Hepatocellular toxicity and pharmacological effect of amiodarone and amiodarone derivatives

Article abstract of DOI:10.1124/jpet.106.108993

The aim of this work was to compare hepatocellular toxicity and pharmacological activity of amiodarone (2-n-butyl-3-[3,5 diiodo-4- diethylaminoethoxybenzoyl]-benzofuran; B2-O-Et-N-diethyl) and of eight amiodarone derivatives. Three amiodarone metabolites

Full text of DOI:10.1124/jpet.106.108993

0
022-3565/06/3193-1413–1423$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
JPET 319:1413–1423, 2006
Vol. 319, No. 3
108993/3154314
Printed in U.S.A.
Hepatocellular Toxicity and Pharmacological Effect of
Amiodarone and Amiodarone Derivatives
Katri Maria Waldhauser, Michael T o¨ r o¨ k, Huy-Riem Ha, Urs Thomet, Daniel Konrad,
Karin Brecht, Ferenc Follath, and Stephan Kr a¨ henb u¨ hl
Division of Clinical Pharmacology and Toxicology and Department of Research, University Hospital Basel, Basel, Switzerland
(
K.M.W., M.T., K.B., S.K.); Cardiovascular Therapy Research Unit, University Hospital of Z u¨ rich, Z u¨ rich, Switzerland (H.-R.H.,
F.F.); and Bsys Ltd., Witterswil, Switzerland (U.T., D.K.)
Received June 5, 2006; accepted September 11, 2006
ABSTRACT
The aim of this work was to compare hepatocellular toxicity of most analogs, confirming their hepatocellular toxicity. Using
and pharmacological activity of amiodarone (2-n-butyl-3-[3,5 freshly isolated rat liver mitochondria, amiodarone and most
diiodo-4-diethylaminoethoxybenzoyl]-benzofuran; B2-O-Et-N- analogs showed a dose-dependent toxicity on the respiratory
diethyl) and of eight amiodarone derivatives. Three amiodarone chain and on ␤-oxidation, significantly reducing the respiratory
metabolites were studied, namely, mono-N-desethylamioda- control ratio and oxidation of palmitate, respectively. The reac-
rone (B2-O-Et-NH-ethyl), di-N-desethylamiodarone (B2-O-Et- tive oxygen species concentration in hepatocytes increased
NH ), and (2-butyl-benzofuran-3-yl)-(4-hydroxy-3,5-diiodophe- time-dependently, and apoptotic/necrotic cell populations
2
nyl)-methanone (B2) carrying an ethanol side chain [(2-butyl- were identified using flow cytometry and annexin V/propidium
benzofuran-3-yl)-[4-(2-hydroxyethoxy)-3,5-diiodophenyl]- iodide staining. The effect of the three least toxic amiodarone
methanone; B2-O-Et-OH]. In addition, five amiodarone analogs analogs on the human ether-a-go-go-related gene (hERG) chan-
were investigated, namely, N-dimethylamiodarone (B2-O-Et-N- nel was compared with amiodarone. Amiodarone, B2-O-acetate,
dimethyl), N-dipropylamiodarone (B2-O-Et-N-dipropyl), B2-O- and B2-O-Et-N-dipropyl (each 10 ␮M) significantly reduced the
carrying an acetate side chain [[4-(2-butyl-benzofuran-3- hERG tail current amplitude, whereas 10 ␮M B2-O-Et displayed
carbonyl)-2,6-diiodophenyl]-acetic acid; B2-O-acetate], B2- no detectable effect on hERG outward potassium currents. In
O-Et carrying an propionamide side chain (B2-O-Et-propi- conclusion, three amiodarone analogs (B2-O-Et-N-dipropyl, B2-
onamide), and B2-O carrying an ethyl side chain [(2-butyl- O-acetate, and B2-O-Et) showed a lower hepatocellular toxicity
benzofuran-3-yl)-(4-ethoxy-3,5-diiodophenyl)-methanone; B2- profile than amiodarone, and two of these analogs (B2-O-Et-N-
O-Et]. A concentration-dependent increase in lactate dipropyl and B2-O-acetate) retained hERG channel interaction
dehydrogenase leakage from HepG2 cells and isolated rat capacity, suggesting that amiodarone analogs with class III anti-
hepatocytes was observed in the presence of amiodarone and arrhythmic activity and lower hepatic toxicity could be developed.
Amiodarone (2-n-butyl-3-[3,5 diiodo-4-diethylaminoe- shown to be at least as efficacious as sotalol in patients with
thoxybenzoyl]-benzofuran; B2-O-Et-N-diethyl) is a class III atrial fibrillation (Singh et al., 2005) and to reduce mortality
antiarrhythmic used in the treatment of a wide spectrum of in patients with a high risk for arrhythmia, e.g., patients
cardiac arrhythmias (Singh, 1996). Amiodarone has been
with severe congestive heart failure (Doval et al., 1994) or
after acute myocardial infarction (1997). Amiodarone is a
class III antiarrhythmic drug that blocks hERG channels,
leading to prolongation of the refractoriness and resulting in
QT prolongation (Singh, 1996). In addition, it has an inhibi-
tory effect on fast sodium as well as on calcium channels
The study was supported by Swiss National Science Foundation Grant SNF
3
10000-112483/1 (to S.K.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.108993.
ABBREVIATIONS: B2-O-Et-N-diethyl, 2-n-butyl-3-[3,5 diiodo-4-diethylaminoethoxybenzoyl]-benzofuran; hERG, human ether-a-go-go-related
gene; B2, 2-butyl-benzofuran-3-yl)-(4-hydroxy-3,5-diiodophenyl)-methanone; B2-O-Et-NH-ethyl, (2-butyl-benzofuran-3-yl)-4-[2-(ethylamino-
ethoxy)-3,5-diiodophenyl]-methanone⅐hydrochloride; B2-O-Et-N-dimethyl, 2-butyl-benzofuran-3-yl)-4-[2-(dimethylamino-ethoxy)-3,5-diiodophe-
nyl]-methanone⅐hydrochloride; B2-O-Et-N-dipropyl, 2-butyl-benzofuran-3-yl)-4-[2-(dipropylamino-ethoxy)-3,5-diiodophenyl]-methanone⅐hydrochloride;
B2-O-Et-NH , [4-(2-aminoethoxy)-3,5-diiodophenyl]-(2-butylbenzofuran-3-yl)-methanone⅐hydrochloride; B2-O-Et-OH, (2-butyl-benzofuran-3-yl)-
2
[4-(2-hydroxyethoxy)-3,5-diiodophenyl]-methanone; B2-O-Et, 2-butyl-benzofuran-3-yl)-(4-ethoxy-3,5-diiodophenyl)-methanone; B2-O-acetate,
[4-(2-butyl-benzofuran-3-carbonyl)-2,6-diiodophenyl]-acetic acid; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography;
NP-40, Nonidet P-40; ROS, reactive oxygen species; DCFH, 2,7-dichlorofluorescein; RCR, respiratory control ratio; CHO, Chinese hamster ovary.
