Doi:10.1016/j.plefa.2006.04.003

Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24 The bioavailability and pharmacodynamics of different concentrations of omega-3 acid ethyl esters M. Bryhna,Ã, H. Hansteena, T. Schancheb, S.E. Aakreb aPronova Biocare, R&D, Vollsveien 6, N-1327 Lysaker, Norway Received 9 March 2006; received in revised form 6 April 2006; accepted 15 April 2006 Omega-3 fatty acids have a long history of use as dietary supplements and more recently for therapeutic applications as prescription pharmaceuticals. Achieving a high concentration is critical for developing convenient, practical therapeuticformulations. The objective of the study was to explore the uptake and effects of different concentrations of omega-3 acid ethylesters. Three different omega-3 concentrations were investigated in a clinical study with 101 subjects. All participants were dosed for14 days with 5.1 g per day of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters provided in threeconcentrations: 62.5%, 80% and 85% of total fatty acids. Key endpoints of the study were serum phospholipids and standardfasting lipid panels at day 14.
Although administered the same quantity of omega-3 fatty acids, the patients taking the more concentrated formulations had higher levels of EPA/DHA in serum phospholipids and greater reductions in serum triglyceride and VLDL cholesterol levels. Totaland non-HDL cholesterol were significantly reduced from baseline with all three formulations.
In conclusion the concentration of omega-3 fatty acids of the formulations studied had independent effects on the uptake and effect outcomes during short-term administration. Very high concentrations of omega-3 acid ethyl esters (X80%) appear to havehigher uptake and are more potent for reducing triglycerides (TGs) and VLDL-cholesterol than formulations with lowerconcentrations.
r 2006 Elsevier Ltd. All rights reserved.
meaningful effects is generally higher than recom-mended dietary supplementation levels With The use of omega-3 fatty acids for health benefits is increasing dose, the number of omega-3 fatty acid well established throughout the world. Various national containing capsules required to achieve therapeutic and international health authorities have also issued effects becomes a key concern. If the number of capsules intake recommendations, including the US Food and to be taken daily is too high, patient compliance may be Drug Administration , American Heart Association jeopardized. In order to create a convenient dosing , European Cardiology Society, United Kingdom regimen of omega-3 containing capsules, concentrating Scientific Advisory Committee on Nutrition and the the omega-3 formulation is a logical step.
International Society for the Study of Fatty Acids and We have developed such a highly concentrated Lipids . Therapeutic use of omega-3 fatty acids to omega-3 fatty acid containing formulation for thera- manage cardiovascular risk factors is also increasing.
peutic applications. This product contains X90% However, the dose needed to reliably achieve clinically omega-3 fatty acids in the ethyl ester form, predomi-nantly (X84%) esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This 90% omega-3 Corresponding author. Tel.: +4722534875; fax: +4722534851.
E-mail address: ethyl ester product has been extensively investigated and 0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
M. Bryhn et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24 documented for cardiovascular indications such as the The study design provided exactly the same amount treatment of hypertriglyceridemia and secondary of omega-3 fatty acids for each treatment group. Study prevention of cardiovascular disease For in- medication was given in identical dark glass bottles stance, the large GISSI Prevenzione study (n ¼ 11; 324) labelled with the subjects number to ensure the subjects showed significant improvement in survival in post- blinding to the treatment group. The least concentrated myocardial infarction patients This product is 62.5% bottles contained 9.20 ml, the 80% bottles now approved in most European countries and the 7.75 ml and the most concentrated EPA/DHA prepara- United States for the treatment of hypertriglyceridemia tion contained the lowest volume 7.25 ml. To prevent and/or secondary prevention of cardiovascular disease.
