Manufacturing and purification processes of Complex protein found in Fraction IV to make a separated Apo, Transferrin, and Alpha 1 Antitrypsin (A1AT) or a combined Transferrin/Apo/Human Albumin/A1AT and all new found proteins

Abstract

Manufacturing and purification processes of complex protein found in Fraction IV to make a separated Apo, Transferrin, and Alpha-1 Antitrypsin (A1AT) or a combined Transferrin/Apo/Human Albumin/A1AT and all new found proteins. A complex of all proteins found currently in plasma, cryoprecipitate, Fraction III and many newly found proteins now being identified or any substances which are known proteins or unknown proteins which contain healthy cells and the combination of any of these known or unknown proteins which contain any one of these healthy cells: neutrophil, lymphocyte, eosinophil, basophil, and macrophage, and their potential applications for treating a wide variety of diseases and other physical conditions and disorders, and for maintaining health.

Claims

1. A method of purifying aplipooprotein A-I (APOA1) from plasma fraction IV comprising: suspending plasma fraction IV in a buffer to obtain a resulting supernatant, wherein the buffer has a pH level in the range of 3.0 to 10.0, and wherein the resulting supernatant contains APOA1; collecting the resulting supernatant; adding a sodium chloride solution to the resulting supernatant and obtaining an APOA1 precipitate solution; centrifuging the APOA1 precipitate solution and collecting a resulting APOA1 paste; suspending the resulting APOA1 paste and obtaining a resulting APOA1 suspension; filtering the resulting APOA1 suspension and obtaining a resulting filtered APOA1 suspension; and subjecting the resulting filtered APOA1 suspension to a chromatography process.

2. The method according to claim 1, wherein the buffer is a sodium acetate solution.

3. The method according to claim 1, wherein the sodium chloride solution has a pH level in the range of 3.0 to 10.0, and wherein the APOA1 precipitate solution is cooled to a temperature range of 1 C. to 1 C.

4. The method according to claim 1, wherein the APOA1 paste is resuspended in a water for injection (WFI) or sodium chloride solution having a pH level in the range of 3.0 to 10.0 and a temperature in the range of 0 C. to 10 C.

5. The method according to claim 1, wherein the resulting APOA1 suspension is filtered with a 0.45 m filter.

6. The method according to claim 1, wherein the chromatography process is diethylaminoethanol (DEAE) ion exchange chromatography and butyl chromatography.

7. The method according to claim 6, further comprising: adjusting the filtered APOA1 suspension to a pH level in the range of 3.0 to 10.0 and the ionic strength in the range of 15 mM to 25 mM; loading the resulting filtered APOA1 suspension into a DEAE chromatography column; washing the DEAE chromatography column in a low salt buffer and a high salt buffer; obtaining an APOA1 elute; adjusting the APOA1 elute to a pH level in the range of 3.0 to 10.0; washing the APOA1 elute in a low salt elute buffer and obtaining a purified APOA1 elute; and washing the purified APOA1 elute in a WFI buffer or an alkaline buffer and collecting a resulting APOA1 enriched elute.

8. The method according to claim 7, wherein the low salt buffer contains Tris having a pH level in the range of 3.0 to 10.0, the high salt buffer contains sodium chloride, the low salt elute buffer contains Tris, and the alkaline buffer contains sodium hydroxide having a pH level in the range of 3.0 to 10.0.

9. The method according to claim 7, further comprising: dialyzing the APOA1 enriched elute; concentrating the APOA1 enriched elute with virus inactivation; adding a stabilizer to the APOA1 enriched elute and obtaining a stabilized high purity APOA1; and lyophilizing the stabilized high purity APOA1.

10. A method of purifying at least one protein from a plasma fraction IV comprising: suspending plasma fraction IV in a buffer to obtain a suspended fraction IV; subjecting the suspended fraction IV to press filtering or centrifugation to remove celite and other impurities and collecting a purified fraction IV suspension; treating the purified fraction IV suspension with solvent/detergent (SD) virus inactivation and obtaining a virus inactivated fraction IV suspension; subjecting the virus inactivated fraction IV suspension to cation chromatography and obtaining a chromatographed suspension; eluting at least one protein from the chromatographed suspension with an eluting agent and obtaining at least one eluted fraction wherein the at least one protein comprises transferrin, human albumin, apolipoprotein A-I (APOA1), or alpha-1 antitrypsin ((A1AT)); and purifying the at least one protein from the eluted plasma fraction.

11. A method according to claim 10, wherein fraction IV is dissolved in a low temperature buffer.

12. A method according to claim 10, wherein suspended fraction IV is cleared by a depth filter.

13. A method according to claim 10, wherein the purified fraction IV suspension is treated with polysorbate 80 and tri(n-butyl)phosphate (TNBP) for virus inactivation at 25 C. for 6 hours.

14. A method according to claim 10, further comprising: dialyzing the at least one eluted fraction; concentrating the at least one eluted fraction; adjusting the pH level of the at least one eluted fraction; and adding a stabilizing agent to the at least one eluted fraction.

15. A method according to claim 10, further comprising subjecting the chromatographed suspension to 20 nm direct flow virus filtration for virus removal.

16. A method according to claim 10, further comprising pasteurizing the chromatographed suspension for virus removal.

17. A method according to claim 10, wherein purifying the at least one eluted fraction further comprises subjecting the at least one eluted fraction to carboxymethyl chromatography.

18. A method according to claim 10, wherein purifying the at least one eluted fraction further comprises subjecting the at least one eluted fraction to butyl chromatography.

19. A method according to claim 10, wherein purifying the at least one eluted fraction further comprises subjecting the at least one eluted fraction to blue chromatography.

20. A method according to claim 10, wherein purifying the at least one eluted fraction further comprises subjecting the at least one eluted fraction to blue chromatography and butyl chromatography.

Description

BRIEF DESCRIPTIONS OF THE DRAWING FIGURES

(1) FIG. 1 is a flowchart of a process of purifying APO from plasma fraction IV according to the present invention;

(2) FIG. 2 is a flowchart of another process of purifying APO from plasma fraction IV according to the present invention;

(3) FIG. 3 is a flowchart of an AFCC process of purifying prothrombin complex from cryopaste in accordance with the present invention;

(4) FIG. 4 is a flowchart of an AFCC process of purifying prothrombin complex from fraction III in accordance with the present invention;

(5) FIG. 5 shows the electrophoresis result of cation chromatography of proteins including transferrin, human albumin, APOA1, PCC and A1AT;

(6) FIG. 6 shows the 2D electrophoresis results of AFOD;

(7) FIG. 7 shows an analysis of a Fraction IV suspension by 2D electrophoresis;

(8) FIG. 8 is a graph showing the relative abundance over time of Q8 Gene Symbol=GC Vitamin D binding protein PrecursorCask isoform 3 of Peripheral plasma membrane protein CASkVIM Vimentin;

(9) FIG. 9 is a graph showing the relative abundance over time of Q13 Gene Symbol=CASK Isoform 3 of Peripheral plasma membrane protein CASKHP HP protein;

(10) FIG. 10 is a graph showing the relative abundance over time of Q15 Gene SymbolCASK Isoform 3 of Peripheral plasma membrane protein CASKIFNA13 IFNA1 Interferon alpha-1/13;

(11) FIG. 11 shows the results of a 2D electrophoresis of prothrombin complex concentrate;

(12) FIG. 12 shows the results of a 2D electrophoresis of Fraction III;

(13) FIG. 13 shows the results of a 2D electrophoresis of cryopaste;

(14) FIG. 14 is a flowchart of a process for purifying AFOD;

(15) FIG. 15 is a graph showing cancer cell proliferation during a 3-day in vitro study of colon and breast cancer cell lines in the presence of varying concentrations of AFOD solution;

(16) FIG. 16 is an image taken on Day 3 after treatment showing the proliferation of Colon cancer cells HCT 116 in 0% AFOD solution;

(17) FIG. 17 is an image taken on Day 3 after treatment showing the proliferation of Colon cancer cells HCT 116 in 2% AFOD solution;

(18) FIG. 18 is an image taken on Day 3 after treatment showing the proliferation of Colon cancer cells HCT 116 in 10% AFOD solution;

(19) FIG. 19 is an image taken on Day 3 after treatment showing the proliferation of Breast cancer cells MCF-7 in 0% AFOD solution;

(20) FIG. 20 is an image taken on Day 3 after treatment showing the proliferation of Breast cancer cells MCF-7 in 2% AFOD solution;

(21) FIG. 21 is an image taken on Day 3 after treatment showing the proliferation of Breast cancer cells MCF-7 in 10% AFOD solution;

(22) FIG. 22 is a graph showing cancer cell proliferation during a 3-day in vitro study of liver and pancreas cancer cell lines in the presence of varying concentrations of AFOD solution;

(23) FIG. 23 is an image taken on Day 3 after treatment showing the proliferation of liver cancer cells HepG2 in 0% AFOD solution;

(24) FIG. 24 is an image taken on Day 3 after treatment showing the proliferation of liver cancer cells HepG2 in 2% AFOD solution;

(25) FIG. 25 is an image taken on Day 3 after treatment showing the proliferation of liver cancer cells HepG2 in 10% AFOD solution;

(26) FIG. 26 is an image taken on Day 3 after treatment showing the proliferation of pancreas cancer cells PAC-1 in 0% AFOD solution;

(27) FIG. 27 is an image taken on Day 3 after treatment showing the proliferation of pancreas cancer cells PAC-1 in 2% AFOD solution;