1413

1
414
Waldhauser et al.
(
Singh, 1996). Similar to its pharmacological action, amiod- et al., 2001a). These studies revealed that the benzofuran
arone’s adverse reaction profile is complex, ranging from ring and the presence of iodines were important for mito-
thyroidal (Harjai and Licata, 1997) to pulmonary (Jessurun chondrial toxicity. More recent studies (Kaufmann et al.,
et al., 1998), ocular (Pollak, 1999), and/or liver toxicity 2005) showed, however, that not the benzofuran ring alone is
(
Morse et al., 1988; Lewis et al., 1989). Amiodarone is a responsible for hepatocellular toxicity of amiodarone but that
mitochondrial toxicant, uncoupling oxidative phosphoryla- a side chain in position 2 and/or 3 of the benzofuran ring was
tion and inhibiting the electron transport chain and ␤-oxida- necessary.
tion of fatty acids (Fromenty et al., 1990a,b; Spaniol et al.,
001a; Kaufmann et al., 2005).
The principal aim of the current study was to find amio-
darone derivatives with minimal mitochondrial toxicity or
2
Amiodarone is composed of a benzofuran ring carrying a without mitochondrial toxicity that still exhibit inhibitory
C H side chain and a highly lipophilic diiodobenzene ring activity toward the hERG channel. We therefore synthesized
4
9
(
B2) with a diethylaminoethoxy side chain (Table 1). It is eight amiodarone derivatives (including three metabolites)
metabolized by dealkylation of the diethylaminoethoxy group with different lipid solubilities (Table 1). All of the deriva-
to mono-N-desethylamiodarone (B2-O-Et-NH-ethyl) (Flana- tives synthesized contained a benzofuran ring carrying a
gan et al., 1982) and to di-N-desethylamiodarone (B2-O-Et- butyl group and differed from each other only by their side
NH ) (Ha et al., 2005), which may be transaminated and chain. B2-O-Et-N-diethyl, B2-O-Et-NH-ethyl, B2-O-Et-NH2,
2
reduced to B2-O-Et-OH (Table 1) (Ha et al., 2005).
B2-O-Et-N-dimethyl, B2-O-Et-N-dipropyl, and B2-O-Et-pro-
In previous studies, we investigated the significance of the pionamide had side chains differing from each other by the
2
-butyl-benzofuran group and O-dealkylation of the amioda- substituents coupled to the nitrogen atom (Table 1). In com-
rone molecule with respect to mitochondrial toxicity (Spaniol parison, B2-O-acetate, B2-O-Et-OH, and B2-O-Et did not
TABLE 1
Compound
R
ClogP
7.3
logP
B2-O-Et-N-diethyl (amiodarone)
B2-O-Et-NH-ethyl
4.92 Ϯ 0.25
6.6
5.9
6.7
8.2
4.47 Ϯ 0.12
3.83 Ϯ 0.10
4.40 Ϯ 0.07
5.51 Ϯ 0.12
B2-O-Et-NH2
B2-O-Et-N-dimethyl
B2-O-Et-N-dipropyl
B2-O-Acetate
6.4
6.3
4.69 Ϯ 0.08
4.31 Ϯ 0.16
B2-O-Et-propionamide
B2-O-Et
7.0
6.2
4.83 Ϯ 0.06
4.18 Ϯ 0.15
B2-O-Et-OH

Toxicity and Pharmacology of Amiodarone and Analogs
1415
carry a nitrogen atom in the side chain coupled to the diio- separated by centrifugation, combined, and evaporated to dryness
dophenyl ring (Table 1). Hepatocellular toxicity was studied under reduced pressure. Two milliliters of 10 N HCl and 15 ml of
toluene were added, and the liquids were removed under reduced
using freshly isolated rat liver mitochondria, primary rat
pressure at 80°C. A white solid was obtained after three additional
treatments with 10 ml toluene. The obtained residue was then crys-
tallized from toluene, yielding 1.55 g (65%) of analytically pure
B2-O-Et-NH-ethyl. The melting point was 176.8–177.7°C.
Synthesis of B2-O-Et-N-dimethyl and B2-O-Et-N-dipropyl.
B2-O-Et-N-dimethyl and B2-O-Et-N-dipropyl were prepared in a
hepatocytes, and HepG2 cells. The effect on the blockade of
the voltage-gated potassium channel hERG was tested for
amiodarone, and the least toxic analogs (B2-O-Et-N-dipropyl,
B2-O-Et, and B2-O-acetate) to estimate their class III anti-
arrhythmic activity.
similar manner as described for B2-O-Et-NH-ethyl, but 2-(dimeth-
Materials and Methods
ylamino)ethyl chloride and 2-(diisopropylamino)ethyl chloride were
used instead of N-ethyl-2-chloroethylamine hydrochloride. For B2-
O-Et-N-dimethyl, the melting point was 89.5–89.7°C, and for B2-O-
Amiodarone and Amiodarone Derivatives
Amiodarone hydrochloride was purchased from Sigma-Aldrich Et-N-dipropyl, the melting point was 146.8–148.7°C.
Buchs, Switzerland). All of the amiodarone metabolites or analogs
Synthesis of B2-O-Et-NH . A mixture of B2 (1.2 g; 2 mmol), 2 ml
(
2
were synthesized starting from B2 as shown in Fig. 1.
(20 mmol) of 2-ethyl-2-oxazoline, and 3 ml of toluene was heated at
reflux for 1 h and cooled to room temperature. The mixture was
taken up in 10 ml of methylene chloride, washed with 4 N potassium
Chemistry and General Methods
hydroxide (3 ϫ 20 ml), dried (Na CO ), and concentrated in vacuo to
All chemicals used in the synthesis work were purchased from
Aldrich (Buchs, Switzerland) and were used without further purifi-
cation. All melting points given are uncorrected. NMR spectra were
obtained for all substances synthesized (data not shown).
