Food and Chemical Toxicology 52 (2013) 121–128

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Food and Chemical Toxicology

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Herb–drug interaction of Fucus vesiculosus extract and amiodarone in rats: Apotential risk for reduced bioavailability of amiodarone in clinical practice

Márcio Rodrigues a,b,c, Gilberto Alves b,c,⇑, João Abrantes a,b, Amílcar Falcão a,b

a Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugalb CNC – Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugalc CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal

a r t i c l e i n f o

Article history:Received 11 September 2012Accepted 8 November 2012Available online 20 November 2012

Keywords:AmiodaroneFucus vesiculosusHerb–drug interactionPharmacokinetics

0278-6915/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.11.012

Abbreviations: AUC, area under the concentration–time zero to the last sampling time; AUC0–1, AUC frolast quantifiable concentration; Cmax, peak concentraDAD, diode array detection; FDA, Food and Drugperformance liquid chromatography; i.p., intraperitonapparent terminal rate constant; LLE, liquid–liquiquantification; MDEA, mono-N-desethylamiodarone;P-gp, P-glycoprotein; SEM, standard error of the meelimination half-life; tmax, time to reach Cmax.⇑ Corresponding author at: CICS-UBI – Health Scien

sity of Beira Interior, Av. Infante D. Henrique, 6200-50275 329002; fax: +351 275 329099.

E-mail address: gilberto@fcsaude.ubi.pt (G. Alves).

a b s t r a c t

Fucus vesiculosus is a seaweed claimed to be useful for obesity management. Therefore, considering therelationship between obesity and cardiovascular diseases, this work aimed to assess the potential foran herb–drug interaction among a standardized F. vesiculosus extract (GMP certificate) and amiodarone(a narrow therapeutic index drug) in rats. In a first pharmacokinetic study, rats were simultaneouslyco-administered with a single-dose of F. vesiculosus (575 mg/kg, p.o.) and amiodarone (50 mg/kg, p.o.);in a second study, rats were pre-treated during 14 days with F. vesiculosus (575 mg/kg/day, p.o.) andreceived amiodarone (50 mg/kg, p.o.) on the 15th day. Rats of the control groups received the correspond-ing volume of vehicle. After analysis of the pharmacokinetic data it deserves to be highlighted the signif-icant decrease in the peak plasma concentration of amiodarone (55.4%) as well as the reduction ofsystemic exposure to the parent drug (�30%) following the simultaneous co-administration of F. vesicu-losus extract and amiodarone. This paper reports, for the first time, the herb–drug interaction between F.vesiculosus and amiodarone, which determined a considerable decrease on amiodarone bioavailability inrats. Therefore, the therapeutic efficacy of amiodarone may be compromised by the concurrent adminis-tration of herbal slimming medicines/dietary supplements containing F. vesiculosus.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Amiodarone [2-n-butyl-3-(3,5-diiodo-4-diethylaminoethoxy-benzoyl)-benzofuran] (Fig. 1) is one of the most commonly pre-scribed antiarrhythmic drugs (Vassallo and Trohman, 2007; vanHerendael and Dorian, 2010; Freemantle et al., 2011). Despite thewell-known safety concerns ascribed to amiodarone and its majormetabolite [mono-N-desethylamiodarone (MDEA); (Fig. 1)],mainly pulmonary and hepatic toxicity as well as thyroid dysfunc-tions, amiodarone has been considered the drug with best efficacyfor the prophylaxis and treatment of a wide range of heart rhythm

ll rights reserved.

time curve; AUC0–t, AUC fromm time zero to infinite; Clast,tion; CYP, cytochrome P450;Administration; HPLC, high-eal; IS, internal standard; kel,d extraction; LOQ, limit ofMRT, mean residence time;an; t1/2el, apparent terminal

ces Research Centre, Univer-6 Covilhã, Portugal. Tel.: +351

disorders (van Herendael and Dorian, 2010; Freemantle et al.,2011). In particular, amiodarone is indicated for the treatment ofsevere rhythm disorders when other treatments are ineffective orhave not been tolerated (van Herendael and Dorian, 2010).

However, amiodarone has some unfavorable and very unusualproperties from the pharmacokinetic viewpoint, which certainlydetermine its pharmacodynamic and toxicological profiles (Fuku-chi et al., 2009; van Herendael and Dorian, 2010). In humans, ami-odarone has shown an erratic gastrointestinal absorption and,consequently, a variable oral bioavailability (Shayeganpour et al.,2008; Wolkove and Baltzan, 2009); this antiarrhythmic agent hasalso a huge body tissue distribution (Ohyama et al., 2000; van Her-endael and Dorian, 2010) and a long elimination half-life (Shaye-ganpour et al., 2008). Moreover, amiodarone has been recognizedas a drug of narrow serum/plasma therapeutic range (0.5–2.0 lg/mL) (Lesne and Pellegrin, 1987; Huy et al., 1991; Manfredi et al.,1995; Iervasi et al., 1997; Pérez-Ruiz et al., 2002; Shayeganpouret al., 2008) and has also been associated to important clinical druginteractions (Edwin et al., 2010; Karimi et al., 2010; Rougheadet al., 2010). Nonetheless, amiodarone has usually been consideredthe precipitant or interacting agent in the majority of pharmacoki-netic-based drug interactions reported in literature, inducing achange (generally an increase) in serum/plasma levels of

Fig. 1. Chemical structures of amiodarone (AM) and its major metabolite mono-N-desethylamiodarone (MDEA).

