Download in vitro biotransformation of sildenafil (viagra) in the male rat: the role

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DRUG METABOLISM AND DISPOSITION
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
DMD 30:655–657, 2002
Vol. 30, No. 6
653/983589
Printed in U.S.A.
IN VITRO BIOTRANSFORMATION OF SILDENAFIL (VIAGRA) IN THE MALE RAT:
THE ROLE OF CYP2C11
JILL S. WARRINGTON, LISA L. VON MOLTKE, RICHARD I. SHADER,
AND
DAVID J. GREENBLATT
From the Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine and Tufts-New England Medical
Center, Boston, MA
(Received December 13, 2001; accepted February 26, 2002)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
2B1/2, 2C11, 2E1, and 3A1/2) revealed that metabolite formation
was inhibited only by an antirat CYP2C11 antibody. Incubation of
sildenafil with a cDNA-expressed CYP2C11 produced 10-fold
higher levels of UK-103,320 than other P450s (CYP1A1, 1A2, 2B1,
2C6, 2C12, 2C13, 2E1, 3A1, and 3A2). Thus CYP2C11 contributes in
a major way to the metabolism of sildenafil in the male rat. P450
isoforms mediating sildenafil biotransformation differ substantially
between humans and the male rat, thereby limiting the applicability
of this species as a model for sildenafil metabolism and drug
interactions in humans.
Sildenafil (Viagra) is a cGMP phosphodiesterase type 5 inhibitor
used in the treatment of erectile dysfunction. Pharmacokinetic studies
of sildenafil demonstrate similarities between the rat and human in
metabolite formation in vivo (Walker et al., 1999). Both species
produce five principal metabolites and form UK-103,320 as the primary circulating metabolite. However, plasma clearance in the male
rat is 8 times greater than that observed in male volunteers. Reflecting
the high clearance of the parent drug, the male rat has a greater
relative exposure to the pharmacologically active metabolite, UK103,320, than to sildenafil. In humans, however, sildenafil is the
principal pharmacologically active compound (Walker et al., 1999).
Factors that contribute to differences in the clearance between the
male rat and human have not yet been identified.
A recent in vitro study using human liver microsomes demonstrates
that 79% of sildenafil biotransformation to UK-103,320 is attributable
to CYP3A (Warrington et al., 2000). A small percentage of metabolite
formation is due to CYP2C9 (20%), CYP2D6 and CYP2C19 (collectively less than 2%) activity. These data are consistent with the
findings of Hyland et al. (2001).
To determine whether differences in clearance between the rat and
human are due to differences in metabolic pathways, we sought to
identify which P4501 isoforms mediate UK-103,320 formation in
male rat liver microsomes.
This work was supported in part by Grants MH-58435, DA-05258, DA13209, DK-58496, DA-13834, MH-34223, MH-01237, and RR-00054 from the
Department of Health and Human Services. Experimental animals were provided by the National Institute on Aging through the Pilot/Dissertation Research Support Program.
1
Abbreviations used are: P450, cytochrome P450; HPLC, high-performance
liquid chromatography.
Address correspondence to: David J. Greenblatt, M.D., Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine,
136 Harrison Avenue, Boston, MA 02111. E-mail: [email protected]
Materials and Methods
Microsomes were prepared from the livers of 10 male Fischer 344 rats (ages
3– 4 months) by differential centrifugation, as previously described (Warrington et al., 2000). Hepatic microsomes from 10 individual rat preparations
were incubated with increasing concentrations of sildenafil (0 – 63 ␮M) in the
presence of an NADPH-regenerating cofactor system. Samples were incubated
for 20 min at 37°C and terminated with the addition of acetonitrile and
exposure to ice. Buspirone (4 ␮M) was added as an internal standard and
samples were centrifuged at 14,000 rpm for 10 min to remove the protein. The
supernatants were transferred to autosampling vials for high-performance
liquid chromatography (HPLC) analysis.
Samples were analyzed by HPLC, as previously described (Warrington et
al., 2000). These conditions were found to be within the linear range with
respect to time and protein for rat hepatic microsomes (data not shown).
Negative controls without protein and cofactor demonstrated no metabolite
formation (data not shown). Since reference samples of UK-103,320 were
unavailable, identification of UK-103,320 was based on chromatographic
retention times, as previously described (Warrington et al., 2000).