oxidation the bottles were sealed under nitrogen gas Although the mechanisms of action of omega-3 fatty flushing. Study medication was taken for 14 consecutive acids are not entirely understood, it is believed that two days, in the morning, in the fasting state, followed by a major effects cause the reduction of triglycerides (TGs) light breakfast meal. During weekdays, the study in the blood. First, omega-3 fatty acids may reduce the medication was taken under observation by study synthesis of TGs in the liver due to inhibition of acyl personnel; while during weekends the subjects self- CoA:1,2-diacylglycerol acyltransferase Because administered the provided medication. Study personnel omega-3 fatty acids such as EPA and DHA have checked to insure that all bottles were completely substantial affinity, but are poor substrates for, the enzymes responsible for triglyceride synthesis, the Weight, height, blood pressure and smoking habits esterification and release of other fatty acids is inhibited were recorded at study entry. All concomitant medica- . Second, omega-3 fatty acids appear to increase tion used during the study was recorded. None of the peroxisomal b-oxidation in the liver due to a high subjects were taking lipid-lowering drugs. Subjects who affinity for PPAR subclasses, thereby up-regulating the used cod liver oil or any other omega-3 food supplement metabolization of fatty acids in the liver .
were excluded. The subjects were instructed to avoid In our efforts to develop this omega-3 fatty acid intake of fish high in omega-3 fatty acids (e.g., mackerel, derived prescription pharmaceutical product, we inves- herring, trout and salmon) during the course of the tigated the uptake and effects of a range of relatively high concentrations of omega-3 ethyl esters. We Venous blood was drawn from fasting individuals in initiated a clinical trial to evaluate the relative uptake the morning at study entry and of study days 7 and 14.
of omega-3 fatty acids and the effect on the lipid profile Adverse events were recorded and were either sponta- with three concentrations of EPA and DHA ethyl esters: neously reported or elicited by non-leading questioning.
62.5%; 80% and 85%. Fish oil was not tested due to the Subjects were withdrawn from the study if they low concentration of omega-3 fatty acids; typically 20– developed an illness that could interfere with the results 35%. This low concentration makes fish oil products of the study, if non-compliance was verified or if the subject decided to discontinue for any reason.
EPA and DHA were measured in the phospholipid fraction of serum by gas chromatography. Plasma total cholesterol, high-density lipoprotein (HDL) cholesteroland TGs were measured using standard methods. Low- The study was approved by the Ethics Committee density lipoprotein (LDL) cholesterol was calculated Region II in Norway. One hundred and one (101) male individuals were randomly allocated to treatment with5.1 g of EPA and DHA provided as 62.5%, 80% or 85% LDL cholesterol in mg=dL ¼ total cholesterol ethyl esters (respectively 71%, 88.5% and 93.5% omega- À HDL cholesterol À triglycerides=5:0.
3 acid ethyl esters in total) in a double-blind fashion. TheEPA and DHA ratio was approximately 1.0:0.8. The Statistical analyses were completed on a per protocol relatively high dose was chosen in order to ensure that we sample. No imputation of missing data-points was would operate at therapeutic dose levels for this study completed. All tests for significance were performed at population with relatively low triglyceride levels. A liquid a ¼ 0:05, two-tailed. Analyses were conducted using formulation was chosen in order to facilitate dosing of SAS version 8.2 (SAS Institute, Cary, NC). Baseline the different concentrations. All subjects were between 18 comparability of treatment groups for age, height, and 60 years of age and gave written informed consent to weight, vital signs measurements and lipid values was participate in the study. Case record forms were provided for the recording of all data. A clinical trial monitor Individual changes from baseline (day 0) to days 7, 14 checked and collected the completed case report forms and 28 were calculated for each subject. Means and and executed source data verification in accordance with standard deviations for the values, the changes, and EU-guidelines for Good Clinical Practice.
percent changes from baseline to each subsequent visit M. Bryhn et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24 were calculated by treatment group for biochemical The values for the lipid parameters and changes Models including treatment as the independent parameters are similar for all treatment groups, except variable were generated to assess differences between for changes in TGs and VLDL-cholesterol. Triglyceride treatment groups for the changes from baseline (day 0) and VLDL-cholesterol reductions compared to baseline to days 7 and 14. Changes within groups were evaluated were significant only for the 80% and 85% EPA/DHA by paired t-tests for the changes and percent changes ethyl ester concentrations as shown in .
from baseline (day 0) to each subsequent visit.