122 M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128

co-administered drugs [e.g. digoxin (Lesko, 1989; Trujillo and No-lan, 2000), warfarin (Lesko, 1989; Trujillo and Nolan, 2000; Yam-reudeewong et al., 2003), phenytoin (Lesko, 1989), theophylline(Trujillo and Nolan, 2000) and simvastatin (Becquemont et al.,2007; Marot et al., 2011)]. Effectively, only few studies have beenpublished describing the interference of other compounds on thepharmacokinetics of amiodarone. Specifically, the metabolism ofamiodarone was dramatically inhibited by grapefruit juice (Libersaet al., 2000); the systemic exposure to amiodarone and MDEA wassignificantly reduced by the simultaneous administration of orli-stat (Zhi et al., 2003); and, more recently, the exposure of rats tob-naphthoflavone (a polycyclic aromatic hydrocarbon) was foundto increase the formation of MDEA probably through cytochromeP450 (CYP) induction (Elsherbiny et al., 2010).

Clinical, pathophysiologic and epidemiological studies haveclearly indicated that overweight and obesity are two of the majorindependent risk factors for coronary heart disease, atrial fibrilla-tion and heart failure (Scaglione et al., 2004; Zalesin et al., 2011).Thus, given the increasing prevalence of obesity, reaching pan-demic proportions in developed countries, it is expected a signifi-cant increase in the incidence of cardiovascular diseases (Bodaryet al., 2007), as well as in the clinical use of drugs such as amioda-rone (Singh, 2008). On the other hand, aiming to improve their car-diovascular status, the patients are also increasingly seekingcomplementary medicines for weight reduction and/or weightmanagement, including herbal dietary supplements. Hence, overthe last years, the consumption of herbal weight loss supplementshas grown at an alarming rate never seen before (Egras et al.,2011).

Fucus vesiculosus, traditionally known as bladderwrack, is amedicinal plant that is claimed to be useful for the treatment ofobesity, mainly due to its high levels in iodine, whose action isthought to be related to the stimulation of the thyroid gland andthe subsequent effect on metabolic rate. Although iodine is consid-ered the most prominent active ingredient of F. vesiculosus, themucilage (dietary fiber), phytosterols and tetraterpenes are alsoimportant constituents responsible for its use in obesity manage-ment (Moro and Basile, 2000; Díaz-Rubio et al., 2009). In fact,based on F. vesiculosus powder, the major components are non-digestible polysaccharides composed in a large extent by both sol-uble (10.52%) and insoluble (48.63%) fractions of dietary fiber(Díaz-Rubio et al., 2009). In addition, besides proteins, minerals,vitamins and fatty acids, F. vesiculosus also contains polyphenols(Zaragozá et al., 2008; Díaz-Rubio et al., 2009) and some of themwere recently found to inhibit drug-metabolizing enzymes likeCYP1A (Parys et al., 2010).

For all the reasons referred to above, and bearing in mind thehigh potential for co-administration of F. vesiculosus medicinal/die-tary preparations and amiodarone, particularly in overweight orobese patients with cardiovascular diseases, it is urgent to generatedata in order to assess the interference of F. vesiculosus on the phar-

macokinetics of amiodarone (a narrow therapeutic index drug).Furthermore, to the best of our knowledge, no study was previ-ously conducted to evaluate the potential of interaction betweenF. vesiculosus and amiodarone. Therefore, the present work was de-signed to investigate whether a commercial standardized F. vesicu-losus extract may influence the pharmacokinetics of amiodarone inrats, after their simultaneous oral co-administration, and followinga 14-day F. vesiculosus pre-treatment period.

2. Materials and methods

2.1. Drugs and materials

Bladderwrack (F. vesiculosus L.) 0.10% dry aqueous extract (certificate of analysisnumber 201003344; provided as Supplementary data) obtained from thallus waspurchased from EPO Istituto Farmochimico Fitoterapico s.r.l. (Milano, Italy). Car-boxymethylcellulose sodium salt for preparation of extract suspension was ob-tained from Sigma (St. Louis, MO, USA). A commercial formulation (ampoules) ofamiodarone 50 mg/mL solution for intravenous injection was used for oral admin-istration to rats after appropriate dilution with 5% glucose intravenous solution forinfusion (B. Braun Medical, Portugal). Other compounds used were sodium chloride0.9% solution for injection (Labesfal, Portugal); heparin sodium 5000 U.I./mL forinjection (B. Braun Medical, Portugal); ketamine for injection (Imalgene 1000)and xylazine for injection (Vetaxilaze 20). Introcan� Certo IV indwelling cannula(22G; 0.9 � 2.5 mm) made of polyurethane from B. Braun (Melsungen AG, Melsun-gen, Germany).

