Metabolism of amiodarone (part III): Identification of rabbit cytochrome p450 isoforms involved in the hydroxylation of mono-N-desethylamiodarone

Article abstract of DOI:10.1080/00498250110046442

1. Amiodarone (AMI) is a potent anti-arrhythmic drug and mono-N-desethylamiodarone (MDEA) is its only known metabolite. It was found recently that in rabbit liver microsomes MDEA was biotransformed to n-3-hydroxybutyl-MDEA (3OH-MDEA). 2. In liver microsomes isolated from the untreated rabbit, the formation of 3OH-MDEA obeyed Michaelis-Menten enzyme kinetics with K_m = 6.39 ± 1.07 μM and V_max = 0.56 ± 0.21 nmol min^-1 mg^-1 protein. 3. Furthermore, (1) among chemicals usually used as inhibitors of cytochrome P450, only midazolam (MDZ), cyclosporin A and ketoconazole inhibited the MDEA hydroxylase activity significantly (> 60% inhibition), (2) MDZ, a substrate of CYP3A, inhibited the 3OH-MDEA formation competitively (K_i = 10 ± 5 μM), (3) the formation rates of 3OH-MDEA correlated positively with those of 1OH-MDZ (r = 0.81; n = 6), and (4) MDEA hydroxylase activity of microsomes isolated from rabbit rifampicin-induced cultured hepatocytes was 4-fold more active than the control. 4. Since CYP3A6 is mainly induced by rifampicin in rabbit-cultured hepatocytes, the data suggest that this isoform is involved in the biotransformation of MDEA to 3OH-MDEA. 5. Since α-naphthoflavone, cimetidine and quinidine also partially inhibited the MDEA hydroxylase activity, it is possible that other CYPs, such as 1A, 2C and 2D, may also be active in the metabolism of amiodarone.

Full text of DOI:10.1080/00498250110046442

2001,
31,
5, 239- 248
no.
xenobiotica,
vol.
Metabolism of amiodarone (Part III):
identiŽ cation of rabbit cytochrome P450
isoforms involved in the hydroxylation of
mono- -desethylamiodarone
N
P. KOZLIK , H. R. HA *, B. STIEGER , L. BIGLER§
{
{
{
and F. FOLLATH
{
Cardiovascular Therapy Research Unit and Clinical Pharmacology and Toxicology
Division, Department of Internal Medicine, University Hospital of Zurich,
Ramistrasse 100, CH-8091 Zurich, Switzerland
{
{
Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland
}
Received 7 November 2000
1. Amiodarone (AMI) is a potent anti-arrhythmic drug and mono- desethylamio-
N-
darone (MDEA) is its only known metabolite. It was found recently that in rabbit liver
microsomes MDEA was biotransformed to -3-hydroxybutyl-MDEA (3OH-MDEA).
n
2. In liver microsomes isolated from the untreated rabbit, the formation of 3OH-
MDEA obeyed Michaelis-Menten enzyme kinetics with
6 39 1 07
and
m
Km
m
ˆ
§
:
:
1
0 56 0 21 nmol min 1 mg protein.
Vmax
ˆ
§
:
:
3. Furthermore, (1) among chemicals usually used as inhibitors of cytochrome P450,
only midazolam (MDZ), cyclosporin and ketoconazole inhibited the MDEA
hydroxylase activity signiŽ cantly (> 60% inhibition), (2) MDZ, a substrate of CYP3A,
inhibited the 3OH-MDEA formation competitively ( 10 ), (3) the formation
A
5
Ki ˆ
m
m
§
rates of 3OH-MDEA correlated positively with those of 10OH-MDZ (
0 81;
6),
r
n
ˆ
ˆ
:
and (4) MDEA hydroxylase activity of microsomes isolated from rabbit rifampicin-
induced cultured hepatocytes was 4-fold more active than the control.
4. Since CYP3A6 is mainly induced by rifampicin in rabbit-cultured hepatocytes, the
data suggest that this isoform is involved in the biotransformation of MDEA to 3OH-
MDEA.
5. Since -naphtho avone, cimetidine and quinidine also partially inhibited the
¬
MDEA hydroxylase activity, it is possible that other CYPs, such as 1A, 2C and 2D, may
also be active in the metabolism of amiodarone.
Introduction
Amiodarone (AMI) has proved to be a potent anti-arrhythmic drug, but
despite its frequent use, data on its metabolism are scarce. In humans and
laboratory animals, mono- desethylamiodarone (MDEA) is known as the
N-
major metabolite of AMI. During long-term therapy, the serum concentration
of this pharmacologically active metabolite is comparable with that of the parent
drug, and cytochrome P4503A4 (CYP3A4) isoforms are involved in AMI deal-
kylation (Ha
1992, 1996, Fabre
1993). Previous data have shown that
et al.
et al.
other metabolites of AMI may be produced. Storey
. (1969) reported that,
et al
unlike in human and rat, the MDEA concentration in blood and tissue of rabbit
1
receiving AMI (i.p. 40 mg kg 1 day for 4 weeks) was very low. It was doubtful
* Author for correspondence; e-mail: huyriem.ha@dim.usz.ch
ISSN 0049-825 4 print/ISSN 1366-5928 online
http://www.tandf.co.uk/journals
2001 Taylor & Francis Ltd
Xenobiotica
#
DOI: 10.1080/0049825011004644 2