(28) FIG. 28 is an image taken on Day 3 after treatment showing the proliferation of pancreas cancer cells PAC-1 in 10% AFOD solution;

(29) FIG. 29 is a graph showing the proliferation of a variety of cancer cells over a 3-day trial period in the presence of varying concentrations of AFOD;

(30) FIG. 30 shows the images of FIGS. 16-21 next to one another for comparison;

(31) FIG. 31 shows the images of FIGS. 23-28 next to one another for comparison;

(32) FIG. 32 is a graph showing cell proliferation during a 3-day in vitro study of cervical cancer cell line Hela in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(33) FIG. 33 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Cervical Cancer line Hela in the presence of 16 different solutions (listed on each photo). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(34) FIG. 34 is a graph showing cell proliferation during a 3-day in vitro study of Gastric cancer cell AGS in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(35) FIG. 35 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Gastric Cancer Cell AGS in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(36) FIG. 36 is a graph showing cell proliferation during a 3-day in vitro study of Breast Cancer Cell Line SK-BR-3 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(37) FIG. 37 contains 9 photos taken on Day 3 after treatment, showing the proliferation of Breast Cancer Cell Line SK-BR-3 in the presence of different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, and TB 5 U/mL;

(38) FIG. 38 contains 7 photographs taken on Day 3 after treatment, showing the proliferation of Breast Cancer Cell Line SK-BR-3 in the presence of different solutions (listed on each photograph). The solutions are AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(39) FIG. 39 is a graph showing cell proliferation during a 3-day in vitro study of Ovarian Cancer Cell Line SK-OV-3 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(40) FIG. 40 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Ovarian Cancer Cell SK-OV-3 in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(41) FIG. 41 is a graph showing cell proliferation during a 3-day in vitro study of Lung Adenocarcinoma Cell Line SPC-A-1 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(42) FIG. 42 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Lung Adenocarcinoma Cell Line SPC-A-1 in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(43) FIG. 43 is a graph showing cell proliferation during a 3-day in vitro study of Espohageal Cancer Cell Line TE-1 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(44) FIG. 44 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Espohageal Cancer Cell Line TE-1 in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(45) FIG. 45 is a graph showing cell proliferation during a 3-day in vitro study of Liver Cancer Cell Line BEL-7402 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(46) FIG. 46 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Liver Cancer Cell Line BEL-7402 in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(47) FIG. 47 is a graph showing cell proliferation during a 3-day in vitro study of Pancreas Cancer Cell Line PANC-1 in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(48) FIG. 48 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Pancreas Cancer Cell Line PANC-1 in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(49) FIG. 49 is a graph showing cell proliferation during a 3-day in vitro study of Leukemia Cancer Cell Line Dami in the presence of 16 distinct solutions, listed on the x axis. The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(50) FIG. 50 contains 16 photographs taken on Day 3 after treatment, showing the proliferation of Leukemia Cancer Cell Line Dami in the presence of 16 different solutions (listed on each photograph). The solutions are CK, HA 10%, HA 2%, IVIG 10%, IVIG 2%, HemoRAAS 25 U/mL, HemoRAAS 5 U/mL, TB 25 U/mL, TB 5 U/mL, AFCC 33 U/mL, AFCC 6 U/mL, rFVIII 65 U/mL, rFVIII 15 U/mL, AFOD 2.5%, AFOD 0.5%. and AFOD 0.1%;

(51) FIG. 51 is a graph showing the summary data for the proliferation of Leukemia Cells (Acute Promyelocytic Leukemia Cell T24) in the presence of 0% protein, 2% protein, and 10% protein of AFOD, and Bladder Cancer cells (Bladder Cancer Cell NB4) in the presence of 0% protein, 2% protein, and 10% protein of AFOD during the 3-day trial period.

(52) FIG. 52 shows 6 pictures taken after the trial which show the proliferation of Leukemia Cells (Acute Promyelocytic Leukemia Cell T24) in the presence of 0% protein, 2% protein, and 10% protein of AFOD, and Bladder Cancer cells (Bladder Cancer Cell NB4) in the presence of 0% protein, 2% protein, and 10% protein of AFOD during the 3-day trial period.

(53) FIG. 53 is a graph showing the summary data for the proliferation of Cervical Cancer Cells (Human Cervical Cancer Cell Line Hela) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(54) FIG. 54 is a graph showing the summary data for the proliferation of Gastric Cancer Cells (Human Gastric Cancer Cell Line AGS) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(55) FIG. 55 is a graph showing the summary data for the proliferation of Ovarian Cancer Cells (Human Ovarian Cancer Cell SK-OV-3) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(56) FIG. 56 is a graph showing the summary data for the proliferation of Breast Cancer Cells (Human Breast Cancer Cell Line SK-BR-3) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(57) FIG. 57 is a graph showing the summary data for the proliferation of Esophageal Cancer Cells (Human Esophageal Cancer Cell Line TE-1) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(58) FIG. 58 is a graph showing the summary data for the proliferation of Liver Cancer Cells (Human Liver Cancer Cell Line BEL-7402) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(59) FIG. 59 is a graph showing the summary data for the proliferation of Lung Cancer Cells (Lung Adenocarcinoma Cell Line SPC-A-1) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(60) FIG. 60 is a graph showing the summary data for the proliferation of Pancreas Cancer Cells (Human Pancreas Cancer Cell Line PANC-) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(61) FIG. 61 is a graph showing the summary data for the proliferation of Leukemia Cells (Human Lymphocyte Leukemia Cell Line Jurkat) in the presence of a variety of solutions, listed on the x-axis, at 0%, 2%, and 10% concentrations of protein, respectively.

(62) FIG. 62 is a photograph of five sample vials of bacteria during a microbe test with AFOD RAAS 1 on Staphylococcus aureus, from left to right, having 8 mL AFOD added, having 10 mL AFOD added, having 12 mL AFOD added, a positive control, and a negative control.

(63) FIG. 63 is a series of photographs of three sample vials of bacteria, taken at different times during a microbe test of AFOD on Staphylococcus aureus.

(64) FIG. 64 is a series of photographs of five sample vials of bacteria, taken at different times during a microbe test of AFCC on Staphylococcus aureus.

(65) FIG. 65 is a photograph showing the aorta of a lab animal given a high fat diet after 10 weeks, with a plaque area of 24.3%.

(66) FIG. 66 is a photograph showing the liver tissue (with fat deposits) of a lab animal after 10 weeks of a high fat diet.

(67) FIG. 67 is a photograph showing the aorta of a lab animal without AFOD RAAS 1 and then a normal diet for 4 weeks, with a plaque area of 45.3%.

(68) FIG. 68 is a photograph showing the aorta of a lab animal without AFOD RAAS 1 and then a normal diet for 8 weeks, with a plaque area of 98.5%.

(69) FIG. 69 is a photograph showing the aorta of a lab animal without AFOD RAAS 1 and then a normal diet for 8 weeks, with a plaque area of 78.94%.

(70) Each of FIGS. 70-76 is a photograph of a container having a mixture of a product with a yellow color and a product with a blue that, unlike yellow and blue chemicals, will not turn green.

(71) FIG. 77 is a photograph showing an artery of a lab animal is given a normal diet for 8 weeks.

(72) FIG. 78 is a photograph showing the aortas of two lab animals tested by AFOD RAAS 1, the aortas having a plaque area of 0.

(73) FIG. 79 is a photograph showing the buildup of plaque to a plaque area of 13.29% in the aorta of a lab animal with AFOD RAAS 1-A1 for 8 weeks.

(74) FIG. 80 is a photograph showing the buildup of plaque to a plaque area of 20.5% in the aorta of a lab animal with AFOD RAAS 1-A1 for 8 weeks.

(75) FIG. 81 is a photograph showing the buildup of plaque to a plaque area of 58.4% in the aorta of a lab animal with AFOD RAAS 1.

(76) FIG. 82 is a photograph showing the buildup of plaque to a plaque area of 82.17% in the aorta of a lab animal with AFOD RAAS 1.

(77) FIG. 83 is a photograph showing the buildup of plaque to a plaque area of 47.27% in the aorta of a lab animal with AFOD RAAS 1 for 11 weeks.

(78) FIG. 84 is a photograph showing the buildup of plaque to a plaque area of 40.32% in the aorta of a lab animal with AFOD RAAS 1 for 11 weeks.

(79) FIG. 85 is a photograph showing the buildup of plaque to a plaque area of 51.13% in the aorta of a lab animal with AFOD RAAS 1 for 11 weeks.

(80) The 3.sup.rd generation of APO purification.

(81) The main difference is that urea is no longer required but need 2 steps of chromatography, as is shown in FIG. 1. 1. A method to purify APO from plasma fraction IV, 1) Fraction IV is resuspended in a buffer with pH 3.00-10.00, and the celite and other impurities were separated by press filter or centrifugation, the resulted supernatant was then collected, 2) The APO in the supernatant was then precipitated by adding NaCl and then was spin to collect the paste, 3) The resulted APO was then resuspended and filtered, 4) The resulted suspension was then underwent DEAE ion exchanging chromatography and butyl chromatography, 2. the fraction IV was resuspended in NaAc buffer with pH 3.00-10.00 3. the APO was precipitated by NaCl, pH 3.0-10.0, cool down to 1 to 1 C 4. the paste of APO can be resuspended in WFI or NaCl solution with pH 3.00-10.00 and 0-10 C 5. The resulted suspension is filtered with 0.45 um filter. 6. The chromatography in step 4 is Canion (DEAE) and butyl 7. The purification of APO by chromatography compromising, Canion chromatography, adjust pH of filtered APO suspension to 3.0-10.0 and ionic strength to 15-25 mM, load on DEAE chromatography, low salt wash the DEAE chromatography, and then high salt elute the DEAE chromatography, collect the resulted APO elute, Butyl chromatography, the resulted APO elute from DEAE chromatography is adjusted to pH 3.0-10.0 and low salt wash for impurities, WFI or alkaline buffer wash to collect APO enriched elute 8. The low salt buffer is a buffer containing Tris with pH 3.00-10.0, the high salt buffer is a buffer containing NaCl, the low salt elute buffer is a buffer containing Tris, the alkaline buffer is a buffer containing NaOH with pH 3.0-10.0 9. The resulted high purity of APO is then dialyzed and concentrated with virus inactivation, adding stabilizer and lyophilized.
Process Nr 4.

