![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Drug Metabolism, Merck & Co., Rahway, New Jersey (D.C.E., R.E.); Department of Medicinal Chemistry, Merck Sharp & Dohme, Harlow, Essex, United Kingdom (D.O'C.); BIBRA International Ltd., Carshalton, Surrey, and Centre for Toxicology, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, United Kingdom (B.G.L.); Department WS3C-90, Merck & Co., Whitehouse Station, New Jersey (C.A.); and Department of Pharmacology, Merck & Co., Westpoint, Pennsylvania (R.H.)
(Received December 23, 2002; accepted April 9, 2003)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). The
pharmacokinetics and metabolism of ETT have been investigated in the rat, dog,
and human. In all three species, ETT was rapidly absorbed and extensively
cleared by metabolism (Milton et al.,
1997; Morgan et al.,
2000). The pathways of ETT metabolism are similar in the rat, dog,
and human, and principal routes include pyrrolidine N-demethylation
to N-desmethyl eletriptan (DETT), together with N-oxidation,
oxidation of the pyrrolidine ring, and formation of tetracyclic quaternary
ammonium metabolites (Morgan et al.,
2000).
|
Members of the cytochrome P450 (P450) superfamily are known to play a major
role in the oxidative metabolism of both xenobiotics and certain endogenous
compounds. Described herein are the first detailed studies to elucidate the
P450 forms responsible for ETT metabolism to DETT in human liver. The
metabolism of ETT to DETT was chosen to be monitored in our studies since DETT
represents a significant metabolite observed to circulate in humans at levels
10 to 20% of those observed for ETT [Product Information for Eletriptan,
Pfizer, Sandwich, UK (2001)]. Reaction phenotyping studies
(Rodrigues, 1999) were
performed by correlation analysis, the use of recombinant human P450 forms,
chemical inhibition, and inhibitory antibodies. In addition, to evaluate the
potential for drug-drug interactions, the effect of ETT on some selected P450
form enzyme activities was also investigated.
It was of interest also to determine whether ETT could serve as a substrate
for P-glycoprotein (P-gp), an efflux transporter expressed on the apical side
of gut epithelia and brain endothelia, which can limit oral absorption and
brain penetration, respectively. The role of P-gp and P450 in the uptake and
metabolism of ETT was investigated because these systems can work in concert
to limit systemic exposure following oral dosing
(Cummins et al., 2002), and
they are also subject to inhibition and/or induction by a variety of
xenobiotics (Wandel et al.,
1999; Dresser et al.,
2003). In addition, the role of P-gp in regulating the uptake of
ETT into the brain was of interest because of the intense speculation about a
central mechanism of action for the triptan class of antimigraine drugs
(Goadsby and Hargreaves, 2000).
In this regard, ETT was considered the ideal tool to investigate this
phenomenon, since it was the first lipophilic agent in this drug class that
would be expected to have good intrinsic brain penetration. Our studies
describe primarily the role of P-gp and P450 in the disposition of ETT from
the perspective of its potential for drug-drug interactions. The role of P-gp
in potentially modulating the dose required for antimigraine efficacy is also
discussed in the context of a comparison to the other triptan drugs,
naratriptan (NARA), rizatriptan (RIZA), sumatriptan (SUMA), and zolmitriptan
(ZOLM).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), and for
CYP2C8/9, also based on in-house studies (unpublished data). Human Liver Microsomes. Washed microsomal fractions were prepared in 0.154 M KCl containing 50 mM Tris-HCl, pH 7.4, using standard methods. Two separate batches, designated pools I and II, of pooled human liver microsomes were prepared. Each batch was prepared by pooling liver samples from five subjects. Pool I comprised liver samples from males aged 2.5 and 58 years and females aged 14, 59, and 74 years, whereas pool II comprised liver samples from male subjects aged 44 and 50 years and female subjects aged 11, 31, and 55 years.
Metabolism of ETT by Human Liver Microsomes. The NADPH-dependent metabolism of ETT was studied in incubation mixtures containing 0.05 to 200 µM ETT (added in 0.25 ml of 0.1 M phosphate buffer, pH 7.4), 10 mM MgCl2, 2 mM EDTA, 85 mM phosphate buffer, pH 7.4, and 0.05 to 0.3 mg of microsomal protein in a final volume of 0.5 ml. After a 5-min preincubation at 37°C in a shaking water bath, the reaction was initiated by the addition of 1 mM NADPH. Incubations were performed in duplicate for 5 to 20 min at 37°C and were terminated by the addition of 0.5 ml of ice-cold acetonitrile containing the L-844151 internal standard for LC/MS-MS analysis (see below). Blank incubations contained all components except NADPH.
ETT metabolism to DETT was quantified by LC/MS-MS analysis. An HTS PAL
autosampler (Presearch Ltd., Hitchin, Hertsfordshire, UK) attached to an
HP1100 series high-performance liquid chromatograph (Hewlett Packard Ltd.,
Bracknell, Berkshire, UK) were used to introduce samples into a Micromass
Quattro LC triple quadrupole mass spectrometer (Micromass UK Ltd., Altrincham,
Cheshire, UK) using an Electrospray Z-spray source. All incubations were
terminated by the addition of an equal volume of ice-cold acetonitrile
containing 200 µM L-844151 internal standard. After vortex mixing, the
tubes were centrifuged at 10,000g for 10 min at 4°C.