Administration of omega-3 fatty acid containing The treatment groups were comparable with respect formulations increased systemic levels of omega-3 fatty to demographic data and other baseline values. No acids. All three treatment groups were administered the protocol violations were recorded. One subject in the same amount of omega-3 fatty acids and, despite this, group given the 62.5% treatment was withdrawn after 1 the EPA increases in plasma phospholipids were week of treatment due to abdominal pain caused by a significantly different between groups. Even on a gall bladder disorder. Six study participants reported relative basis, the treatment group taking the higher adverse events: four in the 62.5% group had diarrhoea 85% concentration demonstrated a statistically signifi- and oesophageal regurgitation. One subject in the 80% cant further increase in phospholipid EPA levels than group had gastrointestinal discomfort, probably caused the treatment group taking the lower 62.5% concentra- by viral infection, as several family members had similar tion. This is an unexpected finding, since the adminis- symptoms. None of the other participants reported any tered dose of omega-3 fatty acids was the same in all One hundred participants completed the study pro- tocol: 35 in the 62.5% group, 35 in the 80% group and30 in the 85% group. The mean values, mean absolute change, mean percent change, and standard deviations for EPA in serum phospholipids are shown in .
There was a significant increase in the EPA content of serum phospholipids versus baseline in all treatment groups after 14 days. However, the increase wassignificantly higher in the group receiving 85% com- pared to the groups receiving 62.5% and 80% as shown Baseline values for serum phospholipid DHA were not significantly different between the groups. Relative 5.1 gr EPA+DHA
5.1 gr EPA+DHA
5.1 gr EPA+DHA
change from day 0 to 14 was 24% in the 62.5% group, 32% in the 80% group, and 30% in the 85% group.
Fig. 1. Relative increase in EPA serum phospholipids versus baseline These changes were not statistically significant.
Table 1Serum phospholipid EPA levels by treatment group Statistical significant changes (absolute and relative) versus baseline within all treatment groups from day 0 to day 14, Po0:001.
Increase (mg/ml) period 0–7: 58% versus 62.5%, Po0:05.
Increase (mg/ml) period 0–14: 85% versus 80%, Po0:05.
Increase (%) period 0–14: 85% versus 62.5%, Po0:05.
M. Bryhn et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24 Table 2Mean lipid parameters by treatment group 5.1 g EPA/DHA in 80% concentration N ¼ 35 5.1 g EPA/DHA in 85% concentration TG ¼ triglycerides; VLDL-C ¼ very low-density lipoprotein cholesterol; CHOL ¼ total cholesterol; LDL-C ¼ low-density lipoprotein cholesterol;Non-HDL-C ¼ non-high-density lipoprotein cholesterol; HDL-C ¼ high-density lipoprotein cholesterol.
aVLDL-C was calculated using the Friedewald Equation where VLD-C in mg/dl ¼ triglcyerides/5.0.
This was not observed for the other omega-3 fatty 5.1 gr EPA+DHA as
5.1 gr EPA+DHA as
5.1 gr EPA+DHA as
acid compound, DHA. In this study, the increase of DHA in plasma phospholipids from baseline to day 14ranged from 24% to 32% and did not clearly differ between the concentrations. The lack of difference inserum phospholipid DHA may be explained by thechoice of determining omega-3 fatty acid absorption by analyzing their incorporation in serum phospholipids.
While EPA typically accumulates in the phospholipid fraction of cells in or near the circulating blood such asblood cells and vascular endothelium, DHA is mainly translocated to the brain, the retina of the eye, lungs and other tissues . Interestingly in a dose-finding studyusing a DHA-preparation with a low dose of 0.75 g/day and a high dose of 1.50 g/day for 6 weeks no significant Fig. 2. Relative decrease in serum triglycerides versus baseline from increase was found between the dosage groups at day 21 day 0 to 14 (P-values versus baseline).
and day 42 even if the difference to baseline values wasstatistically significant for both groups Anotherstudy demonstrated that the washout time for DHA Although the EPA/DHA absorption in the 62.5% group after using omega-3 fatty acids for treatment of was lower compared to 85% and 80% treatment groups, rheumatoid arthritis was significantly longer than for there was still a substantial 308% increase in serum EPA These data suggest that the distribution of phospholipid EPA levels in this group. This is only slightly lower than the 345% increase in serum phospholipid EPA The effect parameters in this study were the blood levels in the 80% group, indicating that the relative uptake lipid fractions for TGs and cholesterol. Concentrated of the fatty acids are not a likely cause for the inferior tri- omega-3 fatty acid formulations are very effective in glyceride reducing potency in the 62.5% treatment group.