(82) A process that is separated the Fraction IV into 4 Proteins, as can be appreciated from FIG. 2:

(83) 1. Transferrin

(84) 2. Human Albumin

(85) 3. APO

(86) 4. Alpha 1 Antitrypsin (A1AT)

(87) 5. Transferrin+Human Albumin+APO+Immunoglobulin

(88) 6. Human Albumin+APO+Alpha 1 AntiStrepsin (A1AT)+Immunoglobulin

(89) 7. Human Albumin+APO+Immunoglobulin

(90) 8. Human Albumin+APO+Immunoglobulin+Transferrin+Antitrypsin

(91) Or all products from Nr 5 to Nr 8 can be processed separately and put together at the Non Sterile Final Bulk-Sterile Filtration-Filling-Final Products.

(92) 1. Re suspension of fraction IV and pretreatment

(93) 1) Fraction IV is resuspended in a buffer with pH 3.00-10.00,

(94) 2) The celite and other impurities were separated by press filter of centrifugation; the resulted suspension was then collected,

(95) 3) The suspension was then treated with SD virus inactivation,

(96) 4) The resulted suspension was then subject to a canion chromatography like DEAE,

(97) 5) Proteins were eluted in different fractions,

(98) 6) The different eluted fractions were then further purified,

(99) 2. The fraction IV was dissolved in low temperature buffer to achieve a in homogenous suspension,

(100) 3. The celite in resulted suspension can be removed by press filter or centrifugation,

(101) 4. the suspension was then cleared by depth filter

(102) 5. the resulted suspension was then treated with Tween-80 and TNBP for virus inactivation at 25 C for 6 hours,

(103) 6. The resulted suspension was adjusted pH and ionic strength and then subjected to a canion chromatography like DEAE. The targeted proteins were then binding to the canion chromatography resin, which are transferrin, human albumin, APO and A1AT. The 1.sup.st elution was salt solution to elude the transferrin. The 2.sup.nd elution was then eluded by a high concentration salt solution, which was APO. The 3.sup.rd eluted fraction was human albumin by a low pH solution. Finally the 4.sup.th elution was A1AT which was eluted by a high concentration salt solution.
7. The resulted various elution was then subjected to different chromatography for further purification to achieve a high purity. The 1.sup.st elution fraction was subjected to a CM chromatography. The 2.sup.nd elution fraction was subjected to a butyl chromatography. The 3.sup.rd elution fraction was subjected to a blue chromatography. The 4.sup.th elution fraction was subjected to a blue chromatography and a subsequent butyl chromatography.
8. The resulted protein fractions were then dialyzed and concentrated. The pH was adjusted and stabilizer was then added.
9. The resulted protein solutions were subjected to DV20 filtration for virus removal except human albumin.
10. The human albumin could be virus inactivated by Double pasteurization.
11. The resulted transferrin, APO, human albumin and A1AT can be filled.

(104) AFCCRAAS 1: These processes of protein containing Healthy Good cells in Process 1 and Process 3 below are specially designed for Hemophilia A, B and WvB who have Been infected by HBV, HCV and specially HIV during the early of 1980 when effective process of inactivation of Enveloped viruses has not been introduced.

(105) 1. Process to separate Factor II, VII, IX, and X Transferrin, Human Albumin, APO and A1AT (ProthoRAAS) from fraction III which also contain at least 20 additional proteins.

(106) 2. Process to separate Factor II, VII, IX, and X (ProthoRAAS)+Human Albumin (AlbuRAAS)+Immunoglobulin (GammaRAAS)

(107) 3. Process to separate Factor II, VII, IX and X from Cryopaste

(108) 4. Process to separate Factor II, VII, IX and X from Cryopaste+ATA1 APO+ Human Albumin (AlbuRAAS) and Immunoglobulin (GammaRAAS)

(109) 5. Process to separate Thrombin (ThrombiRAAS) from Fraction III

(110) 6. Process to combine all protein from Fraction III.

(111) Description

(112) 1. As can be seen in FIG. 3, this process describes a process to purify prothrombin complex from cryoprecipitaiton, which comprises, 1) Re-constitute cryopaste in buffer containing 3,000 U/kg heparin, 2) Adjust pH and temperature 3) Complete mix at room temperature for 3 to 5 hours 4) Filter the resulted suspension with 10CP+90SP filter 5) Solvent detergent virus inactivation of resulted suspension 6) weak anion exchange chromatography of SD virus inactivated suspension 7) Twice washing of weak anion exchange chromatography 8) Elute weak anion exchange chromatography 2 to 3 times 9) Collect the result elution and ultra-filter with 10K membrane 10) Adjust pH of resulted elution 11) Adjust the activity of human FIX in the resulted elution 12) Aseptic filtration and nano filtration for virus removal 13) Filling and lyophilization
Description 2. As can be seen in FIG. 4, this process describes a process to purify prothrombin complex from fraction III, which comprises, 14) Re-constitute cryopaste in buffer, 15) Adjust pH and temperature 16) PEG precipitation of resulted fraction III suspension 17) Centrifugation and collect the supernant 18) Filter the resulted suspension with 10CP+90SP filter 19) Solvent detergent virus inactivation of resulted suspension 20) weak anion exchange chromatography of SD virus inactivated suspension 21) Twice washing of weak anion exchange chromatography 22) Elute weak anion exchange chromatography 2 to 3 times 23) Collect the result elution and ultra-filter with 10K membrane 24) Adjust pH of resulted elution 25) Adjust the activity of human FIX in the resulted elution 26) Aseptic filtration and nano filtration for virus removal 27) Filling and lyophilization
AFOD is High Density Lipoprotein (ApoA1):

(113) Reference is made to FIGS. 5 and 6. The three dots in our analysis of AFOD are all ApoA1. The difference showed in 2D electropherosis of FIG. 6 might be due to different isoform of ApoA1 or Apo in the Apo family. 1. HDL (ApoA1): AFOD RAAS 1 (Trade mark) contains purified ApoA1 in the process described Nr 1 (China Patent granted 200610147503.7) Nr1&N2 (US 2009/0286960 A1) Nr 3,4 (U.S. 61/457,380) with a purity of 96% and the products must be free of all HIV1,2, HCV, HBV viruses and contain very good level of High Density Lipoprotein (HDL) which is GOOD CHOLESTEROL, No value or very low value of VLDL (Very low density Lipoprotein) and no value or very low value of LDL (Low Density Lipoprotein), both of which are BAD CHOLESTEROL. Potential applications: Cholesterol, Angina, hyperlipidemia, Clean plaque, fat on liver, Life span, Control of Obesity, Hypertension, Prevention of Heart attack, Prevention of Stroke, Prevention of Paralysis due to the stroke and other potential indications as described. 2. AFODRAAS 2 (Human Albumin+ApoA1) In addition to current clinical applications for AlbuRAAS Potential applications for trauma management/Arthritis/Schizophrenia/Depression/Certain types of cancers Lung, Pancreas, Kidney, Liver, Prostate, Breast and other potential indications 3. AFODRAAS 3 (Intravenous Immuno Globulin+ApoA1) for current clinical applications for GammaRAAS and for potential applications for all blood (liquid) cancers as described in abstract) 4. AFODRAAS 4 (Factor VIII+ApoA1) for the current clinical applications of HemoRAAS and for potential applications for Hepatitis B, Hepatitis C and HIV 1,2, Bleeding complications/Bone Surgery in Patients with Hemophilia A (orthopedics, liver, pancreas, cancer of the gastrointestinal tube and eventually build up Coagulation so patients will no longer need to use Factor VIII for hemophilia A and all solid tumor and blood cancers/ 5. AFODRAAS5 (Prothombin Complex Concentrate+ApoA1) for the current clinical applications of ProthoRAAS and for potential applications for Hepatitis B, Cirrhosis, and other hepatic trouble like biliary tree obstruction Hepatitis C, HIV 1,2 Bleeding/Bone Complications and surgeries for Hemophilia B and Hemophilia A with inhibitor and eventually build up coagulation so patients will no longer need to use Factor IX or PCC and all solid tumor and blood cancers 6. AFODRAAS6 (Thrombin+ApoA1) for the current clinical applications of ThrombiRAAS and for potential applications for Gastric and duodenal ulcer, ulcers and other problems of colon (large intestine) 7. AFODRAAS7 (Fibrinogen+ApoA1) for the current clinical applications of FibroRAAS and for potential applications for Trauma Management and other potential indications as described 8. AFODRAAS8 (Fibrin Sealant+ApoA1) for the current clinical applications of FibrinGluRAAS and potential TOPICAL APPLICATIONS for ALL SOLID TUMOR CANCERS which can be operated and cancers have not been spread to other parts of body. 9. AFODRAAS 9 (ApoA1+Human Albumin (AlbuRAAS)+Alpha 1 Antitrypsin (A1AT)+Transferrin for all potential indications as described 10. AFODRAAS 10 (ApoA1+Human Albumin+Alpha 1 Antitrypsin (A1AT) for all potential indications as described. 11. AFODRAAS 11 (ApoA1+Human Albumin+Transferrin) for all potential indications as described. 12. AFODRAAS 12 (ApoA1+Alpha 1 Antitrypsin ((A1AT)) for all potential indications as described. 13. AFODRAAS 13 (ApoA1+Transferrin for all potential indications as described. 14. AFODRAAS 14 (Alpha 1 Antitrypsin ((A1AT))+Transferrin) for all potential indications as described. 15. AFODRAAS 15 (Transferrin) for All potential indications as described.