2.2. Animals

Adult male Wistar rats (342 ± 28 g) of approximately 10 weeks old were ob-tained from local animal facilities (Faculty of Health Sciences of the University ofBeira Interior, Covilhã, Portugal). The rats were maintained under controlled envi-ronmental conditions (temperature 20 ± 2 �C; relative humidity 55 ± 5%; 12-hlight/dark cycle). The animals were allowed free access to a standard rodent diet(4RF21, Mucedola, Italy) during almost all experimental procedures and tap waterwas available ad libitum. At night on the day before dosing with amiodarone, a lat-eral tail vein of each rat was cannulated under anesthesia [ketamine (90 mg/kg)/xylazine (10 mg/kg); i.p. injection] by insertion of an Introcan� Certo IV indwellingcannula (22G; 0.9 � 2.5 mm) used for serial blood sampling. The rats fully recov-ered from anesthesia overnight and were fasted for 12–14 h before amiodaroneadministration and maintained with free access to water; an additional fasting per-iod (4 h post-dose) was considered to avoid the effect of food on the oral bioavail-ability of amiodarone. Oral treatments of the rats with F. vesiculosus andamiodarone were performed by gavage. Blood sampling was conducted in con-scious and freely moving rats, which were appropriately restrained only at the mo-ment of blood collection, except for the last blood sampling that was taken by aterminal procedure (decapitation and exsanguination under anesthesia). All theanimal experiments were conducted in accordance with the European Directive(2010/63/EU) for animal experiments and the experimental procedures were re-viewed and approved by the Portuguese Veterinary General Division.

2.3. Experimental design and pharmacokinetic studies

To investigate the effects of F. vesiculosus on the kinetics of amiodarone, twoseparate and independent pharmacokinetic studies were designed: (1) a single oralco-administration study of F. vesiculosus extract and amiodarone; and (2) a 14-dayrepeated oral pre-treatment study with F. vesiculosus extract and on the 15th day asingle oral dose of amiodarone was administered. The dose of F. vesiculosus was se-lected according to the study of Skibola (2004) and taking into account the Food andDrug Administration (FDA) Guidance for Industry on conversion of animal doses tohuman equivalent doses based on body surface area (US DHHS, FDA, CDER, 2005);additionally, a 10-fold potentiating interaction factor was considered. On the otherhand, the oral dose of amiodarone of 50 mg/kg was established because it has pro-vided plasma concentrations of drug in rats within the plasma therapeutic range(Shayeganpour et al., 2005). In each day of the experiments F. vesiculosus extractwas suspended in 0.5% carboxymethylcellulose aqueous solution affording a sus-pension of herbal extract at 57.5 mg/mL. Amiodarone commercial injectable solu-tion (50 mg/mL) was also appropriately diluted with 5% glucose solution toextemporaneously prepare an amiodarone solution at 12.5 mg/mL. At this point,it should be noted that an amiodarone injectable solution was used as starting drugmaterial for oral administration, rather than amiodarone tablets, to avoid the pos-sible interference of tablet excipients on the bioavailability of amiodarone in rats,thus ensuring better reproducibility, practicability and minimizing the presenceof confounding factors that could hinder the interpretation of the results. Appropri-ate volumes of F. vesiculosus extract suspension (10 mL/kg of body weight) and ofamiodarone solution (4 mL/kg of body weight) were orally administered to ratsby gavage.

Fig. 2. Mean plasma concentration–time profiles of amiodarone (AM) and mono-N-desethylamiodarone (MDEA) obtained, over a period of 24 h, from rats simulta-neously treated in single-dose with Fucus vesiculosus extract (575 mg/kg, p.o.), orvehicle (0.5% carboxymethylcellulose aqueous solution), and AM (50 mg/kg, p.o.) byoral gavage. Symbols represent the mean values ± standard error of the mean (SEM)of six determinations per time point (n = 6). ⁄p < 0.05 and #p < 0.005 compared tocontrol (vehicle).

M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128 123

In the first pharmacokinetic study, twelve Wistar rats were randomly dividedinto two groups (experimental and control groups). Rats of the experimental group(n = 6) were concomitantly treated with a single-dose of F. vesiculosus extract(575 mg/kg, p.o.) and a single-dose of amiodarone (50 mg/kg, p.o.); the extract sus-pension was administered right before amiodarone. Rats of the control group (n = 6)received, instead of the F. vesiculosus extract suspension, the corresponding volumeof 0.5% carboxymethylcellulose aqueous solution (vehicle of the extract).

In the second pharmacokinetic study, twelve Wistar rats were also randomly di-vided into two groups. Rats assigned to the experimental group (n = 6) were orallypre-treated with F. vesiculosus extract (575 mg/kg, p.o.) once daily for 14 consecu-tive days (sub-chronic pre-treatment). Rats allocated to the control group (n = 6)were administered with an equivalent volume of vehicle for the same period oftime. During the pre-treatment period, the rats were kept in 12-h light/dark cycleanimal room with controlled temperature and humidity, as indicated above (seeSection 2.2.); free access to a standard rodent diet and tap water was allowed. On15th day, rats of both groups (experimental and control) were gavaged with a singledose of amiodarone (50 mg/kg, p.o.).

In both pharmacokinetic studies, the treatments with F. vesiculosus extract (orvehicle) and/or amiodarone were always carried out on the morning between9:00 am and 11:45 am. At night on the day before amiodarone administration,the rats were anesthetized for cannulation of a lateral tail vein and were fastedovernight as described above (see Section 2.2.). On the day after, multiple serialblood samples (approximately 0.3 mL) were collected through the cannula intoheparinized tubes before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h following ami-odarone administration; then, at 24 h post-dose, blood and tissues (heart, liver, kid-ney and lung) were also harvested after decapitation of the rats. The blood sampleswere centrifuged at 4000 rpm for 10 min (4 �C) to separate the plasma which wasstored at �20 �C until analysis. After exsanguination, liver, kidneys, heart and lungswere excised and stored at �20 �C; the organs were weighed and homogenized indistilled water (3 mL of water per gram of tissue) before analysis of tissue homog-enates samples.