240
et al.
P. Kozlik
Figure 1. Chemical structure of amiodarone (AMI), mono- desethylamiodarone (MDEA) and -3-
N-
n
hydroxybutyl- desethylamiodarone (3OH-MDEA).
N-
that, in Storey’s experiments, MDEA would have been further biotransformed to
unknown product(s). Kannan (1985) analysed the bile of rabbit receiving
et al.
1
AMI (i.p. 20 mg kg for 6 weeks) and found two chromatographic peaks other
than those corresponding to AMI and MDEA. The identity of these compounds
remained unknown. From these data, it is possible that: (1) metabolic pathways
other than dealkylation exist in AMI metabolism; (2) in rabbit the clearance of
MDEA is particularly high; and (3) metabolism of AMI is species dependent.
Recently, the present group has reported that in rabbit liver microsomes, MDEA
is biotransformed to -3-hydroxybutyl-MDEA (3OH-MDEA) (Ž gure 1), and this
n
compound may be detected in the liver, lung, kidney and heart of rat receiving
AMI.HCl (Ha
2001). In this paper, the identity of CYP isoenzyme(s)
et al.
involved in the MDEA hydroxylation is investigated.
Materials and methods
Chemicals
Mono- desethylamiodarone hydrochloride (MDEA, purity > 98%) was a gift from SanoŽ (Mont-
N-
pellier, France). Hydroxylated MDEA (3OH-MDEA, purity 97%) was isolated from the bioreaction
using MDEA and rabbit liver microsomes as described by Ha
(2001). Midazolam (MDZ), 8-
et al.
chloro-6-(2- uorophenyl)-1-hydroxy-methyl-4H-imidazo(1,5-a)(1,4)-benzodiazepine (10OH-MDZ) and
internal standard Ro 05-6669 (7-chloro-1,3-dihydro-5-(4-methoxyphenyl)-1-methyl-2H-1,4-benzodia-
zepin-2-one) were obtained from Ho mann-La Roche (Basle, Switzerland). -Naphtho avone (ANP)
€
¬
was from Aldrich Chemical Co. (Buchs, Switzerland). Coumarin, cimetidine, cyclosporin A, diethyl-
dithiocarbamic acid (DDC), methimazole, mephenytoin, sulphaphenazole, SKF525A (proadifen),
quinidine, quinine, triacetyloleandomycin (TAO), rifampicin (RIF),
-isocitric acid, NADPH and
dl
isocitric dehydrogenase (EC 1.1.1.42) from porcine heart were purchased from Sigma Chemical Co.
(Buchs, Switzerland). Other chemicals, such as diethyl ether, MgCl2.6H2O and Na2HPO4 were from
Fluka Chemical Co. (Buchs, Switzerland).
3OH-MDEA standards
The o -line puriŽ ed 3OH-MDEA (0.15 mg) from Ha
(2001) was dissolved in methanol and its
et al.
UV spectrum was measured between 210 and 360 nm. Its concentration was calculated using the molar
€