(114) In our analysis of Fraction IV suspension by 2 D electropherosis, as shown in FIG. 7, the following proteins were found in the Fraction IV suspension:

(115) The main proteins found in Fraction IV suspension are: Transferrin, Human Albumin, Alpha 1 AntiStrepsin, and ApoA1

(116) The rest of the proteins are: SEMENOGELIN-1, HAPTOGLOBIN, VIMENTIN, NESPRIN-2, INTERFERON ALPHA 1/13, HP PROTEIN, VITAMIN D-BINDING, ALPHA-FETOPROTEIN, CASK, AMYLOID PRECURSOR, NEUREXINS AND SYNDECANS.

(117) SEMENOGELIN-1: The protein encoded by this gene is the predominant protein in semen

(118) HAPTOGLOBIN: In Blood Plasma, Haptoglobin binds free hemoglobin (Hb) released from erythrocytes with high affinity and thereby inhibits its oxidative activity.

(119) VIMENTIN is a member of the intermediate filament family of proteins that is especially found in connective tissue. They, along with microtubules and actin microfilaments, make up the cytoskeleton

(120) THE NESPRINS are a family of proteins that are found primarily in the outer nuclear membrane. Nesprin-1 and Nesprin-2 bind to actin filaments.

(121) VITAMIN-D BINDING PROTEIN belongs to the albumin gene family, together with Human serum albumin and alpha-fetoprotein. It is a multifunctional protein found in plasma, ascetic fluid, cerebrospinal fluid and on the surface of many cell types. It binds to Vitamin D and its plasma metabolites and transport them to target tissues.
CASK PROTEIN Peripheral plasma membrane protein CASK is a protein that in humans is encoded by the CASK gene. This protein is a multi domain scaffolding protein with a role in synaptic transmembrane protein anchoring and ion channel trafficking. It interacts with the transcription factor TBR1 and binds to several cell-surface proteins including amyloid precursor protein, neurexins, and syndecans.

(122) FIG. 8 relates to Q8 Gene Symbol=GC Vitamin D binding protein PrecursorCask isoform 3 of Peripheral plasma membrane protein CASkVIM Vimentin

(123) FIG. 9 relates to Q13 Gene Symbol=CASK Isoform 3 of Peripheral plasma membrane protein CASKHP HP protein

(124) FIG. 10 relates to Q15 Gene SymbolCASK Isoform 3 of Peripheral plasma membrane protein CASKIFNA13; IFNA1 Interferon alpha-1/13

(125) AFCC is Prothombin Complex Concentrate (ProthoRAAS) a combination of blood clotting factors II, VII, IX and X or Factor IX.

(126) Factor IX is one of several factors made in the liver with similar structural properties.

(127) Together with factors II (Prothombin), VII (proconvertin) and X (Stuart-Prower factor), factor IX (antihaemophilic factor B) comprises the so called prothombin complex group of factors, also known as PPSB factors.

(128) Owing to their similarity, the proteins in this group are usually isolated together in fraction III in the Cohn alcohol fractionation process.

(129) They are then purified to give preparations containing all four factors, Prothombin complex concentrates (PCC).

(130) ProthoRAAS are indicated currently for: Haemophilia BProphylaxis, Bleeding, Surgery Haemophilia AInhibitor treatment Liver diseaseAcute and chronicactive hepatitis, cirrhosis Vitamin K deficiencyOral anticoagulants, Obstructive jaundice, Malabsorption, changes in intestinal flora (antibiotics, Morbus Crohn, ulcerative colitis) DIC (Disseminated Intravascular Coagulation)Infection, Liver disease, obstetrical emergencies, surgical complications. 16. AFCC RAAS1 (Trade mark) (ProthoRAAS+ApoA1+AT-III) for the current indications as above together with all potential indications as described. 17. AFCC RAAS 2 (Trade mark) (Prothombin Complex Concentrate (ProthoRAAS)+ApoA1+Human Albumin (AlbuRAAS)), for the current indications as above together with all potential indications as described. 18. AFCC RAAS 3 (Trade mark) (Prothombin Complex Concentrate (ProthoRAAS)+ApoA1+Intravenous Immuno Globulin (GammaRAAS)), for the current indications as above together with all potential indications as described. 19. AFCC RAAS 4 (Trade mark) (Prothombin Complex Concentrate (ProthoRAAS)+ApoA1+Intravenous Immuno Globulin (GammaRAAS), +Human Albumin (AlbuRAAS)), for the current indications as above together with all potential indications as described. 20. AFCC RAAS 5 (Trade mark) (Prothombin Complex Concentrate (ProthoRAAS)+ApoA1+Intravenous Immuno Globulin (GammaRAAS), +Human Albumin (AlbuRAAS) and Fibrinogen (FibroRAAS)), for the current indications as above together with all potential indications as described. 21. AFCC RAAS 6 (Trade mark) (Prothombin Complex Concentrate (ProthoRAAS)+ApoA1+Fibrinogen (FibroRAAS)), for the current indications as above together with all potential indications as described.
2D Electropherosis of PCC:

(131) Results of 2D eletropherosis of PCC not only the above four factors proteins but also show a lot more proteins (Dots) that are being identified, as can be seen in FIG. 11.

(132) 2D Electropherosis of Fr. III:

(133) 2D electropherosis of Fraction III like Fraction IV contains a lot more of proteins other than Thrombin, Prothombin Complex, as can be seen in FIG. 12. All these proteins are also being identified.

(134) 2D Electropherosis of Cryopaste:

(135) 2 D electropherosis of Cryopaste results also show some other proteins which are being identified Beside Fibrinogen and Factor VIII, as can be seen in FIG. 13.

(136) In Vitro/Vivo Studies

(137) 1.sup.st Production of 10 Batches totaling 200 grams for use in vivo study of Rabbits at Fudan University, Shanghai, China.

(138) The 1.sup.st 200 g AFOD was purified in East China University of Science and Technology using Process No. 1, a flow chart for which is presented in FIG. 14. The pilot production capability of that lab is about 5 kg per time. The purification process was modified according to their equipment on site but the flow was same as we have done in this December The major differences are as following 1) The input of production was 5 kg of Fraction IV paste and yield was about 25 g AFOD each time. Dr. Li Chun Zhou produced 10 lots there to get 200 g AFOD; 2) The column used for AFOD purification was about 5 L DEAE chromatography; 3) The centrifugation for collecting AFOD enriched pellet was swing basket rotor centrifuge 4) Standing precipitation was used to get rid of celite; 5) For filling, manual filling was adopted; 6) No virus inactivation was used in the whole process.

(139) The resulted AFOD was about 200 g for total 10 lots and all of AFOD were lyophilized.

(140) The stabilizer used was mannitol and this product has been used to perform clinical studies on 52 rabbits in summer of 2008.

(141) 2.sup.nd Pilot Production of 3 Lots at Shanghai RAAS Blood Products Co Ltd 2000 vials of AFOD RAAS (about 200 g) have been used in the following in vitro studies: 1. Studies of 5 cancer cells line at RuiJin Hospital Shanghai China. 2. Studies of 3 cancer cells line at R/D Lab Shanghai RAAS. 3. Studies of Bacteria at Microbiology Lab, Shanghai RAAS 4. Studies of Viruses HIV1,2 at NAT Lab, Shanghai RAAS

(142) In the most recent pilot production, which was conducted in last December at our Plant, we totally conducted 3 lots of production. It was 25 kg Fraction IV for the 1.sup.st lot, 50 kg for the rest of 2 lots. The total yield was about 2000 vials of AFOD (about 200 g). There were about 440 bottles of lyophilized, 660 vials of liquid with human albumin for stabilizer and another 880 bottles of liquid with mannitol for stabilizer. 1) We use continuous tube centrifugation to collect the AFOD enriched pellet; 2) Using Filtration to get rid of celite; 3) A 15 L DEAD chromatography for AFOD purification; 4) Virus inactivation including SD and DV 20; 5) Automatic filling.

(143) AFCC RAAS 1: A current product of Shanghai RAAS Blood products Co Ltd approved for Sales in China and has been exported to a certain country around the globe. Product has been manufactured at large industrial scale.

(144) Product has been used in the VITRO studies at Shanghai RAAS Blood Products Co Ltd 1. 3 Cancer Cell lines at R/D Lab 2. Bacteria at Microbiology Lab 3. HIV 1+2 Testing at NAT Lab
In Vitro Studies of Cancer Cell Lines:

(145) Procedures to test Cancer cells at RuiJin Hospital in Shanghai, China.