2
3
give a brown oil. The oil was solidified by trituration with petroleum
ether followed by recrystallization from ethyl acetate/hexane to give
a white solid. Thin layer chromatography analysis using Merck
precoated Silica Gel 60-F254 plates and hexane/isopropyl alcohol/25%
Synthesis of B2. This compound was prepared with a yield of
6
1
0% as described previously (Ha et al., 2000). Melting point (146.4– NH [84:15:1 (v/v)] as an eluant revealed only one spot with a
3
46.9°C) and NMR data (not shown) were in agreement with the retention factor value of 0.42 with UV detection at 254 nm. The
previous report. As shown in Fig. 1, B2 is the origin of the synthesis
of all amiodarone metabolites and analogs used in this study.
corresponding retention factor value of B2 was 0.05. This interme-
diate compound corresponded to B2-O-propionamide (Fig. 1) by NMR
Synthesis of B2-O-Et-NH-ethyl. To a mixture of B2 (2 g; 3.66 analysis (data not shown).
mmol) and K CO (1.66 g; 12 mmol) in toluene/water [2:1 (v/v); total
To this compound, 10 ml of 6 N HCl was added. The mixture was
2
3
volume 75 ml] heated to 55–60°C, N-ethyl-2-chloroethylamine hy- heated to 130°C for 3.5 h and then cooled to room temperature.
drochloride (2.66 g; 18.5 mmol) was added portionwise. N-Ethyl-2- Impurities were washed out by diethyl ether until the organic phase
chloroethylamine hydrochloride was prepared by Lasselle’s method was colorless (five washes with 5 ml each). The precipitate was
(
Lasselle and Sundet, 1941). After the addition, the temperature was collected by filtration and washed with water. The compound was
raised to reach reflux over 30 min, until the yellow color of B2
flash-chromatographed using silica gel Merck 60 (40–60 ␮m; 230–
disappeared. The reaction was refluxed for 1 additional hour, and the 400 mesh) with hexane/isopropyl alcohol/25% NH [84:15:1 (v/v)] as
3
phases were separated quickly by a separation funnel at 60°C. The an eluant, yielding 0.6 g (60%) of hydrochloride salt of B2-O-Et-NH
toluene phase was washed three times with 25 ml of water at this as a white solid with a melting point of 200.5–201.4°C. B2-O-Et-NH
2
2
temperature. The organic phase was evaporated to dryness; the was found as a minor metabolite of amiodarone in humans, and its
residue suspended in 10 ml of 5% NH3 and B2-O-Et-NH-ethyl was spectroscopic data have been reported in a previous study (Ha et al.,
extracted three times with 15 ml of toluene. The organic phases were 2005).
Fig. 1. General scheme of the synthe-
sis of amiodarone derivatives. All the
amiodarone metabolites or deriva-
tives used were synthesized from B2.
B2 was synthesized as described pre-
viously (Ha et al., 2000). The deriva-
tives B2-O-Et-NH-ethyl, B2-O-Et-
NH , B2-O-Et-N-dimethyl, and B2-O-
2
Et-N-dipropyl were prepared by
condensing B2 with the respective
2
-chloroethyl amine hydrochloride
salts in the presence of K CO . Using
2
3
the same procedure, B2-O-Et-OH, B2-
O-acetate and B2-O-Et were obtained
from the reaction of B2 with iodoetha-
nol, ␣-bromo-ethyl acetate (subse-
quently hydrolyzed in 0.1 M NaOH),
and iodoethyl, respectively. The com-
pound
was synthesized from B2 and 2-ethyl-
-oxazoline. The yields of the reac-
B2-O-Et-NH-propionamide
2
tions were between 70 and 85%, and
the purity of the final substances was
Ն97% as assayed by HPLC. See Ma-
terials and Methods for more details
on synthesis and characterization of
the compounds.

1
416
Waldhauser et al.
Synthesis of B2-O-Et. To a mixture of B2 (2.0 g; 3.66 mmol) in Isolation of Rat Liver Mitochondria
dry acetone (50 ml), iodoethyl (2.34 g; 15 mmol) was added over 20
min. The reaction was stirred at 50°C for 16 h. The insoluble salt was
removed by filtration, and the filtrate concentrated in vacuo to give
Rat liver mitochondria were isolated by differential centrifugation
according to the method of (Hoppel et al., 1979). The mitochondrial
protein content was determined using the biuret method with bovine
serum albumin as a standard (Gornall et al., 1949). The mean rat
weight was 343 Ϯ 91 g and the mean rat liver weight was 14.12 Ϯ
1
6
.7 g (yield 75%) of B2-O-Et as a white solid. The melting point was
7.0–69.4°C.
Synthesis of B2-O-Et-OH. This compound was prepared in a
2.81 g.
similar manner as B2-O-Et, but iodoethyl was replaced by 2-chloro-
ethanol. The final product was obtained as an oil. When stored in a
closed vial at room temperature, it solidified after 10 days. The
melting point was 69.6–72.9°C. B2-O-Et-OH was found as a minor
metabolite of amiodarone in humans, and its spectroscopic data have
been reported in a previous study (Ha et al., 2005).
Synthesis of B2-O-Acetate. The ethyl ester of B2-O-acetate (not
shown in Fig. 1) was prepared from B2 and ␣-bromoethyl acetate as
described by Carlsson et al. (2002). The hydrolysis of the ester was
performed in the presence of 0.1 M NaOH at 22°C for 16 h. The
melting point of the product was 186.4–187.8°C.
Cell Lines and Cell Culture
HepG2 cells were kindly provided by Professor Dietrich von
Schweinitz (University Hospital Basel, Switzerland). The cell line
was cultured in Dulbecco’s modified Eagle’s medium supplemented
with 10% (v/v) inactivated fetal calf serum, 10 mM HEPES buffer,
pH 7.4, 2 mM GlutaMAX (Invitrogen), nonessential amino acids, and
100 U/ml penicillin/streptomycin. The culture conditions were 5%
CO2 and 95% air atmosphere at 37°C.
Cell Viability
Amiodarone and the analogs were dissolved in dimethyl sulfoxide
(
DMSO). The end concentration of DMSO in the experiments never
Lactate dehydrogenase (LDH) leakage in the supernatant was
exceeded 1% and control incubations contained the same DMSO determined as a measure of cell viability as described by Vassault
concentration.
(1983) and calculated as described by Huang et al. (2000). LDH
leakage from the cells treated with the test compounds was com-
pared with the LDH leakage of cells lysed with the detergent Nonidet
P-40 [0.01% (w/v)]. The activity in the supernatant of lysed cells was
set at 100%.
Determination of the Octanol/Water Partition Coefficient
of Amiodarone and Derivatives
The octanol/water partition of the compounds synthesized was
determined using reversed phase HPLC as described by Braumann Apoptosis and Necrosis Detection by Annexin V Binding
(
1986). HPLC of the substances was performed at 37°C using differ- and Propidium Iodide Uptake
ent 0.005 M phosphate buffer, pH 7.4/methanol mixtures as an
eluant. The octanol/water partition of a substance can be calculated
based on the retention times obtained in the presence of different
concentrations of methanol in the eluant (Braumann, 1986).