Chromatography of 20-µl aliquots of the supernatant was performed with a
150 x 3.0 mm Kromasil C18 5-µm column (Thermo Hypersil-Keystone,
Runcorn, Cheshire, UK) and isocratic elution with a mobile phase of 40% (v/v)
acetonitrile, 6% (v/v) 250 mM ammonium formate, pH 3.0 and 54% (v/v) ultrapure
water, at a flow rate of 0.4 ml/min and a column temperature of 35°C. The
eluate was split volumetrically, with 20% passing directly into the triple
quadrupole mass spectrometer operating in positive electrospray mode, with
probe and source block temperatures of 400 and 100°C, respectively, using
nitrogen for desolvation and nebulization, and a capillary voltage of 2.8 kV
to initiate ionization. The compounds were selectively detected by multiple
reaction monitoring using argon, collision energy 21 V; ETT
m/z 383
84, DETT m/z 36970, and
L-844151 internal standard m/z 354113.
ETT Metabolism Correlation Analysis. Incubation mixtures contained 60 µM ETT (added in 0.25 ml of 0.1 M phosphate buffer, pH 7.4), 10 mM MgCl2, 2 mM EDTA, 85 mM phosphate buffer, pH 7.4, and 0.1 mg of microsomal protein in a final volume of 0.5 ml. After 5 min of preincubation at 37°C in a shaking water bath, the reaction was initiated by the addition of 1 mM (final concentration) NADPH. Incubations were conducted in triplicate with a single blank (no NADPH) for each of the 16 preparations of characterized human liver microsomes and were terminated after 5 min by the addition of 0.5 ml of ice-cold acetonitrile containing the L-844151 internal standard. Incubations were processed for LC/MS-MS analysis as described above.
ETT Metabolism by Recombinant P450 Forms. Incubation mixtures contained 60 µM ETT (added in 0.125 ml of 0.1 M phosphate buffer, pH 7.4) 10 mM MgCl2, 2 mM EDTA, 1 mM NADPH, and 90 mM phosphate buffer, pH 7.4, in a final volume of 0.25 ml. After a 5-min preincubation at 37°C in a shaking water bath, the reaction was initiated by adding 0.1 mg of Blymphoblastoid cell microsomal protein with gentle mixing. The incubations were conducted in duplicate with duplicate blank (no microsomes) for each P450 form preparation. Reactions were terminated after either 5 or 30 min with 0.25 ml of ice-cold acetonitrile containing the L-844151 internal standard. Incubations were processed for LC/MS-MS analysis as described above.
ETT Metabolism Inhibition Studies. Apart from the studies with the
CYP1A2 mechanism-based inhibitor furafylline
(Newton et al., 1995), where
dimethyl sulfoxide was used, methanol was used as the solvent for these
investigations to minimize solvent effects on individual P450 forms.
For the mechanism-based inhibitors requiring preincubation with NADPH, incubation mixtures contained 10 mM MgCl2, 2 mM EDTA, 1 mM NADPH, 84 mM phosphate buffer, pH 7.4, 0.1 mg of microsomal protein, and either 5 to 50 µM furafylline, 5 to 100 µM diethyldithiocarbamate, 2 to 50 µM troleandomycin, or 2 to 50 µM erythromycin in a volume of 0.25 ml. The inhibitors were added in either methanol (5 µl/tube) or dimethyl sulfoxide (furafylline only, 5 µl/tube), and the concentrations refer to the final concentrations in a 0.5-ml incubation. Following a 30-min preincubation at 37°C in a shaking water bath, 60 µM ETT and 1 mM NADPH (final concentration 2 mM) were added in 0.25 ml of 0.1 M phosphate buffer, pH 7.4, at 37°C. Incubations were performed in duplicate for 5 min at 37°C and were terminated by the addition of 0.5 ml of ice-cold acetonitrile containing the L-844151 internal standard. Incubations were processed for LC/MS-MS analysis as described above.
For compounds not requiring extensive preincubation with NADPH, incubation mixtures contained 10 mM MgCl2, 2 mM EDTA, 1 mM NADPH, 84 mM phosphate buffer, pH 7.4, 0.1 mg of microsomal protein, and either 2 to 50 µM sulfaphenazole, 2 to 20 µM quinidine, 50 to 500 µM S-mephenytoin, or 2 to 50 µM miconazole, in a volume of 0.25 ml. All the inhibitors were added in methanol (5 µl/tube), and the concentrations refer to the final concentrations in a 0.5-ml incubation. After a 10-min preincubation at 37°C in a shaking water bath, the reaction was initiated by the addition of 60 µM ETT (added in 0.25 ml of 0.1 M phosphate buffer, pH 7.4, at 37°C). Incubations were performed in duplicate for 5 min at 37°C and were terminated by the addition of 0.5 ml of ice-cold acetonitrile containing the L-844151 internal standard. Incubations were processed for LC/MS-MS analysis as described above.