241
Metabolism of amiodarone
1
extinction coe cient
440000 1 cm at 241 nm (Plomp 1991). The working standards were then
m

ˆ
"
1
prepared by diluting the stock solution with the incubation medium containing 200 g ml heat-
m
deactivated rabbit liver microsomes to give Ž nal concentrations of 3OH-MDEA (base form) in the
range 0.25-2.0 g ml 1. The standards were stable for at least 1 month stored in aliquots of 2 ml at
m
20 C.
8
Quantitative HPLC assay for 3OH-MDEA
The performance of the quantitative HPLC assay being developed for 3OH-MDEA has been
reported by Ha
(2000). Brie y, the separation of compounds was achieved using a LKB liquid
et al.
chromatograph (Bromma, Sweden) connected to an integrator SP 4400 (Spectra-Physics, San Jose, CA,
USA). The assay was operated under the following conditions: Nucleosil 100-5 Protect 1-250 4m
m
£
(Macherey-Nagel, Oensingen, Switzerland) maintained at 45 C; mobile phase, methanol:water:aqueous
8
NH3 25% (300:90:0.25 w/w);  ow, 1.2ml min 1; pressure, 190 bars; detection, 242nm. The coe cients

of variation of the between- and within-assay conditions were < 7%.
Biological materials
Up to 5 g fresh liver of the untreated male New Zealand White rabbits (weighing 3-4kg) was
obtained from Dr H. Cristina, Institute for Laboratory Animal Science, University of Zurich. The
blood in the sample was washed out with 0.25 sucrose at 4 C. A rabbit liver microsome fraction was
m
8
isolated by means of standard di erential centrifugation as described for rat liver microsomes (Meier
et
€
1981). Protein concentrations were measured using the method of Bensadoun
(1976).
al.
et al.
3OH-MDEA hydroxylase activities
Rabbit liver microsomal protein (200 g ml 1) was incubated in 0.1 sodium phosphate bu er, pH
m
m
€
7.4 (PB7.4) at 37 C in a Ž nal volume of 0.2 ml in the presence of a system that generated the reduced
8
form of NADP (4.5 IU isocitrate dehydrogenase ml 1, 5 m isocitrate, 5 m MgCl ). Substrate MDEA
m
m
2
was dissolved in ethanol, diluted with PB7.4 to desired concentrations and then added as a 2-fold
concentrated solution in PB7.4. After a 5-min incubation at 37 C, the reaction was started by addition of
8
40 l 5m NADPH. In the negative control incubation, rabbit liver microsomes were deactivated by
m
m
heating at 90 C for 5 min.
8
After incubation (15 min at 37 C), the reaction was stopped by addition of 1 ml ice-cooled diethyl
8
ether and vortexed. The tube was then capped and shaken horizontally for 10 min (400rpm). The
organic phase was separated by centrifugation and pipetted into another glass test tube. To ensure a
quantitative result (recovery 95%), the extraction was repeated three times. The organic phases were
pooled and evaporated to dryness at 21 C. The residue was then dissolved in 150 l mobile phase, and
8
m
100 l of the aliquot was injected onto the HPLC column.
m
Enzyme kinetics analysis
The linear relationships between 3OH-MDEA formation rates and MDEA concentrations (2-
50 ), microsomal protein (25-3.500 g ml 1) and time (1-120 min) were investigated. Sixteen di er-
m
m
m
€
ent concentrations of MDEA, ranging from 2.5 to 40 , were used in the kinetic experiments. The
apparent enzyme kinetic parameters were Ž rst estimated using Lineweaver-Burk plots (1/ versus 1/ ),
and the values obtained were used as the Ž rst estimate for the non-linear least-squares regression
m
m
V
S
analysis (ProFit software, QuantumSoft, Zurich, Switzerland). The Michaelis-Menten equation:
V¤
S
Km
S
V
S
, where is the velocity of the metabolite formation, is the MDEA concentra-
V
ˆ
…
‡ †
=
tion inmtahx e incubation,
is the a nity constant and
employed for the Ž tting. Eadie-Hofstee plots ( versus / ) were then used to gain information
is the maximum enzyme velocity, was
Km
Vmax

