Metabolism of digoxin and digoxigenin digitoxosides in rat liver microsomes: Involvement of cytochrome P4503A

Article abstract of DOI:10.1080/004982599238722

1. The sequential metabolism of digoxin (Dg3) to digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-digitoxoside (Dg1) and digoxigenin (Dg0) was investigated in rat liver microsomes. 2. Kinetic studies produced results consistent with a single enzyme mechanism describing the successive oxidative cleavages. Formation of Dg2 was catalysed with mean (± SD) K(m) and V(max) of 125 ± 22 μM and 362 ± 37 pmol/min/mg protein, respectively. The corresponding values for the formation of Dg1 were 61 ± 5 μM and 7 ± 1 pmol/min/mg protein. Dg0 formation was catalysed with the apparent values of 30 ± 9 μM and 310 ± 30 pmol/min/mg protein. 3. Chemical inhibition of cytochrome P450 (CYP) 3A subfamily with ketoconazole and triacetyoleandomycin decreased the formation of Dg2 and Dg1 by up to 90%. Antibodies specific to rat CYP3A2 lowered the rate of oxidative cleavage of Dg3 and Dg2 by up to 85%. Inhibition of CYP2E1, CYP2C subfamily and CYP1A2 by chemical and immunoinhibition did not affect initial rates of metabolism of Dg3 and Dg2. In contrast, Dg1 metabolism was not affected by triacetyloleandomycin as well as by antibodies to CYP3A2, CYP2C11, CYP2E1, CYP2B1/2B2 and CYP1A2. It was however inhibited by > 80% by gestodene and 17α-ethynylestradiol (selective inhibitors of human CYP3A). 4. Collectively, these data support the involvement of CYP3A in the cleavage of Dg3 and Dg2 in rat liver microsomes. The enzyme-metabolizing Dg1 remains to be identified.

Full text of DOI:10.1080/004982599238722

xenobiotica , 1999, vol. 29, no. 2, 171±185
M etabolism of digoxin and digoxigenin digitoxosides
in rat liver m icrosomes : involvement of cytochrom e
P4503A
LAURENT SALPHATI and LESLIE Z. BENET*
Department of Biopharmaceutical Sciences, School of Pharmacy, University of
California, San Francisco, CA 94143±0446, USA
Received 16 May 1998
1. The sequential metabolism of digoxin (Dg3) to digoxigenin bis-digitoxoside (Dg2),
digoxigenin mono-digitoxoside (Dg1) and digoxigenin (Dg0) was investigated in rat liver
microsomes.
2. Kinetic studies produced results consistent with
describing the successive oxidative cleavages. Formation of Dg2 was catalysed with mean
lm
a single enzyme mechanism
(‰ SD) K and V
corresponding values for the formation of Dg1 were 61‰ 5
of 125‰ 22
and 362‰ 37 pmol min mg protein, respectively. The
}
}
m
max
lm
and 7‰ 1 pmol min mg
}
}
lm
protein. Dg0 formation was catalysed with the apparent values of 30‰ 9
310‰ 30 pmol min mg protein.
and
}
}
3. Chemical inhibition of cytochrome P450 (CYP) 3A subfamily with ketoconazole and
triacetyoleandomycin decreased the formation of Dg2 and Dg1 by up to 90 %. Antibodies
speci®c to rat CYP3A2 lowered the rate of oxidative cleavage of Dg3 and Dg2 by up to
85%. Inhibition of CYP2E1, CYP2C subfamily and CYP1A2 by chemical and immuno-
inhibition did not aåect initial rates of metabolism of Dg3 and Dg2. In contrast, Dg1
metabolism was not aåected by triacetyloleandomycin as well as by antibodies to CYP3A2,
"
CYP2C11, CYP2E1, CYP2B1 2B2 and CYP1A2. It was however inhibited by
a
gestodene and 17 -ethynylestradiol (selective inhibitors of human CYP3A).
80 % by
}
4. Collectively, these data support the involvement of CYP3A in the cleavage of Dg3
and Dg2 in rat liver microsomes. The enzyme-metabolizing Dg1 remains to be identi®ed.
Introduction
Digoxin is a cardiac glycoside that has been used clinically for
"
200 years
(Antman and Smith 1985, Heller 1990). It is currently utilized as an antiarrhythmic
and is the most commonly prescribed drug in congestive heart failure (Kelly and
Smith 1996).
In man, Dg3 is mostly excreted unchanged by the kidneys (Rodin and Johnson
"
1988). In contrast, in rat, 60 % of a dose is metabolized, while the renally excreted
C
drug accounts for
30 % (Harrison and Gibaldi 1976). Dg3 biotransformations
involve the stepwise cleavage of the digitoxoses to form (®gure 1) the digoxigenin
bis- and mono-digitoxoside (Dg2 and Dg1, respectively) and the aglycone
digoxigenin (Dg0) before conjugation and elimination (Harrison and Gibaldi 1976).
The sequential release of each sugar residue has been shown to be CYP-mediated
(Schmoldt and Ahsendorf 1980). However, kinetic parameters have not been
determined and the isoforms responsible for the metabolism of Dg3, Dg2 and Dg1
have not been identi®ed. In the present study, we investigated the three successive
oxidative cleavages involved in Dg3 metabolism in rat liver microsomes. Kinetic
* Author for correspondence.
Xenobiotica ISSN 0049±8254 print}ISSN 1366±5928 online ’ 1999 Taylor & Francis Ltd
http:}}www.tandf.co.uk}JNLS}xen.htm
http:}}www.taylorandfrancis.com}JNLS}xen.htm