lowering TGs. Even in subjects with essentially normal A review of the literature on the mechanisms of action for omega-3 fatty acids in dyslipidemia provides a 130 mg/dl), the 85% and the 80% EPA/DHA concen- possible explanation for the concentration effect. The trations lowered TGs by about 15%. In contrast, the two major mechanistic pathways for lipid management 62.5% concentration had little effect on TGs. Even for omega-3 fatty acids provided by the literature are: though the subjects in the 62.5% treatment group hadsomewhat higher baseline triglyceride levels (approxi-  omega-3 fatty acids are a poor substrate for synthesis mately 150 mg/dl), this concentration, with the same of TGs and inhibit acyl CoA:1,2-diacylglycerol omega-3 fatty acid content as the 85% and 80% acyltransferase because of their relative high affinity concentrations, did not produce a meaningful impact with this enzyme, coupled with a low catalytic rate M. Bryhn et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24  omega-3 fatty acids increase fatty acid oxidation by with the same quantity, but less concentrated omega-3 up-regulating peroxisomal b-oxidation in the liver, as well as interacting with transcription factors regulat-ing fatty acid oxidation in the mitochondria .
This study was funded by Pronova Biocare, Norway, Particularly the first pathway described above could formerly a wholly owned subsidiary of Norsk Hydro, explain the significant difference between the 62.5% and the 85% concentrations with respect to their triglyceridereducing potency. The 85% EPA/DHA concentration contains 91% omega-3 fatty acids plus minimalamounts of other fatty acids. In contrast, the 62.5% [1] FDA announcement on qualified health claims for omega-3 fatty EPA/DHA concentration contained more than one- acids, September 8, 2004; accessible via [2] P.M. Kris-Etherton, W.S. Harris, L.J. Appel, Fish consumption, third non-omega-3 fatty acids. Because of the high fish oil, omega-3 fatty acids, and cardiovascular disease, Circula- affinity omega-3 fatty acids have with acyl CoA:1,2- diacylglycerol acyltransferase and their poor conversion [3] W.S. Harris, Are omega-3 fatty acids the most important rate into TGs, the omega-3 fatty acids may in effect nutritional modulators of coronary heart disease risk, Curr.
block the enzyme from producing TGs. Other fatty Atherosclerosis Rep. 6 (2004) 447–452.
[4] E. Balk, M. Chung, A. Lichtenstein, et al., Effects of omega-3 acids present in higher amounts in the 62.5% concen- fatty acids on cardiovascular risk factors and intermediate tration would not react in the same manner thereby markers of cardiovascular disease. Evidence report/technology having a lower triglyceride lowering potency.
assessment no. 93 (prepared by Tufts-New England Medical This study also demonstrates the overall beneficial Center Evidence-based Practice Center under Contract No. 290- effects of high dose of omega-3 fatty acid therapy on the 02-0022). AHRQ Publication No. 04-E010-2, Agency for Health-care Research and Quality, Rockville, MD, March 2004.
lipid profile. Despite the relatively low baseline choles- [5] W.S. Harris, H.N. Ginsberg, N. Arunakul, et al., Safety and terol levels for the individuals in this study, statistically efficacy of Omacor in severe hypertriglyceridemia, J Cardiovasc.
significant reductions of total cholesterol, non-HDL cholesterol and LDL-cholesterol were observed. The [6] H.J. Pownall, D. Brauchi, C. Kilinc, et al., Correlation of serum minor reduction in LDL cholesterol in the 85% EPA/ triglyceride and its reduction by omega-3 fatty acids with lipidtransfer activity and the neutral lipid compositions of high-density DHA treatment group may have been the result of a and low-density lipoproteins, Atherosclerosis 143 (1999) 285–297.