V
V S
regarding the involvement of the number of binding sites involved in the biotransformation. The
inhibitory mechanisms of chemical inhibitors were investigated using Dixon plots (1/ versus inhibitor
V
concentrations).
Correlation study
Correlations between the MDEA hydroxylase activities and those of MDZ were investigated by
comparing the formation rates of 3OH-MDEA and 10OH-MDZ in six di erent rabbit liver microsome
€
samples. The concentrations of 10OH-MDZ were measured using the HPLC assay of Ha
(1993).
et al.
The substrate concentrations of MDEA and MDZ were 25 and 20 , respectively.
m
m
Inhibition study
The e ects of various chemical inhibitor or substrate probes were also studied. Selective inhibitors
€
used were: -naphtho avone (ANP) for CYP1A, coumarin (CYP2A6), sulphaphenazole (CYP2C9),
¬
cimetidine (CYP2C11), mephenytoin (2C19), quinidine (CYP2D6), DDC (CYP2E1) and TAO,
ketoconazole (CYP3A). The substrate probes used were quinine and cyclosporin A for CYP3A. Except

242
et al.
P. Kozlik
for DDC, which was dissolved in water, all test substances were dissolved in an organic solvent, such as
ethanol or DMSO, then diluted with PB7.4 to the desired concentration and added (20 l) to the
m
1
standard incubation (10
MDEA, 200 gml microsomal protein, 1 m NADPH, incubated at 37 C
m
m 8
m
m
for 15 min). In the Ž nal mixture, the concentration of the organic solvent was < 1%. The e ect of each
€
compound on the formation of 3OH-MDEA at the respective inhibitor concentrations was compared
with the control value determined from the standard incubation containing the same amount of the
corresponding vehicle. The results were expressed as a percentage of the respective control. All
measurements were performed in triplicate.
For assessing the mechanism of MDZ inhibition, four MDZ concentrations (50, 100, 150, 200
)
m
m
were added to three series of incubation samples containing 2.5, 5.0 and 10
type was analysed using Dixon plots.
MDEA. The inhibition
m
m
E
ect of rifampicin induction
€
Rabbit liver microsomes, which were isolated from cultured hepatocytes [untreated (control; two
samples) and treated with rifampicin (50 for 4 days; two samples) as described by Daujat
m
et al.
m
(1987)], were generously provided by Dr. P. Gallier, Laboratoire de Pharmacologie-Toxicologie INRA,
Toulouse. The samples were diluted with PB7.4 to a Ž nal concentration of 0.1 mg protein ml and
1
incubated with 20
compared with those of controls.
MDEA for 5 min, as described above. The formation rates of 3OH-MDEA were
m
m
Results
, rabbit liver microsomes fortiŽ ed by NADPH possibly biotransform
In vitro
MDEA to 3OH-MDEA and some other minor products. Using the described
HPLC conditions, the retention times of 3OH-MDEA and MDEA were 4.26 and
7.23 min, respectively (Ž gure 2). The formation rates of the metabolite were linear
at 37 C for the incubation time up to 20 min with 5-10
MDEA and up to
m
m
8
1
250 g ml
studies, the conditions of 10
NADPH and 37 C for 15 min in a Ž nal volume of 0.2 ml were chosen for the
microsomal protein (data not shown). Therefore, in the present
m
1
MDEA, 200 g ml microsomal protein, 1 m
m
m
m
m
8
standard incubation. Under these conditions, the 3OH-MDEA formation rates
may be predicted using the classical Michaelis-Menten kinetic law (Ž gure 3), and
the apparent parameters were
6 39 1 07
and
Vmax
0 56 0 21
Km
m
m
ˆ
§
ˆ
§
: :
:
:
1
nmolmin 1 mg
protein. Results shown are the mean SD obtained from
three liver microsomes samples (duplicated determinations). The Eadie-Hofstee
plot (Ž gure 3, insert) suggests that the reaction is monophasic.
§
In order to determine whether CYP or  avin-containing monooxygenase
(FMO) is involved in MDEA hydroxylation, SKF525A (non-speciŽ c CYP in-
hibitor) and methimazole (FMO inhibitor) were co-incubated with MDEA. At a
concentration of 0.2 m , SKF525A abolished the MDEA hydroxylase activity
m
totally, whereas 1 m methimazole suppressed only 20% of it (Ž gure 4) suggesting
m
that CYPs in rabbit liver microsomes are mainly involved in the reaction.
The inhibitory e ect of various chemicals on MDEA hydroxylation was Ž rst
€
investigated. In comparison with human and rat liver microsomes, those of rabbit
are less frequently used for drug metabolism studies. Therefore, the e ects of
€
chemicals that may selectively inhibit the activity of CYPs in rabbit liver are not
well documented. In the present study, these compounds were selected on the
basis of published data showing their putatively selective inhibitors for human
and/or rat CYPs. Among them coumarin, DDC and sulphaphenazole had essen-
tially no e ect (< 20% inhibition) on the MDEA hydroxylation. Thus, CYP
€
isoforms 2A6, 2E1 and 2C9 may be considered as not being involved in the
reaction. Mephenytoin, ANP and cimetidine were more active (30-50% inhibition)
indicating that CYP1A, 2C11 and 2C19 may make a contribution for the MDEA