172
L. Salphati and L. Z. Benet
Figure 1. Chemical structure of digoxin and its metabolites. Digoxigenin (Dg0), digoxigenin mono-
digitoxoside (Dg1), digoxigenin bis-digitoxoside (Dg2) and digoxin (Dg3).
parameters were determined and chemical and immuno-inhibition were used to
identify the enzyme responsible for the metabolism of this drug.
M aterials and m ethods
Chemicals
Digoxin (Dg3), NADPH, isocitrate dehydrogenase, sodium phosphate, sulphinpyrazone, quinidine,
b
diethyldithiocarbamate (DDC), triacetyloleandomycin (TAO), cortisone, corticosterone, 20 -dihydro-
cortisol, 17 -ethynylestradiol (17EE), chlorzoxazone (CLZ) and orphenadrine were purchased from
a
the Sigma Chemical Co. (St Louis, MO, USA). Digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-
digitoxoside (Dg1) and digoxigenin (Dg0) were obtained from Crescent Chemical Co. (Hauppauge, NY,
USA). Dg3 and metabolites were further puri®ed by HPLC to at least 99 %. Ketoconazole (KCZ) and
furafylline were purchased from the US Pharmacopeia (Rockville, MD, USA) and Research Biochemical
International (Natick, MA, USA), respectively. Antibodies to rat CYP were obtained from Gentest
Corp. (Woburn, MA, USA) and gestodene was kindly supplied by Schering, AG (Berlin, Germany).
HPLC-grade methanol and acetonitrile were from Fisher Scienti®c (Pittsburgh, PA, USA).
Animals and preparation of microsomes
Adult male and female Sprague-Dawley rats (280±300 g) were obtained from Charles River
Laboratories (Willmington, MA, USA) and maintained on a 12-h light dark cycle. Rats were fed
}
standard laboratory chow (Formulab 5008) obtained from Deans (Belmont, CA, USA) ad libitum.
Microsomes were prepared from pooled (four to six) fresh livers by homogenization and diåerential
centrifugations following established protocols (Guengerich 1989). All procedures were carried out at
–
m
m
EDTA and 20 % (w v) glycerol. Protein concentrations were determined using the Bio-Rad protein
4 ÊC. The microsomes were stored at 80 ÊC until use in 10 m Tris-HCl (pH 7.4) containing 1 m
}
assay kit (Bio-Rad, Hercules, CA, USA) with BSA as standard.
Incubation conditions
Incubations (®nal incubation volume of 1 ml), containing Dg3, Dg2 or Dg1 as substrates, were
m
m
conducted at 37 ÊC with 0.75 mg protein ml in K HPO (0.1 , pH 7.4), MgCl (5 m ), isocitrate
}
#
%
#
(5 m ) and isocitrate dehydrogenase (10 u ml). Substrates and inhibitors were added in methanol (®nal
m
}
C
methanol concentration :
were chosen (50, 30 and 15
1 %). Substrate concentrations for the incubations of Dg3, Dg2 and Dg1
lm
respectively) based on preliminary estimates of K at about half of the K
.
m
m