2.4. Drug analysis

Plasma and tissue concentrations of amiodarone and its main metabolite(MDEA) were determined by using a liquid–liquid extraction (LLE) procedure fol-lowed by high-performance liquid chromatography-diode array detection (HPLC-DAD) assay previously developed and validated (Rodrigues et al., 2012). Briefly,an aliquot of each plasma sample (150 lL) was diluted with 150 lL of 0.1 M sodiumphosphate buffer (pH 5) and spiked with 20 lL of the IS working solution (50 lg/mL). The mixture was added of 500 lL of n-hexane (used as LLE solvent), vortex-mixed for 30 s and centrifuged at 17000 rpm for 2 min at 4 �C. The upper organiclayer was transferred to a clean glass tube and the sample was re-extracted twomore times with n-hexane (500 lL each time) using the same experimental condi-tions. Then, the whole organic extract was evaporated to dryness under a nitrogenstream at 60 �C and the residue was reconstituted in 100 lL of methanol. Followingthis, an aliquot of the reconstituted extracts (20 lL) was injected into the HPLC sys-tem for analysis.

For the extraction from tissues, each aliquot (400 lL) of tissue (heart, liver, kid-ney and lung) homogenates was spiked with 20 lL of the IS working solution(50 lg/mL); then, the mixture was added of 400 lL of acetonitrile (used as proteinprecipitating agent), vortex-mixed for 1 min and centrifuged at 17000 rpm for10 min at 4 �C in order to precipitate the protein content. The supernatant wastransferred to a new propylene tube and 1 mL of n-hexane (used as LLE solvent)was added. The mixture was vortex-mixed for 1 min and centrifuged at17000 rpm for 5 min at 4 �C. The upper organic layer (n-hexane) was transferredto a clean glass tube and the sample was re-extracted two more times with n-hex-ane (0.8 mL each time) using the same conditions. The organic extract was evapo-rated to dryness, reconstituted, and then injected into the HPLC system using thesame procedures as mentioned above for rat plasma samples. The limit of quantifi-cation (LOQ) was established at 0.100 lg/mL for amiodarone and MDEA in plasmaand in tissue homogenates.

2.5. Pharmacokinetic analysis

The plasma concentration versus time data for amiodarone and MDEA obtainedfrom each individual rat were submitted to a non-compartmental pharmacokineticanalysis using the WinNonlin� version 4.1 (Pharsight Co, Mountain View, CA, USA).The peak concentrations of amiodarone and MDEA in plasma (Cmax) and the time toreach Cmax (tmax) were obtained directly from the experimental data. Other pharma-cokinetic parameters estimated from the individual plasma concentration–timeprofiles were: area under the concentration–time curve (AUC) from time zero tothe last sampling time at which concentrations were at or above the LOQ of themethod (AUC0–t), calculated by the linear trapezoidal rule; AUC from time zero toinfinite (AUC0–1), calculated from AUC0–t + (Clast/kel), where Clast is the last quanti-fiable concentration and kel is the apparent terminal rate constant calculated bylog-linear regression of the terminal segment of the concentration–time profile;apparent terminal elimination half-life (t1/2el) and mean residence time (MRT).The concentrations lower than the LOQ of the assay were taken as zero for allcalculations.

2.6. Effect of the sub-chronic F. vesiculosus treatment on body weight

For the sub-chronic treatment study (a 14-day F. vesiculosus treatment period),the body weight of the rats receiving F. vesiculosus extract (575 mg/kg/day, p.o.;experimental group) or vehicle (control group) was adequately registered on thefirst day and on the last day (14th) of these treatments in order to examine the ef-fect of F. vesiculosus extract on body weight changes.

2.7. Statistical analysis

Data were reported as the mean ± standard error of the mean (SEM). Compari-sons between two groups were usually performed using unpaired two-tailed Stu-dent’s t-test; for body weight change comparisons within the same group thepaired Student’s t-test was employed. A difference was considered to be statisticallysignificant for a p-value lower than 0.05 (p < 0.05).

3. Results

3.1. Effects of the simultaneous co-administration of F. vesiculosus onamiodarone pharmacokinetics

The mean plasma concentration–time profiles (n = 6) of amio-darone and its main metabolite (MDEA) obtained after intragastricco-administration of rats with a single-dose of F. vesiculosus extract(575 mg/kg, p.o.) or vehicle (control group) and a single-dose ofamiodarone (50 mg/kg, p.o.) are shown in Fig. 2. Amiodarone plas-ma concentrations were comparable in both groups at the initialabsorption phase (up to 0.5 h) and at the elimination phase (8–24 h). Conversely, amiodarone plasma concentrations in the grouptreated with F. vesiculosus were significantly lower than those inthe control group over the 1–6 h post-dose time period (at least,p < 0.05). In the case of MDEA, the plasma concentrations weresimilar in both groups, with values near or below the LOQ(0.100 lg/mL) of the method. The main plasma pharmacokineticparameters estimated for amiodarone and MDEA after non-com-partmental analysis of their concentration–time profiles are sum-marized in Table 1. With co-administration of F. vesiculosus themean Cmax of amiodarone was significantly lower than that ob-tained in the control (vehicle) group (p < 0.005), while the meantime to reach tmax was attained later in the experimental group(3.00 ± 1.10 h) comparatively to the control group (1.83 ± 0.48 h).Statistically significant differences were also found for the AUC0–t

Table 1Pharmacokinetic parameters estimated by non-compartmental analysis of the plasma concentration–time profiles of amiodarone (AM) andmono-N-desethylamiodarone (MDEA, major metabolite of AM) obtained in rats after the simultaneous co-administration in single-dose ofFucus vesiculosus extract (575 mg/kg, p.o.), or vehicle (0.5% carboxymethylcellulose aqueous solution), with AM (50 mg/kg, p.o.) by oralgavage (n = 6, unless otherwise noted).