To study the effect of inhibitory antibodies to CYP2C8/9, CYP2D6, and CYP3A4 forms, incubation mixtures contained 10 mM MgCl2, 2 mM EDTA, 84 mM phosphate buffer, pH 7.4, 0.1 mg of microsomal protein, and either 0.025, 0.25, or 2.5 µl of the antibody preparation in a final volume of 0.5 ml. After a 10-min preincubation at 37°C in a shaking water bath, the reaction was initiated by the addition of 60 µM ETT and 1 mM NADPH (added in 0.25 ml of 0.1 M phosphate buffer, pH 7.4, at 37°C). Incubations were performed in duplicate for 5 min at 37°C and were terminated by the addition of 0.5 ml of ice-cold acetonitrile containing the L-844151 internal standard. Incubations were processed for LC/MS-MS analysis as described above.
Effect of ETT on CYP450 Enzyme Activities. The activities of
7-ethoxyresorufin O-deethylase, tolbutamide methylhydroxylase,
debrisoquine 4-hydroxylase, and testosterone 6ß-hydroxylase were
determined as described previously
(Renwick et al., 2000),
employing substrate concentrations of 0.2, 180, 150, and 50 µM,
respectively. For tolbutamide and debrisoquine metabolism, the tubes were
preincubated in duplicate at 37°C in a shaking water bath for 5 min in the
presence of either no ETT (controls) or 0.2 to 100 µM ETT and an
NADPH-generating system, prior to addition of the P450 substrate. The effect
of ETT on 7-ethoxyresorufin metabolism was determined as described for
tolbutamide and debrisoquine metabolism, but with an ETT concentration range
of 0.5 to 100 µM. For testosterone 6ß-hydroxylase, the tubes were
preincubated in duplicate at 37°C in a shaking water bath for 5 min in the
presence of either no ETT (controls) or ETT (concentration range 0.2–100
µM or 1–100 µM) and 50 µM [4-14C]testosterone (0.5
µCi/tube), prior to the addition of 1 mM NADPH. To evaluate the effect of
ETT as a mechanismbased inhibitor of testosterone 6ß-hydroxylase
activity, the tubes were preincubated at 37°C in a shaking water bath for
30 min in the presence of either no ETT (controls) or ETT (concentration range
0.5–50 µM or 0.2–100 µM) and 1 mM NADPH, prior to the
addition of 50 µM [4-14C]testosterone (0.5 µCi/tube) and 1 mM
NAPDH (final concentration 2 mM). Chlorzoxazone 6-hydroxylase was determined
in incubation mixtures containing 45 µM chlorzoxazone, 1 mM NADPH, 0.2 mg
of microsomal protein, and 0.1 M phosphate buffer in a final volume of 1 ml.
Incubations were terminated after 20 min with 0.2 ml of 30% (v/v) perchloric
acid, and the supernatant was analyzed by HPLC. Chromatography was performed
as described previously for tolbutamide methylhydroxylase
(Renwick et al., 2000), except
that the mobile phase consisted of 23% (v/v) acetonitrile and 77% (v/v) 20 mM
sodium perchlorate, pH 2.5, and the eluant was monitored at 287 nm. The
metabolism of S-mephenytoin to both 4'-hydroxymephenytoin and
N-desmethyl S-mephenytoin was determined in incubation
mixtures containing 60 µM S-[4-14C]mephenytoin (0.5
µCi/tube), 1 mM NADPH, 7.5 mM DL-isocitric acid, 2 U/ml
isocitric dehydrogenase, 5 mM MgSO4, 0.5 mg microsomal protein and
0.1 M phosphate buffer pH 7.4 in a final volume of 0.5 ml. Incubations were
terminated after 30 min with 0.1 ml of 30% (v/v) perchloric acid and the
supernatant analyzed by HPLC. Chromatography was performed as described
previously for debrisoquine 4-hydroxylase
(Renwick et al., 2000), except
that the mobile phase consisted of 17% (v/v) acetonitrile and 83% (v/v) 20 mM
sodium perchlorate, pH 2.5, with quantitation by liquid scintillation
counting. The effect of 0.2 to 100 µM ETT on chlorzoxazone and
S-mephenytoin metabolism was studied using a preincubation period of
5 min as described above for tolbutamide and debrisoquine. For all P450 form
enzyme activities studied, plots were constructed of enzyme activity
(percentage of control rate in the absence of added ETT) against ETT
concentration (logarithmic scale). Where inhibition of enzyme activity was
observed, IC50 values (i.e., concentration of ETT to produce a 50%
inhibition of enzyme activity) were calculated by linear regression
analysis.
