New Insights into Omega-3 Fatty Acids and Lipids Using Nanotechnology Approaches

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1 New Insights into Omega-3 Fatty Acids and Lipids Using Nanotechnology Approaches R. Preston Mason, Ph.D. Faculty, Dept. of Medicine, Cardiovascular Division Brigham and Women s Hospital, Harvard Medical School Boston, Massachusetts, USA President and Founder Elucida Research LLC Beverly, Massachusetts, USA

2 Disclosure Information R. Preston Mason, Ph.D. NLA Scientific Sessions, May 20, 2016 Grants/Research Support: Much of this research was conducted with financial support from Amarin Pharma, Inc. Dr. Mason also acknowledges investigator-initiated research support from Novartis and Pfizer.

3 Integrated Perspective on CV Risk Factors and Atherosclerosis Endothelial Dysfunction Oxidative Stress & Inflammation CV Disease Ross R. N Engl J Med. 1999;340: Ross R. N Engl J Med. 1999;340: Mason RP, Jacob RF

4 Effects of Omega-3 Fatty Acids on Inflammation and Oxidation

5 Omega-3 PUFAs are Metabolized into Anti-inflammatory Mediators Omega-6 PUFAs Omega-3 PUFAs Arachidonic acid (AA) O Eicosapentaenoic acid O Docosahexaenoic acid Prostaglandin E2 Leukotriene B4 O O O O O Proinflammatory mediators Resolvin E1 O 3 3 Protectin D1 2 Prostaglandin E3 Leukotriene B5 O O Maresin Less potent mediators Anti-inflammatory mediators Endo J, Arita M. J Cardiol. 2015;67:22-27.

6 Omega-3 Fatty Acids Incorporate into Lipoprotein Particles

7 Lipoprotein Particles Vary in Size and Atherosclerotic Potential Chylomicron 0.95 Density (g/ml) ApoB-Containing Lipoproteins VLDL 1.01 IDL LDL 1.02 Chylomicron remnants HDL VLDL remnants HDL3 3c b 10 3a 2b 2a Triglyceride Lp(a) Cholesteryl ester Free cholesterol ApoA1-Containing Lipoproteins Phospholipids Diameter (nm) Adapted from Ballantyne CM. Clinical Lipidology Saunders, Philadelphia, PA. Apo = apolipoprotein; HDL = highdensity lipoprotein (Lp); IDL = intermediate density Lp; LDL = low-density Lp; VLDL = very-low-density Lp.

8 Lipid Oxidation Markers Predict CV Events in 634 Patients with CAD P < oxlipid levels: lowest quartile oxlipid levels: highest quartile P = HR P = Non-Fatal Vascular Events (Angina/CHF) Major Vascular Procedures (CABG/PTCA) All Vascular Events and Procedures Walter MF, Mason RP, et al. J Am Coll Cardiol. 2008;51:

9 Comparative Effects of TG-Lowering Agents on Human LDL Oxidation In Vitro MDA Equivalents (µm) * Vehicle EPA Fenofib Niacin Gemfib Vit E Fenofib = Fenofibrate; Gemfib = Gemfibrozil; Vit E = Vitamin E *P<0.001 versus vehicle-treated control; P<0.001 versus Fenofib, Niacin, or Gemfib; P<0.001 vs Vit E (Student-Newman- Keuls multiple comparisons test; overall ANOVA: P<0.0001, F=132.38). Values reported are mean ± SD (N=3). Each agent was tested at 10.0 µm. Mason RP, et al. J Cardiovasc Pharm (in press).