Digoxin metabolism by rat CYP3A
173
HPLC Retention time (min)
Figure 2. Separation of Dg3 and metabolites by reverse phase HPLC. (A) Representative analysis
after Dg3 incubation. Internal standard (IS) : corticosterone. Mobile phase : 26 % acetonitrile in
water. (B) Representative chromatogram after incubation of Dg2. IS : cortisone. Mobile phase:
b
23% acetonitrile in water. (C) Representative chromatogram after Dg1 incubation. IS : 20 -
dihydrocortisol. Mobile phase : 20 % acetonitrile in water.
m
Reactions were initiated by adding NADPH (to 1 m ) after a 5-min preincubation and quenched after
15 min by addition of 5 ml methylene chloride containing the internal standard. The samples were
vortexed, centrifuged (2000g for 15 min) and an aliquot of the organic phase (3 ml) was evaporated under
l
l
nitrogen. The residue was dissolved in 200 l HPLC mobile phase and 100 l were analysed by HPLC.
For mechanism-based inhibitors, catalysis-dependent inactivation was initiated by the addition of
NADPH. After 30 min, substrate and NADPH were added and the incubation was carried out for 15

174
L. Salphati and L. Z. Benet
Table 1. Kinetic parameters for the formation of Dg2, Dg1 and Dg0 in male rat liver microsomes.
lm
l
( l min mg
m
Reaction
V
(pmol min mg protein)
}
K
(
)
V
K
}
}
}
}
max
m
max
protein)
Dg3 ! Dg2
Dg2 ! Dg1
Dg1 ! Dg0
362‰ 37
7‰ 1
310‰ 30
125‰ 22
61‰ 5
30‰ 9
2.92‰ 0.42
0.11‰ 0.03
10.95‰ 2.99
Data are the mean‰ SD of three independent experiments from six pooled livers.
Figure 3. Metabolism of Dg3, Dg2 and Dg1 in female rat liver microsomes as compared with the male.
Incubations were conducted as described in the Materials and methods. The concentrations of
lm
Dg3, Dg2 and Dg1 were 50, 30 and 15
respectively. Data represent the ratio of the rate of
metabolism in females males and are reported as the average of duplicate determinations‰ half
}
the range. *No metabolite detected.
min. The reactions were stopped and the samples processed as described above. In control incubations,
the same volume of methanol was added and the preincubation and incubation were conducted as in the
presence of inhibitors.
In immuno-inhibition experiments, microsomes were incubated for 30 min at room temperature with
various amounts of antibodies speci®c to rat CYP. Thereafter, substrates and NADPH were added and
the reactions conducted as described above. In control incubations, the same amounts of preimmune sera
were added and the preincubation and incubation were conducted as described for the speci®c antibodies.
Assay for digoxin and its metabolites
l
Digoxin, its metabolites and the internal standards were resolved on a Beckman C (5 m3 4.6 mm
)
i.d. 3 150 mm) column with a Shimadzu SCL-10A system controller and a LC-10AD pump equipped
with an SIL-10A autoinjector. The absorbance was monitored at 220 nm with a UV-VIS spectro-
photometric detector SPD-10AV and the compounds were quanti®ed by comparison of the ratio of
their peak area (measured with a Hewlett-Packard 3392A integrator) to those of the internal standards.
C
Digoxin and metabolites were eluted isocratically over 20 min at room temperature. The ¯ow rate was
1 ml min and the mobile phases used to analyse the products of the incubations of Dg3, Dg2 and Dg1
}