243
Metabolism of amiodarone
Figure 2. HPLC chromatogram (a) showing that in the standard incubation (10
MDEA,
m
m
1
200 g ml microsomal protein, 1 m NADPH, 0.1 phosphate bu er, pH 7.4, incubated at
m
m
m
€
37 C for 15 min) MDEA was biotransformed to 3OH-MDEA and other minor products (?).
8
Their retention times were 4.26, 5.67 and 6.07min, respectively. The retention time of MDEA
was 7.23 min. Negative control incubation using heat-deactivated microsomes (b).
Figure 3. Representative enzyme kinetics of 3OH-hydroxylation of MDEA in rabbit liver micro-
somes. MDEA concentrations were in the range 2.5-40 . Other conditions (see Ž gure 2) were
m
m
unchanged. The estimates of Michaelis-Menten parameters were:
6 45
:
;
m Vmax
m
Km
ˆ
ˆ
0 51 nmol min 1 mg protein. Eadie-Hofstee plot (insert) suggests that the reaction is mono-
1
:
phasic.

244
et al.
P. Kozlik
Figure 4. E ect of methimazole (FMO inhibitor) and SKF 525A (CYPs inhibitor) on MDEA-3-
€
hydroxylase activity.
hydroxylation. Our data (Ž gure 5) also showed that cyclosporin A, MDZ,
quinidine, quinine and ketoconazole suppressed the initial formation rates of
3OH-MDEA by > 60%, suggesting that MDEA hydroxylase belonged to
CYP3A subfamily. Remarkably, the well-known inhibitor TAO for CYP3A iso-
forms in human and rat showed only a weakly inhibitory e ect (< 30% inhibition)
€
in the present study. This may be explained by the fact that, in contrast to rat, in
rabbit TAO does not form signiŽ cant amounts of the CYP3A-TAO nitroso-
complex and, therefore, does not exhibit decreased monoxygenase activities
(Wrighton
1985).
et al.
Furthermore, quinidine is well known as an e cient inhibitor of the CYP2D6

isoform (Dayer
1987). In the current studies, quinidine and its optical
et al.
isomer, quinine, had comparable inhibitory e ects on MDEA hydroxylation.
€
Thus, the inhibitory e ect of quinidine may not only have an e ect on
€
€
CYP2D6, but also have a result on non-speciŽ c inhibition of CYP3A, since
quinidine (as well as quinine) is a known substrate for human CYP3A (Guengerich
1986). The antimycotic agent ketoconazole is a well-known inhibitor of
et al.
CYP3A isoforms (Balwin
1995). Its estimated
inhibition constant
in vitro
at al.
(
) with use of human liver microsomes for 10OH-MDZ is < 0.01
(von
m
Ki
Moltke
m
1996). Moreover, (1) the present liver microsome samples prepared
et al.
from untreated rabbit also biotransformed MDZ to its 10OH-MDZ (
2 9
:
)
m
Km
m
ˆ
(data not shown); (2) the Dixon plot (Ž gure 6a) for the inhibition of MDEA
hydroxylase activity by MDZ had an intersection above the abscissa [suggesting
that MDZ inhibits competitively (
Ki
10
5
) the 3OH-MDEA formation];
m
m
ˆ
§
and (3) a positive correlation (
0 81) between the formation rates of 10OH-
r
ˆ
:
MDZ and those of 3OH-MDEA measured in six di erent rabbit liver microsomes
€
was observed (Ž gure 6b).