After an 18-h incubation with the test compounds, hepatocytes
were stained with 0.5 ␮l of Alexa Fluor 633-labeled annexin V and 2
l of propidium iodide (final concentration 1.5 ␮g/l). After 15 min of
incubation at 37°C, the cells were analyzed by flow cytometry
FACSCalibur flow cytometer; BD Biosciences). As positive controls
␮
(
Other Chemicals
for apoptosis, 100 ␮M deoxycholic acid was used. As a positive control
for necrosis, cells were treated with the surfactant Nonidet P-40
14
1
-[ C]Palmitic acid was purchased from Amersham Pharmacia
[
final concentration 0.1% (v/v)].
Biotech (D u¨ bendorf, Switzerland), and collagenase type 2 was from
BioConcept (Allschwil, Switzerland). Propidium iodide was from Mo-
lecular Probes (Eugene, OR), and Alexa Fluor 633-labeled annexin V
was a generous gift from Dr. Felix Bachmann (Aponetics Ltd., Wit-
ATP Content of the Cells
Freshly isolated hepatocytes were seeded on a 12-well plate
terswil, Switzerland). All other chemicals were purchased from Sig- (200,000 cells/well). Subsequently, they were treated for 18 h with
ma-Aldrich (Buchs, Switzerland) and were of best quality available the test compounds. After the incubation, cells were trypsinized, the
when not otherwise stated. All cell culture media, all supplements, pellet was resuspended in 600 ␮l of reagent grade water and snap-
and fetal calf serum were from Gibco (Paisley, UK), except for Wil-
frozen in liquid nitrogen (Yang et al., 2002). Intracellular ATP con-
liams E, which was purchased from Cambrex Bio Science Verviers centration of the cells was determined using a luciferin-luciferase
S.p.r.l. (Verviers, Belgium). The 96-well plates and the 12-well plates assay (Sigma Chemie, Deissenhofen, Germany).
were purchased from BD Biosciences (Franklin Lakes, NJ).
Measurement of Reactive Oxygen Species
A fluorescence-based microplate assay (Wang and Joseph, 1999)
Animals
was used for the evaluation of oxidative stress in primary hepato-
Male Sprague-Dawley rats were purchased from Charles River
cytes treated with the test compounds. DCFH-diacetate is a mem-
Laboratories (L’Arbresle, France) and kept in the animal facility of
brane-permeable, nonpolar, and nonionic molecule. In the cytoplasm,
the University Hospital Basel (Basel, Switzerland). Rats were kept
it is hydrolyzed by intracellular esterases to nonfluorescent DCFH,
in a temperature-controlled environment with a 12-h light/dark cycle
which is oxidized to fluorescent dichlorofluorescein in the presence of
and food and water ad libitum. The study protocol had been accepted
by the Animal Ethics Committee of the Canton Basel Stadt.
.
2
reactive oxygen species (H O2 and O ). Hepatocytes were simulta-
2
neously exposed to test compounds and to DCFH-diacetate dissolved
in ethanol (final concentration 5 ␮M) and incubated for 2, 4, 6, and
18 h. The fluorescence was measured using a microtiter plate reader
Isolation of Rat Hepatocytes
(
HTS 700 Plus Bio Assay Reader; PerkinElmer, Beaconsfield, Buck-
The isolation of rat hepatocytes by a two step collagenase perfu-
sion was based on a method described by Berry (1974) and performed
as described previously (Kaufmann et al., 2005). Cell viability was
determined by trypan blue exclusion and was always greater than
inghamshire, UK) in incubations containing cells and exposure me-
dium at an excitation wavelength of 485 nm and an emission wave-
length of 535 nm.
8
0%. Cells were seeded into cell culture dishes in Williams E medium
Oxygen Consumption and ␤-Oxidation of Intact
Mitochondria
supplemented with 10% heat-inactivated fetal calf serum, 10 mM
HEPES buffer, pH 7.4, 2 mM GlutaMAX (Invitrogen, Basel, Swit-
zerland), and 1000 U/ml penicillin/streptomycin. The mean rat
weight was 395 Ϯ 115 g.
Oxygen consumption by intact mitochondria was measured in a
chamber equipped with Clark-type oxygen electrode (YSI, Yellow

Toxicity and Pharmacology of Amiodarone and Analogs
1417
Springs, OH) at 30°C (Hoppel et al., 1979). The final concentration of until the steady-state level of current block was reached. At least
L-glutamate was 20 mM. The mitochondrial respiratory control ratio three individual measurements were run per test compound. The
(
RCR) as well as state 3 and state 4 of respiration were determined steady-state level currents were compared with those from control
according to Estabrook (1967) and as described by Kr a¨ henb u¨ hl et al. conditions (0.01% DMSO), and the residual current was calculated
1991). Uncoupling of the oxidative phosphorylation was investi- as percentage of control.
(
gated by monitoring oxygen consumption in the presence of test
substances and oligomycin, an inhibitor of F F -ATPase (final con- Statistical Methods
1
0
centration of 5 ␮g/ml) (Kaufmann et al., 2005).
Data are presented as mean Ϯ S.E.M. of at least three individual
experiments. Differences between groups (control and test compound
incubations) were analyzed by analysis of variance and Dunnett’s
post hoc test was performed if analysis of variance showed signifi-
cant differences. A p value Յ0.05 was considered to be significant.
Mitochondrial ␤-Oxidation
14
Mitochondrial ␤-oxidation of [1- C]palmitic acid in the presence
of test compounds was determined using freshly isolated liver mito-
chondria according to the method of Freneaux et al. (1988) with some
modifications as described by Spaniol et al. (2001a).
Results
Oxygen Consumption. Oxygen consumption by isolated
mitochondria can roughly be divided into two states. In state
, a substrate donates electrons to the electron transport
chain and ADP is being phosphorylated to ATP. In state 4,
ADP is depleted; therefore, no ATP can be produced. Since
the RCR is the ratio between these two states, a decrease in
Effect of Amiodarone, B2-O-Et-N-dipropyl, B2-O-Acetate,
and B2-O-Et on the Inhibition of hERG Currents
3
Chinese Hamster Ovary hERG Cells. The three least toxic
compounds (B2-O-Et-N-dipropyl, B2-O-acetate, and B2-O-Et) and
amiodarone were chosen for examination concerning their antiar-
rhythmic effect. The interaction of the test substances (final concen-
tration 10 ␮M) with the hERG channel was examined using CHO the RCR can be due to reduced state 3 of respiration (impair-
cells stably expressing this potassium channel. In brief, two separate ment of the function of electron transport chain) or to an
human cardiac plasmid cDNA libraries were prepared from freshly increase in state 4 of respiration (in most cases, uncoupling of
isolated tissue and the hERG ␣ subunit PCR product released from
the pCR2.1-TOPO vector (Invitrogen) for ligation into a modified
pcDNA5/FRT/TO vector (Invitrogen) with excluded BGH site. Re-
striction analysis and complete sequencing confirmed the correct
oxidative metabolism on isolated rat liver mitochondria was
composition and expression of the hERG ␣ subunit in the plasmid.