Cell Proliferation Assay by Using CCK 8 Test Kit

Check all Reagents at Least 3 Days Before Assay

(146) Reagents:

(147) 1. Cancer cell line: Human colon cancer cell line (HCT-116), Human Breast cancer cell line (MCF-7), Human liver cancer cell line (HepG2), Human pancreatic cancer cell line (PAC-1) 2. CCK 8 (cell counting kit-8): Dojindo molecular technologies, Inc. (Maryland, US), product code # CK04-11 3. Cell culture medium:

(148) TABLE-US-00002 DMEM - high glucose 4.5 gm/L Gibco (invitorgen) 11965084 ANTIBIOTIC ANTIMYCOTIC Invitrogen 15240062 FBS Hyclone SH30071.03 4. AFOD: lyophilized formulation
Procedures:
Day 3 Before Treatment 1. Pre-Warm up cell culture medium to 37 C (DMEM/10% FBS/antibiotics) 2. Seed cells to a 15-cm dish and let cells grow 2-3 days to reach 2*10.sup.8
Day 1 Before Treatment 1. Pre-Warm up cell culture medium to 37 C (DMEM/2% FBS/antibiotics) 2. Seed cell at density of 2000/well in a 96-well plate. Triplicate every treatment condition. 1) 0% AFOD 2) 2% AFOD 3) 10% AFOD 3. Leave cells for overnight growth
Day 0 of Treatment 1. Pre-Warm up cell culture medium to 37 C (DMEM/2% FBS/antibiotics) 2. Change fresh medium in each for cells as following 1) 0% AFOD: Cancer cell+fresh DMEM/2% FBS/antibiotics 2) 2% AFOD: Cancer cell+1-4 diluted 10% AFOD with fresh DMEM/2% FBS/antibiotics 3) 10% AFOD: Cancer cell+lyophilized AFOD dissolved in fresh DMEM/2% FBS/antibiotics 4) Negative control: lyophilized AFOD dissolved in fresh DMEM/2% FBS/antibiotics only 3. Incubate cells for 2-3 days and observe cell growth everyday

(149) Day 1-2 after Treatment

(150) Observation the proliferation of cells under microscopy

(151) Day 3 after Treatment 1. Discard cell culture medium 2. Take picture of cell 3. Conduct the CCK8 assay according to manufacturer's instruction

(152) The antibiotics added in cell culture medium is basically to prevent the potential bacterial or fugal contamination during the culture. The antibiotics they added contains penicillin, streptomycin, and amphotericin B. Basically it won't kill cell and it is a routine recipe in animal cell culture. And they also include a control in which cells are cultured with DMEM/FBS/antibiotics only.

(153) Reference is made to FIGS. 15-28.

The AFOD Anti-Tumor In Vitro Test Result

(154) Procedures of Testing of cancer cells LS 174T (Colon), AGS (Gastric), and 45 (Gastric cancer cells at Shanghai RAAS R/D Lab procedures for both product AFODRAAS 1 and AFCCRAAS 1

(155) The dose dependent cell killing effect of AFOD by CCK8 assay is shown in FIG. 29.

(156) Pictures of the cancer cells are shown in FIGS. 30 and 31.

(157) Method and materials used at Shanghai RAAS Blood Products Co Ltd R/D Dept.

(158) Reagents:

(159) 1. Cancer cell line: Human colon cancer cell line (HCT-116), Human Breast cancer cell line (MCF-7), Human liver cancer cell line (HepG2), Human pancreatic cancer cell line (PAC-1)

(160) 2. CCK 8 (cell counting kit-8): Dojindo molecular technologies, Inc. (Maryland, US), product code # CK04-11

(161) 3. Cell culture medium:

(162) TABLE-US-00003 DMEM - high glucose 4.5 gm/L Gibco (invitorgen) 11965084 ANTIBIOTIC ANTIMYCOTIC Invitrogen 15240062 FBS Hyclone SH30071.03
4. AFOD: lyophilized formulation
Procedures:
Day 3 Before Treatment
5. Pre-Warm up cell culture medium to 37 C (DMEM/10% FBS/antibiotics)
6. Seed cells to a 15-cm dish and let cells grow 2-3 days to reach 2*10.sup.8
Day 1 Before Treatment
4. Pre-Warm up cell culture medium to 37 C (DMEM/2% FBS/antibiotics)
5. Seed cell at density of 2000/well in a 96-well plate. Triplicate every treatment condition. 4) 0% AFOD 5) 2% AFOD 6) 10% AFOD
6. Leave cells for overnight growth
Day 0 of Treatment
4. Pre-Warm up cell culture medium to 37 C (DMEM/2% FBS/antibiotics)
5. Change fresh medium in each for cells as following
7. 0% AFOD: Cancer cell+fresh DMEM/2% FBS/antibiotics
8. 2% AFOD: Cancer cell+1-4 diluted 10% AFOD with fresh DMEM/2% FBS/antibiotics
9. 10% AFOD: Cancer cell+lyophilized AFOD dissolved in fresh DMEM/2% FBS/antibiotics
10. Negative control: lyophilized AFOD dissolved in fresh DMEM/2% FBS/antibiotics only
6. Incubate cells for 2-3 days and observe cell growth everyday

(163) Day 1-2 after Treatment

(164) Observation the proliferation of cells under microscopy

(165) Day 3 after Treatment

(166) 11. Discard cell culture medium

(167) 12. Take picture of cell

(168) 13. Conduct the CCK8 assay according to manufacturer's instruction

(169) Further in vitro studies of more cancer cell lines are shown in FIGS. 32-61.

(170) In vitro studies of bacteria performed at Shanghai RAAS Microbiology Lab Due to the large dosage to kill bacteria while product available are limited and saved for animal study of other diseases and cancers, this study does not completely kill all bacteria.

(171) We performed bacteria experiments on AFOD RAAS 1 by both increasing the dosage of AFOD RAAS 1 and decreasing the density of testing bacteria. The current results will be listed in the following tables:

(172) The amount of + in the following tables doesn't represent the accurate number of the testing bacteria remained in the medium after incubation; it represents the relative amount of them.

(173) 1. The microbe test with AFOD RAAS 1 on Staphylococcus aureus (a kind of aerobes)

(174) TABLE-US-00004 AFOD added (ml) 16 hours 24 hours 40 hours 72 hours 0.5 ml Staphylococcus None ++ ++++ ++++++++ aureus + 0 ml AFOD 0.5 ml Staphylococcus None ++ ++++ ++++ aureus + 8 ml AFOD 0.5 ml Staphylococcus None ++ ++++ ++++ aureus + 10 ml AFOD 0.5 ml Staphylococcus None None ++++ ++++ aureus + 12 ml AFOD

(175) In this experiment, we increased the dosage of AFOD (8 ml; 10 ml; 12 ml) to the medium. We found that the liquid culture medium converted to solid medium when we added 8 ml or more AFOD to the liquid medium and incubated for 24 hours. I think the main reason is the concentration of AFOD we added was too high. So maybe it's a little difficult to increase the dosage of AFOD anymore (such as 14 ml; 16 ml) even if the testing Staphylococcus aureus can still grow up when we added 12 ml AFOD to the medium.

(176) 2. The microbe test with AFOD RAAS 1 on Micrococcus luteus (a kind of aerobes)

(177) TABLE-US-00005 AFOD added (ml) 16 hours 24 hours 40 hours 72 hours 0.5 ml Micrococcus None None ++++ ++++++++ luteus + 0 ml AFOD 0.5 ml Micrococcus None None ++ ++++++++ luteus + 8 ml AFOD 0.5 ml Micrococcus None None ++ ++++++++ luteus + 10 ml AFOD 0.5 ml Micrococcus None None ++ ++++++++ luteus + 12 ml AFOD
3. The microbe test with AFOD RAAS 1 on Candida albicans (a kind of fungus)

(178) TABLE-US-00006 AFOD added (ml) 48 hours 72 hours 96 hours 120 hours 0.5 ml Candida ++ ++++ ++++++++ ++++++++ albicans + 0 ml AFOD 0.5 ml Candida ++ ++++ ++++++++ ++++++++ albicans + 4 ml AFOD 0.5 ml Candida ++ ++++ ++++++++ ++++++++ albicans + 8 ml AFOD
4. The microbe test with AFOD RAAS 1 on Aspergillus niger (a kind of fungus)

(179) TABLE-US-00007 48 AFOD added (ml) hours 72 hours 96 hours 120 hours 0.5 ml Aspergillus ++ ++++ ++++++++ ++++++++ niger + 0 ml AFOD 0.5 ml Aspergillus ++++ ++++++++ ++++++++ ++++++++ niger + 4 ml AFOD 0.5 ml Aspergillus ++++ ++++++++ ++++++++ ++++++++ niger + 8 ml AFOD

(180) Reference is made to FIGS. 62-64.

(181) HIV NAT Testing of AFODRAAS 1 and AFCC RAAS 1

(182) TABLE-US-00008 Total HCV years samples positive % positive Percentage positive Percentage positive Percentage positive Percentage positive Percentage 2006 633480 78 0.0123% 162 0.0256% 160 0.0253% 5 0.0008% 2 0.0003% 7 0.0011% 2007 345620 70 0.0203% 50 0.0145% 93 0.0269% 0 0.0000% 5 0.0014% 6 0.0017% 2008 428422 23 0.0054% 43 0.0100% 108 0.0252% 0 0.0000% 1 0.0002% 18 0.0042% 2009 524299 17 0.0032% 45 0.0086% 110 0.0210% 0 0.0000% 3 0.0006% 0 0.0000% 2010 554297 53 0.0096% 31 0.0056% 131 0.0236% 0 0.0000% 5 0.0009% 0 0.0000% 2006~ 2486118 241 0.0097% 331 0.0133% 602 0.0242% 5 0.0002% 16 0.0006% 31 0.0012% 2010

(183) The above table show HCV, HIV1,2 and HBsAg on the left are Elisa testing HCV-RNA HIVRNA, HBV DNA are results of NAT Testing at Shanghai RAAS NAT Laboratory for A total of units of plasma 2,486,188 during five years period from 2006 to 2010. Out of these number of units, there were 241 tested positive HCV by Elisa, confirmed HCV positive by NAT is only 5 units so a total of 236 units are false positive by Elisa Method. From 2007 to 2010 there is no HCV positive from our donor population therefore we have No Hepatitis B Virus to test for our study. Further study will be done when receiving samples from Infectious disease hospital in Shanghai which will also carry out animal study for all HIV, HCV and HBV.