Parameter AMFucus AMVehicle

AM MDEA AM MDEA

tmax (h) 3.00 ± 1.10 8.00 ± 2.00a 1.83 ± 0.48 7.20 ± 1.36b

Cmax (lg/mL) 0.615 ± 0.060* 0.114 ± 0.004a 1.378 ± 0.179 0.125 ± 0.012b

AUC0–t (lg h/mL) 8.995 ± 0.725* ND 12.774 ± 0.688 NDAUC0–1 (lg h/mL) 15.675 ± 1.722# ND 21.431 ± 2.077 NDkel (h�1) 0.0426 ± 0.0070 ND 0.0433 ± 0.0082 NDt1/2el (h) 18.29 ± 2.47 ND 20.73 ± 5.74 NDMRT (h) 27.24 ± 3.45 ND 28.64 ± 7.74 ND

ND, not determined.a n = 3.b n = 5.* p < 0.005, significantly different from the control group.

# p = 0.059, versus control group.

Fig. 3. Ratios for the main plasma pharmacokinetic parameters (Cmax, AUC0–t andAUC0–1) estimated for amiodarone (AM) in rats simultaneously treated in single-dose with Fucus vesiculosus extract (575 mg/kg, p.o.), or vehicle (0.5% carboxy-methylcellulose aqueous solution), and AM (50 mg/kg, p.o.) by oral gavage.

Fig. 4. Mean plasma and tissue (heart, lung, liver and kidney) concentrations ofamiodarone (AM) and mono-N-desethylamiodarone (MDEA) obtained, at 24 h post-dose, from rats simultaneously treated in single-dose with Fucus vesiculosus extract(575 mg/kg, p.o.), or vehicle (0.5% carboxymethylcellulose aqueous solution), andAM (50 mg/kg, p.o.) by oral gavage. Data are expressed as the mean values ± stan-dard error of the mean (SEM) of six determinations (n = 6).

124 M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128

pharmacokinetic parameter (p < 0.005) calculated from the plasmaconcentration–time data obtained for amiodarone in both groups;in contrast, these differences were not so evident for the AUC0–1parameter (p = 0.059) (Table 1). Taking into consideration theinformation derived from Fig. 3, it is clear that following the simul-taneous co-administration of F. vesiculosus and amiodarone aremarkable decrease (55.4%) in the Cmax of drug was observed, aswell as a reduction of 29.6% in the extent of systemic drug expo-sure (as assessed by AUC0–t). As suggested by the visual inspectionof the elimination phase of plasma pharmacokinetic profiles ofamiodarone (Fig. 2), the mean values estimated for the eliminationpharmacokinetic parameters (kel, t1/2el and MRT) are comparablebetween experimental (F. vesiculosus) and control (vehicle) groups.Considering the scarcity of quantifiable plasma concentrations forMDEA obtained in both groups, only the Cmax and tmax pharmaco-kinetic parameters are presented in Table 1.

On the other hand, to investigate some aspects related to thebiodistribution of amiodarone and MDEA in rats, the animals werekilled at 24 h after dosing either in the presence or absence of theco-administration of F. vesiculosus and several tissues were excisedand analyzed. The mean concentrations of amiodarone and MDEAdetermined in heart, lung, liver and kidney tissues, and also theirplasma concentrations at the same time point (24 h), are shown

in Fig. 4. As indicated in Fig. 4, the tissue concentrations of amioda-rone and MDEA were markedly higher than those determined inplasma, and were absolutely noteworthy the levels found for bothcompounds (amiodarone and MDEA) in the lung tissue. However,no significant differences were found in the concentrations of ami-odarone and MDEA in tissues (heart, liver, kidney and lung) col-lected from experimental (F. vesiculosus) and control (vehicle)groups (p > 0.05) at 24 h post-dose.

3.2. Effects of the sub-chronic pre-treatment with F. vesiculosus onamiodarone pharmacokinetics

Rats were administered for 14 days with F. vesiculosus extract(575 mg/kg, p.o.) or vehicle (control group) in order to investigatea possible interference of the F. vesiculosus sub-chronic treatmenton the pharmacokinetics of amiodarone. The animals were given50 mg/kg amiodarone (p.o.) one day after the last treatment withF. vesiculosus or vehicle, and the mean plasma concentration–timeprofiles (n = 6) of amiodarone and its main metabolite (MDEA) aredepicted in Fig. 5. The corresponding pharmacokinetic parameters,calculated by using non-compartmental analysis, are listed in Ta-ble 2. Overall, it was observed a close overlap between the plasma

Fig. 5. Mean plasma concentration–time profiles of amiodarone (AM) and mono-N-desethylamiodarone (MDEA) obtained, over a period of 24 h, from rats submitted toa 14-day pre-treatment period with Fucus vesiculosus extract (575 mg/kg/day, p.o.),or vehicle (0.5% carboxymethylcellulose aqueous solution), and treated on the 15thday with a single-dose of AM (50 mg/kg, p.o.) by oral gavage (n = 6). Symbolsrepresent the mean values ± standard error of the mean (SEM) of six determinationsper time point (n = 6).