CHO cells were transfected with the calcium phosphate precipitation
method (Invitrogen). Clones were selected with 700 ␮g/ml hygromy-
cin B (Invitrogen) and checked back electrophysiologically for the
the activity of electron transport chain from production of ATP).
To get an overview about the effect on mitochondria, the
influence of 1, 10, and 100 ␮M amiodarone and analogs on
tested in the presence of L-glutamate as a substrate (Table 2).
For all test compounds, the RCR decreased dose dependently,
and for all compounds, except for B2-O-Et, the decrease was
presence of sufficient potassium current, long-term recording stabil- statistically significant in comparison with control incuba-
ity, and sealing properties. Using limited dilution, clone CHO hERG tions. B2-O-Et did not have a significant effect on mitochon-
K2, which displayed an average tail current amplitude of approxi- drial respiration at any concentration.
mately 700 to 1500 pA, was isolated. This clone was successfully
Most of the analogs and amiodarone were associated with
cultivated over 40 passages without detectable loss of current den-
an increase in state 4 respiration, suggesting uncoupling
sity and was used in the current studies. The cells were generally
properties. To test this possibility, oxygen consumption was
maintained and passaged in Ham’s F-12 with GlutaMAX I (GIBCO)
studied in the presence of test compounds and oligomycin.
supplemented with 9% fetal bovine serum (GIBCO), 0.9% penicillin/
Oligomycin is an inhibitor of the F F -ATPase, and induces a
1
0
streptomycin solution (GIBCO), and 500 ␮g/ml hygromycin B (In-
vitrogen). For electrophysiological measurements, the cells were
seeded onto 35-mm sterile culture dishes containing 2 ml of culture
so-called state 4u when incubated with mitochondria. Under
these conditions, an increase in oxygen consumption can only
medium (without antibiotics or antimycotics). Confluent clusters of reflect uncoupling and not production of ADP by other mech-
CHO cells are electrically coupled. Because responses in distant cells anisms. After an incubation in the presence of oligomycin,
are not adequately voltage clamped and because of uncertainties amiodarone, B2-O-Et-N-dimethyl, B2-O-Et-N-dipropyl, and
about the extent of coupling, cells were cultured at a density enabling
single cells (without visible connections to neighboring cells) to be
used for the experiments.
Electrophysiology. hERG currents were measured by means of
the patch-clamp technique in the whole-cell configuration. The incu-
B2-O-acetate significantly increased oxygen consumption at
10 ␮M, and B2-O-Et-N-dipropyl and B2-O-Et-propionamide
at 100 ␮M (data not shown). In contrast, B2-O-Et-OH did not
change the state 4u significantly up to 100 ␮M. B2-O-Et was
not investigated, since it did not change the RCR (Table 2).
These data match well with the observed effect on state 4
respiration (compare with data in Table 2).
bation buffer contained 137 mM NaCl, 4 mM KCl, 1.8 mM CaCl , 1
2
mM MgCl , 10 mM D-glucose, and 10 mM HEPES, pH (with NaOH)
2
7
.40. The pipette solution consisted of 130 mM KCl, 1 mM MgCl , 5
2
mM MgATP, 10 mM HEPES, and 5 mM EGTA, pH (with KOH) 7.20.
Mitochondrial ␤-Oxidation. Since amiodarone is known
After formation of a Gigaohm seal between the patch electrodes and to impair mitochondrial ␤-oxidation (Fromenty et al., 1990b;
individual hERG stably transfected CHO cells, the cell membrane Kaufmann et al., 2005), the effect on the metabolism of
across the pipette tip was ruptured to ensure electrical access to the
palmitate was investigated. In comparison with the control
cell interior. All solutions applied to cells were continuously perfused
incubations, amiodarone and most of the derivatives signifi-
(
1 ml/min) and maintained at room temperature. As soon as a stable
cantly decreased palmitate metabolism in a dose-dependent
manner (Fig. 2). The exceptions were B2-O-Et-N-dipropyl
and B2-O-Et, which did not affect mitochondrial ␤-oxidation
up to 100 ␮M.
seal could be established, hERG outward tail currents were mea-
sured upon depolarization of the cell membrane to ϩ20 mV for 3 s
(
activation of channels) from a holding potential of Ϫ80 mV and
subsequent partial repolarization to Ϫ40 mV for 4 s. This voltage
protocol was run a minimum of 10 times at intervals of 10 s before
Production of ROS. Since increased ROS formation
application of the test compound. After equilibration (typically tak- can be one of the consequences of the inhibition of the
ing approximately 3 min), the voltage protocol was run continuously electron transport chain (Kaufmann et al., 2005), this was

1
418
Waldhauser et al.
TABLE 2
Effects of amiodarone and amiodarone metabolites and analogs on oxidative metabolism in isolated rat liver mitochondria
The values represent mean Ϯ S.E.M. of at least three experiments. Units for states 3 and 4 oxidation are nanoatoms/milligram of mitochondrial protein/minute. All
incubations contained 1% DMSO; *, p Ͻ 0.05 and **, p Ͻ 0.01 versus control incubations.