(184) For HIV 1,2 by Elisa had 331 positive and confirmed by NAT is only 16 so false positive is 315 units. Luckily our R/D still had 1 sample of positive HIV1,2 to conduct this study. For HBV we had a total of 602 positive by Elisa and 31 confirmed by NAT total false positive by Elisa is 571 samples. In 2009 and 2010 we had no positive sample confirmed by NAT.

(185) To avoid the large dosage of limited AFODRAAS and AFCCRAAS, NAT Lab has diluted the positive plasma sample to weaken the presence of HVI1,2 virus to the level of 1:3200 and the first test result is as below for the HIV Positive Plasma to use as A CONTROL.

(186) HIV positive plasma (60051215) 5.9E+6 IU/ml

(187) the plasma (1:100) 3E+5 IU/ml

(188) the plasma (1:200) 1.1E+5 IU/ml

(189) the plasma (1:400) 5.4E+4 IU/ml

(190) the plasma (1:800) 2.9E+4 IU/ml

(191) the plasma (1:1600) 1.4E+4 IU/ml

(192) the plasma (1:3200) 5.7E+3 IU/ml

(193) Sample incubation at room temperature until Day 3 Concentration

(194) 20 ml of the plasma (1:800)+2 bottles of AFODRAAS1 2.3E+4 IU/ml

(195) 20 ml of the plasma (1:1600)+2 bottles of AFODRAAS 1 5.9E+3 IU/ml

(196) 20 ml of the plasma (1:3200)+2 bottles of AFODRAAS1 1.9E+3 IU/ml

(197) 20 ml of the plasma (1:800)+2 bottles of AFCCRAAS 1 1.3E+4 IU/ml

(198) 20 ml of the plasma (1:1600)+2 bottles of AFCC RAAS 1 2.9E+3 IU/ml

(199) 20 ml of the plasma (1:3200)+2 bottles of AFCC RAAS 1 56 IU/ml

(200) As shown in the table above, we have observed HIV1,2 Positive Sample mixed With AFODRAAS 1 and AFCC RAAS 1 have reduced Number of IU/MI

(201) The second test results show as below there is a significant drop from 29000 IU/ml of Positive Plasma Control down to 4500 IU/ml due to the decade of HIV1,2 Virus in the plasma; Thus we conduct further test to assure by using NON DILUTED SAMPLES even though we have to increase dosage of AFOD RAAS1 and AFCCRAAS 1 to cope with high concentration of HIV1,2 virus. However it is an experimental test that needs further STUDIES.

(202) TABLE-US-00009 Initial Sample concentration Day 3 Concentration the positive 29000 IU/ml 4500 IU/ml plasma(1:800) the positive 14000 IU/ml 2100 IU/ml plasma(1:1600) the positive 5700 IU/ml 670 IU/ml plasma(1:3200) 20 ml of the positive 29000 IU/ml 13000 IU/ml plasma(1:800) + 2 bottles of AFODRAAS 1 20 ml of the positive 14000 IU/ml 6300 IU/ml plasma(1:1600) + 2 bottles of AFODRAAS 1 20 ml of the positive 5700 IU/ml 2500 IU/ml plasma(1:3200) + 2 bottles of AFODRAAS1 20 ml of the positive 29000 IU/ml 4600 IU/ml plasma(1:800) + 2 bottles of AFCC RAAS1 20 ml of the positive 14000 IU/ml 620 IU/ml plasma(1:1600) + 2 bottles of AFCC RAAS 1 20 ml of the positive 5700 IU/ml 130 IU/ml plasma(1:3200) + 2 bottles of AFCC RAAS1 20 ml of the positive 29000 IU/ml 22000 IU/ml plasma(1:800) + 1 bottle of AFODRAAS + 1 bottle of AFCC 20 ml of the positive 14000 IU/ml 2800 IU/ml plasma(1:1600) + 1 bottle of AFODRAAS + 1 bottle of AFCC 20 ml of the positive 5700 IU/ml 110 IU/ml plasma(1:3200) + 1 bottle of AFODRAAS + 1 bottle of AFCC

In Vivo Study

Experimental Design and Results of Pilot Scale

Pre-clinical Animal Test of AFOD RAAS 1

For the Antiatherogenic and Cholesterol-Lowing Properties

(203) 1. Purpose of the Experiments:

(204) 1.1 To test the effects of AFOD RAAS 1 for the suppression of fatty streak lesions.

(205) 1.2. To test the efficiency of making animal models for atherosclerosis

(206) 1.3 To test the efficacy and dosage for AFOD RAAS 1 in the suppression of fatty streak lesions and cholesterol-related plasma indicators

(207) 2. Experimental Design

(208) 2.1 Experimental Animals.

(209) Male New Zealand white-ear or other strain healthy rabbits (2.0 kg body weight, 4 in each group).sup.were used in the experiment.

(210) 2.2 Experimental Model Construction

(211) The rabbits were fed with normal diet under regular lab conditions for 5-10 days. The rabbits were fasted for 12 hrs before the beginning of the experiments. Blood parameters were then tested as the normal level of plasma indicators. The animals were then randomly grouped for the experiment.

(212) 2.3 Treatments

(213) After grouping of the experimental animals, they were switched to high-fat diet. Body weight and plasma parameters were tested and recorded once every two weeks until indicators shown to have lipid metabolism disorders and the formation of obvious fatty streak lesions in blood vessels. The animals were switched from high-fat diet to normal diet. These are the grouping of the experimental animals: (1) positive control group, (2) AFOD RAAS 1-Al treatment group was further divided into high, medium and low three dose sub-groups. During the first 4 weeks of the AFOD RAAS 1-treatment, plasma parameters and animal general conditions were carefully monitors and recorded. At the end of the experiments, the lab animals were sacrificed for pathological and anatomical analysis.

(214) 2.4 Parameters Tested:

(215) 1) blood cholesterol-related parameters:

(216) TC: total cholesterol TG: tri-glyceride LDL-C: low density lipoproteinLDL VLDL-C: very low density lipoproteinVLDL HDL-C: high density lipoproteinHDL TC/HDL-C or (LDL-C+VLDL-C)/HDL-C: ratio
2) pathological tests pathology in aorta (main artery)
3) Liver index
3. Experimental Process

(217) A total of 52 rabbits were purchased at different time, four of them were used as normal control and fed with normal diet the whole time during the experiments. There rest of the animals was switched to high fat diet.

(218) Some of the lab animals showed stomach symptoms after switched to high-fat diet and died within 4 weeks. From week 7 to week 10, 6 more lab animals died for the same reason. At week 10 and 11, two animals were sacrificed as animal model control. These two animals were dissected to obtain aorta, heart and liver tissue samples. Observation: the appearance of the aorta and liver were observed and recorded. The inside surface of aorta has obvious fatty streak deposit and lesion. The liver tissue has white fatty tissue deposit. Taken together, the construction of the animal models was successful. Two lab animals from the normal control group were also sacrificed and dissected. No abnormality of aorta and liver tissues were observed.

(219) The rest 34 rabbits were then switched to normal diet and grouped into animal-model group, positive control group and low, medium and high dosage AFOD RAAS 1-Al treatment groups.

(220) Animal-model group (no AFOD RAAS 1 and normal diet): 4

(221) Positive control: 5

(222) AFOD RAAS 1-Al treatment group:

(223) High dosage group (100 mg/each), average body weight 2.8 kg: 11

(224) Medium dosage group (50 mg/each): 8

(225) Low dosage group (25 mg/each): 6

(226) After 4 weeks of treatment, two lab animals from the high-dosage group were sacrificed and dissected. No significant changes of fatty streak lesions were observed. Based on this observation, all of the lab animals from the experimental groups were switched to high-dosage AFOD RAAS 1 treatment, which is 100 mg/each.

(227) 4. Experimental Results

(228) 4.1 Duration of the Animal Model Construction

(229) After feeding of the lab animals with high-fat diet (1.5% cholesterol, 3% lard, and normal feed) for four weeks, all cholesterol-related plasma parameters were increased significantly (see attached data sheet). At week 4, one of the lab animals were sacrificed and showed limited amount of fatty streak lesions. At week 10 and week 11, five lab animals were sacrifices and dissected. Obvious fatty streak lesions can be observed on the inside surface of the aorta. Fat deposit can also be observed on the liver tissues.

(230) Conclusion: At week 10-11, fatty streak lesions were formed.

(231) 4.2 Successful Rate for Model Construction

(232) During the animal model construction, 7 animals died during the first 4 weeks of high-fat diet due to stomach symptoms. Between week 7-10, 6 more lab animals died because of high-fat. The mortality rate is 16.7%. These lab animals were also dissected and 90% of them the aorta tissue showed fatty streak lesions occupied 20% of the total area.

(233) Conclusion: the successful rate of model construction is 60%.

(234) Models (high fat diet, execute at 10 weeks, the picture of the aorta, with a Plaque area=24.3%) is shown in FIG. 65. Reference is also made to FIG. 66.

