PROGRESS TOWARDS POLYUNSATURATED FATTY ACID BASED THERAPEUTICS FOR CARDIOVASCULAR DISEASES: TURNING A MILLSTONE INTO A MILESTONE?

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1 PROGRESS TOWARDS POLYUNSATURATED FATTY ACID BASED THERAPEUTICS FOR CARDIOVASCULAR DISEASES: TURNING A MILLSTONE INTO A MILESTONE? Antonio Ferrante*,, and Charles S. Hii*, *Department of Immunopathology, Children, Youth and Women s Health Service, Adelaide, South Australia; School of Pediatrics and Reproductive Medicine, University of Adelaide, South Australia; Sansom Institute, School of Pharmacy and Medical Sciences Samson Institute, University of South Australia Introduction Although dietary supplementation with ω3 polyunsaturated fats from fish meal or fish oil capsules has found general acceptance as a means to help curtail the increasing incidence of cardiovascular diseases, including atherosclerosis, several gaps in our knowledge have limited the ability of nutritionists to make appropriate recommendations to the community for such application [1]. Recent indications of potential adverse effects of fish oil, especially on prolonged use [2,3] have created unease among health professionals and the community. Achieving the next milestone through polyunsaturated fatty acid-based therapeutics [4] is our response to these disturbing reports. The Inuit people of Greenland provided the first demonstration that fish oil has protective effects against cardiovascular disease but since then we have come to realize the complexity of applying such fats. Their beneficial effects on the cardiovascular system are through their ability to reduce risk factors and their anti-arrhythmic actions [5]. These desired properties have to be balanced against toxic effects from contaminants in fish and fish oil, including methylmercury, dioxins, and polychlorinated biphenyls which may increase the risk of cancer, myocardial infarction [6], and neurological damage. While the benefits of ω3 fats in coronary heart disease were recognized in a meta-analysis study [7], a large intervention study contradicts other studies in not finding a beneficial effect of ω3 fats [8]. Whether some differences are related to the consumption of fish versus fish oil capsules or the type of ω3 fatty acid remains to be clarified but such uncertainties give rise to a burning desire to not only conduct more appropriate clinical trials but to also identify alternative ways of using polyunsaturated fats. Targets of ω3 Fatty Acids in Atherogenesis Multiple factors and processes are involved in atherogenesis [9]. These include elevated lowdensity lipoprotein (LDL), monocyte and lymphocyte infiltration into the intima, oxidative stress, autoimmunity, complement activation, and proteases. A contribution by neutrophil products such as elastase, myeloperoxidase, and H 2 O 2 is also likely to occur in some stages of this disease. Responses initiated by oxidized LDL are perpetuated by the generation of several inflammatory mediators, including cytokines, e.g. tumor necrosis factor (TNF), chemokines, and eicosanoids. These mediators control both the infiltration and activation of the inflammatory mononuclear cells, and directly or indirectly contribute to fibrosis and hardening of the blood vessel wall [9]. Over the last few decades several targets for preventing atherosclerosis have been identified. One approach is to alter the levels of cholesterol e.g. by using statins. Most attractive

2 has been the use of ω3 polyunsaturated fatty acids found in plants and fish oils, α linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) [10]. The relevant targets are the eicosanoid pathways. By rebalancing the ω3 to ω6 ratio in tissues, a reduction in the levels of highly potent inflammatory eicosanoids (leukotrieneb4, thromboxaneb2, and prostaglandine2) and replacement with less active ω3 metabolites of the cyclooxygenase and lipoxygenase pathways (leukotrieneb5, prostaglandine3) are achieved [10]. Eicosanoid pathways can be targeted in the endothelial cells and leukocytes, producing quite dramatic reduction in cell adhesion, migration into tissues, and activation. However in the last two decades other targets of ω3 fats on inflammatory processes have been identified. Individuals and animals fed ω3-rich diets exhibit decreased production of a range of cytokines, including TNF, IL1β and IL-6 by macrophages and IL-2 and IFNγ by T lymphocytes [10], consistent with protection against atherosclerosis by ω3 fats [9,10]. Such effects appear to be distinct from their actions on lipoxygenases and cyclooxygenases and are most likely a consequence of the inhibition of transcription factor activation by ω3 fats [4,10] through the activation of peroxisomal proliferator-activated receptor α (PPAR α) [11]. Thus, ω3 fats inhibit the activation of the transcriptional factor NFκB and the up-regulation of cell adhesion molecule expression on vascular endothelium. Another recently described distinction between the anti-inflammatory ω3 and the pro-inflammatory ω6 fatty acids is their effects on TNF receptor expression on neutrophils, the latter but not the former up-regulating TNFR1 and TNFR2, thereby increasing the cellular activation induced by TNF [12]. A Novel Approach Recent evaluation of the risk and benefits of omega 3 fats reveals that long chain and shorter chain omega 3 fats do not have a clear effect on total mortality, combined cardiovascular events or cancer [2]. However, using a different set of criteria in data analysis, a more recent evaluation of published studies have found evidence of beneficial effects, especially in secondary prevention trials [13]. Nevertheless, clinical trials continue to question the protective effects of ω3 polyunsaturated fats in various conditions including cardiovascular disease. There is not enough evidence to say that people should stop taking rich sources of omega 3 fats, but further high quality trials are needed to confirm the previously suggested protective effect of omega 3 fats for those at increased cardiovascular risks [3]. Perhaps, the inconsistent results between the studies are caused, in part, by differences in the environmental factors and lifestyles to which the subject groups are exposed [14]. These include differences in dietary antioxidants, the types and amounts of other fats consumed, the level of physical activity, and the level of toxic contaminants consumed, especially in those with higher intake of fish or fish oil capsules. There are also no agreed levels at which the amount of ω3 fat intake is effective and safe. Evidently more data is needed to fill the gaps in order of developing specific recommendations for the community in the use of ω3 fats in a beneficial manner [14] Several years ago we considered that this may in part be related to our lack of clear understanding of the spectrum of biological effects of polyunsaturated fatty acids, where EPA and DHA were more pro-inflammatory than ω6 under certain conditions [15]. These included the ability of ω3 fats to stimulate neutrophil and macrophage oxygen radical production and adhesion. Recently this has been reported in individuals receiving fish oil supplement and includes increases in production of pro-inflammatory cytokines [16].