State 3
State 4
RCR
Control (1% DMSO)
78.9 Ϯ 15.0
2.30 Ϯ 0.37
7.91 Ϯ 1.28
B2-O-Et-N-diethyl (amiodarone) (␮M)
1
72.9 Ϯ 9.3
72.2 Ϯ 7.7
64.8 Ϯ 8.1
2.5 Ϯ 0.5
4.17 Ϯ 1.53*
9.83 Ϯ 2.25**
6.60 Ϯ 0.53
4.21 Ϯ 1.38**
1.52 Ϯ 0.31**
1
1
0
00
B2-O-Et-NH-ethyl (␮M)
1
68.5 Ϯ 16.9
69.7 Ϯ 8.0
3.7 Ϯ 1.3**
3.67 Ϯ 2.25
3.33 Ϯ 1.44
3.7 Ϯ 1.3
5.06 Ϯ 2.17*
5.10 Ϯ 1.42*
1.0 Ϯ 0.0**
1
1
0
00
B2-O-Et-NH2 (␮M)
1
76.7 Ϯ 3.2
75.2 Ϯ 8.8
3.4 Ϯ 1.6**
2.50 Ϯ 0.5
2.50 Ϯ 0.5
3.4 Ϯ 1.6
7.07 Ϯ 1.5
7.01 Ϯ 2.09
1.0 Ϯ 0.0**
1
1
0
00
B2-O-Et-N-diemethyl (␮M)
1
75.1 Ϯ 12.3
66.8 Ϯ 6.0
5.2 Ϯ 1.3**
2.67 Ϯ 0.58
3.67 Ϯ 0.58*
5.2 Ϯ 1.3**
6.40 Ϯ 0.66
4.15 Ϯ 0.67**
1.0 Ϯ 0.0**
1
1
0
00
B2-O-Et-N-dipropyl (␮M)
1
77.3 Ϯ 11.4
72.1 Ϯ 10.1
68.4 Ϯ 3.4
3.17 Ϯ 0.29
3.67 Ϯ 1.26
5.17 Ϯ 2.08**
5.55 Ϯ 1.24*
4.70 Ϯ 1.31**
3.32 Ϯ 1.27**
1
1
0
00
B2-O-Acetate (␮M)
1
69.1 Ϯ 8.9
49.1 Ϯ 10.2*
9.7 Ϯ 5.2**
2.50 Ϯ 0.87
3.67 Ϯ 0.58*
5.13 Ϯ 1.15**
6.50 Ϯ 1.39
3.00 Ϯ 0.38**
1.25 Ϯ 0.43**
1
1
0
00
B2-O-Et-propionamide (␮M)
1
63.2 Ϯ 26.1
63.9 Ϯ 9.3
47.6 Ϯ 6.8*
2.50 Ϯ 2.71
3.83 Ϯ 0.29*
47.6 Ϯ 6.8**
5.67 Ϯ 2.34*
3.77 Ϯ 0.79**
1.0 Ϯ 0.0**
1
1
0
00
B2-O-Et (␮M)
1
84.7 Ϯ 21.3
81.0 Ϯ 15.2
73.6 Ϯ 19.8
2.33 Ϯ 0.29
2.50 Ϯ 0.25
2.67 Ϯ 0.29
8.40 Ϯ 3.22
7.27 Ϯ 1.36
6.12 Ϯ 0.99
1
1
0
00
B2-O-Et-OH (␮M)
1
72.9 Ϯ 13.4
75.8 Ϯ 7.7
20.0 Ϯ 3.2**
2.50 Ϯ 0.50
2.67 Ϯ 0.29
20.0 Ϯ 3.2**
6.55 Ϯ 0.18
6.37 Ϯ 0.40
1.0 Ϯ 0.0**
1
1
0
00
Fig. 2. Effect of amiodarone and its analogs on
-oxidation by isolated rat liver mitochondria.
␤
With the exception of B2-O-Et-N-dipropyl and
B2-O-Et, amiodarone (B2-O-Et-N-diethyl), and
all amiodarone derivatives impaired mitochon-
drial ␤-oxidation at a concentration of 10 to 100
␮
M. Incubations were performed as described
under Materials and Methods. Results are ex-
pressed as acid-soluble products produced from
1
4
[
C]palmitic acid and represent mean Ϯ
S.E.M. of at least four experiments. All incuba-
tions contained 1% DMSO; ء, p Ͻ 0.05 and ءء,
p Ͻ 0.01 versus control incubations.
determined using isolated rat hepatocytes. ROS formation these possibilities, HepG2 cells or primary rat hepatocytes
was measured after incubation for 2, 4, 6, or 18 h with 100 were treated for 18 h with amiodarone or its analogs in rising
␮
M amiodarone or its analogs. The emitted fluorescence concentrations (1, 10, and 100 ␮M) and the extracellular
increased time-dependently for all derivatives and for ami- LDH activity was measured in the supernatant as a marker
odarone as shown in Fig. 3. After 18 h of incubation, ROS of cell death (see Fig. 4 for HepG2 cells). Amiodarone, its
production was significantly increased in comparison with metabolites B2-O-Et-NH-ethyl, B2-O-Et-NH , and B2-O-
2
control incubations for all compounds except for B2-O-Et- Et-OH as well as B2-O-Et-N-dimethyl, B2-O-acetate, and
N-dipropyl and B2-O-Et.
B2-O-Et-propionamide showed a dose-dependent toxicity,
Cell Viability. Impaired mitochondrial ␤-oxidation and/or which, at a concentration of 10 and/or 100 ␮M, was signifi-
impaired function of oxidative phosphorylation with ROS cant in comparison with control incubations. A similar pat-
production can be associated with necrosis and apoptosis of tern was obtained when this experiment was repeated with
the affected cells (Kaufmann et al., 2005). To investigate primary rat hepatocytes (data not shown).

Toxicity and Pharmacology of Amiodarone and Analogs
1419
Fig. 3. ROS formation by primary
hepatocytes. ROS formation by primary
hepatocytes treated with 100 ␮M ami-
odarone and analogs increased time-
dependently for all compounds tested,
reaching statistical significance for
amiodarone, B2-O-NH-ethyl, B2-O-Et-
NH2, B2-O-Et-N-dimethyl, B2-O-ace-
tate, B2-O-Et-propionamide, and B2-O-
EtOH. Incubations were performed as
described under Materials and Meth-
ods. Results are shown as mean Ϯ
S.E.M. of at least three individual ex-
periments in quadruplicate. All incu-
bations contained 1% DMSO; ء, p Ͻ
0
.05 and ءء, p Ͻ 0.01 versus control
incubations.
Fig. 4. Cytotoxicity of amiodarone and amiodarone derivatives. Cytotoxicity was studied using HepG2 cells under the conditions described under
Materials and Methods. Amiodarone (B2-O-Et-N-diethyl), B2-O-Et-NH-ethyl, B2-O-Et-NH , B2-O-Et-N-dimethyl, B2-O-acetate, B2-O-Et-propi-
2
onamide, and B2-O-Et-OH increased the LDH leakage into the cell culture media dose-dependently after an incubation for 18 h. In contrast, for
B2-O-Et-N-dipropyl and B2-O-Et, the cell membrane remained tight under each concentration used for the incubations. Similar data were obtained
using primary rat hepatocytes. Data are given as mean Ϯ S.E.M. of at least three individual experiments in triplicates. All incubations (except “no
treatment”) contained 1% DMSO; ء, p Ͻ 0,05 and ءء, p Ͻ 0.01 versus control incubations.