10 Small Dense LDL-C Alone Predicts CVD Risk of CHD Over Time by SD LDL-C Quartiles Risk of CHD Over Time by LB LDL-C Quartiles st SD LDL-C quartile 2 nd SD LDL-C quartile 3 rd SD LDL-C quartile 4 th SD LDL-C quartile st LB LDL-C quartile 2 nd LB LDL-C quartile 3 rd LB LDL-C quartile 4 th LB LDL-C quartile Risk Follow-up (years) SD LDL-C = small, dense LDL-C; LB LDL-C = large, buoyant LDL-C. Hoogeveen RC, et al. Arterioscler Thromb Vasc Biol. 2014;34:

11 Comparative Effects of TG-Lowering Agents on Human sdldl Oxidation In Vitro 14 MDA Equivalents (µm) * Vehicle EPA Fenofib Niacin Gemfib Vit E *P<0.001 versus vehicle-treated control; P<0.001 versus Fenofib, Niacin, or Gemfib; P<0.001 vs Vit E (Student-Newman- Keuls multiple comparisons test; overall ANOVA: P<0.0001, F=1268.1). Values reported are mean ± SD (N=3). Each agent was tested at 10.0 µm. Mason RP, et al. J Cardiovasc Pharm (in press).

12 Schematic of Proposed Protective Effects of Omega-3 Fatty Acids on sdldl Lipid Oxidation Adapted from: Mason RP, Jacob RF. Diabetes. 2015; 64, Suppl 1:A178-A179

13 Omega-3 Fatty Acids Differentially Inhibit Human sdldl and VLDL Oxidation In Vitro sdldl Oxidation VLDL Oxidation MDA Equivalents (µm) 14 Vehicle 12 EPA DHA * * * * * * * 20 Vehicle EPA 16 DHA * * * * * * * Time Point (hr) Time Point (hr) *P<0.001 versus vehicle-treated control; P<0.05 and P<0.001 versus DHA (Student-Newman-Keuls multiple comparisons test; overall ANOVA sdldl data: P<0.0001, F=391.88; VLDL data: P<0.0001, F=1074.8). Values reported are mean ± SD (N=3). Each agent was tested at 10.0 µm (sdldl) and 2.5 µm (VLDL). Mason RP, et al. J Cardiovasc Pharm (in press).

14 Effects of TG-lowering Agents on Atherosclerosis and Cholesterol Crystal Formation

15 Small Angle X-ray Diffraction of Cell Membranes Membrane suspension Lucite Sedimentation Cell Interbilayer water space Aluminum foil substrate water space d Centrifugatio n 35,000 g 2 d sin curved glass support sedimentation cell is dismantled and sample removed Membrane multibilayer sample is mounted on curved glass support

16 Oxidative Stress, Cholesterol Domains, and Endothelial Dysfunction with Atherosclerosis Mason RP, Jacob RF. Circulation. 2003;107:

17 SEM Photomicrograph of Cholesterol Crystal and Macrophage Foam Cells Kellner-Weibel G et al. Arterioscler Thromb Vasc Biol. 1999;19:1891

18 Cholesterol Crystals Associated with Apoptotic Cell Death Kellner-Weibel G, Mason RP, et al. Arterioscler Thromb Vasc Biol. 1999;19:

19 Characterizing Model Membrane Cholesterol Crystalline Domains by X-ray Diffraction Mason RP, et al. J Biol Chem. 2006;281:

20 Effects of Omega-3 Fatty Acid EPA and Vitamin E on Cholesterol Domain Formation Photons (Log Scale) Mason RP, Jacob RF. Biochim Biophys Acta. 2015;1848: Space -1 (Å -1 )

21 Effects of TG-Lowering Agents on Cholesterol Domain Formation Photons (Log Scale) Mason RP, Jacob RF. Biochim Biophys Acta. 2015;1848: Space -1 (Å -1 )

22 Comparative Effects of TG-lowering Agents on Cholesterol Domain Formation Adapted from: Mason RP, Jacob RF. Biochim Biophys Acta. 2015;1848:

23 Effects of Omega-3 Fatty Acids with Statins on Endothelial Function with Dyslipidemia

24 Nitric Oxide Is a Key Mediator of Vascular Protection Vessel lumen NO Platelet inhibition Subendothelium NO GUANYLATE CYCLASE NO Cell growth/proliferation Matrix formation Leukocyte migration GTP cgmp Relaxation Vascular smooth muscle cells Behrendt D, Ganz P. Am J Cardiol. 2002;90(10C):40L-48L. Vita JA. J Card Fail. 2003;9(5 Suppl Nitric Oxide):S199-S204.

