in-part of U.S. 10 On a cellular level, this is essentially the healthy tissue patent application Ser. No. 12/383.212, filed Mar. 20, 2009, maintenance process: damaged cells are detected and now U.S. Pat. No. 8,367,120 B1, which is a Continuation repaired or replaced by healthy cells. This cellular repair and in-Part of U.S. patent application Ser. No. 12/290,398, filed regeneration process is constantly taking place, millions of Oct. 30, 2008, now abandoned, which claims priority to U.S. times an hour, in all parts of the body. Provisional Patent Application Ser. No. 61/001010, filed 15 It has long been known that the electrolysis of fluids can Oct.
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