(235) Control group (build up the animal models, without AFOD RAAS 1, then normal diet for 4 weeks) Plaque area=45.3%, as can be appreciated from FIG. 67.

(236) Control groups (build up the animal models, without AFOD RAAS 1, then normal diet for 8 weeks). FIG. 68 shows a Plaque area=98.5%, and FIG. 69 shows a plaque area=78.94%.

(237) 4.3 Plasma Parameters

(238) 1) First 8 weeks of treatment (n=7, for the first 4 weeks, AFOD RAAS 1 was administered once a week and 100 mg/each; in the following 4 weeks, 50 mg/each were administered twice a week)

(239) TABLE-US-00010 TC/ HDL- Weight TG TCH VLDL-C HDL-C LDL-C C Start 2.164 0.967 1.152 0.870 0.748 0.282 1.938 Before 2.7 5.191 36.153 14.996 8.261 21.157 6.560 After 2.79 1.17 3.69 1.09 1.46 2.60 3.000
2) At week 11 of the experiment (n=7, for the first 4 weeks, AFOD RAAS 1 was administered once a week and 100 mg/each; in the following 4 weeks, 50 mg/each were administered twice a week, for the last 3 weeks, 100 mg/each were administered once a week)

(240) TABLE-US-00011 TC/ HDL- Weight TG TCH VLDL-C HDL-C LDL-C C Start 2.2 0.93 1.430 0.958 0.432 0.472 4.185 Before 2.45 4.507 34.683 15.443 10.168 19.24 3.667 After 2.65 1.94 3.322 1.14 1.17 2.19 3.844
3) Positive control (Crestor, administered 4 weeks)

(241) TABLE-US-00012 TC/ HDL- Weight TG TCH VLDL-C HDL-C LDL-C C Start 2.25 0.450 0.946 0.509 0.539 0.437 1.844 Before 2.85 9.122 20.339 9.710 8.404 10.911 4.511 After 3.1 0.474 8.535 3.675 1.25 4.86 6.811
4) control (statin) (n=4)

(242) TABLE-US-00013 TC/ HDL- Weight TG TCH VLDL-C HDL-C LDL-C C Start 2.113 0.843 1.444 0.885 0.684 0.559 2.108 Before 2.742 2.666 32.42 7.467 5.657 24.953 9.459 End 3.1 1.207 5.277 1.961 0.759 3.316 6.458
5) Summary of plasma parameter data

(243) TABLE-US-00014 Weight TG TCH VLDL-C HDL-C LDL-C TC/HDL-C Model Group Week 8 0.050 0.343 31.114 9.225 8.484 21.889 2.003 0.020 1.995 21.839 4.053 5.763 17.786 0.345 0.020 1.632 26.320 0.570 4.698 25.750 2.918 0.250 1.866 29.300 8.175 4.648 21.125 0.303 Sum 0.160 5.836 108.573 22.023 23.593 86.550 5.568 Ave 0.023 0.834 15.510 3.146 3.370 12.364 0.795 AFOD RAAS 1 Group Week 8 0.200 19.259 28.873 5.730 17.483 23.143 1.863 0.250 4.061 25.631 12.718 9.323 12.913 0.034 0.200 0.487 26.677 14.607 5.932 12.070 2.164 0.100 0.677 31.438 13.629 4.971 17.809 2.739 0.100 0.211 39.804 15.867 3.983 23.937 6.655 0.200 3.394 41.059 23.550 1.933 17.509 13.858 0.000 1.432 33.792 11.265 3.955 22.527 4.051 Sum 0.650 28.167 227.274 97.366 47.580 129.908 27.638 Ave 0.093 4.024 32.468 13.909 6.797 18.558 3.948 ApoAI 0.0500 0.1658 1.1028 1.8167 31.4684 5.7427 13.6172 1.7595 5.6328 2.2182 17.8512 5.4016 3.1286 2.4466 (n = 5) Model 0.0400 0.1407 1.4590 0.7590 27.1432 4.0510 5.5058 3.9762 5.8982 1.7989 21.6375 3.2697 1.3920 1.2890 group P value 0.926 0.727 0.246 0.004 0.852 0.26 0.013

(244) Conclusion: After 8 weeks of AFOD RAAS 1 treatment, all cholesterol-related plasma parameters decreased. There is also a decrease in the model control group. Significant changes can only be observed in VLDL-C and TC/HDL-C (p<0.05). There is no significant change for the rest of the parameters.

(245) TABLE-US-00015 AFOD RAAS 1 group Week 11 Weight TG TCH VLDL-C HDL-C LDL-C TC/HDL-C 0.200 4.241 30.064 8.388 10.183 21.676 1.294 0.300 3.740 31.052 7.169 8.799 23.883 2.282 0.000 0.092 29.618 19.679 11.844 9.939 0.164 0.300 2.374 34.708 21.989 5.151 12.719 1.978 Sum 0.800 10.263 125.442 57.225 35.977 68.217 3.129 Ave 0.200 2.566 31.361 14.306 8.994 17.054 0.782 Model 0.0400 0.1407 1.4590 0.7590 27.1432 4.0510 5.5058 3.9762 5.8982 1.7989 21.6375 3.2697 1.3920 1.2890 AFOD 0.2000 0.1414 2.5658 1.9396 31.3605 2.3107 14.3062 7.6126 8.9942 2.8486 17.0542 6.7679 0.7824 1.6706 RAAS 1 11 wk P value 0.689 0.99 0.532 0.11 0.658 0.583 0.795

(246) Conclusion: All cholesterol-related parameters showed significant decrease. But compared with the control group, there is no statistical significance.

(247) 6) Changes of HDL-C:

(248) TABLE-US-00016 AFOD RAAS 1 Fold normal treatment increase increase 8 wks 0.748 1.464 0.716 1.436 11 wks 0.432 1.423 0.992 3.078 control 0.684 0.759 0.074 0.102

(249) Compare the HDL-C values before and after the treatment, it indicated that HDL-C is elevated after AFOD RAAS 1-Al treatment

(250) Conclusion, iv infusion of AFOD RAAS 1 could lower blood cholesterol through the formation of HDL.

(251) The invention reveals that all healthy good cells have eaten all fats (BAD CELLS and damaged cells as described in the function of the liver not through the formation of HDL. In this preliminary and small study, The Inventor has found that even with the longer And higher dosage of AFOD RAAS 1 Group 2, the formation of HDL has been reduced to 54.15% from 92.36% of AFODRAAS 1 Group 1 and the Total of TC/HDL-C is 53.55% much higher than to compare with AFODRAAS 1 Group 2 which has TC/HDL-C 40.48% To prove it, Atorvastatin was also used in this study when comparing with control group, it has significantly reduced TG 60.73% and increased HDL to 64.69% however

(252) TABLE-US-00017 Weight TG TCH VLDL-C HDL-C LDL-C TC/HDL-C control 3.1 1.207 5.277 1.961 0.759 3.316 6.458 AFOD 1 2.79 1.17 3.69 1.09 1.46 2.6 3 compare to control 10.00% 3.07% 30.07% 44.42% 92.36% 21.59% 53.55% AFOD 2 2.65 1.94 3.322 1.14 1.17 2.19 3.844 compare to control 14.52% 60.73% 37.05% 41.87% 54.15% 33.96% 40.48% Atorvastatin 3.1 0.474 8.535 3.675 1.25 4.86 6.811 compare to control 0.00% 60.73% 61.74% 87.40% 64.69% 46.56% 5.47%

(253) TCH have increased to 61.74%, VLDL-C increased 87.40% LDL-C increased 46.56% and TC/HDL-C is 5.47%. In conclusion it can reduces TG and increases HDL but will NOT LOWER Bad Cholesterol VLDL-C, LDL-C, TCH and TC/DHL-C

(254) Drugs like Atorvastatin cannot remove FATS from PLAQUE whereas AFODRAAS1 and AFCCRAAS1 CAN REMOVE FATS from PLAQUE, CLEAN the arteries.

(255) In the first year of high school, we began to learn that when the YELLOW COLOR Mixed with BLUE, it turns GREEN. In a drug if you have to have a YELLOW COLOR and BLUE COLOR for your final product, It is IMPOSSIBLE as the final product will turn GREEN. This is the reason why Chemicals REACT that is why most of drugs from Chemicals have SIDE EFFECTS.

(256) There is only ONE MANUFACTURER, MODERATOR, REGULATOR and DISTRIBUTOR which can produce the product which maintains the YELLOW COLOR and BLUE COLOR is THE LIVER producing PILE approximately 0.5-0.9 liter of PILE per day. PILE is a LIQUID which has YELLOW COLOR and BLUE COLOR.

(257) Reference is made to FIGS. 70-76.

(258) Therefore in this case, ATORVASTATIN is one among thousands of drugs available can be combined with AFODRAAS1-85 or AFCC RAAS1-85 to enhance the EFFICACY of the drugs. Before this invention, Drugs like ATORVASTATIN and LIPITOR have helped a lot of people with HIGH CHOLESTEROL.