3 The Relationship between Biological Activity and Specific Structural Elements on Polyunsaturated Fatty Acids An extensive effort was made to gain insights into how the structure of long chain fatty acids may direct specific biological functions [14]. This showed that the pro-and anti-inflammatory properties were related to different structural elements on the fatty acids. This characteristic could well explain many of the failures to consistently observe a beneficial effect of ω3 fats. We pointed out that the metabolites of polyunsaturated fatty acid have highly potent antiinflammatory properties [17,18], a finding which was recently confirmed by other groups [11]. This is also consistent with the reports by Serhan and colleagues [19] of anti-inflammatory lipids being generated from the oxidation of ω3 fatty acids. We exploited this knowledge by discovering a novel approach to introduce an oxygen into the beta position of polyunsaturated fatty acids [19] and synthesized new types of fatty acids (Figure 1) which could display greater potency and more selectivity for the key targets outlined above [4,20,21]. Four key improvements on the biological properties of the ω3 fats were achieved. Firstly, these novel fats did not significantly stimulate oxygen radical production by phagocytes. Secondly, they showed greater selectivity for inflammatory targets. Thirdly, they had greater potency for the selected targets and lastly, the fats had increased stability in tissues. One of these, β-oxa 21:3n-3, for example, is particularly potent in inhibiting the 5-lipoxygenase [20] but has little effect on either the phagocyte respiratory burst or the ability of activated endothelial cells to express adhesion molecules and bind leukocytes, a property of another novel fatty acid, β-oxa23:4n-6 (4). Figure 1. A ball model of β-oxa-23:4n-6. Note the oxygen atom in the β position of the long chain fatty acid. One of a number of possible conformations is depicted. Shutting the Gate to Inflammatory Cells The endothelium is the gateway to inflammation in diseases such as atherosclerosis whereby monocytes, T lymphocytes, and neutrophils undergo a series of interactions with the endothelium to extravasate and accumulate in tissues, where they cause destruction and abnormal tissue