Brain Penetration of the 5-HT1B/1D Agonists in
Mdr1a–/– and
Mdr1a+/+ CF-1 Mice. The
impact of the drug efflux pump, P-gp
(Umbenhauer et al., 1997) on
the brain entry of the triptan drugs was investigated in vivo using male CF-1
mice. ETT was formulated in 25% polyethylene glycol 300/water at 0.5 mg free
base equivalents/ml, and NARA, RIZA, ZOLM, and SUMA were formulated as aqueous
solutions at the same concentration. All drugs were dosed at 1 mg free base
equivalents/kg as a bolus injection into the tail vein (dose volume 2 ml/kg)
to 24 male CF-1 mice (12 Mdr1a+/+, 12
Mdr1a–/–), approximate weight 35
g. Blood (by cardiac puncture into heparinized containers) and brain samples
were taken at either 0.5, 2, 4, or 6 h after dosing (n = 3 per time
point per genotype). Samples were stored frozen (–20°C) until
analysis.
To blood (0.1 ml) was added internal standard (10 µl of a 10 ng/µl solution), 1 M sodium hydroxide (0.05 ml), water (1 ml), and ethyl acetate (4 ml). Samples were vortex mixed and centrifuged. Supernatant was removed and evaporated to dryness (70°C, under nitrogen), and the residue was dissolved in mobile phase (0.1 ml) and transferred to a HPLC vial. Calibration standards covering appropriate ranges were prepared by spiking solutions of analyte at appropriate concentrations into control blood (0.1 ml).
To weighed samples of brain cortex were added internal standard (10 µl of a 10 ng/µl solution) and water (2 ml). After homogenization with an ultrasonic probe, 1 M sodium hydroxide (0.2 ml) and ethyl acetate (4 ml) were added. Samples were vortex mixed and centrifuged. Supernatant was removed and evaporated to dryness (70°C, under nitrogen), and the residue was dissolved in mobile phase (0.15 ml) and transferred to a HPLC vial. Calibration standards covering appropriate ranges were prepared by spiking solutions of analyte at appropriate concentrations into control brain cortex.
Typically 25-µl injections were made onto a KR100-5C8 HPLC column (150 x 3.2 mm i.d., Hichrom Ltd., Reading, UK) with a mobile phase consisting of acetonitrile (A) and 25 mM ammonium formate, adjusted to pH 3 with formic acid, at a flow rate of 0.4 ml/min, with the following time program: 0 min, 20% A; 2 min, 20% A; 6 min, 50% A; 7 min, 20% A; 10 min, 20% A. Detection was for appropriate fragmentation transitions as determined by infusion of 1 ng/µl solutions at 10 ml/h into the mass spectrometer (Micromass Quattro).
Since three animals per time point were used to construct blood and brain
concentration versus time profiles, Bailer's method for determining area under
the curve (AUC) confidence intervals during sparse sampling was applied
(Bailer, 1988;
Nedelman et al., 1995).
Standard error of the mean (S.E.M.) for the brain/blood ratio was determined
using the following equation: S.E.M. = (Brain AUC S.E.M.2/Blood
AUC2) + (Brain AUC2 · Blood AUC
S.E.M.2/Blood AUC4). An unpaired two-tailed t
test was performed to determine the significance of the difference between
brain/blood AUC0.5–6h ratios determined in
Mdr1a–/– and
Mdr1a+/+ mice.
Human MDR1 and Mouse Mdr1a P-Glycoprotein Bidirectional Transport Assay. The extent of P-gp-mediated transport of ETT, NARA, RIZA, ZOLM, and SUMA was also evaluated in LLC-PK1 wild-type, LLC-MDR1 (human), and LLC-Mdr1a (mouse) cell lines (kindly provided by Dr. A. H. Schinkel, The Netherlands Cancer Insitute, Amsterdam, The Netherlands). Cells were cultured in 199 medium as described. For transport experiments, cells were cultured in 24-well Transwell plates (Falcon) at a density of 1.5 x 105 cells/well with 0.3 ml of medium in the upper compartment and 35 ml of medium in the lower nonsubdivided compartment. After culturing for 5 days, the transport wells were transferred to a 24-well plate. The incubation and sampling procedure during the transport experiment was performed on a Tecan Genesis Workstation 150 Robot (Tecan, Zürich, Switzerland). Cells were washed three times with Hanks' balanced salt solution containing Hepes (10 mM). Subsequently, analyte in Hanks' balanced salt solution containing Hepes (500 µl) was added either to the apical or basolateral compartment. Analyte concentrations used were: 10 µM ETT, NARA, RIZA, ZOLM, and SUMA, 10 µM dexamethasone, 5 µM ritonavir, and 0.5 µM verapamil. After incubating for 4 h at 37°C, samples (100 µl) were taken from both the apical and basolateral compartment and transferred to a 96-well microtiter plate. Samples were analyzed by positive ion single ion monitoring atmospheric pressure chemical ionization on a SCIEX API 2000 triple quadrupole mass spectrometer (PerkinElmerSciex, Concord, ON, Canada). The HPLC system used was a PerkinElmer Series 200 system with 2 Micro pumps (PerkinElmer Instruments, Shelton, CT) and a Leap HTS PAL autosampler (LEAP Technologies, Carrboro, NC). Samples were chromatographed using an Aquasil C18, 2 x 20 mm, 5-µm HPLC column (Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA). The mobile phase used was 0.1% acetic acid in water (A) or in acetonitrile (B) at 2.0 ml/min with a 4:1 split. The HPLC run time was 2 min/sample. Standard curves were prepared by a 2-fold serial dilution in water/methanol (60:40, v/v) of a standard prepared at 2 times the dosing solution concentration for an 11-point standard curve. Percentage of transport was calculated by dividing the concentration of the compound appearing in the receiver compartment by the sum of the compound concentrations measured in the receiver and donor compartment (x 100%). The BA/AB ratio was calculated by dividing the concentration of compound measured in the apical compartment (resulting from introduction of compound in the basolateral well) by the concentration of compound measured in the basolateral compartment (resulting from introduction of the compound in the apical well).