Digoxin metabolism by rat CYP3A
175
were 26, 23 and 20 % acetonitrile in water, respectively. The internal standards used in Dg3, Dg2 and
b
Dg1 incubations were corticosterone, cortisone and 20 -dihydrocortisol, respectively. The limits of
detection for Dg2, Dg1 and Dg0 were 20, 16 and 12 ng respectively.
!
Data analysis
lm
Metabolite formation was characterized using precursor concentrations up to 400
and kinetic
parameters were estimated by nonlinear regression analysis (Minim 3.0.8) assuming single enzyme
Michaelis-Menten kinetics. No evidence of biphasic kinetics was observed in Eadie-Hofstee plots.
Michaelis-Menten parameters are presented as mean‰ SD of three independent experiments whereas
the other results are reported as average‰ half the range of duplicate experiments.
Results
Each step of the sequential metabolism of Dg3 was investigated in separate
incubations using authentic standards of each metabolite. Figure 2 shows three
representative chromatograms obtained from microsomal incubations of Dg3 (A),
Dg2 (B) and Dg1 (C). Preliminary experiments showed that metabolite formation
from each substrate (Dg3, Dg2 and Dg1) was linear up to 1.5 mg protein ml and 20
}
min. Thus, subsequent incubations were performed for 15 min with a protein
concentration of 0.75 mg ml.
}
Table 1 summarizes the Michaelis-Menten parameter estimates for the oxidative
cleavage of Dg3, Dg2 and Dg1. The mean apparent K and V
for the formation
m
max
of Dg2 were 125‰ 22 lm and 362‰ 37 pmol min mg protein, respectively. Par-
}
}
ameter estimates were 61‰ 5 lm and 7‰ 1 pmol min mg protein for the formation
}
}
of Dg1 and 30‰ 9 lm and 310‰ 30 pmol min mg protein for the formation of Dg0.
}
max
}
Calculation of the intrinsic clearances (V
K ) for each step of the metabolism of
C
}
m
Dg3 revealed that the rate of elimination of Dg2 was
the rates of elimination of Dg3 and Dg1, respectively.
30- and 100-fold lower than
The metabolism of Dg3, Dg2 and Dg1 was also evaluated in female rat liver
microsomes (®gure 3). Dg1 was metabolized to the same extent in microsomes from
the male and female whereas Dg3 metabolism in females represented only 8 % of
that observed in the male. No metabolite of Dg2 could be detected following
incubation in female rat liver microsomes.
Eåects of chemical inhibitors on metabolite formation
We measured the eåects of competitive (KCZ, sulphinpyrazone, quinidine and
CLZ) and mechanism-based (DDC, furafylline and TAO) inhibitors on the
metabolism of Dg3, Dg2 and Dg1 in male rat liver microsomes. KCZ, a selective
inhibitor of CYP3A, caused a 90 % inhibition of Dg3 metabolism to Dg2 when used
at 0.5 lm (®gure 4A) whereas it reduced Dg2 oxidative cleavage to Dg1 by 70 %
when incubated at 1 lm (®gure 5A). Higher concentrations of KCZ reduced the
formation of Dg1 to non-detectable levels. In contrast, 10 lm was necessary to
C
inhibit Dg1 metabolism by
80 % (®gure 6A). Quinidine (an inhibitor of CYP2D)
and sulphinpyrazone (an inhibitor of CYP2C) up to 100 lm did not aåect Dg3, Dg2
and Dg1 metabolism (®gures 4B, 5B and 6B). CLZ (an inhibitor of CYP2E1)
showed no inhibition of Dg3 and Dg1 metabolism at the concentrations tested
(®gures 4B and 6B). Its eåects on Dg2 metabolism could not be measured because
6-OH CLZ (the metabolite of CLZ) co-eluted with Dg1 under the HPLC conditions
used. The importance of CYP2E1 in Dg2 metabolism was, however, investigated
using other types of inhibition (see below).

