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Mn aha 05

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Mn aha 05

  1. 1. Presenter Disclosure Information DISCLOSURE INFORMATION: The following relationships exist related to this presentation: The Speaker is a principal in Bruin Pharma Mohamad Navab, Ph.D. An Oral ApoJ Peptide Renders HDL Anti-inflammatory in Mice and Monkeys and Dramatically Reduces Atherosclerosis in ApoE Null Mice
  2. 2. ApoA-I Peptide mimetics - An emerging strategy for CVD prevention is the development of apoA-I mimetic peptides. This strategy is based on the hypothesis that apoA-I mimetic peptides could be capable of stimulating the first step in the RCT pathway in a manner similar to native apoA-I, which is 243 amino acid residues in length. In comparison, the apoA-I mimetic peptide D-4F comprises only 18 amino acids.
  3. 3. 18 AA peptide Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 D-4F
  4. 4. -D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase. - 18 AA peptide Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 D-4F
  5. 5. -D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase. - It markedly reduced lesion formation on LDL r-/- mice on Western diet and in apoE-/- mice 18 AA peptide Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 D-4F
  6. 6. D-4F Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused: - the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL. -D-4F in humans.
  7. 7. D-4F Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused: - the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL. -D-4F in humans: Phase 1, a, b, c
  8. 8. Negative Stain EM of D-4F in PBSD-4F, negative stain EM
  9. 9. Proposed Mechanism of Action of D-4F LOOH & Ox-PAPC and cholesterol CholesterolPhospholipids Pre-β HDL ABCA1 α HDL ApoA-I cycles through pre-β HDL Cholesterol ABCA1 Phospholipids D-4F
  10. 10. Proposed Mechanism of Action of D-4F LOOH & Ox-PAPC and cholesterol POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CE-OOH HPETE HPODE MM-LDL O O PAPC m/z 782.4 LDL CH3(CH2 )14 CH2 Phosphorylcholine - 12-LO Chol.18:2 CH2 HC O O - CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - CH3(CH 2 )14 CH2 Phosphorylcholine CH2 HC O O CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - O O OH OO O OH O O 12-LOHPETE HPODE HPETE HPODE O O O POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CholesterolPhospholipids Pre-β HDL ABCA1 α HDL ApoA -I cycles through pre -β HDL Cholesterol ABCA1 Phospholipids D-4F
  11. 11. Proposed Mechanism of Action of D-4F LOOH & Ox-PAPC and cholesterol POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CE-OOH HPETE HPODE MM-LDL O O PAPC m/z 782.4 LDL CH3(CH2 )14 CH2 Phosphorylcholine - 12-LO Chol.18:2 CH2 HC O O - CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - CH3(CH 2 )14 CH2 Phosphorylcholine CH2 HC O O CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - O O OH OO O OH O O 12-LOHPETE HPODE HPETE HPODE O O O POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CholesterolPhospholipids Pre-β HDL ABCA1 α HDL ApoA-I cycles through pre-β HDL Cholesterol ABCA1 Phospholipids D-4F
  12. 12. Proposed Mechanism of Action of D-4F LOOH & Ox-PAPC inactivated and/or cleared and reverse cholesterol transport is also increased LOOH & Ox-PAPC and cholesterol POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CE-OOH HPETE HPODE MM-LDL O O PAPC m/z 782.4 LDL CH3(CH2 )14 CH2 Phosphorylcholine - 12-LO Chol.18:2 CH2 HC O O - CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - CH3(CH 2 )14 CH2 Phosphorylcholine CH2 HC O O CH3(CH2)14 CH2 Phosphorylcholine - CH2 HC O O - O O OH OO O OH O O 12-LOHPETE HPODE HPETE HPODE O O O POVPC m/z 594.3 PGPC m/z 610.2 PEIPC m/z 828.6 CholesterolPhospholipids Pre-β HDL ABCA1 α HDL ApoA-I cycles through pre-β HDL Cholesterol ABCA1 Phospholipids D-4F
  13. 13. D-4F Plus Pravastatin Increased ApoA-I in both Alpha and Pre-beta HDL Particles CHOW D-4F PRAVASTATIN D-4F+PRAVASTATIN Pre-β alpha
  14. 14. Lesion Prevention 0 50,000 100,000 150,000 200,000 250,000 300,000µm2/Section CHOW D-4F PRAVASTATIN D-4F+ PRAVASTATIN
  15. 15. A Comparison of 4F and [113-122]apoJ Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa) Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa)
  16. 16. D-[113-122]apoJ Increases Paraoxonase 1 Activity in ApoE Null Mice PON Activity per unit Cholesterol 0.00 0.50 1.00 1.50 2.00 2.50 3.00 CHOW HDL D-[113-122]apoJ HDL 0.00 0.50 1.00 1.50 2.00 2.50 3.00 CHOW D-[113-122]apoJ HDL p=0.0001
