Abstract
AIM
To evaluate the bioequivalence of two omega-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA) ethyl ester preparations, previously shown not to be bioequivalent in healthy subjects, with the objective of providing a guideline for future work in this area.
METHOD
A randomized double-blind crossover protocol was chosen. Volunteers with the lowest blood concentrations of n-3 LC-PUFA were selected. They received the ethyl esters in a single high dose (12 g) and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) blood concentrations were analyzed after fingerprick collection at intervals up to 24 h.
RESULTS
Differently from a prior study, the pharmacokinetic analysis indicated a satisfactory bioequivalence: for the AUC(0,24 h) 90% CI of the ratio between the two formulations were in the range for bioequivalence (for EPA 0.98, 1.04 and for DHA 0.99, 1.04) and the same was true for Cmax and tmax (90% CI were 0.95, 1.14 and 1.10, 1.25 for EPA and 0.88, 1.02 and 0.84, 1.24 for DHA).
CONCLUSION
This study shows that, in order to obtain reliable bioequivalence data of products present in the daily diet, certain conditions should be met. Subjects should have low, homogeneous baseline concentrations and not be exposed to food items containing the product under evaluation, e.g. fish. Finally, as in the case of omega-3 fatty acids, selected doses should be high, eventually with appropriate conditions of intake.
Keywords: bioequivalence, clinical, omega-3 fatty acids, pharmacokinetics, pharmacology
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT
Omega-3 fatty acids are dietary components, present in the body with variable blood concentrations.
Bioavailability evaluations of ethyl ester preparations are hampered by the difficulty in achieving similar concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the preparations being compared. This may require questionable corrections for baseline concentrations.
If repeated doses are given, this may lead to errors because of variable dietary fish intake. If a single dose is selected, this needs to be large, since omega-3 LC-PUFA are present in many compartments.
WHAT THIS STUDY ADDS
We selected subjects with uniform omega-3 background concentrations, to obtain comparable results at the end of treatment.
Testing bioequivalence of two formulations with different EPA : DHA ratios led to single dose intakes of 12 g, which were well tolerated.
In spite of clear differences in EPA : DHA ratios between the two preparations, plasma ratios did not differ and bioequivalence could be well ascertained.
Introduction
Dietary supplements are defined as products that improve health and in some instances prevent disease. Despite their increasing use by the general population, clinical pharmacological data on dietary supplements are often missing. A number of recent reports have stressed the need for more precise and reliable clinical studies [1]–[3]. Pharmacokinetic studies, on the other hand, may represent a challenge for clinical pharmacologists, in particular in the case of compounds already present in the daily diet, although in variable amounts. Dietary supplements are administered as tablets, capsules or other formulations in order to reach recommended intakes [4], in some cases, in high doses, in order to reach ‘pharmacological’ activities [5]. Although these supplements may not be intended necessarily to treat, cure, or mitigate disease, still consumers want to be assured of their quality, and adequate absorption is a crucial factor.
A special case is represented by the long chain polyunsaturated fatty acids (LC-PUFA) of the omega-3 series, i.e. eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3) acids, that are increasingly used with a wide range of clinical objectives [6]–[8]. After administration these fatty acids (FA), especially DHA, are incorporated into selected lipids in biomembranes of specialized cells (cardiomyocytes, synapses and retinal cells, immune cells, etc) where they modulate functional and structural properties [9]. Moreover, they must be ingested preformed mainly as fish and fish derived products, since they cannot be synthesized de novo, and, in most populations, intakes of these FA are way lower than the recommended amounts, i.e. 250 mg day−1 EPA + DHA [10]. In addition, far higher intakes are needed for a triglyceride lowering effect, a major indication of omega-3 FA [11].
For these reasons large daily intakes, i.e. 1 g or more, are generally suggested by medical societies [12]. These are available in formulated preparations as triglycerides as well as, more recently, EPA and DHA ethyl esters (EE). These latter are formulated in capsules and distributed mainly through pharmacies. In countries such as Italy, omega-3 EE are provided free of charge by the National Health Service for specific cardiovascular indications.
Since blood concentrations of omega-3 FA are highly dependent upon daily intakes of fish-derived products [13], evaluation of plasma kinetics and, in particular, of bioequivalence between different formulations may be of special complexity. In fact, in order to apply criteria properly for bioequivalence evaluation [14], relatively high doses of the compounds and assessment of changes of circulating concentrations over a relatively short time period (24 h) are required [15].