25 Nanotechnology Approaches Used to Measure Endothelial Function NO 300 mm Time (s) Calcium ionophore Malinski T, Taha Z. Nature. 1992;358: Mason RP, et al. Circulation. 2005;112:

26 Normal Endothelial Function Is a Balance Between NO and ONOO Release Levels ONOO Mason RP, Jacob RF. Vasoconstriction Inflammation High Angiotensin II Pro-thrombotic Trophic Fibrotic enos NO Vasodilation Anti-inflammatory Natriuresis Anti-fibrotic Anti-thrombotic

27 CV Risk Factors Lead to Loss of NO Release from Rat Aortic and Glomerular Endothelium 500 Control Hypertension Diabetes + Hypertension 400 NO Release (nm) * * * * 0 Aortic ECs Glomerular ECs *P<0.001 versus control; P<0.05 versus NO release measured from hypertensive animals (Student-Newman-Keuls multiple comparisons test; overall ANOVA aortic EC data: p<0.0001, F=89.991; glomerular EC data: p<0.0001, F=74.629). Values are mean ± SD (N=5-6). Spontaneously hypertensive (SH) rats: BP = 167 ± 5; STZ-induced blood glucose = 354 ± 83. ECs = endothelial cells. Mason RP, et al. Am J Hypertens. 2009;22:

28 Omege-3 Fatty Acid and Atorvastatin Pretreatment Inhibits the Effects of Oxidized LDL on Human Endothelial Cell Function NO/ONOO Release Ratio *** * *** 0 Vehicle oxldl oxldl + oxldl + oxldl + EPA ATM EPA Atorvastatin active metabolite (ATM) was used in this study. Values are mean ± SD (N=3-6). *P<0.05 and ***P<0.001 vs. oxidized LDL (oxldl); P<0.01 vs. oxldl + EPA; P<0.001 vs. oxldl + Atorv (Student-Newman-Keuls multiple comparison test; overall ANOVA: P<0.0001, F=25.827). Mason RP, et al. J Clin Lipidol. 2014;8: ATM

29 Illustration of Postulated Combined Effects of EPA and Statin on Reversal of Endothelial Dysfunction Mason RP et al. J Am College Cardiol. (abstract presented April, 2016).

30 Cellular and Molecular Mechanisms of Atherosclerosis and Effects of O3FA on Early Lesion Development Antioxidant effects Cholesterol crystalline domains Ox-LDL RLP-C Improved endothelial function Adhesion of monocytes Macrophages Foam cells Borow KM, Nelson JR, Mason RP. Atherosclerosis. 2015;242:

31 Fish Oil Supplements as Source of Omega-3 Fatty Acids Fish oil is the most commonly used dietary supplement among US adults. 1 Based on the 2012 National Health Interview Survey, about 7.8% of adults (19 million) had taken a fish oil supplement in the previous 30 days. 2 Although numerous dietary supplements containing OM3FA are widely available, their integrity and efficacy remain unverified Barnes PM, et al. National Health Statistics Reports. 2008;12: NIH NCCIH Mason RP, et al. Poster presented at the AMCP 2015 Nexus, Orlando, FL.

32 Can Fish Oil Supplements be Used to Treat Patients? Most common dietary supplements report 30% of their contents as omega-3 fatty acids (OM3FAs) Each 1 gram capsules may contain 300 mg of EPA/DHA In order to reach 4 g/day of OM3FAs, a patient would need a large number of capsules High potency Rx OM3FA (4) 1.0 g capsules EPA + DHA Dietary Supplement (13) 0.5 g capsules of EPA + DHA Dietary Supplement (27) Mason RP, et al. Poster presented at the AMCP 2015 Nexus, Orlando, FL.

