P450-Mediated Ring Opening of a 1,3,4-Oxadiazole
765
water-18O, male RLMs (2 mg/ml), setileuton (100 M), and 1 mM NADPH. initial condition was 20% B and was maintained for 1 min. The gradient then
Samples were incubated for 30 min at 37°C in a shaking water bath. At the end
of the incubation, two volumes of acetonitrile were added to the mixtures to
terminate the reaction. Samples were centrifuged, and the supernatants were
transferred into glass culture tubes and evaporated to dryness under a nitrogen
stream. The sample residues were reconstituted in 20% acetonitrile in aqueous
0.1% formic acid before analysis by high-pressure liquid chromatography
coupled with mass spectrometry (LC-MS).
Incubations containing 0.1 M potassium phosphate buffer (pH 7.4) prepared in
either deionized water or water-18O; male RLMs (1 mg/ml); meperidine (50 M),
a known esterase substrate; and 1 mM NADPH were prepared as a positive control
for esterase activity. These samples were incubated for 40 min at 37°C in a shaking
water bath. At the end of the incubation, two volumes of acetonitrile were added
to the mixtures to terminate the reaction. Samples were centrifuged, and the
supernatants were transferred into glass culture tubes and evaporated to dryness
under a nitrogen stream. The sample residues were reconstituted in 5% acetonitrile
in aqueous 0.1% formic acid before LC-MS analysis.
was increased linearly to 90% B over 39 min and maintained at 90% B for 5
min. The column was re-equilibrated under the initial conditions for 5 min
before the next injection.
Meperidine incubation samples were injected onto a Phenomenex Synergi
Polar-RP column (250 ϫ 4.6 mm, 4 m). The mobile phase composition and
flow rate were the same as those described above. The initial condition was 5%
B and was held for 5 min. The gradient then was increased linearly to 90% B
at 15 min and held at 90% B for 5 min. The column was re-equilibrated under
the initial conditions for 5 min before the next injection.
In all cases, column effluent was split such that ϳ150–200 l/min flowed to the
mass spectrometer source and the remainder to waste. All mass spectrometric
analyses were performed with electrospray ionization in the positive mode.
Kinetic Analysis. Data from incubations of setileuton with RLMs,
CYP3A1/2, and CYP1A1/2 for kinetic studies were used to fit equations
describing the classic hyperbolic (eq. 1) or the sigmoidal (eq. 2) kinetic models
used previously (Huang et al., 2004b):
Incubation under Oxygen-18 Gas Atmosphere. Incubation conditions
were modified from Stearns et al. (1995) as follows. Degassed 0.1 M potas-
sium phosphate buffer was prepared through five cycles of freezing under an
argon atmosphere followed by thawing under a vacuum. The degassed buffer
was stored under a positive argon atmosphere. Incubation mixtures were
prepared in a 15-ml three-neck round-bottom flask that was evacuated at room
temperature and refilled with argon two times. The flask then was placed on
ice, and the incubation mixture was added through a septum using a syringe.
The incubation mixture consisted of degassed 0.1 M potassium phosphate
buffer (pH 7.4), male RLMs (2 mg/ml), and setileuton (100 M). The reaction
flask then was evacuated and filled with argon three times. The argon atmo-
sphere was replaced by 18O-labeled oxygen. The incubation mixture then was
preincubated at 37°C for 3 min with gentle agitation using a magnetic stir bar.
The reaction was initiated by the addition of NADPH (1 mM, in degassed 0.1
M potassium phosphate buffer) and incubated at 37°C. After 30 min, an aliquot
of the reaction mixture was transferred to a test tube containing two volumes
v ϭ Vmax͓S͔/͑Km ϩ ͓S͔͒
v ϭ Vmax͓S͔n/͑S50 ϩ ͓S͔ ͒
(1)
(2)
n
n
where v is the initial velocity, Vmax is the maximum velocity, [S] is the
substrate concentration, Km is the Michaelis constant (substrate con-
centration at which velocity is 50% Vmax), n is the Hill coefficient, and
50 is the substrate concentration at which velocity is 50% Vmax. The
kinetic parameters were estimated using SigmaPlot (version 9.01;
Systat Software, Inc., Point Richmond, CA).
S
Results
Metabolism to M5. After incubation of setileuton with NADPH-
fortified rat liver microsomes, several oxidative metabolites were
of acetonitrile. Samples were centrifuged, and the supernatants were trans- formed (data not shown). Mass spectrometric analysis revealed that
ferred into glass culture tubes and evaporated to dryness under a nitrogen
stream. The sample residues were reconstituted in 20% acetonitrile in aqueous
0.1% formic acid and analyzed by LC-MS.