Lipophilicity Measurements. Octanol-pH 7.4 buffer partition
coefficients (logD7.4) were determined by the shake flask method
with HPLC-UV analysis of the aqueous and organic phases after equilibration
(Hansch and Leo, 1979). Results
were derived from the mean of two partition coefficient determinations
performed at overall concentrations of 0.5 and 0.25 mg/ml in 1:1 pH 7.4
buffer/octanol mixtures.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Kinetics of ETT Metabolism in Human Liver Microsomes. The kinetics of the NADPH-dependent metabolism of 0.05 to 200 µM ETT to DETT was examined with both the pool I and pool II human liver microsomal preparations. Pooled, rather than individual, liver microsomal preparations were used for these studies since the aim was to identify average Km (i.e., the concentration of substrate giving half-maximal velocity) values to select a suitable substrate concentration for the reaction phenotyping studies. With both liver microsome pools, DETT formation was detected at all ETT substrate concentrations examined.
The kinetics of ETT metabolism to DETT was examined with Michaelis-Menten and Eadie-Hofstee plots. Kinetic analysis of data for both liver microsome pools revealed mean Km and Vmax (i.e., the maximal velocity) values of 27.1 µM (pool I and pool II individual values were 24.3 and 29.9 µM, respectively) and 78 pmol/min/mg protein (pool I and pool II individual values were 105.4 and 50.5 pmol/min/mg protein, respectively). The mean intrinsic clearance (CLint; i.e., Vmax/Km) value was 3.02 µl/min/mg protein (pool I and pool II individual values were 4.34 and 1.69 µl/min/mg protein).
An ETT substrate concentration of 60 µM was selected for the reaction phenotyping studies described below. This substrate concentration was selected as being approximately 2 times the Km value for DETT formation.
ETT Metabolism Correlation Analysis. The metabolism of 60 µM ETT to DETT was examined with a characterized panel of 16 human liver microsomal preparations. ETT was metabolized to DETT by all 16 human liver microsomal preparations examined. Microsomal ETT metabolism varied from 58 to 635 pmol/min/mg protein, with most of the individual human liver microsomal preparations being more active than the two pooled human liver microsomal preparations.
The rates of ETT metabolism to DETT were correlated with data for total
P450 content and a range of P450 form enzyme activities. A good correlation
(r2 = 0.932) was observed between ETT metabolism to DETT
and CYP3A4-catalyzed testosterone 6ß-hydroxylation. Although some
correlation (r2 = 0.541) was observed between ETT
metabolism to DETT and total P450 content, only low correlations were observed
(r2 = 0.000–0.361) with P450 form enzymatic markers
for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and
CYP4A9/11. These enzyme activities (with r2 values in
parentheses) comprised 7-ethoxyresorufin O-deethylase (0.023,
CYP1A2), coumarin 7-hydroxylase (0.022, CYP2A6), S-mephenytoin
N-demethylase (0.310, CYP2B6), paclitaxel 6
-hydroxylase
(0.361, CYP2C8), diclofenac 4'-hydroxylase (0.104, CYP2C9),
S-mephenytoin 4'-hydroxylase (0.145, CYP2C19), dextromethorphan
O-demethylase (0.098, CYP2D6), chlorzoxazone 6-hydroxylase (0.004,
CYP2E1), and lauric acid 12-hydroxylase (0.000, CYP4A9/11).
Metabolism of ETT by Recombinant P450 Forms. The metabolism of 60 µM ETT to DETT by rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1, and rCYP3A4, together with microsomes from control cells (which contain native CYP1A1) was studied. Incubations were performed for 5 and 30 min. Only trace amounts of DETT were observed in incubations with the control cell microsomes (data not shown) and with rCYP2A6, rCYP2B6, and rCYP2E1 preparations (Fig. 2). Low rates of DETT formation were observed with the rCYP1A2 and rCYP2C8 preparations, with higher rates being observed with rCYP2C9 and rCYP2C19 preparations. The highest rates of ETT metabolism to DETT were observed with the rCYP2D6 and rCYP3A4 preparations (Fig. 2).
|
Inhibition of ETT Metabolism. The effect of some human P450 form
inhibitors and one P450 form substrate (S-mephenytoin) on the
metabolism of ETT to DETT in human liver microsomes was studied. For the
mechanism-based inhibitors (Newton et al.,
1995) furafylline (CYP1A2), diethyldithiocarbamate (CYP2E1),
erythromycin (CYP3A4), and troleandomycin (CYP3A4), the compounds were
preincubated for 30 min at 37°C with liver microsomes and NADPH prior to
the addition of ETT and another aliquot of NADPH. In the studies with the
inhibitors sulfaphenazole (CYP2C9) and quinidine (CYP2D6), and the substrate
S-mephenytoin (CYP2C19), the compounds were preincubated with liver
microsomes for 10 min at 37°C prior to the addition of NADPH and ETT.