33 Fish Oil Supplements Contain Various Levels of OM3FA and Saturated Fat EPA (20:5) DHA (22:6) DHA Palmitic Acid DS1 DS2 DS3 EPA DHA Palmitic Acid (16:0; Saturated FA) Palmitic Acid EPA 8:0 9:0 10:0 11:0 11:1 12:0 13:0 13:1 14:0 14:1 15:0 16:0 16:1 17:0 17:1 18:0 18:1 cis 18:1 trans 18:2 18:3 (6,9,12) 18:3 (9,12,15) 19:0 20:0 20:1 20:2 20:3 (5,8,11) 20:3 (8,11,14) 20:4 20:5 22:0 22:1 22:2 22:3/22:5n6 22:4 22:5n3 22:6 23:0 24:0 24:1 Mason RP, et al. Poster presented at the AMCP 2015 Nexus, Orlando, FL. DS = Dietary Supplement.

34 Saturated Fat Content in Fish Oil Supplement Leads to Solid Mass following Isolation Rx of Pure Omega-3 Fatty Acids is a Clear Fluid Fish Oil Dietary Supplement forms Solid Mass Sherratt, CR, Mason RP

35 International Fish Oil Supplements Exceed Recommended Levels of Oxidation Markers Albert BB, et al. Sci Rep. 2015;5:7928. DOI: /srep Recommended international thresholds are indicated by dotted lines in each panel. 83% of fish oil products tested exceeded recommended PV threshold 25% exceeded recommended AV 50% exceeded recommended TOTOX Only 3 of 36 (8%) met the international recommendations, not exceeding any of these indices Best-before date, cost, country of origin, and exclusivity were all poor markers of supplement quality

36 U.S. Leading Fish Oil Supplements Exceed Recommended Levels of Oxidation Markers PV (med/l) Anisidine Value TOTOX Value Normalized to Rx OM-3 FA GOED standard * <5 meq/kg <20 meq/kg <26 meq/kg Rx DS 1 DS 2 DS 3 DS 4 Fish Oil Products Primary oxidative product Secondary oxidative products *Global Organization for EPA and DHA Omega-3s (GOED). Available at: Mason RP et al. Poster presented at the AMCP 2015 Nexus, Orlando, FL.

37 Effects of a DS Fatty Acid Extract vs Non-Oxidized and Partially Oxidized Preparations of EPA and DHA on Human sdldl Oxidation In Vitro 12 MDA Equivalents (μm) * * * 0 Vehicle EPA + DHA DS oxepa + oxdha Each agent was tested separately at 10.0 µm or in combination at 5.0 µm against vehicle-treated controls. *p<0.001 versus vehicle alone; p<0.001 versus DS; p<0.001 versus oxepa + oxdha (Student-Newman-Keuls multiple comparisons test; overall ANOVA: p<0.0001, F=993.26). Values are mean ± S.D. (N = 3). Mason RP et al. Poster presented at the AMCP 2015 Nexus. Orlando, FL.

38 Conclusions Lipids modify cell function through association with specific domains that regulate signal transduction. At high levels, cholesterol forms domains that lead to cytotoxic crystals. Omega-3 FA such as EPA inhibit oxidative damage to ApoB-containing particles and cellular membranes leading to reduced inflammation, endothelial dysfunction and cholesterol crystals, potentially improving LDL clearance. Supplements contain variable amounts of omega-3 fatty acids, saturated FAs and elevated peroxidation products that limit their benefit. Given the lack of oversight, supplements should not be used as a replacement for Rx.

39 Take Home Message Using nanotechnology approaches, we have characterized novel effects of omega-3 fatty acids like EPA on pathogenic mechanisms associated with CV disease, including oxidative stress, inflammation, and endothelial dysfunction.

40 Research Team R. Preston Mason, Ph.D. Robert F. Jacob, Ph.D. Samuel C.R. Sherratt, B.S.