176
L. Salphati and L. Z. Benet
Figure 4. For legend see opposite.

Digoxin metabolism by rat CYP3A
177
Figure 4. Inhibition of Dg2 formation by chemical and immuno-inhibition. Chemical inhibitors and
antibodies were incubated with male rat liver microsomes, as described in the Materials and
lm
methods. Dg3 concentration was 50
. Data represent the average of duplicate determina-
tions‰ half the range. (A) Inhibition of Dg2 formation by ketoconazole; (B) eåects of quinidine
(E ), chlorzoxazone (CLZ) (D ) and sulphinpyrazone ( ) on Dg2 formation ; (C) eåects of the
x
mechanism-based inhibitors diethyldithiocarbamate (DDC), furafylline and triacetyloleando-
mycin (TAO) on Dg2 formation ; (D) immuno-inhibition of Dg2 formation with antibodies to
CYP3A2 (E ), CYP2E1 ( ), CYP1A2 (^) and preimmune sera (D ).
x

178
L. Salphati and L. Z. Benet
Figure 5. For legend see opposite.

Digoxin metabolism by rat CYP3A
179
Figure 5. Inhibition of Dg1 formation by chemical and immuno-inhibition. Chemical inhibitors and
antibodies were incubated with male rat liver microsomes as described in the Materials and
lm
methods. Dg2 concentration was 30
. Data represent the average of duplicate determina-
tions‰ half the range. (A) Inhibition of Dg1 formation by ketoconazole; (B) eåects of quinidine
(E ), and sulphinpyrazone (* ) on Dg1 formation ; (C) eåects of the mechanism-based inhibitors
diethyldithiocarbamate (DDC), furafylline and triacetyloleandomycin (TAO) on Dg1 formation ;
(D) immuno-inhibition of Dg1 formation with antibodies to CYP3A2 (E ), CYP2E1 (D ),
CYP1A2 (^) and preimmune sera ( ).
x

180
L. Salphati and L. Z. Benet
Figure 6. For legend see opposite.

Digoxin metabolism by rat CYP3A
181
Figure 6. Inhibition of Dg0 formation by chemical and immuno-inhibition. Dg1 concentration was
lm
15
. Data represent the average of duplicate determinations‰ half the range. (A) Inhibition of
Dg0 formation by ketoconazole; (B) eåects of quinidine (E ), chlorzoxazone (CLZ) (+ ) and
sulphinpyrazone (D ) on Dg0 formation ; (C) eåects of the mechanism-based inhibitors
diethyldithiocarbamate (DDC), furafylline and triacetyloleandomycin (TAO) on Dg0 formation ;
(D) immuno-inhibition of Dg0 formation with antibodies to CYP3A2 ( ), CYP2E1 (D ),
x
CYP1A2 () ), CYP2C11 (^), CYP2B1 (* ) and preimmune sera (D ).