ETT metabolism to DETT was not markedly affected by 5 to 50 µM furafylline, 2 to 50 µM sulfaphenazole, 50 to 500 µM S-mephenytoin, and 2 to 20 µM quinidine (Fig. 3, A and B). Whereas 100 µM diethyldithiocarbamate produced some inhibition of ETT metabolism, little effect was observed with lower concentrations of this inhibitor (Fig. 3A).
|
The effect of erythromycin, miconazole, and troleandomycin on ETT metabolism to DETT was studied with both pools of human liver microsomes. ETT metabolism to DETT was markedly inhibited to 3 to 13% of control (mean of both liver microsome pools) by 2 to 50 µM miconazole and to 20 to 35% of control by 2 to 50 µM troleandomycin (Fig. 4). Erythromycin also produced a concentration-dependent inhibition of ETT metabolism, with enzyme activity being reduced to 23% of control by 50 µM erythromycin.
|
The effect of inhibitory antibodies to CYP2C8/9, CYP2D6 and CYP3A4 on ETT metabolism to DETT in human liver microsomes was studied. With both pools of human liver microsomes treatment with 0.05–5 µl/ml incubation of either the CYP2C8/9 or the CYP2D6 antibodies had little effect on ETT metabolism (Fig. 5A and B). In contrast, the CYP3A4 antibody produced a marked inhibition of the metabolism of ETT to DETT in human liver microsomes.
|
Effect of ETT on P450 Form Enzyme Activities. The effect of addition of ETT at concentrations of up to 100 µM on some P450 form enzyme activities in human liver microsomes was studied. ETT produced a weak inhibition of CYP2D6-dependent debrisoquine 4-hydroxylase activity, with a calculated IC50 value (i.e., concentration of ETT to produce a 50% inhibition of enzyme activity) of 84 µM (Table 1). The addition of ETT had no effect (IC50 values all >100 µM) on 7-ethoxyresorufin O-deethylase (CYP1A2), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methylhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), and chlorzoxazone 6-hydroxylase (CYP2E1) activities. ETT produced a weak inhibition of CYP3A4-dependent testosterone 6ß-hydroxylase activity, with a calculated IC50 value (mean of two liver microsome pools) of 95 µM (Table 1). When ETT was evaluated as a mechanism-based inhibitor of testosterone 6ß-hydroxylase activity, an observed IC50 value (mean of two liver microsome pools) of 40 µM was obtained. Using a 30-min preincubation period with NADPH, 20 µM troleandomycin (positive control) inhibited testosterone 6ß-hydroxylase activity to 18% of control levels.
|
Brain Penetration of the 5-HT1B/1D Agonists in
Mdr1a–/– and
Mdr1a+/+ CF-1 mice. The
brain penetration of ETT, NARA, RIZA, SUMA, and ZOLM was investigated in
Mdr1a+/+ and Mdr1a
–/– mice
(Umbenhauer et al., 1997). Of
the 5-HT1B/1D agonists investigated, ETT was shown to be the best
substrate for P-gp; the brain/blood AUC ratio was 13 in
Mdr1a–/– mice, which lack the
brain endothelial Mdr1a P-gp efflux pump, and 0.3 in
Mdr1a+/+ mice, which express Mdr1a P-gp. The
mouse P-gp efflux pump therefore has the effect of reducing brain exposure to
ETT by approximately 40-fold (Fig.
6A). The effect for all the other triptan molecules, NARA, RIZA,
SUMA, and ZOLM, was smaller and less that 5-fold. The brain/blood AUC ratio
for inulin, a poorly brain-penetrant reference compound, was approximately
0.16 in both mutant and wild-type CF-1 mice, indicating that the integrity of
the blood-brain barrier was not compromised by the absence of P-gp. The brain
penetration of 5-HT1B/1D agonists in
Mdr1a–/– mice appeared to be in
line with their lipophilicity (Fig.
6B).
|
Human MDR1 and Mouse Mdr1a P-Glycoprotein Bidirectional Transport Assay. The transport of NARA, RIZA, SUMA, and ZOLM across LLC-PK1 parent cell monolayers was poor, with <5% of each drug being transported in either the B to A or A to B direction over a 4-h period. This diffusion rate was too low to reliably test whether these compounds were substrates for P-gp. In contrast, the transport of ETT was >10% in this parent cell line, a value consistent with the extent of transport observed for the positive control compounds used: dexamethasone, ritonavir, and verapamil (Table 2). In transport experiments across LLC-MDR1 and LLC-Mdr1a cell monolayers, the BA/AB ratio for ETT was in the range 9 to 11, highlighting it to be a good substrate of human and mouse P-gp in vitro, and similar to the positive control compounds studied.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Goadsby and Hargreaves, 2000;
Goadsby et al., 2002), factors
that regulate the brain penetration of ETT, relative to NARA, RIZA, SUMA, and
ZOLM, were investigated also and included measurements of lipophilicity and
brain penetration in CF-1 wild-type
(Mdr1a+/+) and P-gp-deficient
(Mdr1a–/–) mice.