Formation of UK-103,320 in the male rat was consistent with a singleenzyme Michaelis-Menten kinetic model. The following equation was fitted to
UK-103,320 data points by nonlinear regression:
V⫽
V max 䡠 S
Km ⫹ S
in which V is the rate of UK-103,320 formation and S represents the concentration of the substrate, sildenafil (Venkatakrishnan et al., 2001). Since pure
samples of UK-103,320 were not available, absolute values of Vmax could not
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To assess the suitability of the male rat model for human studies
on sildenafil metabolism, we examined the biotransformation of
sildenafil in male rat liver microsomes and identified the role of
specific cytochrome P450s (P450) using inhibitory antibodies and
cDNA-expressed P450s. Rates of formation of the major circulating metabolite of sildenafil, UK-103,320, were 11-fold greater in the
male rat than in human liver microsomes at 36 ␮M sildenafil,
whereas substrate concentration corresponding to 50% Vmax (Km
values) were 2.9-fold lower in the male rat. Although sildenafil is
largely metabolized by CYP3A isoforms in humans, coincubation of
rat liver microsomes with immunoinhibitory antibodies (CYP1A1/2,
656
WARRINGTON ET AL.
TABLE 1
Comparison of rat to human Km values for the formation of UK-103,320
Species
Human
mean ⫾ S.E.; n ⫽ 4 livers
mean ⫾ S.D.; n ⫽ 3 livers
Rat
mean ⫾ S.E.; n ⫽ 4 livers
Km values
Reference
14.4 ⫾ 2.0 ␮M
6 ⫾ 3 ␮M for Km1a
22 ⫾ 9 ␮M for Km2b
Warrington et al., 2000
Hyland et al., 2001
5.0 ⫾ 0.2 ␮M
a
Km1 refers to the high-affinity component.
b
Km2 describes the low-affinity component.
Sildenafil biotransformation in 10 rat livers (mean ⫾ S.E.) is indicated by dashed
lines and square symbols. Denoted by solid lines and circle symbols, sildenafil
biotransformation in a representative human liver is provided for comparison
(Warrington et al., 2000).
be calculated. Therefore, the amount of UK-103,320 formed was expressed in
arbitrary units reflecting the ratio of the chromatographic peak height of
UK-103,320 relative to that of the internal standard.
Using five specific immunoinhibitory antibodies, we assessed the involvement of CYP1A1/2, CYP2B1/2, CYP2C11, CYP2E1, and CYP3A1/2 in the
biotransformation of sildenafil in male rat hepatic microsomes. Hepatic microsomes from 10 rats were pooled, and samples of pooled microsomes were
incubated in triplicate. At 37°C, sildenafil (36 ␮M) was incubated for 30 min
with 10 ␮g of microsomes and either 50 mM potassium phosphate buffer, 100
␮g of control serum (goat or rabbit), or 100 ␮g of each polyclonal antirat P450
antibody (Gentest, Woburn, MA). A NADPH-regenerating cofactor system
was then added, and samples were incubated for an additional 20 min, as
described above. The samples were then subjected to HPLC analysis, as
previously described (Warrington et al., 2000). The degree of inhibition was
determined by the peak height ratio of the antibody-containing sample divided
by the peak height ratio of the appropriate serum control.
The role of specific P450s in sildenafil biotransformation was further
examined using a screen of rat cDNA-expressed P450s. Sildenafil (15 and 132
␮M) was incubated with either a rat cDNA-expressed P450 (CYP1A1, 1A2,
2B1, 2C6, 2C11, 2C12, 2C13, 2E1, 3A1, or 3A2; Gentest) or a vector control
(from an AHH-1 TK⫹/⫺ human lymphoblastoid or Hi5 insect cell line).
Positive controls with either human or rat liver microsomes were incubated in
parallel. Negative controls were also performed in which either the protein,
cofactor, or sildenafil was omitted from the incubation. As recommended by
the manufacturer, P450s expressed in an insect cell line (Supersomes; Gentest)
were incubated at 50 pmol of cytochrome P450/ml, whereas enzymes expressed in lymphoblastoid cells (CYP2E1) were incubated at 1 mg/ml. Samples were incubated for 30 min and performed in duplicate.
The results from the immunoinhibitory study were analyzed by Student’s t
test, in which samples with an immunoinhibitory antibody were compared with
the appropriate serum control.