Mechanism of Cell Death. The mechanism of cell death pressing hERG channels. As shown in Fig. 6, 10 ␮M amio-
was investigated using staining with annexin V/propidium darone rapidly and robustly blocked hERG tail currents
iodide, which can differentiate between early apoptosis and (6.28 Ϯ 4.05% tail current relative to control, mean Ϯ S.E.M.
late apoptosis/necrosis (Kaufmann et al., 2005). After 18 h of of n ϭ 3 experiments). The derivatives B2-O-acetate (10 ␮M)
incubation, hepatocytes showed a significant increase in late and B2-O-Et-N-dipropyl (10 ␮M) also significantly inhibited
apoptosis/necrosis for all substances investigated, except for the tail current but less so than amiodarone (remaining tail
B2-O-Et-N-dimethyl, B2-O-Et-N-dipropyl, B2-O-acetate, and current 81.9 Ϯ 2.6 and 76.2 Ϯ 6.5%, respectively, relative to
B2-O-Et (Fig. 5). In agreement with these findings, the ATP control values, mean Ϯ S.E.M. of n ϭ 3 experiments). No
content of the hepatocytes was reduced at this time point in significant effect was observed for 10 ␮M B2-O-Et (94.6 Ϯ
the incubations containing amiodarone, B2-O-Et-NH-ethyl, 3.5% tail current relative to control, mean Ϯ S.E.M. of n ϭ 3
B2-O-Et-NH2, B2-O-Et-propionamide, and B2-O-Et-OH experiments). As expected, at higher concentrations (Ն30
(
(
data not shown). With the exception of B2-O-Et-N-dimethyl ␮M) the inhibitory effects of B2-O-acetate and B2-O-Et-N-
significant LDH release and nonsignificant increase in late dipropyl was more pronounced with the exception of B2-O-Et,
apoptosis/necrosis), these findings agree well with the results which again displayed no significant hERG blockage (data
of the LDH leakage assay. The discrepancy between LDH not shown). In addition, the vehicle (0.01% DMSO) did not
release and annexin V/propidium iodide staining may be interfere with hERG channel activity (96.70 Ϯ 0.49%, rela-
associated with extent of cell damage needed to obtain a tive tail current, mean Ϯ S.E.M. of n ϭ 3 experiments).
positive result.
؉
Effects on the Cardiac Rapid Delayed Rectifier K
Discussion
Current. Since one of the important antiarrhythmic mech-
anisms of amiodarone is the inhibition of hERG channels, the
In our current studies, amiodarone and most of its analogs
effect of amiodarone and of the three least toxic amiodarone demonstrated a similar toxicity pattern toward hepatic mi-
derivatives (B2-O-Et-N-dipropyl, B2-O-acetate, and B2-O-Et) tochondria as described in similar investigations (Fromenty
ϩ
on the K current was investigated in CHO cells overex- et al., 1990a,b; Spaniol et al., 2001b; Kaufmann et al., 2005).

1
420
Waldhauser et al.
Fig. 5. Mechanism of cell death in primary hepatocytes. Cell death was assessed using staining with annexin V/propidium iodide followed by flow
cytometry. The assays were carried out as described under Materials and Methods and quantified as described previously (Kaufmann et al., 2005).
Deoxycholate (100 ␮M) was used as a positive control for early apoptosis and the detergent NP-40 [0.01% (v/v)] as a positive control for late
apoptosis/necrosis. During apoptosis, phosphatidylserine is externalized and can be bound by annexin V, which can be detected by flow cytometry.
During late apoptosis or necrosis, propidium iodide is able to enter the cells across disintegrated membranes and to stain DNA, which can be
differentiated by flow cytometry from early apoptosis (Kaufmann et al., 2005). After 18 h of incubation with the compounds shown in figure, the
positive control (100 ␮M deoxycholate) was associated with early apoptosis, but none of the test compounds investigated. In comparison, late
apoptosis/necrosis was detected in the presence of the positive control [NP-40 0.01% (v/v)] and also in the presence of amiodarone (B2-O-Et-N-diethyl),
B2-O-Et-NH-ethyl, B2-O-Et-NH , B2-O-Et-propionamide, and B2-O-Et-OH (all at a concentration of 100 ␮M). The results are presented as mean Ϯ
2
S.E.M. of three individual experiments. With the exception of the incubation labeled “no treatment”, all incubations contained 1% DMSO; ء, p Ͻ 0.05
and ءء, p Ͻ 0.01 versus control incubations.
Fig. 6. Inhibition of potassium current. Representative current traces of potassium currents through hERG channels stably expressed in CHO cells
are shown. Measurements were accomplished in the whole-cell patch-clamp configuration at room temperature. Outward currents were activated upon
depolarization of the cell membrane from Ϫ80 to ϩ20 mV for 3 s. Partial repolarization to Ϫ40 mV for 4 s evoked large tail currents. At least three
cells were recorded per test compound. The vehicle (0.1% DMSO) as well as 10 ␮M B2-O-Et had no significant effect on hERG channel activity (traces
at the top). In contrast, amiodarone, B2-O-acetate, and B2-O-Et-N-dipropyl had a clear inhibitory effect on the hERG channels. The top line in the
figures depicts the control incubations (0.1% DMSO), and the bottom line depicts the incubations containing the test compounds.

Toxicity and Pharmacology of Amiodarone and Analogs
1421
As shown in Table 3, amiodarone and its analogs inhibited Pessayre, 1995). It can therefore be predicted that beside
the function of the respiratory chain, impaired mitochondrial amiodarone, also its metabolites B2-O-Et-NH-ethyl, B2-O-
␤
-oxidation, and/or uncoupled oxidative phosphorylation. Et-NH , and B2-O-Et-OH as well as the other amiodarone
2
Most substances were cytotoxic; exceptions were B2-O-Et analogs synthesized and tested (all of them except B2-O-Et
and B2-O-Et-N-dipropyl. and B2-O-Et-N-dipropyl) will probably be associated with
Regarding B2-O-Et and the B2-O-Et-NR derivatives, the microvesicular steatosis. For amiodarone, it has been shown
2
pattern of cytotoxicity (strong cytotoxicity of the amiodarone that inhibition of carnitine palmitoyltransferase I is a mech-
metabolites B2-O-Et-NH-ethyl and B2-O-Et-NH , lower cy- anism for the inhibition of ␤-oxidation (Kennedy et al., 1996).