4 growth. Our strategy was to interfere with the leukocyte-endothelial cell interaction using the synthesized fatty acids. Of the β-oxa compounds synthesized, β-oxa 23:4n-6 was found to be the most potent in inhibiting neutrophil adherence to the endothelium [4]. At equivalent concentrations the ω3 fats were weak inhibitors. This β-oxa fatty acid also inhibited the adherence of peripheral blood monocytes to activated endothelial cells, an effect which was caused by the suppression of the ability of endothelial cells to upregulate the expression of the cell adhesion molecules, VCAM-1, ICAM-1, and E-selectin in vitro and in vivo by the fatty acid. This correlated with the inhibition of both acute and chronic inflammatory responses [4]. A major intracellular target of β-oxa 23:4n-6 is the IκB kinase - NFκB pathway which is required for the transcription of CAM genes. This work further demonstrates that metabolism of the β-oxa fatty acid occurred in the endothelial cells. Quite intriguingly, we found that a 12- lipoxygenase product, formed only in small quantities, was required for this inhibition. This result implies that a product with high potency was generated. Whether or not this is a resolvinlike product [19] remains to be established. The β-oxa compounds are in many ways like natural fats. These are esterified in the sn-2 position of the phospholipids, they can be delivered orally and have preferences for certain tissues. They continue to be omega oxidizable, representing a source of energy. It is therefore tempting to speculate that these fats may represent a considerable improvement in our approach to treat cardiovascular disease with polyunsaturated fatty acids. Conclusions In consideration of the recently expressed uncertainties in the ability to make specific recommendations on the use of ω3 fats to the community, we believe that the strategy of developing polyunsaturated fatty acid based anti-inflammatory agents provides a new approach to the prevention and treatment of cardiovascular diseases and indeed needs to undergo consideration in the coming years. Acknowledgements We are indebted to our organic chemist team for assistance with the fatty acid model. This research received financial support from the National Heart Foundation and National Health and Medical Research Council of Australia. References 1. Deckelbaum RJ, Akabas SR. n-3 fatty acids and cardiovascular diseases: navigating toward recommendations. Am J Clin Nutr Editorial 2006;84: Hooper L, Thompson RL, Harrison RA, et al. Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review. Brit Med J 2006;332: Hooper L, Thompson RL, Harrison RA, et al. Omega 3 fatty acids for prevention and treatment of cardiovascular diseases. Cochrane Database System Rev 2004;4:CD Ferrante A, Robinson BS, Singh H, et al. A novel β-oxa polyunsaturated fatty acid down regulates the activation of the IκB kinase/nuclear factor κb pathway, inhibits expression of endothelial cell adhesion molecules, and depresses inflammation. Circ Res 2006;99: Leaf A, Albert CM, Josephson M, et al. Prevention of fatal arrhythmias in high-risk subjects by fish oil n-3 fatty acid intake. Circulation 2005;112:

5 6. Butcher JF, Hengestler P, Schindler C, Meier G. N-3 polyunsaturated fatty acids in coronary heart disease: a meta-analysis of randomised controlled trials. Am J Med 2002;112: Guallar E, Sanz-Gallardo I, van t Veer P, et al. Mercury, fish oils and the risk of myocardial infarction. N Engl J Med 2002;347: Burr ML, et al Lack of benefit of dietary advice to men with angina: results of a controlled trial. Eur J Clin Nutr 2003;57: Hanson G, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol 2006;6: Calder PC. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr 2006;83:105S-19S. 11. Sethi S, Ziouzenkova O, Ni H, Wagner DD, Plutzky J, Mayadas TN. Oxidised omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPARα. Blood 2002;100: Wang C, Harris WS, Chung M, et al. n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid, benefit cardiovascular disease outcomes in primary- and secondaryprevention studies: a systematic review. Am J Clin Nutr 2006;84: Mogddahami, N, Costabile M, Grover PK, et al. Unique effect of arachidonic acid on human neutrophil TNF receptor expression: Up regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A 2. J Immunol 2003;171: Akabas SR, Deckelbaum RJ. Summary of a workshop on n-3 fatty acids: current status of recommendations and future directions. Am. J. Clin. Nutr 2006;83(Suppl):153S-8S. 15. Ferrante A., Hii CS, Costabile M. Regulation of neutrophil functions by long chain polyunsaturated fatty acids. In: Gabrivich DI, editor. The neutrophil: new outlook for old cells. London: Imperial College Press, 2005: Gorjao R, Verlengia R, de Lima TM, et al. Effect of docosahexaenoic acid-rich fish oil supplementation on human leukocyte function. Clin Nutr May [Epub ahead of print]. 17. Ferrante JV, Huang ZH, Nandoskar M, et al. Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid and arachidonic acid: inhibition of TNF production. J Clin Invest 1997;99: Huang ZH, Bates EJ, Ferrante JV, Poulos A, Robinson BS, Ferrante A Inhibition of stimuliinduced endothelial cell intercellular adhesion molecule -1, E-selectin and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy- and hydroperoxyderivatives. Circ Res1997;80: Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2006;6: Pitt MJ, Easton CJ, Ferrante A, Poulos A, Rathjen DA. Synthesis of polyunsaturated β-thia and γ- thia fatty acids from naturally derived polyunsaturated fatty alcohols and in vitro evaluation of their susceptibility to β-oxidation. Chem Phys Lipids 1998;92: Robinson BS, Rathjen DA, Trout NA, Easton CJ, Ferrante A. Inhibition of neutrophil leukotriene B 4 production by a novel synthetic n-3 polyunsaturated fatty acid analogue. J Immunol 2003;171: Please address correspondence to: Antonio Ferrante, FRCPath, Ph.D. Professor and Head of Immunopathology Professor of Immunopharmacology Children, Youth and Women s Health Service North Adelaide, South Australia 5006 Australia antonio.ferrante@adelaide.edu.au