shift from VLDL cholesterol to LDL cholesterol as a [7] M.I. Mackness, D. Bhatnagar, P.N. Durrington, et al., Effects of consequence of the substantial triglyceride reduction a new fish oil concentrate on plasma lipids and lipoproteins in In fact, in subjects with higher baseline triglyceride patients with hypertriglyceridemia, Eur. J. Clin. Nutr. 48 (1994) levels, this shift may actually cause an increase in LDL [8] L. Borthwick, The effects of an omega-3 ethyl ester concentrate cholesterol. However, the total amount of cholesterol on blood lipid concentrations in patients with hyperlipidemia, carried by artherogenic particles, often measured as the Clin. Drug Invest. 15 (5) (1998) 397–404.
non-HDL cholesterol (LDL-C+VLDL-C) level, will [9] H. Grundt, D.W.T. Nilsen, O. Hetland, et al., Improvement of typically decrease when patients with very high TGs are serum lipids and blood pressure during intervention with n-3 fatty treated with the pharmaceutical omega-3 derived acids was not associated with changes in insulin levels in subjectswith combined hyperlipidemia, J. Intern. Med. 237 (3) (1995) product . In addition, omega-3 fatty acids may have a positive effect on the distribution of LDL cholesterol [10] GISSI-Prevenzione Investigators, Dietary supplementation with particle size, inducing a shift from small, dense n-3 polyunsaturated fatty acids and vitamin E after myocardial triglyceride-rich particles to more buoyant, cholesterol- infarction: results of the GISSI-Prevenzione Trial, Lancet 354 rich particles . Analysis of particle size was, however, [11] R. Marchioli, et al., on behalf of the GISSI-Prevenzione not completed as part of this study.
Investigators. Early protection against sudden death by n-3 The key finding of this study is that formulations with polyunsaturated fatty acids after myocardial infarction, Circula- same amount of omega-3 fatty acids, but with different concentrations of the active ingredients, yield differences [12] J.B. Marsh, D.L. Topping, P.J. Nestle, Comparative effects of fish in the uptake and effect properties. Furthermore, a high oil and carbohydrate on plasma lipids and hepatic activities ofphosphatidate phoshorylase, diacyl glycerol acyltransferase and concentrated omega-3 preparation facilitates dosing neutral lipase activities in the rat, Biochim. Biophys. Acta 922 compared to lower concentrates a fact which translates into greater convenience and associated compliance.
[13] A.C. Rustan, J.O. Nossen, E.N. Chrisriansen, et al., Eicosapen- These findings are important when omega-3 fatty acid- derived compounds are used therapeutically.
triacylglycerol by decreasing the activities of acyl-coenzyme A:1,2-diacylglycerol-acyl-transferase, In conclusion, the uptake and lipid lowering proper- ties of highly concentrated omega-3 fatty acid-derived [14] H. Sampath, J.M. Ntambi, Polyunsaturated fatty acid regulation formulations are superior compared to formulations of gene expression, Nutr. Rev. 62 (9) (2004) 333–339.
M. Bryhn et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 19–24 [15] S.S. Lee, W.Y. Chan, C.K. Lo, et al., Requirement of PPAR- non-esterified fatty acids in subjects of Asian Indian background, alpha in maintaining phospholipid and tri-acylglycerol home- ostasis during energy deprivation, J. Lipid Res. 45 (11) (2004) [19] P.S. Sastry, Lipids of nervous tissue: composition and metabo- 2025–2037 (Epub 2004 September 1).
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[16] D.B. Jump, Fatty acid regulation of gene transcription, Crit. Rev.
[20] W.S. Harris, N-3 fatty acids and serum lipoproteins: human Clin. Lab. Sci. 41 (1) (2004) 41–78.
studies, Am. J. Clin. Nutr. 65 (5, Suppl) (1997) 1645S–1654S.
[17] A.P. Simopolous, Omega-3 fatty acid in health and disease and in [21] A.F.H. Stalenhoef, J. de Graaf, M.E. Wittekoek, et al., The effect of growth and development, Am. J. Clin. Nutr. 70 (1999) S560–S569.
concentrated n-3 fatty acids versus gemfibrozil on plasma lipoproteins, [18] J.A. Conquer, B.J. Holub, Effects of supplementation with low density lipoprotein heterogeneity and oxidizability in patients with different doses of DHA on the levels of circulating DHA as hypertriglyceridemia, Atherosclerosis 153 (2000) 113–129.

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