Fig. 6. Ratios for the main plasma pharmacokinetic parameters (Cmax, AUC0–t andAUC0–1) estimated for amiodarone (AM) in rats submitted to a 14-day pre-treatment period with Fucus vesiculosus extract (575 mg/kg/day, p.o.), or vehicle(0.5% carboxymethylcellulose aqueous solution), and treated on the 15th day with asingle-dose of AM (50 mg/kg, p.o.) by oral gavage.

M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128 125

pharmacokinetic profiles and no significant differences (p > 0.05)in the pharmacokinetic parameters were detected for amiodaroneand its main metabolite (MDEA) among the two groups (F. vesicu-losus versus vehicle pre-treatment). Once again, the plasma concen-trations of MDEA were near or below the LOQ (0.100 lg/mL) of themethod in both groups of rats. Regarding the data shown in Fig. 6,it is clear that the rate (as assessed by Cmax) and the extent (as as-sessed by AUC) of systemic exposure to amiodarone is similaramong experimental and control groups (AMFucus versus AMVehicle

ratios near to unity).To examine the influence of a 14-day pre-treatment period with

F. vesiculosus or vehicle (control group) on the distribution andmetabolism of amiodarone in rats, the concentrations of amioda-rone and its major metabolite (MDEA) were also determined invarious tissues (additionally to plasma) at 24 h post-dose and theresultant mean concentrations are represented in Fig. 7. As pointedout in Fig. 7, the concentrations of both compounds (amiodaroneand MDEA) in tissues were distinctly greater than those measuredin plasma, and the concentration levels found in lung tissue wereexceptionally high in experimental (F. vesiculosus) and control(vehicle) groups. However, taking into account all the comparisonsperformed, only significant differences were detected between

Table 2Pharmacokinetic parameters estimated by non-compartmental analysismono-N-desethylamiodarone (MDEA, major metabolite of AM) obtainevesiculosus extract (575 mg/kg/day, p.o.), or vehicle (0.5% carboxymethsingle-dose of AM (50 mg/kg, p.o.) by oral gavage (n = 6, unless otherw

Parameter AMFucus

AM MDEA

tmax (h) 1.00 ± 0.00 7.00 ± 1Cmax (lg/mL) 1.066 ± 0.199 0.108 ±AUC0–t (lg h/mL) 10.555 ± 0.674 NDAUC0–1 (lg h/mL) 13.786 ± 0.720 NDkel (h�1) 0.0594 ± 0.0051 NDt1/2el (h) 12.15 ± 1.12 NDMRT (h) 17.10 ± 1.45 ND

ND, not determined.a n = 2.b n = 3.

both groups for amiodarone concentrations in liver tissue, beingsuch concentrations lower in liver tissue of rats treated with F. ves-iculosus extract (p < 0.05).

3.3. Effect of the sub-chronic F. vesiculosus treatment on body weight

The resulting changes in body weight of the rats submitted to a14-day treatment period with F. vesiculosus extract (575 mg/kg/day, p.o.) or vehicle are demonstrated in Fig. 8. From the analysisof the data it is clear that the increase in body weight of rats trea-ted with F. vesiculosus (p < 0.005) or treated with vehicle(p < 0.0005) was statistically significant between the 1st and 14thday. On the other hand, the increase in body weight of the rats ofboth groups (F. vesiculosus versus vehicle) was comparable.

4. Discussion

Complementary and alternative therapies such as herbal or nat-ural products are finding increasing use around the world despitethe paucity of scientific evidence about their safety and efficacy.Accordingly, unexpected and significant herb–drug interactionsmay occur and put individuals at risk, particularly those peoplewho use multiple medicines for co-morbid conditions. These inter-

of the plasma concentration–time profiles of amiodarone (AM) andd in rats submitted to a 14-day pre-treatment period with Fucus

ylcellulose aqueous solution), and treated on the 15th day with aise noted).

AMVehicle

AM MDEA

.00a 2.17 ± 0.60 7.33 ± 2.40b

0.002a 0.952 ± 0.157 0.119 ± 0.014b

10.532 ± 0.889 ND15.325 ± 0.949 ND0.0533 ± 0.0082 ND

14.36 ± 1.85 ND20.97 ± 2.42 ND

Fig. 7. Mean plasma and tissue (heart, lung, liver and kidney) concentrations ofamiodarone (AM) and mono-N-desethylamiodarone (MDEA) obtained, at 24 h post-dose, from rats submitted to a 14-day pre-treatment period with Fucus vesiculosusextract (575 mg/kg/day, p.o.), or vehicle (0.5% carboxymethylcellulose aqueoussolution), and treated on the 15th day with a single-dose of AM (50 mg/kg, p.o.) byoral gavage. Data are expressed as the mean values ± standard error of the mean(SEM) of six determinations (n = 6). ⁄p < 0.05 compared to control (vehicle).