(259) Liver

(260) 1) Liver surface: When the animal models were first made, the liver surface of the lab animals from the animal-model group showed abnormal white colored spots. Histological analysis showed that it is???. The surface of the liver feels harder than normal tissue. The liver samples taken from the Al treated group has fewer???. The surface is not as tough as when the animal model was first made. The un-treated group also showed relief in the??? and softened. The probable reason is that because the high cholesterol and atherosclerosis model is made in a short period of time, the switch to normal diet also helped to relief the symptoms.
2) Liver index

(261) TABLE-US-00018 weight Liver (g) index ApoAI 8 weeks 0.09 0.033 ApoAI 11 weeks 0.117 0.044 model control 0.111 0.036

(262) The liver index did not show any changes after the AFOD RAAS 1 treatment. 6 fatty streak lesions

(263) TABLE-US-00019 Compare Compare to Increase to model Decrease Area control % control % AFOD RAAS 1 43.84 19.03 77 27.36 38.43 8 wk AFOD RAAS 1 50.51 25.71 104 20.69 29.05 11 wk Model control 71.20 46.39 187 Model first 24.81 made
1) Fatty streak lesion appearance: the tissue from the non-AFOD RAAS 1-group (model control) has bumps on the surface. The tissue feels tender and hard as touched with bare hand. Dissection of the blood vessels showed fat deposit in the cross-section of the tissue. The fatty streak lesion decreases as the aorta descends. Compare with the model control group, the AFOD RAAS 1-group do not have bumps in the blood vessel surface. The tissue feels soft as touched with bare hand.
2) Area measurement of the fatty streak lesion: Compare with the model-control group, the surface area of the fatty streak lesion increased 77% and 104%, and the non-AFOD RAAS 1-treated group increased by 187%. Compare with the non-AFOD RAAS 1-Al treated group, the fatty streak lesion of the AFOD RAAS 1-group decreased by 38.43% at week 8 and decreased by 29.05% at week 11.

(264) Conclusion: Administration of AFOD RAAS 1 to the lab animals with atherosclerosis obviously suppresses the further development of fatty streak lesion.

(265) Drugs like Atorvastatin cannot remove FATS from PLAQUE whereas AFODRAAS1-85 and AFCCRAAS1-85 CAN REMOVE FATS from PLAQUE, CLEAN the arteries. FIG. 77 shows Normal (normal diet 8 weeks) and FIG. 78 shows Plaque area=0. FIGS. 79 and 80 show build up the animal models, with AFOD RAAS 1-Al 8 weeks. FIG. 79 shows Plaque area=13.29%, and FIG. 80 shows Plaque area=20.5%. FIGS. 81 and 82 show build up the animal models, with AFOD RAAS 1 8 weeks (another rabbit). FIG. 81 shows Plaque area=58.4%, and FIG. 82 shows Plaque area=82.17%.

(266) FIGS. 83-85 show Group with AFOD RAAS 1 11 weeks. FIG. 83 shows Plaque area=47.27%, FIG. 84 shows Plaque area=40.32%, and FIG. 85 shows Plaque area=51.13%.

(267) 3) Analysis of lipid content at dissected aorta

(268) TABLE-US-00020 lipid con. (umol/mg) sig 8 weeks (n = 7) 0.025 0.0095 0.006 p < 0.01 11 weeks (n = 4 0.0267 0.0054 0.015 p < 0.05 positive control (n = 4) 0.0274 0.006 0.046 p < 0.05 Control (n = 4) 0.0736 0.014

(269) Conclusion: Comparing with the model control group, the triglyceride content at dissected aorta of the AFOD RAAS 1 treated group is significantly lowered. The decrease is significant statistically. (p<0.05).

(270) 5. Experimental Summary

(271) The purpose of this pilot-scale preclinical animal test of AFOD RAAS 1 is the successful rate and time estimate of making animal model of atherosclerosis, dose and effects human AFOD RAAS 1-Al administration on the blood cholesterol-related levels and the suppression of development of fatty streak lesion.

(272) Based on data collected from the experiment, successful making of a high cholesterol rabbit model need 4-5 weeks. The formation of atherosclerosis fatty streak lesion need more than 10 weeks of high-fat diet (at week 10-11, the average surface area of fatty streak lesion is 24%). The successful rate for model making is 60%. After intravenous infusion of human AFOD RAAS 1 at 100 mg/wk for 8-11 weeks, the hypercholesterolemia and liver lesion improved dramatically, but the does not stop the formation of fatty streak lesion at aorta. Thus, the hypercholesterolemia and liver lesion can slowly regress after switch to low fat diet, but the atherosclerosis fatty streak lesion progresses and need to be treated.

(273) The experiment shows that the administration of AFOD RAAS 1 to hypercholesterolemia lab animals reduces the surface area of fatty streak lesion at aorta and decreases the triglyceride content in the lesion tissue, thus; AFOD RAAS 1 has the potential to be developed to be an antiatherogenic and cholesterol-lowing medicine.

(274) Table 1. The Change of Plasma Lipid Parameter

(275) 1. Wk0, wk10 and wk18 mean the actual value of each parameter. 2. Wk18wk0 (or wk21wk0) means the change calculated by comparing the value of wk 18 (or wk21) to the value of wk 0. This means the overall results during the whole process, which means (high fat diet+switch to normal diet+different treatment). It is calculated by % of change=(value of wk18value of wk 0)/value of wk 0. 3. Wk18wk10 (or wk21wk10) means the change calculated by comparing the value of wk 18 (or wk21) to the value of wk 10. This represents the results during the second half process, which means (switch to normal diet+different treatment). It is calculated by % of change=(value of wk18value of wk 10)/value of wk 10. 4. Please refer to table 2 for the experiment design

(276) TABLE-US-00021 Weight TG TCH VLDL-C HDL-C LDL-C TC/HDL-C AFODRAAS 1 treatment for 8 weeks wk 0 2.164 0.967 1.152 0.87 0.748 0.282 1.938 wk 10 2.7 5.191 36.153 14.996 8.261 21.157 6.56 wk 18 2.79 1.17 3.69 1.09 1.46 2.6 3 wk 18-wk 0 28.93% 20.99% 220.31% 25.29% 95.19% 821.99% 54.80% wk 18-wk 10 3.33% 77.46% 89.79% 92.73% 82.33% 87.71% 54.27% AFODRAAS 1 treatment for 11 weeks Wk 0 2.2 0.93 1.43 0.958 0.432 0.472 4.185 Wk 10 2.45 4.507 34.683 15.443 10.168 19.24 3.667 Wk 21 2.65 1.94 3.322 1.14 1.17 2.19 3.844 wk 21-wk 0 20.45% 108.60% 132.31% 19.00% 170.83% 363.98% 8.15% wk 21-wk 10 8.16% 56.96% 90.42% 92.62% 88.49% 88.62% 4.83% Atorvastatin Atorvastatin for 4 weeks Wk 0 2.25 0.45 0.946 0.509 0.539 0.437 1.844 Wk 10 2.85 9.122 20.339 9.71 8.404 10.911 4.511 Wk 18 3.1 0.474 8.535 3.675 1.25 4.86 6.811 wk 18-wk 0 37.78% 5.33% 802.22% 622.00% 131.91% 1012.13% 269.36% wk 18-wk 10 8.77% 94.80% 58.04% 62.15% 85.13% 55.46% 50.99% control Wk 0 2.113 0.843 1.444 0.885 0.684 0.559 2.108 Wk 10 2.742 2.666 32.42 7.467 5.657 24.953 9.459 Wk 18 3.1 1.207 5.277 1.961 0.759 3.316 6.458 wk 18-wk 0 46.71% 43.18% 265.44% 121.58% 10.96% 493.20% 206.36% wk 18-wk 10 13.06% 54.73% 83.72% 73.74% 86.58% 86.71% 31.73%

(277) TABLE-US-00022 TABLE 2 The experiment design Wk 0 Wk 10 Wk 14 Wk 18 Wk 21 Control group Establish animal model Normal diet without APOAI with high fat diet APOAI group 1 Establish animal model APOAI APOAI No APOAI with high fat diet 100 mg/animal 50 mg/animal once a week twice a week APOAI group 2 Establish animal model APOAI APOAI APOAI with high fat diet 100 mg/animal 50 mg/animal 100 mg/animal once a week once a week once a week Atorvastatin group Establish animal model Atorvastatin with high fat diet

(278) TABLE-US-00023 TABLE 3 change of fatty streak lesions Fatty Compare to Compare to streak wk 10 of wk 18 of Time lesions control control point area (%) group Increase % group Decrease % AFODRAAS 1 Wk 18 43.84 19.03 77 27.36 38.43 Group 1 AFODRAAS 1 Wk 21 50.51 25.71 104 20.69 29.05 Group 2 Atrovastatin Wk 18 71.20 46.39 187 Control group Wk 10 24.81

(279) TABLE-US-00024 TABLE 4 Analysis of lipid content at dissected aorta P value (compared Lipid con. (umol/mg) to control group) AFODRAAS 1 Group 1 0.025 0.0095 0.006 (n = 7) AFODRAAS 1 Group 2 0.0267 0.0054 0.015 (n = 4) Atorvastatin group (n = 4) 0.0274 0.006 0.046 Control group (n = 4) 0.0736 0.014

(280) TABLE-US-00025 Weight TG TCH VLDL-C HDL-C LDL-C TC/HDL-C control 3.1 1.207 5.277 1.961 0.759 3.316 6.458 AFOD 1 2.79 1.17 3.69 1.09 1.46 2.6 3 compare to 10.00% 3.07% 30.07% 44.42% 92.36% 21.59% 53.55% control AFOD 2 2.65 1.94 3.322 1.14 1.17 2.19 3.844 compare to 14.52% 60.73% 37.05% 41.87% 54.15% 33.96% 40.48% control Atorvastatin 3.1 0.474 8.535 3.675 1.25 4.86 6.811 compare to 0.00% 60.73% 61.74% 87.40% 64.69% 46.56% 5.47% control