182
L. Salphati and L. Z. Benet
Figure 7. Inhibition of Dg0 formation in male and female rat liver microsomes. Inhibitors were
preincubated with male and female rat liver microsomes in the presence of NADPH as described
the in Materials and methods. Data represent the average of duplicate determinations‰ half the
range.
TAO, a mechanism-based inhibitor of CYP3A, markedly inhibited Dg3 and
Dg2 metabolism whereas DDC (an inhibitor of CYP2E1) and furafylline (an
inhibitor of CYP1A2) had no signi®cant eåect (®gures 4C and 5C). The minor
inhibition (10±20 %) observed with DDC was likely to be due to a slight inhibition
of CYP3A at these concentrations (Chang et al. 1994, Newton et al. 1995). Dg1
metabolism was not aåected by any of these mechanism-based inhibitors in male rat
liver microsomes (®gure 6C).
Immunoinhibition
C
Antibodies to CYP3A2 strongly inhibited ( 80 %) Dg3 and Dg2 metabolism as
shown in ®gures 4D and 5D. We assessed the eåects of antibodies to CYP2E1 to
verify the involvement of this enzyme, since DDC caused a slight inhibition in our
studies. We also tested antibodies to CYP1A2 because furafylline, although speci®c,
is not a very potent inhibitor of the rat CYP1A2 (Sesardic et al. 1990). These
antibodies, as well as preimmune sera, had no eåect on Dg3 and Dg2 metabolism
(®gures 4D and 5D). Unexpectedly, Dg1 oxidative cleavage to Dg0 was not
decreased by any of the antibodies tested in male rat liver microsomes (®gure 6D).
Dg1 metabolism
Inhibition of Dg1 metabolism was further investigated in both male and female
rat liver microsomes. Figure 7 shows that TAO did not inhibit Dg1 cleavage in the
male or in female. However, gestodene and 17EE, mechanism-based inhibitors of
the human CYP3A4 (Guengerich 1988, 1990), decreased to a great extent Dg1

Digoxin metabolism by rat CYP3A
183
metabolism in hepatic microsomes from both the male and female. Orphenadrine,
which forms a metabolite complex with CYP3A, CYP2C and CYP2B (Roos and
Mahnke 1996), also inhibited Dg1 metabolism in the male and female.
Discussion
In the present study we have investigated the metabolism of digoxin and
digoxigenin digitoxosides in rat liver microsomes. Digoxin is metabolized by
stepwise cleavage of the sugar residues (Harrison and Gibaldi 1976) to form Dg2,
Dg1 and Dg0. Digoxin has been shown to be readily taken up by hepatocytes
(Hedman and Meijer 1998, Olinga 1998). Each step was studied in separate
C
incubations. We observed that the cleavage of Dg2 to Dg1 was much slower ( 20-
fold) than the formation of Dg2 from Dg3. This is consistent with in vivo data
(Klaassen 1974, Wirth and Frolich 1974) showing that Dg2 is the main metabolite
of Dg3 whereas only traces of Dg1 and Dg0 can be found, despite the very high rate
of formation of Dg0. After being formed, Dg2 is most likely conjugated and excreted
at a much faster rate than it can be further metabolized. The cleavage of the terminal
digitoxosyl group from Dg3 and its digitoxosides is thought to involve two steps:
®rst, a dehydrogenation of the axial hydroxyl group from the terminal digitoxose,
which is CYP-mediated, then the terminal dehydro-digitoxosyl is split oå by a non-
enzymatic reaction (Schmoldt and Ahsendorf 1980). The apparent Michaelis-
Menten parameters describe the complete reaction leading from each digitoxose to
the next excretable metabolite (i.e. from Dg3 to Dg2, Dg2 to Dg1 and Dg1 to Dg0).
Consequently, these parameters incorporate the two steps required for the cleavage
of each digitoxose.
Chemical inhibition of CYP3A by TAO and KCZ resulted in signi®cant
inhibition of Dg3 metabolism. This was further con®rmed using speci®c antibodies
to rat CYP3A2. Consistent with these results, incubation with inhibitors and
antibodies directed to other CYP isoforms did not aåect Dg3 metabolism. Dg2
oxidative cleavage was also inhibited by chemical and immuno-inhibition of CYP3A.
Moreover, the absence of signi®cant metabolism of Dg3 and Dg2 in female rat liver
microsomes corroborates the involvement of CYP3A2 in the male. These results
contrast with what is observed in man where Dg3 is mostly excreted unchanged
(Rodin and Johnson 1988) and no metabolism can be detected after incubation of
Dg3 with liver microsomes or hepatocytes (Lacarelle et al. 1991). In addition, when
stepwise cleavage of Dg3 does occur in man, this reaction is mediated by intragastric
hydrolysis and its extent varies depending on gastric pH (Gault et al. 1980, 1981).
The involvement of CYP3A also provides an explanation for the increased
metabolism of Dg3 in rat following chronic administration of PCN or spironolactone
(Klaassen 1974, Wirth and Frolich 1974), which are inducers of this enzyme.
Moreover, digoxin interaction studies performed in rat with amiodarone (Wein-
house et al. 1988) and verapamil (Weinhouse et al. 1985) should be re-evaluated in
terms of a metabolism interaction, since all three drugs are CYP3A substrates
(Wacher et al. 1995). However, the use of rat (all the studies mentioned above used
males) as a model to predict or explain Dg3 drug interactions in man does not seem
appropriate given the diåerences in the handling of this drug between the two
species.
In male rat liver microsomes, Dg1 was metabolized at the highest rate of
elimination as compared with Dg3 and Dg2. Inhibition by KCZ required