245
Metabolism of amiodarone
Figure 5. E ect of chemicals used as speciŽ c inhibitors and/or substrates on 3OH-MDEA hydroxy-
€
lase. The tested compound was added to the standard incubation (see Ž gure 2). The absolute
1
value of the 100% activity (control) was 0.32nmol min 1 mg protein and results shown are the
mean of three measurements.
In summary, the experiments performed using chemical inhibitors indicate
that, in rabbit, isoforms belonging to the subfamily CYP3A are mainly responsible
for the hydroxylation of MDEA. It is possible, however, that other CYPs such as
1A, 2C and 2D are also involved in this biotransformation.
In order to strengthen the probability that CYP3A isoforms are mainly
involved in the studied reaction, a further experiment performed using biological
reagents was carried out. The rabbit liver microsomes isolated from RIF-induced
hepatocytes were used. The results in Ž gure 7 show clearly that the RIF-induced
microsomes are at least 4-fold more active than those of untreated samples. It is
well documented that in cultured rabbit hepatocytes, the CYP3A6 isoform is RIF
inducible (Daujat
1991); it is therefore possible that in our reaction this
at al.
isoform is the one mainly involved. Moreover, (1) rat CYP3A2 polyclonal antisera
(Gentest, Woburn, MA, USA) inhibited the formation of 3OH-MDEA in

246
et al.
P. Kozlik
Figure 6. Dixon plot (A) for the inhibition of MDEA hydroxylase activity by midazolam (MDZ) has
an intersection above the abscissa. This pattern is indicative of substrate competitive inhibition
with
10-15 . Correlation (
0 81) between MDEA 3-hydroxylase activity and that of
Ki ˆ
m
r
m
ˆ
:
10OH-MDZ in rabbit liver microsomes (B).
Figure 7. Representative in uence of rifampicin treatment on MDEA 3-hydroxylase activity.
incubation using rat liver microsomes; and (2) human cDNA-expressed CYP3A4
isoforms purchased from the same source catalysed the hydroxylation of MDEA
(submitted for publication). These observations support the notion that in
mammals, CYP3A isoforms could mainly correspond to MDEA hydroxylase.
Discussion
Experiments performed with chemical inhibitors suggested that the CYP3A
isoform in rabbit liver microsomes may be mainly involved in the MDEA
hydroxylation. This assumption is supported by observations that: (1) substrates
of CYP3A isoforms, such as MDZ, quinidine, quinine and cyclosporin A, inhibit
the MDEA hydroxylase; (2) MDZ inhibits competitively the 3OH-MDEA
hydroxylation; and (3) good correlation between the formation rates of 3OH-