The case of a new formulation of omega-3 EE is analyzed in the present report. Bioequivalence data from a study by other investigators, based on parallel, repeated dose design, vs. a marketed compound, were questionable and did not completely meet bioequivalence criteria [14]. Nevertheless results were later published in a major clinical pharmacology journal [16].
Based on criticisms on the methodology used by the previous investigators [16], we here provide new data aimed to compare properly bioequivalence of two omega-3 FA preparations, and indicate guidelines to be followed for testing bioequivalence. The present study provides a good case in point for future evaluations of dietary supplements, based on compounds already present in blood and tissues and potentially absorbed from the daily diet.
Methods
Materials
The new formulation of EPA and DHA EE was provided by IBSA Institut Biochimique SA (Manno, CH) and the comparator was a preparation already on the market (Seacor, SPA Soc. Prod. Antibiotici, Milan, Italy). The relative composition of EPA and DHA in the two formulations is reported in Table 1.
Table 1.
Composition of the two formulations used in the study
| EPA | DHA | Total | EPA/DHA | |
|---|---|---|---|---|
| Seacor | 485 | 348 | 833 | 1.32 |
| Ibsa | 434 | 434 | 868 | 1 |
Data are expressed as mg per capsule.
Subjects, study design and determination of kinetic parameters
The protocol of the study was approved by the Ethics Committee of the University of Milan (Italy). The study details were explained to all the volunteers, and written informed consent was obtained. We elected to analyze whole blood FA, since omega-3 FA are incorporated into different lipid classes with different distributions in serum and red blood cells, thus making this type of sample more representative than just plasma, red blood cells or phospholipids, as assessed in previous studies [17].
A randomized double-blind crossover protocol with at least an 8 weeks washout between treatments was selected, on the basis of the time required for the return of fasting concentrations of EPA and DHA to basal pre-treatment values. The crossover protocol was justified by the highly individual pattern of absorption/metabolism/elimination, making a parallel study more difficult to evaluate. It was finally decided to test a single, very high dose of each omega-3 preparation, as discussed below.
The analytical method applied for the determination of blood FA profiles was based on fingerprick blood collection on a special adsorbent, embedded with the anti-oxidant butyl hydroxyl toluene (BHT) provided in a specific kit (Sigma-Aldrich, St Louis, MO). The FA analysis of whole blood was then carried out as previously described [17]. Briefly FA methyl esters were directly prepared by transesterification and, after extraction, they were analyzed by gas liquid chromatography (Shimadzu FAST GC 2010), equipped with a PTV injector, a FID detector and an Agilent DB-FFAB capillary column. Oven temperature was programmed from 150°C to 248°C. Peaks were identified using pure reference compounds and FA from 16 to 24 carbon atoms were detected and expressed as relative percentages of total FA.
Selection of the omega-3 dose for the bioequivalence study
Since circulating blood lipids are in constant exchange with major tissues containing omega-3 FA, in particular intestine, liver and peripheral organs, we assumed that there was a clear need for elevated doses of these FA in order to achieve detectable changes in blood concentrations after a single oral dose. After several tests we decided to base our choice on a report by Nordoy et al. [15] indicating that a dose of at least 12 g of EE should be used, in order to evaluate bioavailability over a short time period. Since omega-3 LC-PUFA represent a minor component of circulating FA and quantitative analysis of EPA and DHA was based on total blood FA at time 0, we had to keep FA concentrations as constant as possible during the observation period, so we decided to follow the procedure proposed by Raatz et al. [18]. These authors indicated that the intake of fish oil capsules together with a low fat breakfast or lunch, can lead to a significant increase of EPA and DHA concentrations. Thus, 12 × 1 g capsules of omega-3 EE together with this low fat breakfast were given to the volunteers. Breakfast and lunch provided 1100 total calories, of which 10% were from lipids, 78% from carbohydrates and 12% from proteins. Potential sources of omega-3 FA were avoided.
After the intake of the capsules, blood was collected by fingerpricking over 24 h at intervals 0, 2, 4, 6, 8, 12 and 24 h.
In order to obtain absolute quantitative data on omega-3 FA concentrations, concentrations of each FA were calculated following their determination, in the presence of an internal FA standard, in total lipids extracted from a venous blood sample drawn at time 0.