126 M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128

actions may lead to therapeutic failure and/or toxic effects, espe-cially for drugs characterized by a narrow therapeutic index (e.g.amiodarone) (Tachjian et al., 2010). Undoubtedly, the informationnow available on herb–drug interactions is scarce and, in manycases, inappropriate. Indeed, the information published aboutherb–drug interactions has derived mainly from in vitro studies;however, as suggested by some authors, numerous herb–druginteractions reported in literature are irrelevant and misleadingdue to the use of inappropriately high concentrations of the ex-tracts or their constituents lacking, therefore, in vivo relevancewhen their bioavailability is considered (Cott, 2008; Markowitzet al., 2008). Since the in vitro data on herb–drug interactions can-not usually be directly extrapolated to the in vivo conditions, it isimportant to conduct well-designed in vivo non-clinical studieswith enough potency for human extrapolation.

Although safety issues are always important in the areas ofpharmaceutical and food research, some safety data are alreadyavailable in the literature for F. vesiculosus extracts. Indeed, acutetoxicity studies performed by Zaragozá et al. (2008) using two F.vesiculosus extracts indicated LD50 values in rats of 1000–2000(or >2000) mg/kg after oral dosing; on the other hand, the overall

Fig. 8. Effects on the body weight of the rats induced by the sub-chronic treatment(14-day period) with Fucus vesiculosus extract (575 mg/kg/day, p.o.) and vehicle(0.5% carboxymethylcellulose aqueous solution) by oral gavage. ⁄p < 0.01 and⁄⁄p < 0.001, 1st day versus 14th day.

results obtained from a 4 week (sub-chronic) toxicity study con-ducted in rats with two oral doses of 200 mg/kg/day (low dose)and 750 mg/kg/day (high dose) indicated that no relevant signsof toxicity occurred even at the daily dose of 750 mg/kg (Zaragozáet al., 2008). Moreover, Leite-Silva et al. (2007) demonstrated theabsence of F. vesiculosus extract-mediated genotoxicity in culturedhuman lymphocytes and also evidenced its antigenotoxic activityagainst doxorubicin-induced DNA damage. Parys et al. (2010) alsoreported potential chemopreventive activity of three fucophlore-thols from F. vesiculosus. Thus, no safety issues were expectedand none was observed at the dose of F. vesiculosus extract selectedfor the present study (575 mg/kg/day).

The current work was planned to investigate in vivo the poten-tial of interaction between F. vesiculosus extract and amiodarone,using adult male Wistar rat as a whole-animal model. The use offemale rats was also hypothesized during the experimental designof these studies; however, only male Wistar rats were included be-cause it has been reported that the pharmacokinetics of amioda-rone is not gender-dependent and also to avoid the potentialinterference of menstrual cycle hormones (possible confoundingfactors) (Sanofi-Synthelabo Pfizer Canada Inc., 2010). Bearing inmind that drug–drug or herb–drug interactions mainly occur atthe level of absorption and/or metabolic (inhibition or induction)pathways, the pharmacokinetic studies reported herein were de-signed to examine the interference of F. vesiculosus extract on thegastrointestinal absorption (simultaneous co-administrationstudy) and metabolism of amiodarone (14-day F. vesiculosus pre-treatment study).

Our results clearly evidenced a significant decrease (55.4%) inthe peak plasma concentration (Cmax) of drug following the simul-taneous co-administration of the F. vesiculosus extract and amioda-rone, as well as a reduction of approximately 30% in the extent ofsystemic drug exposure (as assessed by AUC0–24 h). On the otherhand, no important effects were detected either on the rate or ex-tent of systemic exposure to amiodarone after the administrationof the drug to pre-treated rats one day after the last treatment withF. vesiculosus extract or vehicle. Hence, taking these findings to-gether, it is apparent that F. vesiculosus extract or its componentsinteract with amiodarone in the gastrointestinal tract, reducingsignificantly the bioavailability of the drug; actually, this interac-tion was identified as relevant only after the simultaneous co-administration in single-dose of F. vesiculosus extract and amioda-rone. Moreover, the similarity in plasma pharmacokinetic profilesof amiodarone obtained from rats pre-treated for 14 days with F.vesiculosus extract or vehicle excludes the impact of F. vesiculo-sus-induced metabolism on the bioavailability and also supportsthe significance of the interaction with amiodarone at the levelof the gastrointestinal tract. Zhi et al. (2003) also observed inhealthy volunteers a significant reduction of the absorption of ami-odarone induced by orlistat; this drug, a lipase inhibitor, signifi-cantly reduced the systemic exposure to amiodarone byapproximately 25% and a decrease of similar magnitude (�25%)was detected in the generation of the metabolite MDEA (the majormetabolite of amiodarone). According to Zhi et al. (2003) theabsorption of highly lipophilic drugs such as amiodarone may de-pend on the presence of a lipid phase in the gastrointestinal envi-ronment, which may be affected by the pharmacological action oforlistat. In fact, there are strong evidences supporting the effects offood upon the bioavailability of amiodarone; effectively, the rate(Cmax) and extent (AUC0–t) of absorption of amiodarone enhancedby 3.8 and 2.4-fold, respectively, in healthy volunteers who re-ceived a single-dose of the drug immediately after consuming ahigh-fat meal versus following an overnight fast (Meng et al.,2001). The effects of food on the pharmacokinetics of amiodaronewere also studied in rats by Shayeganpour and collaborators(2005) and the results obtained concerning the interference of lip-

M. Rodrigues et al. / Food and Chemical Toxicology 52 (2013) 121–128 127

ids on the oral bioavailability of amiodarone corroborated those re-ported in humans.