247
Metabolism of amiodarone
MDEA and those of 10OH-MDZ is found. However, since other chemicals such as
ANP, cimetidine and quinidine also have the ability to inhibit partially the MDEA
hydroxylase activity, the contribution of other CYPs (1A, 2C, 2D) for the reaction
cannot be completely ruled out. Furthermore, the present results also revealed that
in accordance with previous observations (Wrighton
not a speciŽ c inhibitor of the CYP3A isoform.
1985) in rabbit, TAO is
et al.
Finally, the identity of CYP3A6 involved in the MDEA-3 hydroxylation can be
determined by comparing the catalytic activities of microsomes isolated from RIF-
induced rabbit hepatocytes with those from untreated cells. In rabbit, the gene
coding for this isoform is on chromosome 6 in the q-ter region (Daujat
1991).
et al.
This enzyme, being investigated in the literature under several other names
[P450IIIA4 (Daujat 1987), P-450 LM (Fabre 1988), LM , (Koop
et al.
1981), P-450A7 (Pichard
et al.
1990)], is known to be involved in the
et al.
3c
3c
et al.
metabolism of many compounds such as cyclosporin A (Bertault-Peres
1987),
et al.
1988) and diltiazem
steroid (Waxman
1988), midazolam (Fabre
et al.
et al.
1990).
(Pichard
et al.
It is evident that the results of
experiments must be used with great
in vitro
data. However, the Ž ndings in the present paper
prudence to interpret the
in vivo
are in accordance with the increase of MDEA (and AMI) clearance, when AMI
and RIF were co-administered to a patient (Zarembski 1999). This drug
et al.
interaction may be explained at least in part by the increase of the MDEA
hydroxylase activity of RIF-induced enzyme(s). If so, in humans, the hydroxyla-
tion pathway would also be an important elimination route for AMI derivatives.
In conclusion, in rabbit the CYP3A6 isoform is mainly involved in the
hydroxylation of MDEA. However, the contribution of other CYPs such as 1A,
2C and 2D to the reaction cannot be completely ruled out.
Acknowledgements
P. K. thanks the Cardiovascular Therapy Research Foundation, Department of
Internal Medicine, University Hospital Zurich, for Ž nancial support. The authors
thank Dr P. Gallier, Laboratoire de Pharmacologie-Toxicologie INRA, Toulouse,
for supplying the rifampicin-treated liver microsomes and Dr H. Cristina,
Institute for Laboratory Animal Science, University of Zurich, for supplying
the rabbit liver. The authors also thank Dr Aileen McAinsh, Nashville, TN, for
reviewing the manuscript. This publication represents, in part, the requirements
of P. K.’s PhD thesis at the Swiss Federal Institute of Technology Zurich
(ETHZ).
References
and
1995,
Chenery, R. J.,
Baldwin, S., Bloomer, J. C., Smith, G. J., Ayrton, A. D., Clarke, S. E.
Ketoconazole and sulphaphenazole as the respective selective inhibitors of P450 3A and 2C9.
, 25, 261-270.
Xenobiotica
and
Analytical Biochemistry
1976, Assay of protein in the presence of interfering materials.
Bensadoun, A.
Weinstein, D.,
, 70, 241-250.
Bertault Peres, I., Bonls, C., Fabre, G., Just, S., Cano, J. P.
cyclosporin A. II. Implication of macrolide antibiotic inducible cytochrome P-450 3c from rabbit
liver microsomes. , 15, 391-398.
-
and
1987, Metabolism of
Maurel, P.,
Drug Metabolism and Disposition