Results
Selection of volunteers
The omega-3 FA status in 50 young male individuals, since the metabolism of omega-3 FA is different in men and women [19], was determined. All subjects were in good health, with no known fish allergy. As shown in Figure 1, there were very wide ranges of EPA and DHA percentage levels in these individuals.
Figure 1.
Basal concentrations of EPA and DHA (% of total blood FA) in a population of young healthy men, screened for the study. Lines represent the median values for this population. EPA (
); DHA (
)
Out of the 50 volunteers the 10 subjects with the lowest omega-3 concentrations were selected and they were instructed not to eat any fish or vegetable sources rich in omega-3 FA in the 4 weeks preceding the initiation of the study. This approach allowed the direct comparison of the kinetics of the two omega-3 FA preparations, each lasting 24 h.
The selected subjects had normal blood lipids, liver and kidney function as well as haematological variables (Table 2). Their average age was 25 years with BMI in the normal range. EPA and DHA blood concentrations were in the very low range for a normal population [20], [21], i.e. mean ± SD 0.37 ± 0.12 for EPA and 1.90 ± 0.31 for DHA (% of total blood FA).
Table 2.
Characteristics of the subjects selected for the study (mean ± SD)
| Age (years) | 25.1 ± 3.0 |
| BMI | 23.8 ± 6.5 |
| Total cholesterol (mg dl−1) | 165.4 ± 27.9 |
| Triglycerides (mg dl−1) | 105.8 ± 48.5 |
| HDL-cholesterol (mg dl−1) | 53.3 ± 10.4 |
| LDL-cholesterol (mg dl−1) | 91.0 ± 24.8 |
| ALT (IU l−1) | 17.6 ± 7.3 |
| AST (IU l−1) | 18.4 ± 2.7 |
| GGT (IU l−1) | 27.5 ± 13.9 |
| EPA (% of total blood FA) | 0.37 ± 0.1 |
| DHA (% of total blood FA) | 1.90 ± 0.31 |
Pre-treatment blood concentrations of EPA and DHA were substantially comparable for each period of the cross-over study (Table 3).
Table 3.
Pre-treatment blood concentrations (mean ± SD) of EPA and DHA for each period of the crossover study
| EPA (mg l−1) | DHA (mg l−1) | |
|---|---|---|
| Period 1 | 6.64 ± 6.83 | 36.67 ± 29.17 |
| Period 2 | 8.71 ± 6.91 | 40.15 ± 18.90 |
All differences were not statistically significant.
At the different points of the time course, blood concentrations of EPA and DHA were expressed as incremental values with respect to t0.
Pharmacokinetic findings
As shown in Figure 2, the intake of 12 g of omega-3 supplements led to a clear rise of both EPA and DHA. The very similar shapes of the curves for test and reference products, both for EPA and DHA, indicated a substantial bioequivalence of the two preparations. Pharmacokinetic parameters derived from blood DHA and EPA concentrations, measured during the 24 h period and after subtraction of the values at t0, are reported in Table 4.
Figure 2.
Time-course of (A) eicosapentaenoic acid (EPA) and (B) docosahexaenoic acid (DHA) after intake of the two preparations (test (●) and reference (
)) in a single oral dose of 12 g. Data represent means ± SEM) expressed as variations of the blood concentrations in comparison to t0
Table 4.
Pharmacokinetic parameters (mean ± SD) derived from the incremental blood concentration of EPA and DHA
| EPA | DHA | |||
|---|---|---|---|---|
| Parameter | Test | Reference | Test | Reference |
| AUC(0,24 h) (mg l−1 h) | 92.80 (106.30) | 98.75 (129.84) | 136.42 (217.24) | 128.0 (185.63) |
| Cmax (mg l−1) | 5.70 (6.57) | 7.07 (10.50) | 9.19 (9.84) | 11,01 (13.60) |
| tmax (h) | 11.11 (7.36) | 10.44 (7.80) | 11.78 (9.35) | 11,56 (9.37) |
Data analyzed after logaritmic transformation with univariate anova (AUC and Cmax) or with Wilcoxon's non parametric test (tmax) were not statistically different.
Continuous variables, analyzed with univariate anova, after logarithmic transformation, indicated no statistical difference between test and reference formulation for both AUC(0,24 h) and Cmax. For tmax the Wilcoxon's non-parametric test was applied and also for this parameter no statistical difference was observed between the two preparations.