2
totoxicity for amiodarone and very low or lacking cytotoxicity This may also be the case for the amiodarone metabolites and
for B2-O-Et) was very similar to the toxicity found on alveo- analogs, but so far formal proof is lacking.
lar macrophages, as reported in a recent investigation (Qua-
For amiodarone, the toxicity found in the current investi-
glino et al., 2004). As shown for B2-O-Et-N-dipropyl, uncou- gations (strong uncoupling activities and inhibition of mito-
pling of the respiratory chain was not sufficient to induce chondrial ␤-oxidation) is in agreement with previous inves-
cytotoxicity. Incomplete uncoupling (mitochondria are still tigations (Fromenty et al., 1990a; Spaniol et al., 2001a;
able to produce some ATP) and extramitochondrial produc- Kaufmann et al., 2005). Since the two metabolites B2-O-Et-
tion of ATP (e.g., by glycolysis) could serve as explanations NH-ethyl and B2-O-NH are strong inhibitors of the respira-
2
for this finding. As evidenced e.g., by amiodarone and by the tory chain and are both associated with ROS production
amiodarone metabolites B2-O-Et-NH-ethyl and B2-O-Et- (which may be a consequence of the inhibition of the respi-
NH , cytotoxicity is associated primarily with substances ratory chain; Kaufmann et al., 2005) and with a remarkable
2
that affect the function of the respiratory chain and/or mito- cytotoxicity, it seems to be possible that they are at least
chondrial ␤-oxidation. Inhibition of the respiratory chain can partially responsible for the hepatic toxicity in patients
be associated with ROS production (Kaufmann et al., 2005), treated with amiodarone. If this were the case, induction of
which can trigger opening of the mitochondrial permeability CYP3A4, the main cytochrome P450 isozyme responsible for
transition pore, leading to the release of cytochrome c and amiodarone deethylation (Fabre et al., 1993), may be a risk
other substances into the cytoplasm and triggering apoptosis factor for hepatotoxicity associated with amiodarone. Al-
and/or necrosis, depending on the ATP content of the cell though a high dosage and/or high plasma levels of amioda-
(
Eguchi et al., 1997; Leist et al., 1997). In combination with rone are considered to represent risk factors for hepatotoxic-
the concomitant drop in the cellular ATP content, the results ity associated with this drug (Pollak et al., 1990; Bravo et al.,
in Fig. 4 therefore indicate that after 18 h of incubation most 2005), induction of CYP3A4 has so far not been reported to be
hepatocytes had undergone necrosis in the presence of 100 a risk factor for amiodarone-associated liver injury (Rigas et
␮
M amiodarone, B2-O-Et-NH-ethyl, B2-O-Et-NH , B2-O-Et- al., 1986; Flaharty et al., 1989; Lewis et al., 1989). Since
2
propionamide, or B2-O-Et-OH.
CYP3A4 inducers (e.g., antiepileptics such as phenytoin, phe-
Amiodarone and the amiodarone metabolites B2-O-Et-NH- nobarbital and carbamazepine as well as rifampicin) are used
ethyl and B2-O-NH2 were shown to be strong inhibitors of quite frequently and since hepatotoxicity associated with
mitochondrial ␤-oxidation and of the respiratory chain (B2- amiodarone is potentially fatal (Lewis et al., 1989), this ques-
O-Et-NH-ethyl and B2-O-NH ) or uncouplers of oxidative tion is clinically important and should therefore be investi-
2
phosphorylation (amiodarone). Regarding amiodarone, mito- gated and answered.
chondrial toxicity explains the histological findings in liver
As shown in Table 3, the toxicity of the investigated sub-
biopsies from patients (Lewis et al., 1990) and mice (Fro- stances showed tendencies but no clear correlation with their
menty et al., 1990b) with amiodarone-associated hepatotox- lipophilicity profile. Inhibition of the respiratory chain (state
icity, revealing micro- and macrovesicular accumulation of 3 respiration), of mitochondrial ␤-oxidation, ROS production
fat in hepatocytes as a hallmark of their toxicity. Accumula- and cytotoxicity were preferentially associated with less li-
tion of small lipid droplets in hepatocytes (microvesicular pophilic substances. In contrast, uncoupling of oxidative
steatosis) is considered to be a consequence of the inhibition phosphorylation was associated preferentially with sub-
of ␤-oxidation in hepatocellular mitochondria (Fromenty and stances showing a higher lipophilicity. Since all substances
TABLE 3
Overview of the toxicological properties of amiodarone and analogs and of their effect on hERG channels. The compounds are listed with
increasing lipophilicity
The graduation was as follows. For states 3, 4, and 4u, ␤-oxidation and cytotoxicity: ϩ ϭ p Ͻ 0.05 at 100 ␮M; ϩϩ ϭ p Ͻ 0.01 at 100 ␮M; and ϩϩϩ ϭ p Ͻ 0.05 at 10 ␮M.
For ROS production: ϩ ϭ p Ͻ 0.05 at 18 h; ϩϩ ϭ p Ͻ 0.05 at 6 h; and ϩϩϩ ϭ p Ͻ 0.05 at 4 h. For the effect on hERG channels: ϩ ϭ p Ͻ 0.05 at 10 ␮M; ϩϩ ϭ p Ͻ 0.01
at 10 ␮M; and ϩϩϩ ϭ p Ͻ 0.001 at 10 ␮M.
logP
State 3
State 4
State 4u
␤-Oxi-dation
ROS
Citotoxicitya
Effect on hERG Channels
B2-O-Et-NH2
3.83
4.18
4.31
4.40
4.47
4.69
4.83
4.92
5.51
ϩϩ
0
ϩϩϩ
ϩϩ
ϩϩ
ϩ
ϩϩ
0
0
0
N.D.
0
ϩϩ
ϩϩ
ϩϩϩ
ϩϩ
ϩϩ
ϩϩ
0
ϩϩϩ
ϩ
ϩ
ϩ
ϩ
ϩϩϩ
0
ϩ
ϩϩ
ϩϩ
ϩ
ϩϩ
ϩϩϩ
ϩ
0
ϩϩ
0
N.D.
N.D.
N.D.
N.D.
N.D.
ϩ
B2-O-Et-OH
0
B2-O-Et-propionamide
B2-O-Et-N-dimethyl
B2-O-Et-NH-ethyl
B2-O-Acetate
B2-O-Et
B2-O-Et-N-diethyl (amiodarone)
B2-O-Et-N-dipropyl
ϩϩϩ
ϩϩϩ
0
ϩϩ
ϩϩ
ϩϩϩ
ϩϩ
ϩϩ
ϩϩϩ
N.D.
ϩϩϩ
N.D.
ϩϩϩ
ϩϩϩ
0
ϩϩ
0
ϩϩϩ
ϩ
0
N.D., not determined.
a
Cytotoxicity was determined using the data of Fig. 4.

1
422
Waldhauser et al.
investigated showed some mitochondrial toxicity (at least amiodarone analogs with activity against hERG channels
uncoupling of oxidative phosphorylation or inhibition of ␤-ox- but with a lower mitochondrial toxicity than amiodarone,
idation), all of the compounds studied had to be able to potentially offering the possibility to find safer antiar-
penetrate the inner mitochondrial membrane. Therefore, rhythmic drugs.
lack of penetration of the mitochondrial membranes is no
probable explanation for a low toxicity.
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Address correspondence to: Dr. Stephan Kr a¨ henb u¨ hl, Division of Clinical
Pharmacology and Toxicology, University Hospital, CH-4031 Basel, Switzer-
land. E-mail: kraehenbuehl@uhbs.ch