248
Metabolism of amiodarone
and
1991,
Maurel, P.,
Daujat, M., Clair, P., Astier, C., Fabre, I., Pineau, T., Yerle, M., Gellin, J.
Induction, regulation and messenger half-life of cytochromes P450 IA1, IA2 and IIIA6 in
primary cultures of rabbit hepatocytes. , 200, 501-510.
European Journal Pharmacology
Daujat, M., Pichard, L., Dalet, C., Larroque, C., Bonls, C., Pompon, D., Li, D., Guzelian, P. S.
1987, Expression of Ž ve forms of microsomal cytochrome P450 in primary cultures
and
Maurel, P.,
of rabbit hepatocytes treated with various classes of inducers.
3597-3606.
, 36,
Biochemical Pharmacology
and
1987, Enzymatic basis of the
Meyer, U. A.,
Dayer, P., Kronbach, T., Eichelbaum, M., Covo, J.
debrisoquine/sparteine-type genetic polymorphism of drug oxidation. Characterization of
bufuralol 10-hydroxylation in liver microsomes of
phenotyped carriers of the genetoc
in vivo
, 36, 4145-4152.
deŽ ciency.
Biochemical Pharmacology
Fabre, G., CrevatPisano, P., Dragna, S., Covo, J., Barra, Y.
the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by
microsomal fractions prepared from rabbit liver. , 37, 1947-1953.
and
1988, Involvement of
Cano, J. P.,
Biochemical Pharmacology
and 1993, Evidence for CYP3A-
mediated deethylation of amiodarone in human liver microsomal fractions.
-
Fabre, G., Julian, B., Sant Aubert, B., Joyeux, H.
Berger, Y.,
N-
Drug Metabolism
, 21, 978-985.
and Disposition
-
and
1986, Oxidation of quinidine by human liver
Blair, I. A.,
Guengerich, F. P., Mueller Enoch, D.
cytochrome P-450.
, 30, 287-295.
Molecular Pharmacology
and
2001,
Follath, F.,
Ha, H. R., Bigler, L., Binder, M., Kozlik, P., Hesse, M., Altorfer, H. R.
Metabolism of amiodarone (Part I): identiŽ cation of a new hydroxylated metabolite of
amiodarone. , 29, 152-158.
Drug Metabolism and Disposition
and
1996, Interaction between
, 26, 533-539.
Ha, H. R., Candinas, R., Stieger, B., Meyer, U. A.
Follath, F.,
amiodarone and lidocaine.
Journal of Cardiovascular Pharmacology
and
2000, Metabolism of amiodarone
Ha, H. R., Kozlik, P., Stieger, B., Bigler, L.
Follath, F.,
(Part II): high-performance liquid chromatographic assay for mono- desethylamiodarone
N-
hydroxylation in liver microsomes.
press, April 2001).
(in
Journal of Chromatography B, Biomedical Application
and
1993, Determination of midazolam
Vonderschmitt, D. J.,
Ha, H. R., Rentsch, K. M., Kneer, J.
and its -hydroxy metabolite in human plasma and urine by high-performance liquid
¬
chromatography.
, 15, 338-343.
Therapeutic Drug Monitoring
Ha, H. R., Wyss, P., Stieger, B., Meyer, U. A.
and
1992, Amiodarone metabolism in
Follath, F.,
human liver microsomes.
and
chronic administration in rabbits.
, 6, A1845 (abstract).
FASEB Journal
1985, Tissue uptake and metabolism of amiodarone after
Kannan, R., Miller, S.
Singh, B. N.,
Drug Metabolism and Disposition
1981, Properties of electrophoretically homogeneous
, 13, 646-650.
and
Koop, D. R., Persson, A. V.
Coon, M. J.,
constitutive forms of liver microsomal cytochrome P-450.
10704-10711.
, 256,
Journal of Biological Chemistry
and
1981, Isolation and characterization of rough
Meyer, U. A.,
Meier, P. J., Spycher, M. A.
endoplasmic reticulum associated with mitochondria from normal rat liver.
, 46, 283-297.
Biophysica Acta
Biochemica et
-
and
1990,
Maurel, P.,
Pichard, L., Gillet, G., Fabre, I., Dalet Beluche, I., Bonls, C., Thenot, J. P.
IdentiŽ cation of the rabbit and human cytochromes P-450IIIA4 as the major enzymes involved
in the
Plomp, T. A.,
demethylation of diltiazem.
, 18, 711-719.
Drug Metabolism and Disposition
N-
1991, Analytical proŽ les of amiodarone. In K. Florey (ed.),
Analytical ProŽ les of Drug
, vol. 20 (San Diego: Academic Press), pp. 2-120.
Substances
and
1969, High-performance liquid
Holt, D. W.,
Storey, G. C. A., Adams, P. C., Campbell, R. W. F.
chromatographic measurement of amiodarone and desethylamiodarone in small tissue samples
after enzymatic digestion. , 177, 340-352.
Journal of Clinical and Pathology
von Moltke, L. L., Greeblatt, D. J., Schmider, J., Duan, S. X., Wright, C. E., Harmatz, J. S.
1996, Midazolam hydroxylation by human microsomes in vitro: inhibition by
and
Shader, R. I.,
 uoxetine, nor uoxetine and by azole antifungal agents.
, 36,
Journal of Clinical Pharmacology
783-791.
and
1988, Human liver microsomal
Lapenson, D. P.,
Waxman, D. J., Attisano, C., Guenguerich, F. P.
steroid metabolism: identiŽ cation of the major microsomal steroid hormone 6-hydroxylase
cytochrome P450 enzyme. , 263, 424-436.
Archives of Biochemistry and Biophysics
and
1985,
Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., Young, B.
IdentiŽ cation of the cytochrome P-450 induced by macrolide antibiotic in rat liver as the
glucocorticoid responsive cytochrome P-450p. , 24, 2171-2178.
Guzelian, P. S.,
Biochemistry
Zarembski, D. G., Fischer, S. A., Santucci, P. A., Porter, M. T., Costanzo, M. R.
1999, Impact of rifampicin on serum amiodarone concentrations in a patient with
and
Trohman,
R. G.,
congenital heart disease.
, 19, 249-251.
Pharmacotherapy

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