184
L. Salphati and L. Z. Benet
concentrations 20 and 10 times higher than those needed to inhibit Dg3 and Dg2,
respectively. In addition, neither the mechanism-based inhibitors initially tested
nor antibodies to CYP3A2, CYP1A2, CYP2B1 2B2, CYP2C11 and CYP2E1 had
}
any eåect on Dg1 oxidative cleavage. Furthermore, contrary to Dg3 and Dg2, Dg1
was metabolized to the same extent in female as in male rat liver microsomes (®gure
3). This is consistent with the absence of inhibition by antibodies to CYP3A2 and
CYP2C11 in the male, assuming that Dg1 is metabolized by the same enzyme in
the male and female. Mechanism-based inhibitors speci®c to human CYP3A4,
gestodene (Guengerich 1990) and 17EE (Guengerich 1988) decreased Dg1 metab-
"
olism by 80 %, whereas TAO, a mechanism-based inhibitor of human CYP3A4
and rat CYP3A1 3A2, had no eåect. The speci®city of gestodene and 17EE in rat
}
has not been assessed ; however, given the marked similarities between rat 3A
enzymes and their human CYP3A4 orthologue, it is likely that these two drugs
inhibit a rat CYP3A isoform. Orphenadrine could also decrease Dg1 metabolism to
!
20 % of control activity in male and female rat liver microsomes. This compound
seems to form a complex with rat CYP2C11, CYP3A and CYP2B (Roos and
Mahnke 1996). The speci®c CYP3A isoform was not identi®ed by these latter
investigators and, in our study, antibodies to CYP2C11 and CYP2B1 B2 did not
}
have any inhibitory eåect on Dg1 oxidative cleavage.
Taken together, these data seem to implicate a CYP3A isoform expressed in the
male and female rat in the metabolism of Dg1. Five isoforms of CYP3A have been
characterized in rat: CYP3A2 is not expressed in adult female rat, CYP3A1 is
inducible in the male and female (Correia 1995) and CYP3A9, CYP3A18 and
CYP3A23 are present at the mRNA or protein levels in the male and female rat liver
(Mahnke et al. 1997, Wang and Strobel 1997). Among these last three isoforms, only
CYP3A9 has been studied functionally and seems to possess common substrate
preferences with CYP3A2 (Wang and Strobel 1997). Although the data presented
here do not allow a conclusion to be made on the enzyme responsible for Dg1
metabolism, the diåerences in the inhibitory patterns of TAO on one hand, and
gestodene and 17EE on the other, may be useful to discriminate functionally these
CYP3A isoforms.
In summary, these results demonstrate that CYP3A, most likely CYP3A2, is the
primary enzyme responsible for Dg3 and Dg2 metabolism in rat. Studies are
currently ongoing in our laboratory to identify the CYP metabolizing Dg1.
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