Results/Discussion
Rat liver microsomes formed UK-103,320 at higher rates than
human liver microsomes under the same conditions (Fig. 1). At 36
␮M sildenafil, UK-103,320 formation rate was 11-fold greater in the
rat than in the human. In addition, reactions in the rat exhibited lower
substrate concentration corresponding to 50% Vmax (Km values), reflecting a higher affinity of the rat enzyme for sildenafil biotransformation via the UK-103,320 metabolite pathway (Table 1). Although
FIG. 2. Immunoinhibition of sildenafil biotransformation in male rat hepatic
microsomes using a screen of antirat P450 antibodies.
Sildenafil (36 ␮M) was incubated with either antirat CYP3A1/2 (3A), CYP2E1
(2E), CYP1A1/2 (1A), CYP2B1/2 (2B), CYP2C11 (2C), 50 mM potassium phosphate buffer (B), or goat or rabbit serum controls. Histograms represent UK-103,320
formation rate relative to serum controls (mean ⫾ S.E.) for microsomal samples
pooled from 10 rats. Samples were performed in triplicate. 多, p ⬍ 0.05 and 多多, p ⬍
0.001, as evaluated by Student’s t test.
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FIG. 1. Comparison of sildenafil biotransformation with its major circulating
metabolite, UK-103,320, in human and rat liver microsomes.
samples from the rat were compared to previously published human
studies (Fig. 1; Table 1), comparable results were obtained when rat
and human experiments were performed in parallel (data not shown).
These in vitro experiments correlate well with in vivo human and rat
studies (Walker et al., 1999). Studies with both inhibitory antibodies
and cDNA-expressed enzymes identified the role of CYP2C11 in
UK-103,320 formation in the rat liver (Figs. 2 and 3). As shown in
Fig. 2, the antirat CYP2C11 antibody inhibited UK-103,320 formation
by 87%, while the remaining antibodies (antirat CYP1A1/2, 2B1/2,
2E1, and 3A1/2) did not cause inhibition. When two concentrations of
sildenafil (15 and 132 ␮M) were incubated with rat cDNA-expressed
P450s (CYP1A1, 1A2, 2B1, 2C6, 2C11, 2C12, 2C13, 2E1, 3A1, and
3A2), only CYP2C11 resulted in substantial UK-103,320 formation
(Fig. 3). Incubation with CYP3A1 and 3A2 resulted in 6 and 10% of
the metabolite formation observed with CYP2C11, respectively.
The differences in clearance between species may be attributable, in
part, to differences in the affinities of the enzymes controlling the
metabolic pathways. While UK-103,320 formation is largely mediated by CYP3A in humans, it appears to be largely dependent on
CYP2C11 in the male rat.
Many CYP3A substrates in humans are likely to be largely CYP3A
substrates in rats such as cyclosporine (Smith, 1991). However, several CYP3A-specific reactions in humans such as nifedipine oxidation
and lidocaine N-deethylation demonstrate overlapping substrate specificity with CYP2C in the rat (Smith, 1991). It has been suggested
SILDENAFIL BIOTRANSFORMATION IN THE MALE RAT
657
References
FIG. 3. Sildenafil biotransformation in rat cDNA-expressed cytochromes.
Sildenafil at 15 ␮M (A) and 132 ␮M (B) was incubated with either a rat
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Since sildenafil appears to be predominantly metabolized by
CYP2C11, its N-demethylation to UK-103,320 may serve as a useful
index for analysis of CYP2C11 activity. As CYP2C11 represents one
of the most abundantly expressed P450s in the rat liver (Guengerich
et al., 1982), a specific index reaction may be particularly important.
The involvement of CYP2C11 in UK-103,320 formation is likely to
explain differences in clearance found between the male and female
rat. Plasma clearance in the female rat is comparable to that found in
the human and, hence, is significantly lower than clearance in the
male rat (Walker et al., 1999). While CYP2C12 is expressed in
females, CYP2C11 and CYP2C13 are male-specific P450s (Imaoka et
al., 1991). Thus, if CYP2C11 is the primary enzyme involved in
sildenafil biotransformation in male rat liver microsomes and this
enzyme is largely absent in the female, the metabolic pathways in the
female rat are likely to be different.
These findings have two important implications. First, it is possible
that sildenafil may be a useful CYP2C11 index substrate in the male
rat. Second, this study, along with several others (Ring et al., 1994;
Eagling et al., 1998; Perloff et al., 2000), emphasizes that caution is
required in extrapolating rat metabolic data to humans.