The NADPH-dependent N-demethylation of ETT to DETT was
investigated in two preparations of pooled human liver microsomes and in 16
individual liver microsome preparations, and was concluded to be mediated by
CYP3A, a good correlation (r2 = 0.932) being observed
between DETT formation and testosterone 6ß-hydroxylase activity. The
effect of erythromycin, miconazole, and troleandomycin on ETT metabolism to
DETT was also studied with both preparations of pooled human liver microsomes.
No significant differences were noted between the two human liver microsome
pools in the effects of the three compounds on DETT formation. The
mechanism-based inhibitors troleandomycin and erythromycin, together with
miconazole, produced a marked concentration-dependent inhibition of ETT
metabolism to DETT in human liver microsomes
(Fig. 4). Previous studies have
demonstrated that troleandomycin is a specific inhibitor of human hepatic
CYP3A4 (Newton et al., 1995).
In addition, both erythromycin and miconazole are also known to inhibit
CYP3A4-catalyzed reactions in human liver microsomes
(Thummel and Wilkinson,
1998).
Prototypical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1
(Newton et al., 1995;
Ono et al., 1996;
Rodrigues, 1999) had little
effect on the metabolism of ETT to DETT in human liver microsomes
(Fig. 3). Although DETT
formation was inhibited by high concentrations of diethyldithiocarbamate, this
is unlikely to reflect metabolism by CYP2E1 but, rather, that this compound
also inhibits other P450 forms (Ono et
al., 1996).
The NADPH-dependent metabolism of 60 µM ETT to DETT by recombinant P450 forms was also studied. The highest rates of DETT formation were observed with rCYP2D6 and rCYP3A4 preparations (Fig. 2). Although ETT was also metabolized to DETT by rCYP2C9 and rCYP2C19 preparations, rates of DETT formation were either much lower or essentially undetectable with the rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, and rCYP2E1 preparations.
The observation that ETT is extensively metabolized to DETT by rCYP3A4 is
in agreement with the results of the correlation analysis, chemical
inhibition, and inhibitory antibody studies. The finding that ETT is also
extensively metabolized by rCYP2D6 appears to have little significance when
other competing enzymes are present. This finding is borne out by the
correlation analysis study, and the lack of effect of quinidine and the
CYP2D6-inhibitory antibody on DETT formation in liver microsomes. This result
is probably attributable to the relatively higher levels of CYP3A4 compared
with those of CYP2D6 in human liver. Levels of CYP3A/CYP3A4 in human liver
microsomes have been reported to range from 44 to 250 pmol/mg protein, whereas
those for CYP2D6 have been reported to range from only 5 to 25 pmol/mg protein
(Shimada et al., 1994;
Rodrigues, 1999).
Many examples of drug-drug interactions in human subjects have been
described, and these may be due to either the induction or inhibition of
P450-dependent and other xenobiotic metabolizing enzyme activities
(Lin and Lu, 1998). The
implication of our in vitro data, that of ETT dependence on CYP3A4 for
metabolic clearances and its potential for drug interactions when
coadministered with CYP3A4 inhibitors or inducers, is that plasma
concentrations of ETT in patients would be expected to either increase or
decrease, respectively. With regard to coadministration of ETT (Relpax) with
CYP3A4 inhibitors, this is borne out in the Product Information for Eletriptan
[Pfizer, Sandwich, UK (2001)]. In clinical studies with erythromycin (1000 mg)
and ketoconazole (400 mg), which are both strong inhibitors of CYP3A4,
significant increases in ETT Cmax (2- and 2.7-fold) and
AUC (3.6- and 5.9-fold), respectively, were observed. This increased exposure
was associated with an increase in ETT half-life from 4.6 h to 7.1 h for
erythromycin and from 4.8 h to 8.3 h for ketoconazole. Coadministration of ETT
with CYP3A4 inhibitors is therefore discouraged [Product Information for
Eletriptan, Pfizer, Sandwich, UK (2001);
Tepper et al., 2003). No
information is provided with regard to coadministration of ETT with strong
CYP3A4 inducers such as phenytoin, rifampicin, and others
(Smith, 2000), but a reduction
in ETT plasma concentrations would be expected following such a dosing
regimen.
In our studies, the potential for ETT to produce drug-drug interactions as a consequence of inhibiting P450-mediated reactions was also evaluated. Overall, ETT at concentrations of up to 40 µM had little effect on the enzymatic markers of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 studied (Table 1). These data suggest that ETT is unlikely to produce any significant inhibitory interactions in vivo with other drugs that are metabolized by these P450 forms.
P-gp plays a role in modulating oral exposure and brain penetration. Unlike
the other triptans investigated, ETT was an excellent substrate for human and
mouse P-gp in vitro, with BA/AB ratios in bidirectional transport studies
across LLC-MDR1 and LLC-Mdr1a cell monolayers in the range 9 to 11
(Table 2). This finding has
significance clinically since it was reported recently that administration of
both ETT and verapamil resulted in a 2.7- and a 2.2-fold increase in ETT AUC
and Cmax, respectively
(Humphrey, 2002). These data
also have significance preclinically, since brain exposure to ETT was reduced
40-fold in Mdr1a+/+ relative to
Mdr1a–/– mice
(Fig. 6A).