Chemical Hydrolysis. Approximately 10 mg of setileuton was added to 1
ml of water (deionized or 18O-labeled) in a glass screw-cap tube, capped, and
agitated on a vortex mixer forming a saturated aqueous solution of setileuton.
The samples were stressed in an oven at 80°C for approximately 6 days. A
200-l aliquot of each tube was placed in an autosampler vial, and 50 l of
acetonitrile was added before LC-MS analysis.
Sample Analysis. Concentrated samples from each study were reconstituted as
described previously. LC-MS was performed by using an Agilent 1100 HPLC (Agilent
Technologies, Santa Clara, CA) coupled to a Finnigan LTQ linear ion trap or Quantum
mass spectrometer (Thermo Fisher Scientific, Waltham, MA).
Reconstituted samples for metabolite profiling were injected onto a Prodigy
ODS3 column (250 ϫ 4.6 mm, 5 m) (Phenomenex). The mobile phase
consisted of a 0.1% formic acid (solvent A) and acetonitrile (solvent B)
gradient with a flow rate of 1 ml/min. The initial condition was 10% B and was
maintained for 5 min. After 5 min, the gradient was increased linearly to 90%
B at 40 min and held at 90% B for 5 min. The column was re-equilibrated
under the initial conditions for 5 min before the next injection.
Samples for quantitation were injected onto a Phenomenex Luna C18(2)
column (50 ϫ 4.6 mm, 5 m). The mobile phase composition and flow rate
were the same as those described in the previous paragraph. The initial
condition was 20% B and was maintained for 1 min. The gradient then was
increased linearly to 90% B over 4 min and held at 90% B for 1.5 min. The
column was re-equilibrated under the initial conditions for 2.5 min before the
next injection. Data were acquired in selected reaction monitoring mode using
the following transitions: M5, m/z 482 (MHϩ) 3 296 (collision energy, 18 V;
retention time, 4.7 min); and internal standard, m/z 482 (MHϩ) 3 145
(collision energy, 42 V; retention time, 6.2 min). The calibration standards
covered a concentration range of 12.5 to 750 nM.
the metabolite M5 (Fig. 2) had a molecular ion that was 18 Da higher
than that of setileuton, and accurate mass analysis supported the
conclusion that this was due to the net addition of the elements of
water. The product ion spectrum of metabolite M5 (Fig. 3) contained
the m/z 253 ion, which was detected in the product ion spectrum of
setileuton, resulting from the neutral loss of the oxadiazole portion of
the molecule. This suggested that the structural modification occurred
to the oxadiazole portion of the molecule. Structural identification was
confirmed by matching the retention time and product ion spectrum to
the synthetic standard of M5. Metabolite M5 subsequently was iso-
1
lated from rat urine, and the H-NMR spectrum of the isolated M5
was compared to the synthetically prepared compound (Fig. 4). The
1
two one-dimensional (1D) H-NMR spectra in Fig. 4 have identical
chemical shifts and coupling patterns for all proton signals, which
indicate that the two samples have the same chemical structure.
To distinguish between oxidation and either chemical or enzymatic
hydrolysis, setileuton was incubated with rat liver microsomes in the
presence and absence of NADPH, and the formation of M5 was
shown to be NADPH dependent (Table 1). This suggested that the
formation was oxidative and led to an investigation of the enzymes
and mechanism responsible for metabolite formation.
Enzymes in RLMs Responsible for M5 Formation. Of the 15
recombinant rat P450 enzymes studied (CYP1A1, 1A2, 2A1, 2A2,
2B1, 2C6, 2C11, 2C12, 2C13, 2D1, 2D2, 3A1, 3A2, 2J3, and 2J4),
only four (CYP1A1, CYP1A2, CYP3A1, and CYP3A2) catalyzed the
formation of M5 at appreciable levels (data not shown). Kinetic
studies were conducted in male rat liver microsomes as well as
recombinant rat CYP1A1, CYP1A2, CYP3A1, and CYP3A2 to de-
termine the Km and Vmax (Fig. 5). Although the metabolic formation
of M5 presumably involves two biotransformation steps, it was as-
Labeled oxygen experiment samples were injected onto an Allure PFP
Propyl column (250 ϫ 4.6 mm, 5 m) (Restek, Bellefonte, PA). The mobile
phase composition and flow rate were the same as those described above. The sumed that one step was rate limiting and the other step was rapid