Nevertheless, in this case, taking into account the great contentof soluble dietary fiber present in F. vesiculosus, we hypothesize theoccurrence of a physical–chemical interaction between dietary fi-ber (or other extract components) and amiodarone in the gastroin-testinal tract to explain the considerable decrease in the systemicexposure/bioavailability of amiodarone in the rats simultaneouslyco-administered in single-dose with F. vesiculosus extract. How-ever, further studies are needed to understand the mechanismassociated to the herb–drug interaction reported herein for thefirst time (F. vesiculosus extract/amiodarone). At this point it shouldbe highlighted the overview recently published by Colalto (2010)about the herbal interactions on absorption of drugs and underly-ing interaction mechanisms. Dietary fiber may reduce the drugabsorption, when both are assumed nearly, by a mechanism of ac-tion similar to bile sequestration; herb–drug interactions atabsorption level associated with dietary fiber have been reported,as instance, for lovastatin (Richter et al., 1991), digoxin (Brownand Juhl, 1976; Brown et al., 1978), metformin (Gin et al., 1989)and glibenclamide (Neugebauer et al., 1983). In addition, Lodeiroet al. (2012) recently reported that F. vesiculosus interacts with alu-minum of acidic waters by a mechanism of adsorption, suggestingthat these physicochemical data may be of interest in modelingdrug–food interactions, particularly those referring to aluminum-containing antacids-food pharmacokinetic process produced inthe gastrointestinal tract. Therefore, similar physicochemicaladsorption mechanisms may occur between F. vesiculosus andamiodarone.

It is well-recognized the central role that CYP or P-glycoprotein(P-gp) induction or inhibition play on drug-drug and herb–druginteractions. In fact, amiodarone is a substrate of P-gp (Shapiroand Shear, 2002) and is metabolized by several inducible CYP iso-enzymes in rat (Elsherbiny et al., 2010). Hence, to check the possi-ble interference of F. vesiculosus extract on the CYP or P-gp activity,the extract was administered for 14 days (575 mg/kg/day, p.o.) un-til 24 h before applying amiodarone; however, no significant influ-ence was observed on the systemic (plasma) pharmacokinetics ofamiodarone in these circumstances. In these experimental condi-tions, it is only worthy of note the lower concentrations of amioda-rone in liver tissue, at 24 h post-dose, in rats pre-treated with F.vesiculosus extract (Fig. 7). Although this finding has not had in thiscase a significant impact on the magnitude of systemic exposure toamiodarone, it is an interesting aspect to explore in further studiesdirected to evaluate the potential of enzyme induction by F. vesicu-losus. Up to date, there is no evidence suggesting the hepatic met-abolic induction of CYPs mediated by F. vesiculosus. On thecontrary, the inhibition of CYP1A by trifucodiphlorethol A and trif-ucotriphlorethol A, compounds extracted from F. vesiculosus, wasrecently reported by Parys et al. (2010).

Overall, considering the rat plasma data generated in the pres-ent work and that reported from clinical studies following oraladministration of amiodarone, it is evident that MDEA is the majormetabolite of amiodarone in both species, even though differenceswill exist in their metabolite-to-parent ratios. Indeed, in our phar-macokinetic studies, the plasma concentrations of MDEA found inrat were significantly lower than those of amiodarone, and werefound at levels near or below the LOQ (0.100 lg/mL) of the bioan-alytical assay. Furthermore, amiodarone and MDEA were found inconcentrations considerably lower in plasma than in tissues (heart,liver, lungs and kidneys) at 24 h post-dose, supporting their greatplasma/tissue distribution; these differences were absolutelyremarkable for plasma/lung tissue. These rat tissues were selectedfor bioanalysis of amiodarone and MDEA because they representimportant targets from therapeutic (heart), toxicological (liverand lungs) and pharmacokinetic (liver and kidneys) viewpoints.

Based on data of herb–drug interaction between F. vesiculosusextract and amiodarone, derived from this non-clinical investiga-tion in rat, it is suggested that patients who are taking amiodaroneshould avoid the concurrent administration of herbal slimmingmedicines/dietary supplements containing F. vesiculosus. In addi-tion, in our experimental conditions, the sub-chronic administra-tion of F. vesiculosus extract to rats (575 mg/kg/day, 14-dayperiod, p.o.) not evidenced efficacy on weight reduction (Fig. 8).It is also true that results from animal experiments cannot be di-rectly extrapolated to humans; however, bearing in mind the stud-ies of Shayeganpour et al. (2005) and Meng et al. (2001) the ratappears to be an appropriate animal model for man in thissituation.

5. Conclusions

In conclusion, to best of our knowledge, this work is the first re-port documenting the interaction of F. vesiculosus and amiodarone;particularly, a reduced bioavailability of amiodarone in the pres-ence of F. vesiculosus extract was observed in rats. Further clinicalstudies should be performed to reliably assess the clinical rele-vance of the interaction between F. vesiculosus extract and amioda-rone. Thus, considering the currently available information, it isprudent not take amiodarone concomitantly with herbal slimmingmedicines/dietary supplements containing F. vesiculosus.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors thank the support of Fundação para a Ciência e aTecnologia (FCT, Portugal) through the fellowships (SFRH/BD/61901/2009) and (SFRH/BPD/46826/2008).

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