Bioequivalence findings
The assessment of pharmacokinetic parameters for bioequivalence is reported in Table 5. For the AUC(0,24 h), calculated after subtraction of the values at t0, 90% CI for the ratio of geometric means between the two formulations were mainly in the range for bioequivalence both for EPA (0.98, 1.04) and for DHA (0.99, 1.04). For Cmax and tmax 90% CI for the test : reference ratio were also within the acceptability range for both EPA and DHA (Table 5).
Table 5.
Pharmacokinetic parameters for bioequivalence based on the blood concentrations of EPA and DHA after subtraction of the values at t0
| EPA | DHA | ||||
|---|---|---|---|---|---|
| Test | Reference | Test | Reference | ||
| AUC(0,24 h) (mg l−1 h) | Geometric mean | 66.00 | 64.21 | 65.01 | 63.01 |
| Ratio test : reference | 1.03 | 1.03 | |||
| 90% C.I. | 0.98, 1.04 | 0.99, 1.04 | |||
| Cmax (mg l−1) | Geometric mean | 4.09 | 4.20 | 6.75 | 7.57 |
| Ratio test : reference | 0.97 | 0.89 | |||
| 90% C.I. | 0.95, 1.14 | 0.88, 1.02 | |||
| tmax (h) | Geometric mean | 9.58 | 8.59 | 8.59 | 8.70 |
| Ratio test : reference | 1.12 | 0.99 | |||
| 90% C.I. | 1.01, 1.25 | 0.84, 1.24 | |||
Discussion
In the previous report of a bioequivalence study between two omega-3 EE formulations the authors relied on parallel study design with a repeated dose, long term (28 days) intake of the new omega-3 EE formulation vs. one already on the market [16]. The pharmacokinetic parameters were than evaluated after the morning dose on the last day of treatment. In view of the different relative proportions of EPA and DHA in the two formulations tested, the study design compelled the authors to correct the data for this difference. Under our experimental conditions no recalculation of experimental data was required. In the previous report [16] only a clinical characterization of the participating volunteers was provided. In particular, it was not tested whether their concentrations of omega-3 were high or low. In addition, dietary intake of omega-3 was not monitored, a variable of special relevance, since it has been reported by our group that three portions per week of salmon, providing a few hundred mg day−1 of EPA and DHA, led to comparable concentrations of circulating omega-3 FA as the daily intake of grams of omega-3 EE in capsules [22]. The lack of selection of the volunteers according to omega-3 blood levels, may thus have led to the enrolment of individuals with highly scattered circulating concentrations of these FA, compelling the authors to correct final kinetic data for pretreatment values of EPA and DHA for each subject. In addition a potentially variable intake of fish in the course of the study was not monitored, contributing to the probable inadequacy of the study for the objective of testing bioequivalence. We tested the same new formulation of omega-3 EE vs. the same comparator [16] with a number of preliminary evaluations as well as with a different protocol, leading to different bioequivalence findings.
The results of this bioequivalence study are of particular interest today, when clinical pharmacologists are confronted more and more with formulations of natural products, present in the diet, e.g. lactotripeptides for blood pressure management [23], creatine for sports [24] or natural molecules present in the body, e.g. carnitine or derivatives for cardiac indications [25] or for Lp(a) lowering [26] and numerous others.
Although at first glance the results of our study may seem of little clinical relevance, a precise knowledge of the bioavailability of nutraceuticals plays a significant interest in defining appropriate doses and dosage regimens. The case of omega 3 is peculiar in view of their indication also in clinically relevant conditions such as hypertriglyceridaemia [11] and prevention of cardiovascular disease [27], [28]. In these cases specific dosage regimens as well as monitoring of adherence to treatment is essential to define clinical outcomes [29].
Careful avoidance of potential sources of error, in this new area of nutraceutical bioavailability, should be the care of clinical pharmacologists responsible for the investigation.
In order to carry out reliable bioavailability studies, certain conditions should be met, as clearly reported in the present study. Participating subjects must be selected among individuals with low and homogeneous baseline concentrations of the variables to be determined. Volunteers should not be exposed in the study to food items containing the product under evaluation. Since a large background of molecules may be present in the body, as in the case of omega-3 FA, selected doses should be high, as in the present report and associated with optimized dietary conditions.
Competing Interests
Funding for this study was provided by IBSA Institut Biochimique SA (Manno, CH) with a research contract with Università di Milano.
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