  17. 17. D-[113-122]apoJ Renders HDL Anti- inflammatory in ApoE Null Mice No Addition Cont. LDL Cont. LDL + HDL 0 2 4 6 8 Water MigratedMonocytes/Field Autologous HDL+ LDL from treated ApoE Null Mice * Assay Controls D [113-122] apoJ No Addition Cont. LDL Cont. LDL + HDL 0 2 4 6 8 Assay Controls - -
  18. 18. Comparison of D-4F and D-[113-122]apoJ 8 Br - cAMP-Induced Efflux (ABCA1-Mediated) 0 10 20 30 40 50 %Efflux HDL D-[113- 122]apoJ 8 Br - -Induced Efflux (ABCA1-Mediated) VehicleStd. (5% plasma) D-4FD-4F
  19. 19. D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (Aortic Root Sinus) 0 100,000 200,000 300,000 400,000 500,000 CHOW n=21 CHOW+ D-[113-122]apoJ n=23 395893 +63371 117685 +107571 p=4.3x 10-13 70.2% Aorticsinuslesionarea CHOW n=21 CHOW+ D-[113-122]apoJ n=23 395893 +63371 117685 +107571 p=4.3x 10-13 µm2 /section
  20. 20. D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (En Face) 0 1.0 2.0 3.0 4.0 CHOW n=27 CHOW +D- [113-122]apoJ n=23 %SURFACEAREAWITHLESIONS 1.7+/- 0.8 0.5+/- 0.5 70.5% p=1.5x10-6
  21. 21. D-[113-122]apoJ Decreases Lipoprotein Lipid Hydroperoxides in Monkeys 0 100 200 300 400 Time 0 3 hrs HDL 0 400 800 1200 Time 0 3 hrs LDL p=0.001 ngLOOHper50µgcholesterol ngLOOHper50µgcholesterol p=0.001 0 100 200 300 400 0 100 200 300 400 Time 0 3 hrs HDL 0 400 800 1200 0 400 800 1200 Time 0 3 hrs LDL gcholesterol gcholesterol
  22. 22. D-[113-122]apoJ Renders HDL Anti-inflammatory in Monkeys MigratedMonocytes/Field 0 5 10 15 20 0 5 10 15 20 No Addition LDL+ Monkey HDL at Time 0 Monkey # 1 2 3 4 1 2 3 4Std. LDL LDL+ Std. HDL Assay Controls LDL+ Monkey HDL at 3 hrs P<0.001
  23. 23. A Comparison of ApoA-I, 4F, [113- 122]apoJ, and Tetrapeptides ApoA-I (243 aa) Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa) Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa) Boc-K(eBoc)-R-E-S(tBu)-OtBu KRES (4aa) Boc-F-R-E-L-OtBu FREL (4aa)
  24. 24. Making the Good Cholesterol Better TM Oral Amphipathic Peptides with Anti- atherosclerotic Properties •D-[113-122]apoJ is one of three classes of peptides that associate with HDL after an oral dose •Peptides with a Class A amphipathic helix (D-4F) •Peptides with a G* amphipathic helix (D-[113-122]apoJ) •Tetrapeptides that are too small to form a helix (KRES; FREL)
  25. 25. 14 C-ApoJ 125 I-D-4F
  26. 26. Biologically Active Biologically Inactive
  27. 27. 14 C-FREL
  28. 28. Exploring the Differences in the Association of the Peptides with HDL •We hypothesized that given the differing temporal association of the peptids with HDL a combination of the three compounds ( D-4F and D-[113-122]apoJ and FREL) might extend the anti-inflammatory properties of HDL after a single oral dose.
  29. 29. HDL Inflammatory Properties 6 Hours After Dosing 0 5 10 15 20 25 F+J F+J 0.5 D- 4F MigratedMonocytes/Field F+J 1.0 D- 4F p<0.001 p<0.001 LDL+HDLNo Addition Std LDL +Std HDL
  30. 30. 24 Hours After Dosing 0 5 10 15 MigratedMonocytes/Field No Addition Std LDL +Std HDL F+J F+J 0.5 D-4F F+J 1.0 D - 4F p<0.001p<0.001 LDL+HDL
  31. 31. 48 Hours After Dosing 0 5 10 15 20 25 30 MigratedMonocytes/Field p<0.001 p<0.01 No Addition Std LDL +Std HDL F+J F+J 0.5 D- 4F F+J 1.0 D- 4F LDL+HDL
  32. 32. 72 Hours After Dosing 0 5 10 15 20 25 30 MigratedMonocytes/Field No + HDL + HDL + HDL+F+J + HDL + HDL+F+J p<0.001 p<0.001 No Addition Std LDL +Std HDL F+J F+J 0.5 D- 4F F+J 1.0 D- 4F
  33. 33. D-4F versus D-[113-122]apoJ versus Tetrapeptides Property D-4F 113-122 Tetrapeptides Stimulate Pre-β HDL Yes No No Stim HDL Chol Efflux Yes Yes No Stim. Reverse Chol Transport Yes ND No Reduce LOOH Yes Yes Yes Increase PON Yes Yes Yes Improve HDL Yes Yes Yes Inflammatory Properties
  34. 34. D-4F versus D-[113-122]apoJ versus Tetrapeptides Property D-4F 113-122 Tetrapeptides Improve LDL-MCA Yes Yes Yes Prevent Lesions ~75% ~70% ~50% L-Active Orally No Not likely Yes Statin Synergy Yes ND ND Regress Lesions Yes ND ND
  35. 35. Summary •Oral amphipathic peptides from different classes associate with HDL differently but share certain common properties: •Reduce HDL-lipid hydroperoxides •Increase HDL-anti-oxidant enzymes (e.g. Paraoxonase) •Render HDL anti-inflammatory •These peptides provide unique tools for dissecting and understanding HDL metabolism and its relationship to the innate immune system.
  36. 36. UCLA UAB Alan M. Fogelman G.M. Anantharamaiah Srinivasa T. Reddy David W. Garber Brian J. Van Lenten Manjula Chaddha Greg P. Hough Geeta Datta Susan Y. Hama Shaila Handattu Alan Wagner Vinod K. Mishra Joy Frank Mayakonda N. Palgunachari Victor R. Grijalva Vannakambadi K. Ganesh Jan Danciger

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