The finding that ETT is a good mouse and human P-gp substrate led us to
investigate four other triptans in these models. This was undertaken to enable
us to speculate on the role of P-gp in modulating a central mechanism of
action for the triptan class of antimigraine drug. Three potential mechanisms
of action have been proposed for 5-HT1B/1D receptor-selective
agonists in the treatment of acute migraine attacks. The first two invoke
peripheral mechanisms: vasocon-striction of dilated, pain-producing meningeal
blood vessels and inhibition of meningeal perivascular trigeminal nerves,
which reduces the release of, for example, the powerful vasodilator calcitonin
generelated peptide. The third mechanism, for brain-penetrant compounds, is
antinociceptive activity within the brain stem sensory trigeminal nuclei;
anatomical studies having shown a high concentration of 5-HT1B/1D
receptors in the trigeminal ganglion of animals and humans. It might be that
each of these mechanisms operates in a complementary manner to reduce the
intense central trigeminal input that occurs during a migraine headache
(Goadsby and Hargreaves, 2000).
If the central site is important for antimigraine efficacy, then P-gp might be
expected to limit the accessibility of ETT to central sites of action. An
analysis of the available pharmacokinetic data for lead triptans indeed
indicates that higher free drug levels of ETT, the most potent of all the
triptans at 5HT1B/1D receptors in vitro, are required for clinical
efficacy when compared with ZOLM or RIZA at doses that provide equivalent
efficacy (Table 3;
Goadsby et al., 2002). These
higher plasma levels of ETT may reflect a requirement to provide sufficient
free drug levels in the brain for adequate occupancy of central
5-HT1B/1D receptors. The potential theoretical gains of increased
potency and lipophilicity on improved efficacy and barrier transport (gut and
brain) for ETT may, therefore, have been effectively offset by relatively high
plasma protein binding and good substrate specificity for P-glycoprotein
(Fig. 6B,
Table 3).
|
It is recognized that the triptan class of compounds do generally have poor
brain penetration characteristics when compared with typical CNS marketed
drugs (Doan et al., 2002). The
extent of brain penetration is, however, a poor guide to central activity,
especially with potent agonist drugs such as the triptans, since they, in
contrast to most other CNS agents that are antagonists, will require only low
fractional receptor occupancy to exert central effects. Further studies are
warranted to relate plasma and brain concentrations of the triptans to their
occupancy of central antimigraine 5-HT1B/1D receptors in vivo to
evaluate the potential contribution of central sites to their mechanism of
action.
In summary, the results of this study demonstrate that ETT can be metabolized to DETT by human liver microsomal preparations, and this biotransformation is primarily catalyzed by CYP3A4. ETT was determined not to be a potent inhibitor of a number of P450-dependent enzyme activities, although ETT metabolism was markedly reduced in vitro by prototypical CYP3A4 inhibitors. These findings are in agreement with the clinical data inasmuch as plasma levels of ETT are increased in human subjects when ETT is coadministered with inhibitors of CYP3A. ETT was shown to be a substrate of human P-glycoprotein, although the importance of this in the context of a central mechanism of action remains inconclusive.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Address correspondence to: Dr. David C. Evans, Department of Drug Metabolism, Merck & Co., Inc., 126 East Lincoln Avenue (RY80E-200), Rahway, NJ 07065. E-mail: david_c_evans{at}merck.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. C. Kalvass, T. S. Maurer, and G. M. Pollack Use of Plasma and Brain Unbound Fractions to Assess the Extent of Brain Distribution of 34 Drugs: Comparison of Unbound Concentration Ratios to in Vivo P-Glycoprotein Efflux Ratios Drug Metab. Dispos., April 1, 2007; 35(4): 660 - 666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Uhr, C. Namendorf, M. T. Grauer, M. Rosenhagen, and M. Ebinger P-glycoprotein is a factor in the uptake of dextromethorphan, but not of melperone, into the mouse brain: evidence for an overlap in substrate specificity between P-gp and CYP2D6 J Psychopharmacol, December 1, 2004; 18(4): 509 - 515. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) and 5 to 100 µM diethyldithiocarbamate (
) (A), together with
2 to 50 µM sulfaphenazole (
), 2 to 20 µM quinidine (
), and
50 to 500 µM S-mephenytoin (
) (B) were evaluated on the
metabolism of 60 µM ETT to DETT. Incubations were performed with 0.1 mg
protein of the pool I human liver microsomal preparation for 5 min. Each point
represents the mean of a duplicate test incubation.
),
together with 2 to 50 µM miconazole (
, 
) were evaluated on the
metabolism of 60 µM ETT to DETT. Incubations were performed with 0.1 mg
protein of either the pool I (filled symbols) or pool II (open symbols) human
liver microsomal preparations for 5 min. Each point represents the mean of a
duplicate test incubation.
